Characterization of Xylan Degradation Systems in Streptomyces ©

Khalil Thompson

A Thesis Submitted in Partial Fulfillment

of the Requirements for the Degree of

Master of Science

in

The Faculty of Science

Applied Bioscience

University of Ontario Institute of Technology

July 2012

© Khalil Thompson, 2012

CERTIFICATE OF APPROVAL

Submitted by Khalil Thompson

In partial fulfillment of the requirements for the degree of

Master of Science in Applied Bioscience

Date of Defence: 2012/07/10

Thesis title: Characterization of Xylan Degradation Systems in Streptomyces

The undersigned certify that the student has presented his thesis, that the thesis is acceptable in form and content and that a satisfactory knowledge of the field covered by the thesis was demonstrated by the candidate through an oral examination. They recommend this thesis to the Office of Graduate Studies for acceptance. Examining Committee: __________________________________ Dr. Jean-Paul Desaulniers Chair of Examining Committee __________________________________ Dr. Julia Green-Johnson External Examiner __________________________________ Dr. Janice Strap Research Supervisor __________________________________ Dr. Dario Bonetta Examining Committee Member __________________________________ Dr. Ayush Kumar Examining Committee Member

As research supervisor for the above student, I read and approved the changes required by the final examiners and recommend the thesis for acceptance: __________________________________ Dr. Janice Strap

iii

Abstract

Plant biomass serves as a carbon and energy source for Streptomyces spp. which

secrete degradative enzymes capable of breaking down the complex plant biomass into

simple saccharides. Hemicellulose is a major component of plants and is composed of

five and six carbon sugars, such as xylose and glucose. Enzymatic degradation of

hemicellulose to obtain desired sugars has been a cornerstone of many industries, as well

as the subject of worldwide research for additional sources of efficient enzymes for

substrate conversion. In this study, environmentally-derived Streptomyces isolates were

screened for their ability to hydrolyze oat-spelt and birchwood xylan in agar-based high

throughput activity screens. Of the isolates tested, eight displayed high levels of

substrate-degrading activity and were chosen for further characterization which included

16S rRNA gene analysis, microscopic analysis from both liquid and agar grown cultures,

xylanase-specific activity, lignin peroxidase production and indole acetic acid

production.Qualitative assessment of extracellular lactone signalling for all eight isolates

was also performed. Putative lactone signalling was observed for Streptomyces isolates

JLS1-C4, JLS1-A6, JLS2-D6 and KT1-B1 which exhibited xylanase-specific activities of

0.622 µmol/min/mg, 0.0243 µmol/min/mg, 0.721 µmol/min/mg, and 0.706 µmol/min/mg

respectively. Streptomyces isolates JLS1-F12 and JLS1-C12 did not exhibit lactone

signalling but did exhibit xylanase-specific activities of 0.125 µmol/min/mg and 0.0688

µmol/min/mg respectively. No xylanase-specific activity was detected for isolates JLS2-

C7 and KT1-B8; however lactone signalling was observed for isolate KT1-B8.

Streptomyces isolate JLS1-A6 degraded birchwood xylan optimally at pH 4 and 28°C

with a maximal xylanase activity of 1.56 x10-3 µmol/min/mg.

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Keywords: xylanase, Streptomyces, xylan, actinomycete, xylose

v

Acknowledgements

I would like to thank Dr. Janice Strap for her guidance, support, caring and

encouragement over the course of my research. Working under the supervision

Dr. Strap has been a privilege to which I would like to extend my deepest

gratitude. Dr. Strap has taught me to believe in myself and I stand now forever

changed by her confidence in my abilities. Strap lab will forever be a home away

from home. I am very grateful to my committee members Dr. Dario Bonetta, Dr.

Ayush Kumar and my external Dr. Julia Green – Johnson for providing me with

considerable guidance, understanding and inspiration during my research. I would

also like to extend my gratitude and acknowledgment to my fellow colleagues for

their support, and understanding such as: Andrew Latos, Sandy Clark and Osama

Qureshi.

Finally I would like to thank the Faculty of Science for allowing me to

pursue my dreams and strive for my future.

vi

Table of Contents ABSTRACT…………………………………………………………………..……...iii

ACKNOWLEDGMENTS………………………………………………………...…..v

TABLE OF CONTENTS……………………………………….………………..…..vi

LISTS OF TABLES……………………………………………………..…………..xii

LISTS OF FIGURES……………………………………………………………......xiv

LISTS OF ABBREVIATIONS…………………..………………………………...xvii

RESEARCH AIMS…………………………………………………………..............xx

I. CHAPTER 1, INTRODUCTION

I.1. Microbiology of Streptomyces

I.1.1. Composition of Lignocellulose and Streptomyces Life Cycle...………..….1

I.1.2. Streptomyces Carbon Cycling in the Environment……………………...….3

I.2. Importance of the Classification of Xylanases from Streptomyces……………....3

I.3. Xylanase Families……………………………………………………………..….4

I.4. Xylan Degradation………………………………………………………..………5

I.5. Industrial Application of Xylanases……………………………………………...8

I.6. Peroxidase Systems in Streptomyces……………………………………………10

II. CHAPTER 2, MATERIALS AND METHODS

II.1. Materials……………………………………………………………………...…12

II.2. Bacterial Strains, Plasmids and Oligonucleotides………………………………12

II.2.1. Bacterial Strains……………………………………………………….…12

II.2.1.1. Maintenance of Bacterial Strains……………………………..….12

II.2.1.2. Spore Stock Standardization……………………………….…….14

II.2.1.3. Media Preparation and Growth Conditions……………….……..14

vii

II.3. Cultural, Phenotypic and Taxonomic Characterization of Environmental

Isolates…………………………………………………………………………..15

II.3.1. Selection of Highly Degradative Streptomyces Strains……………….…15

II.3.1.1. Qualitative Identification of Strain Degradation………………....15

II.3.1.2. Degradative Capability during Streptomyces Isolate Interaction...16

II.3.1.3. Well Diffusion………………………………………………...…18

II.3.1.4. 16S rDNA Sequencing and Analysis…………………….………20

II.3.2. Phylogenetic Analysis……………………………………………………20

II.3.3. Liquid Culture Conditions……..………………………………………...21

II.3.4. Antibiotic Susceptibility of Environmental Streptomyces Isolates………21

II.3.5. Microscopic Analysis…………….………………………………………21

II.3.5.1. Cell-to-cell Communication of Environmental Isolates…….…...22

II.3.5.1.1. Indole-3- Acetic Acid Production by Environmental

Isolates…………………………………………………………..22

II.3.5.1.2. Lactone Assay…………………………………….……...23

II.4. Characterization of Environmental Isolates……………………………………..25

II.4.1. Liquid Culture…………………………………………………………....25

II.4.2. Protein Purification and Precipitation...……………………………….....26

II.4.2.1. Protein Purification………………………………………….…...26

II.4.2.1.1. Acetone Precipitation………………………………….....26

II.4.2.1.2. Ammonium Sulfate Precipitation………………………..27

II.4.2.2. Protein Analysis………………………………………………….27

II.4.2.2.1. Quantitation by Bradford Assay…………………………27

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II.4.3. Non-Denaturing Gel Electrophoresis…………………………………….27

II.4.4. Denaturing Gel Electrophoresis………………………………………….28

II.4.5. Protein Staining…………………………………………………………..29

II.4.5.1. Coomassie Blue Staining………………………………………...30

II.5. Enzymatic Activity of Environmental Isolates………………………………….30

II.5.1. Peroxidase Activity ……………………………………………………...30

II.5.2. Xylanase Activity………………………………………………………...31

II.5.2.1. Amended Growth Assay…………………………………………31

II.5.2.2. Assay to Determine Constitutive Versus Induced Xylanase

Activities……………………………………………………………...31

II.5.2.3. Induced Xylanase Assay…………………………………………32

II.5.2.4. Quantitation of Xylan Degradation………………………………32

II.5.2.4.1. Effect of Temperature and Time on Xylanase Activity….33

II.5.2.5. Xylanase Production during Growth…………………………….33

II.6. Isolation of Nucleic Acids………………………………………………………37

II.6.1. Genomic DNA Extraction...……………………………………………...37

III. CHAPTER 3, RESULTS…………………………………………………………….40

III.1. Phenotypic and Taxonomic Characterization of Environmental

Streptomyces Isolates……..……………………………………………………..40

III.1.1. Phylogenetic Determination………………………………………….…..40

III.1.2. Microscopy………………………………………………………………40

III.1.3. Antibiotic Susceptibility of the Environmental Streptomyces Isolates…..40

III.1.4. Quorum Sensing…………………………………………….……………45

ix

III.1.4.1. Cross Hatch Assessment for Enhanced Growth of Isolates.……..54

III.1.4.2. The Effects of Streptomyces Extracellular Supernatant on

Neighbouring Isolates…………………………………….…………..68

III.1.4.3. Extracellular Signalling and the Effect on Xylan Degradation by

Streptomyces Isolates…………………………………………………72

III.1.5. Extracellular Protein Analysis of Streptomyces Isolates using PAGE…..75

III.1.6. Assessment of Lignin Peroxidase using B-dye Decolourization Assay...77

III.1.6.1. Enzymatic Activity………………………………………………81

III.1.6.2. Constitutive or Inducible Nature of the Xylanase Enzymes of

Streptomyces Isolate JLS1-A6………………………………………..82

III.1.6.3. Temperature and pH Effects on Xylanase Activity……….……..85

IV. CHAPTER 4, DISCUSSION………………………………………………………...96

V. CONCLUSION…………………………………………………………………….110

VI. FUTURE DIRECTIONS…………………………………………………………...111

VII. APPENDIX…………………………………………………………………………112

VII.1. INTRODUCTION……………………………………………………...112

VII.1.1. Transposon Mutagenesis………………………………………..112

VII.2. METHODS..……………………………………………………………113

VII.2.1. Maintenance of Bacterial Strains……………….………………113

VII.2.2. Assessment of Mutagenized Streptomyces Environmental Isolate

JLS1-A6……………………………………………………………….......113

VII.2.3. Growth Media and Storage Conditions………………………....113

VII.2.4. Antibiotic Resistance of Environmental Isolates……………….114

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VII.2.5. Plasmid DNA Extraction……………………………….………114

VII.2.6. DNA Transformation…………………………………….……..115

VII.2.6.1. Culture Conditions……………………………………….……..115

VII.2.6.2. Electrocompetent Cell Preparation………………………….….115

VII.2.6.3. Electroporation…………………………………………….……115

VII.2.7. Sucrose Utilization Assay of Putative Tn Mutants……………..116

VII.2.8. Enhanced Degradation Capability Assay………………………119

VII.2.9. Enhanced Mutant Degradation…………………………………119

VII.2.10. DNA Analysis…………………………………………………..119

VII.2.10.1. Amplification of Gentamycin Resistance Cassette in pBT20.….119

VII.2.10.2. Restriction Digest……………………………………………….120

VII.2.10.3. Locating the Inserted Transposon……………………………....120

VII.2.10.4. PCR Optimization………………………………………………121

VII.2.10.5. Semi-random PCR……………………………………….….….121

VII.2.11. Bi-parental Mating (Conjugation)………………………………125

VII.3. RESULTS……………………..………………………………..……....126

VII.3.1. Quality Control of Streptomyces Isolate JLS1-A6……………..126

VII.3.2. PCR Amplification of the pBT20 Transposon…………………126

VII.3.3. pBT20 Extraction…………………………………….…………126

VII.3.4. Putative Mutant Patch Plating……………………………….…132

VII.3.5. Enzymatic Activity of Enhanced Degradation Mutants…….….132

VII.3.6. Putative Transposon Mutant Screening………………………...133

VII.3.7. Sucrose Utilization Assay of Putative Tn Mutants………….….133

xi

VII.3.8. Locating the Inserted Transposon………………………………134

VII.4. DISCUSSION………………..…………………………………………135

VII.5. CONCLUSION………………………………………………..…….….137

VIII. REFERENCES……………………………………………………………………..138

xii

LIST OF TABLES

Table II.1 Source and Characteristics of eight Streptomyces isolates of interest…….….13

Table III.1 16S rDNA Analysis of Streptomyces isolates used in this study……..……..42

Table III.2 Antibiotic susceptibility assessment of Streptomyces isolates when plated on

LB agar plates supplemented with gentamycin, kanamycin, trimethoprim and

streptomycin incubated at 28°C for a growth period of 1 week compared to LB agar

control plates without antibiotic…..…………………………………………………..46

Table III.3 Putative quorum sensing assessment of simultaneously inoculated

Streptomyces isolates in close proximity to E. coli MT102 containing plasmid

pJBA132 on LB agar observed over a 72 h period at an incubation temperature of

28°C……………………………………………………………………………….…..49

Table III.4 Putative quorum sensing of 48 h cultures of Streptomyces isolates inoculated

with E. coli MT102 containing plasmid pJBA132 on LB agar over an observation

period of 1 week at an incubation temperature of 28°C ……...…………..…………..51

Table III.5 Indole-3- acetic acid (IAA) production by Streptomyces isolates grown on

minimal media supplemented with 0.1% (w/v) birchwood xylan and 0.1% (w/v)

glucose, or grown on yeast extract-malt extract-dextrose (YEMED) medium and

YEMED supplemented with 2 mg/ml tryptophan…………..………………………...52

Table III.6 Inhibition and degradation observed by Streptomyces isolates on 0.1% (w/v)

birchwood xylan agar medium for a growth period of 10 days at 28°C in a cross-hatch

assay…………………………………………………………………………….…….57

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Table III.7 Streptomyces isolates displaying enhanced growth or degradation on a cross-

hatch assay. Cultures were grown for 10 days at 28°C on 0.1% (w/v) xylan agar

medium……..………………………………………………………………...……….64

Table III.8 Extracellular lignin peroxidase activities of eight environmental Streptomyces

isolates as determined by the Azure B dye decolourization assay……….....………...78

Table III.9 Comparative analysis of assay data for characteristics of eight Streptomyces

isolates of interest. …………………….…………………………………………..….80

Table VII.3.1. Average clearing zone sizes created by 196 putative JLS1-A6 transposon

mutants on 0.1% (w/v) birchwood xylan, cellulose and carboxymethyl

cellulose.……………………………………………………………………….….…127

Table VII.3.2 T – test statistical analysis of clearing zones created by mutants #33, 62,

102 in LB and YDA supplemented with 0.1% (w/v) birchwood xylan compared to

JLS1-A6 wild type………………………………………...……………..………….128

Table VII.3.3 Plasmids investigated for use in the transposon mutagenesis of JLS1-A6

environmental isolate.……………...………………………………………………..129

Table VII.3.4 Donor strains of E. coli utilized during transposon mutagenesis of

Streptomyces environmental isolate JLS1-A6……..……..…………………………130

Table VII.3.5 Oligonucleotide primers used in Gm cassette amplification and arbitrary

PCR of the flanking regions surrounding the pBT20 genomic insertion.…….……..131

xiv

LIST OF FIGURES

Figure I.1 Life cycle of Streptomyces on a nutritive medium………………….………….2

Figure I.2 The sequential enzymatic breakdown of the homopolymeric xylan backbone of

β-1,4-linked D-xylose units…..……………….………..……………..……..….……...7

Figure II.1 Cross hatch assay pattern layout……..……………………………….……...17

Figure II.2 Schematic representation of a paired interaction degradation assay…….......19

Figure II.3 Crude enzymatic time course experiment flow chart of Streptomyces strain

JLS1-A6 liquid culture 0.1% (w/v) xylan minimal media.………...………………....36

Figure III.1 Neighbour-Joining tree based on partial 16S rRNA gene sequence of eight

environmental Streptomyces isolates…………………..……………………..….…...41

Figure III.2 Morphology of eight environmental Streptomyces isolates on 0.1% (w/v)

birchwood xylan agar medium…...…..…………………………………………….....43

Figure III.3 Cellular morphology of the eight Streptomyces isolates used in this study...44

Figure III.4 Gamma-butyrolactone assay (GBL) of eight Streptomyces isolates………..48

Figure III.5 Wimpenny plate assay for gamma-butyrolactone signalling………….…....50

Figure III.6 Interaction assay of Streptomyces isolates……………………...…………..55

Figure III.7 Streptomyces isolates in cross hatch streaking assay……………………….56

Figure III.8 Degradation produced by the eight isolates on 0.1% (w/v) birchwood and

0.1% (w/v) oat-spelt xylan agar media……...……….………………………………..65

Figure III.9 Degradation of 0.1% (w/v) birchwood xylan by Streptomyces isolates…….66

Figure III.10 Streptomyces isolate growth inhibition……………………………...…….69

Figure III.11 Pair morphology study of Streptomyces isolates…………………………..70

xv

Figure III.12 The effects of Streptomyces extracellular supernatant on neighbouring

isolates………………………………………………………………….…………….71

Figure III.13 Assessment of putative extracellular signalling molecule characteristics

from Streptomyces isolates…………..………....……………………………….….…73

Figure III.14 Distance assessment of Streptomyces isolate interactions affecting

degradation and colony growth. Streptomyces environmental isolates JLS2-D6 and

JLS2-C7 were observed for differences in growth and degradation capability in

relation to the distance between the colonies……..………………………..…………74

Figure III.15 Extracellular protein profiles of Streptomyces isolates grown in Minimal

Medium containing 0.1% (w/v) birchwood xylan………………………..….……….76

Figure III.16 Xylanase activity of partially purified extracellular Streptomyces protein..79

Figure III.17 Assessment of the inducible nature of the xylanase enzyme of Streptomyces

isolate JLS1-A6 using well diffusion...…………………………………..……..…….83

Figure III.18 Induction of xylan degradation by eight Streptomyces isolates with varying

carbon sources………………………………………………………………….……..84

Figure III.19 pH and temperature optimization for Streptomyces extracellular xylanase

degradative activity in 0.1% (w/v) birchwood xylan agar…..….………………….....87

Figure III.20 Specific-xylanase activity of Streptomyces isolate JLS1-A6 on 0.1% (w/v)

birchwood xylan substrate………………..………………………………………..….88

Figure III.21 Growth curve and protein content of Streptomyces strain JLS1-A6……....89

Figure III.22 Crude extracellular xylanase activity observed during a time course

experiment of Streptomyces isolate JLS1-A6 grown in Minimal Medium with 0.1%

(w/v) birchwood xylan and assessed using a reaction carried out at pH 4……….......90

xvi

Figure III.23 Crude extracellular xylanase activity observed during a time course

experiment of Streptomyces isolate JLS1-A6 grown in Minimal Medium with 0.1%

(w/v) birchwood xylan and assessed using a reaction carried out at pH 5………...….91

Figure III.24 Crude extracellular xylanase activity observed during a time course

experiment of Streptomyces isolate JLS1-A6 grown in Minimal Medium with 0.1%

(w/v) birchwood xylan and assessed using a reaction carried out at pH 6…....…..….92

Figure III.25 Crude extracellular xylanase activity observed during a time course

experiment of Streptomyces isolate JLS1-A6 grown in Minimal Medium with 0.1%

(w/v) birchwood xylan and assessed using a reaction carried out at pH 7………..…..93

Figure III.26 Crude extracellular xylanase activity observed during a time course

experiment of Streptomyces isolate JLS1-A6 grown in Minimal Medium with 0.1%

(w/v) birchwood xylan and assessed using a reaction carried out at pH 8………..….94

Figure III.27 Crude extracellular xylanase activity observed during a time course

experiment of Streptomyces isolate JLS1-A6 grown in Minimal Medium with 0.1%

(w/v) birchwood xylan and assessed using a reaction carried out at pH 9……..….….95

Figure IV.1 The Lux quorum sensor…………………………………………………....105

Figure VII.2.1 Morphological assessment of Tn mutants 33, 62, 102, 70 and 81

for sporulation…………………….……………………..…………..………………117

Figure VII.2.2 Steps taken for confirmation that mutagenized strains are from JLS1-A6

strain and not contaminants………………..………….………………………..……118

Figure VII.2.3 pBT20 plasmid…………………….……………………………………123

Figure VII.2.4 Arbitrary PCR of pBT20 transposon using specific and degenerate

oligonucleotide primers……………..……………………………………………….124

xvii

LIST OF ABBREVIATIONS

Abbreviation Explanation

AHL N-acyl homoserine lactone(s)

Amp Ampicillin

BLAST Basic Local Alignment Search Tool

bp base pair(s)

BSA bovine serum albumin

CMC carboxymethyl cellulose

CTAB hexadecyltrimethylammonium bromide

CRM callus regeneration media

dH2O distilled water

DDT Dithiothreitol

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DNSA dinitrosalicylic acid

dNTPs deoxyribonucleoside triphosphate(s)

E. coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

EtOH Ethyl alcohol

F forward primer

g gravity

gDNA genomic deoxyribonucleic acid

GBL gamma-butyrolactone

GFP green fluorescent protein

Gm gentamycin

xviii

h hour(s)

IAA indole-3- acetic acid

kb kilobase(s)

kDa kilodalton(s)

L litre(s)

LiP lignin peroxidase

LB Luria Bertani

Mbps Megabase pairs

min minute(s)

mg milligram(s)

MM Minimal Medium

MW Molecular weight

MWCO molecular weight cut off

mL millilitre(s)

nm nanometer(s)

ng nanogram(s)

PAGE polyacrylamide gel electrophoresis

PCR polymerase chain reaction

PDA potato dextrose agar

PEG polyethylene glycol

PBS phosphate buffered saline

PMSF phenylmethylsulfonyl fluoride

RNase Ribonuclease

rpm revolutions per minute

rRNA ribosomal ribonucleic acid

xix

R reverse primer

SDS sodium dodecyl sulphate

sec second(s)

STE sodium chloride-Tris-EDTA, buffer

TAE tris-acetate-EDTA

TE Tris-EDTA, buffer

TEMED tetramethylethylenediamine

Tc tetracycline

Tn transposon

Tp trimethoprim

Trp tryptophan

Tris Tris (hydroxymethyl) aminomethane

µg microgram(s)

µL microlitre(s)

µM micromolar

µmol micromole

U units

YDA yeast dextrose agar

YEME yeast extract malt extract

wt weight

vol volume

xx

RESEARCH AIMS

Streptomyces a genus of the phylum Actinobacteria are ubiquitous in nature where

they are found in soil and decaying plant material. Streptomyces play a role in the

conversion of carbon-rich lignocellulosic biomass to energy and carbon (Titirici, 2007;

Benner 1987) by enzymatic hydrolysis (Hodgson et al., 2000; Tuomela et al., 2000).

Given the importance of this genus to carbon recycling in the environment, the purpose of

this study was to characterize the enzymes involved in xylan degradation by select

Streptomyces environmental isolates. To gain a better understanding of how xylan

degradation occurs, optimal conditions for xylanase activity were investigated, including

how nutrient conditions and the presence of neighbouring isolates affected degradation.

This knowledge has potential application in industrial processes such as increasing the

digestibility of feedstock, bleaching of pulp and paper, obtaining fermentable sugars for

use in biofuels and decreasing the variability in the nutritional quality of baked goods

(Marques et al., 2000; Polizeli et al., 2005; Dornez et al., 2007).

1

I. CHAPTER 1, INTRODUCTION

I.1. Microbiology of Streptomyces

I.1.1. Composition of Lignocellulose and Streptomyces Life Cycle

In the environment, plants acquire energy by photosynthesis and store this energy

in lignocellulosic polymers of cellulose, hemicellulose and lignin which comprise the cell

wall and vascular tissues (Rubin, 2008; Benner et al., 1987). Due to both inter and

intramolecular hydrogen bonding, lignin is difficult to hydrolyze (Weil et al., 1994) and

requires the coordination of multiple enzymes (Grethlein, 1984). Enzymatic hydrolysis

by Streptomyces occurs during primary metabolism beginning at the germination stage of

its life cycle (Hesketh et al., 2002). The Streptomyces life cycle begins as a unigenomic

spore. Once a nutritive substrate is found the spore will germinate causing the formation

of substrate mycelia (Figure I.1) which consists of a network of syncytial, nutrient-

gathering hyphae (Del Sol et al., 2007; Kelemen & Buttner, 1998). When nutrients are

depleted, the substrate mycelia are cannibalized to support the growth of aerial hyphae

which grow and extend upward.

The multigenomic hyphae septate into a string of unicellular compartments and

these compartments become separated by cell walls and remodelled to form unigenomic

spores (McCormick, 2009) (Figure I.1). By extracting nutrients from substrates for their

growth and converting these substrates to simpler substances Streptomyces facilitate the

decomposition of organic matter and participate in the carbon cycle.

2

Figure I.1. Life cycle of Streptomyces on a nutritive medium. The Streptomyces life cycle

begins as a unigenomic spore. Once a suitable nutritive substrate is found, germination

occurs allowing substrate mycelia to grow into the solid medium. Nutrient depletion leads

to cannibalization of substrate mycelia to provide the nutrients required for the formation

of aerial hyphae which septate into unigenomic compartments to form unigenomic

spores. Adapted from Jakimowicz, 2007.

3

I.1.2. Streptomyces Carbon Cycling in the Environment

Much of the degradation of plant cell wall-derived carbohydrates can be attributed

to Streptomyces in the environment due to mycelia-associated cellulolytic and xylanolytic

systems (Sternberg & Mandels, 1980; Henrissat et al., 1998) comprised of glucanases,

peroxidases and xylanases (Caspi et al., 2008). The endoglucanase 1,4-β-D-glucan

glucanohydrolase is responsible for the random cleavage of cellulose and cellulose

derivatives, while the exoglucanase 1,4-β-D-glucan cellobiohydrolase cleaves cellobiose

units from the non-reducing ends in cellulose. β-D-glucoside glucohydrolase, a β-

glucosidases, hydrolytically catalyzes the degradation of cellobiose and

cellooligosaccharides to glucose (Wachinger et al., 1989). The complete hydrolysis of

hemicellulose may require the activity of the aforementioned enzymes, in addition to

glycoside hydrolases of which xylanases are an example (Coughlan, 1985). Once

degradative enzymes are able to penetrate into internal xylan polymers, Streptomyces

continue to utilize xylanases to breakdown xylotriose and xylobiose units to xylose, a

simple and fermentable sugar.

I.2. Importance of the Classification of Xylanases from Streptomyces

Streptomyces degrade hemicellulose through the coordination of multiple enzymes

whose activities are controlled by partially degraded oligosaccharides and sugar

monomers (Bertrand et al., 1989).

Carbohydrate mediated regulation is due to carbon source depletion within the

environment (Ellaiah & Srinivasulu, 1996). Induction and regulation of xylanase

secretion by Streptomyces is not completely understood (Beg et al., 2001). Classification

of xylanases secreted by diverse environmental Streptomyces isolates is vital in order to

4

exploit a desired product such as xylose, and to gain a greater understanding of carbon

cycling in the environment. Classification of the mycelial associated degradation systems

of Streptomyces begins with the characterization of xylanases based on glycosyl

hydrolase families of differing specificities. These families are described below.

I.3. Xylanase Families

Xylanases which cleave the β-1-4 glycosidic linkage are found in many glycoside

hydrolase families such as 5, 7, 8, 10, 11 and 43 (Coutinho & Henrissat, 1999). In most

cases the structure of xylanases is comprised of a single or multiple catalytic domain

connected to a non–catalytic carbohydrate domain, using a linker region made from

flexible glycine and proline amino acids (Rahman et al., 2003). Xylanases cleave the β-

1,4-glycosidic linkage of both glucans and xylans including xylooligosaccharides using

an endo-type of action (Rahman et al., 2003). Although the majority of the enzymes in

these families exhibit an endo-type of action, β-D-xylosidases attack

xylooligosaccharides at the non-reducing ends (Rahman et al., 2003; Collins et al., 2005).

Glycoside hydrolase families are classified in terms of the similarity of their primary

structure, the carboxylic acid residues at their catalytic site, similar three-dimensional

folding and molecular mechanism of action (Henrissat & Bairoch, 1993). Xylanase

families can also differ in their physiochemical properties. For example, family 5 has a

diverse array of substrates and are the largest xylanase family. Notably, only eight

members of this family display a high degree of activity on xylan (Nolling et al., 2001).

Family 8 is mainly comprised of cellulases, but does contain endo-1,4-β-xylanases

while families 10 and 11 consist of predominantly endo-xylanases with

cellobiohydrolases and true xylanases respectively (Coutinho, 1999). The majority of

5

xylans belong to families 10 or 11 and function at temperatures between 35°C and 85°C

(Collins et al., 2005; Paes et al., 2012). Xylanases of glycosidic hydrolase families 10 and

11 exist as multi-domain structures which contain a catalytic domain and carbohydrate-

binding molecule (CBM). These CBMs degrade insoluble polysaccharides by appending

to glycoside hydrolases (Boraston et al., 2004).

Family 10 can cleave xylobiose, xylotriose and cellobiose glycosides at either the

β-1,4 or β-1,3-glycosidic linkages resulting in activity on short xylooligosaccharides

(Biely et al., 1993; Biely et al., 1997; van Tilbeurgh, 1985). It is characteristic of family

10 xylanases to have four to five substrate binding sites, high molecular mass and low

isoelectric point (pI) (Biely et al., 1993; Derewenda et al., 1994).

Family 11 are known as true xylanases because they can only act on substrates

which contain D-xylose. Due to the fact that they cannot cleave β-1,3-glycosidic

linkages, they usually result in large xylooligosaccharide hydrolysis products (Biely et

al., 1997). There are two methods by which enzymatic hydrolysis occurs: either by

double displacement, which results in retention of an anomeric configuration; or a direct

displacement; which results in the inversion of the anomeric configuration (Withers,

2001). Families 7, 8, 10 and 11 use the former, making double displacement the main

method of hydrolysis, while xylanase families 5 and 43 use the latter (Jeffries, 1996). In

Streptomyces coelicolor, whose genome has been sequenced (Bentley et al., 2002), the

majority of enzymes involved in primary metabolism are linked to glycolysis (Hesketh et

al., 2002).

6

I.4. Xylan Degradation

Xylan is a main component of hemicellulose where it is cross-linked to lignin using

diferulate bridges (Mackenzie et al., 1987; Murkwalder, 1976). It contains acetyl, α-

arabinofuranosyl and 4-O-methyl-D-glucuronosyl residues joined to a β-1,4-linked xylan

backbone, (Zhou et al., 2008). Streptomyces-derived xylanases can be used to improve

the digestibility of feedstock, nutritional quality of baked goods, deinking recycled paper,

and biofuels (Vazquez et al., 2000; Dornez et al., 2007; Marques et al., 2003; Polizeli et

al., 2005). There are many types of xylan, such as birchwood and oat-spelt xylan used in

this study, containing different degrees of substitution and acetylation which dictates the

enzymatic hydrolysis patterns observed during degradation (Dekker & Richards, 1975).

Oat-spelt xylan is more substituted containing 75% xylose, 10% arabinose and 15%

glucose while birchwood xylan is less substituted containing greater than 90% xylose

(Chandra & Chandra, 1996). The coordinated activity between enzymes, whether acting

in an endo, exo or oligosaccharide-specific mode of action during lignocellulosic

degradation, is common amongst many microbes including Streptomyces (Tomme et al.,

1995).

Streptomyces can secrete xylanases that enzymatically break down lignocellulosic

biomass resulting in xylobiose and xylose hydrolysis products. Coordinated multi-

enzyme degradation has been observed in thermoalkaliphilic Streptomyces sp. PC22 by

way of α-L-arabinofuranosidase, a xylan debranching enzyme, interacting with

endoxylanase (Raweesri et al., 2008).

7

Figure I.2. The sequential enzymatic breakdown of the homopolymeric xylan backbone

of β-1,4-linked D-xylose units. Xylanase enzymes α-L-arabinosfuranosidase, xylanase

and β-xylosidase cleave the xylan backbone before backbone hydrolysis by endo-1,4-β-

xylanase. Adapted from Zhou et al., 2008.

8

Reactions which occur during xylan degradation are often sequential in that the reactions

work more efficiently with co-treatments of enzymes with differing specificities due to

the enzymatic product of one reaction serving as a substrate for another enzyme. Such is

the case with the enzymes α-L-arabinosfuranosidase, xylanase and β-xylosidase which

work together to sequentially cleave the branch chains off the xylan backbone before

backbone hydrolysis is performed (Rahman et al., 2003). For example, Raweesri et al.

(2008) reported a 1.25 fold rate increase of substrate hydrolysis and release of reducing

sugars from oat-spelt xylan by Streptomyces sp. PC22 could be achieved through the

simultaneous presence of the xylan backbone-degrading enzymes xylanase and β-

xylosidase, preceded by the simultaneous presence of debranching enzymes acetyl

esterase and α-L-arabinosidase. The increased rate of hydrolysis was not observed in the

absence of any of these enzymes suggesting that a coordinated effort was required for

efficient degradation.

A reason for lignocellulosic hydrolysis resistance is the large amounts of

acetylation present within hemicellulose. Cooperation of acetyl esterase, an enzyme

which liberates acetyl groups from acetylated polysaccharides, with xylanase and β-

xylosidase during the hydrolysis of birchwood xylan has also been documented

(Chungool, 2008) (Figure I.2).

Effective hydrolysis of hemicellulose is dependent on the coordination of glycosyl

hydrolase families 10 and 11 (Biely et al., 1997).

I.5. Industrial Application of Xylanases

In the last thirty years, the use of xylanases in large scale industrial applications has

increased dramatically (Poutanen, 1987). The extensive use of xylanases in the

9

modification of animal feed (Vasquez et al., 2000), baking (Dornez et al., 2007), and pulp

and paper industries (Marques et al., 2000) has warranted further investigation into

different sources of xylanases.

There are many advantages to using xylanases, including the reduction of large

amounts of harsh acids which persist in the environment as industrial wastes (Bajpai &

Bajpai, 1996). The hydrolysis of lignin is limited in large scale production due to the

expense of acidic pretreatments, such as that for chlorine bleaching in pulp and paper

industries (Viikari, 1994). The benefits of xylanases in many industrial mills includes

reduced effluent released into the environment from excessive bleaching, a decrease in

production cost due to less bleach being utilized, an increased capacity for production and

the ease with which xylanases can be incorporated into the sequence of pulp bleaching

(Bajpai, 1999).

The genus Streptomyces is the most prolific and xylanolytic of the actinomycetes

(Li, 2009), and therefore may be useful for industrial applications. Currently used enzyme

preparation methods used in the pulp and paper industry involve the growth of the

bacteria for several days in an agitated fermentation vessel which maintains the optimal

pH, oxygen and temperature conditions required for growth (Bajpai, 1999). Extracellular

enzymes are separated from the living cell mass with the remaining liquid being

packaged and sent to pulp mills (Bajpai, 1999).

Streptomyces also secrete lignin peroxidase which degrades lignin increasing access

to xylan-derived saccharides. There is a lack of knowledge pertaining to the use

peroxidases by Streptomyces able to degrade lignocelluloses due to the fact that

extracellular peroxidases have only been partially characterized in Streptomyces

10

(Ramachandra et al., 1988). Fungal sources of lignin peroxidases, including

Phanerochaete chrysosporium, have been extensively studied and found to produce

lignin degrading activity as part of a secondary metabolism. The secretion of lignin

peroxidases by Phanerochaete chrysosporium requires nutrient starvation in order to

activate lignin degradation (Keyser et al., 1978). However, Streptomyces have been

shown to be good producers of lignin peroxidases as part of their primary metabolism

(Godden et al., 1992). Due to the non-specific nature of lignin peroxidase activity,

Streptomyces can serve as an alternative source of these enzymes (Godden et al., 1992;

Spiker et al., 1992; Macedo et al., 1999; Gottschalk et al., 2008). The variety of

degradative enzymes produced by diverse Streptomyces during lignocellulosic

degradation makes Streptomyces isolates suitable candidates for lignocellulosic

degradation.

I.6. Peroxidase Systems in Streptomyces

The genus Streptomyces has demonstrated extracellular lignin degrading systems

primarily aimed at breaking down the three major components of lignin which are p-

coumaryl, coniferyl and sinapyl alcohols (Demont-Caulet et al., 2010; Rubin, 2008).

These alcohols are interconnected by a variety of carbon–carbon bonds and ether

linkages. Studies of the actinomycete, Thermomonospora mesophila (McCarthy et al.,

1987), and various Streptomyces spp. (Crawford et al., 1983) have shown the release of

single-ring aromatic phenols during the degradation of lignocelluloses confirming alcohol

involvement (Crawford et al., 1983).

The function of a peroxidase enzyme is to catalyze the hydrogen peroxide-

dependent oxidation of phenolic compounds (Ramachandra et al., 1988). During these

11

reactions acid precipitatable polymeric lignin (APPL) is produced (Ramachandra et al.,

1987). While it has been reported that the lignin peroxidase enzyme of Streptomyces

viridosporus belongs to a heme family of enzymes based on its absorption spectra of 280

nm and 408 nm which is characteristic of heme proteins (Ramachandra et al., 1988),

Mason et al., (2001) countered that the observed peroxidase activity was caused by a non-

heme porphyrin protein involved in metal detoxification. Regardless of the nature of the

protein involved, lignin degradation by Streptomyces has been observed.

12

II. CHAPTER 2, MATERIALS AND METHODS

II.1. Materials

Enzymes and culture media components were purchased from Bioshop (Burlington,

ON) while agarose and PCR Clean-Up Kits were purchased from Biobasic (Missassauga,

ON). All chemical reagents were from Bioshop with the exception of birchwood xylan

and oat-spelt xylan (Sigma, Oakville, ON) unless otherwise stated.

It should be noted that during the course of this study, oat-spelt xylan was

discontinued and unavailable from any supplier. Therefore, birchwood xylan was used to

complete the study.

II.2. Bacterial Strains, Plasmids and Oligonucleotides

II.2.1. Bacterial Strains

The eight bacterial strains used in this study were isolated from bulk soil, forest

rhizosphere and decaying birchwood. The isolates were chosen for their degradative

ability (Table II.1). E. coli MT102 was used as a biosensor strain utilizing the pJBA132

plasmid (Anderson et al., 2001).

II.2.1.1. Maintenance of Bacterial Strains

For long term storage of bacterial spore stocks an equal volume of 40% (v/v)

sterile glycerol was added to 500 µL aliquots of the stock, preventing damage of the

spores from the formation of ice crystals, and transferred to a cryovial in replicate. Each

replicate of the stock was vortex-mixed and stored at -20°C.

Working plates of E. coli MT102 were cultured on LB agar supplemented with 10

µg/mL tetracycline at 37°C and stored at 4°C.

13

Table II.1. Source and Characteristics of eight Streptomyces isolates of interest.

Isolate Source of Origin

Physiological Characteristics Spore Colour

Mycelia Colour

Odour Colony Size

JLS1-A6 Bulk soil Brown Beige Dirt odour

Small

JLS1-C4 Bulk soil Grey White or grey colour

Dirt odour

Small

JLS1-F12 Bulk soil Beige Grey or light beige colour

Dirt odour

Small

JLS1-C12 Bulk soil Brown Beige or green colour

No odour Small

JLS2-C7 Forest rhizosphere soil

Dark brown

Beige No odour Large

JLS2-D6 Forest rhizosphere soil

Green or grey

Green or grey

Dirt odour

Medium

KT1-B1 Decaying Birch

Dark brown

Grey Dirt odour

Large

KT1-B8 Decaying Birch

Black or dark green

Light grey Dirt odour

Small

14

II.2.1.2. Spore Stock Standardization

Sporulating isolates were maintained as frozen glycerol spore stocks. For the

preparation of spore stocks, sporulating actinomycete colonies were separately excised

from agar plates and homogenized with a sterile pestle in 1.5 mL microfuge tubes

containing 300 µL of 0.85% saline. The homogenized samples were plated in 100 µL

aliquots onto oatmeal agar plates and incubated at 28°C until well sporulated. Spores

were scraped from the agar surface and aseptically transferred into a 50 mL screw-capped

tube containing 2-5 mL of sterile water. The mixture was vortex-mixed for 5 min at 3000

rpm (Sorvall) to disconnect mycelia from the spores. This mixture was filtered through

sterile cotton to separate the mycelia from the spores. The resulting purified spore

suspension was centrifuged at 3000 rpm at 4°C (Sorvall) for 15 min. The supernatant was

discarded and the pellet was resuspended in an equal volume of sterile 40% glycerol and

stored at -20°C. Spore stocks of individual isolates were dispensed in aliquots in

duplicate 96-well plates. The working plate was kept at -20°C while the archival plate

was kept at -80°C (Thermo Electrocorp). Spores were quantified using a hemocytometer.

II.2.1.3. Media Preparation and Growth Conditions

Oatmeal Agar was prepared by adding 60 g of ground oatmeal (BeachNut brand,

Superstore, ON) to a 2.0 L flask. A 1.0 L volume of 18Ω distilled H2O was added in 200

mL increments and mixed vigorously until smooth. Finally, 15 g of agar (food grade;

C.L.T Intertrade Co. LTD) was added while swirling the flask. The medium was then

autoclaved (Steris) for 90 minutes and placed in a 55°C water bath to temper before

pouring plates. All remaining growth media were autoclaved (Steris) at 121°C for 20 min

15

prior to use. During instances where agar plates were supplemented with antibiotics all

antibiotics were added after the agar media had tempered in a 55°C water bath.

Yeast Extract Malt Extract (YEME) agar plates were used as a growth medium

during morphological characterization of the eight environmentally isolated Streptomyces

strains. One litre of YEME contains 4 g yeast extract, 10 g malt extract, 8 g glucose, and

was adjusted to pH 7 at room temperature.

Potato dextrose agar (PDA) plates were used as a growth medium during

morphological characterization of the eight environmentally isolated Streptomyces

strains. One litre contains 24 g of Potato Dextrose Broth and 15 g agar adjusted to pH 7 at

room temperature.

Yeast Dextrose Agar (YDA) plates were used to assess the morphological

characteristics of each strain, as well as to culture strains for spore stock production of all

environmental isolates. One litre contains 1 g Yeast Extract and 3 g dextrose pH adjusted

to pH 7 at room temperature.

II.3. Cultural, Phenotypic and Taxonomic Characterization of Environmental

Isolates

II.3.1. Selection of Highly Degradative Streptomyces Strains

II.3.1.1. Qualitative Identification of Strain Degradation

To determine whether the select environmental streptomycete isolates could

interact with one another causing changes in substrate degradation capability, a cross

hatch streaking assay was performed on a variety of agar media each containing one of

three substrates: 1% (w/v) carboxymethyl cellulose (CMC), 1% (w/v) cellulose or 1%

(w/v) birchwood xylan. CMC is a highly soluble, synthetic derivative of cellulose. The

16

assay was set up as follows: near the long edge of an omni plate containing one of the

three media, 1.0 x 104 spores from each of eleven selected Streptomyces strains was

spotted in a row 1 cm apart. The procedure was repeated on the opposite side of the plate.

Each spot was streaked across the plate to its analogous spot in order to create a line

across the plate and then streaked back to the original spot to create a line of spores which

was of similar concentration throughout. Subsequently ~6 spots of 1 x 104 spores of

differing Streptomyces strains were spotted along either side of the short edges and

streaked across the plate. This formed the cross hatching pattern across 6 test plates to

test for interaction between Streptomyces isolates (Figure II.1). All observable

physiological characteristics of interactions from each strain combination, such as

cooperation, antagonism, pigment release, and morphology, were recorded (Table III.6).

Based on the results of this assay eight isolates displaying a positive or negative

interaction with an intersecting strain while still possessing a high degree of substrate

degradation were selected for further investigation.

II.3.1.2. Degradation Capability during Streptomyces Isolate

Interaction

An in vitro assay was performed to confirm increased or decreased oat-spelt and

birchwood xylan degradation of the eight selected isolates. Paired combinations of

isolates were spotted using 1.0 x 104 spores from spore stocks onto a large petri dish (150

x 15 mm) containing 0.1% (w/v) oat-spelt xylan medium and incubated at 28°C for 3

days.

17

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18

The xylan medium was selected due to the greatest number of observed interactions

occurring on xylan medium during the cross hatch assay and since the focus of this study

was xylan degradation. Degradation zones were visualized by staining with Gram’s

Iodine (Kasana et al., 2008) (Figure II.2).

The growth and degradation capability of individual strains were tested by

individually spotting 1 x 104 spores of each isolate onto the center of eight individual

petri dishes containing 0.1% (w/v) birchwood xylan medium and 0.1% (w/v) oat-spelt

xylan medium. The plates were incubated at 28°C for 3 days and degradation zones were

visualized as described above. The distance from the center of the colony to outer edge of

the degradation zone was measured and recorded. Measurements were statistically

analyzed using a one-way ANOVA.

II.3.1.3. Well Diffusion

In order to confirm the presence of enzymatic activity in culture supernatants, a

well diffusion assay was performed using 0.1% (w/v) birchwood xylan agar in small petri

dishes (60 mm) in which 0.5 mm wells had been excised. Aliquots equalling 45 µg of

partially purified protein from each strain were placed within each well and incubated for

48 h. Degradation was observed as clear zones surrounding the well upon staining with

Gram’s Iodine.

19

Figure II.2 Schematic representation of a paired interaction degradation assay. Paired

combinations of eight degradative isolates were organized to enhance degradation

potential of each strain. Aliquots of 1.0 x 104 spores were pipetted at a distance of 0.8

cm from each other.

20

II.3.1.4. 16S rDNA Sequencing and Analysis

The 16S rRNA gene sequence of each of the eight Streptomyces isolates was

determined by polymerase chain reaction using universal bacterial primers 27F (5’-

AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-

TACGGYTACCTTGTTACGACTT-3’) (Aljanabi et al., 1997) where M = A or C and Y

= C or T. Internal primers used for sequencing were 338F (5'-

ACTGAGACACGGTCCAGAC-3’) and 907R (5’CGTCHATTCMTTTRAGTTT-3’)

where H = A, C or T (Lane et al., 1991; Stackebrandt & Liesack, 1993). The PCR

reaction conditions were as follows: 1 cycle (denaturing at 94°C, 1 min), 30 cycles

(denaturing at 94°C, 30 sec; annealing at 55°C, 30 sec; extension at 72°C, 30 sec), and 1

cycle of 72°C for 5 min followed by a hold step at 4°C. PCR amplified 16S rRNA gene

samples were diluted 1:50 (v/v) with sterile H2O for sequencing which was performed at

TCAG (Sick Kids Hospital, Toronto) with 3-5pmol of the appropriate primers (27F,

338F, 907R, or 1492R). Contiguous sequences were assembled using CLC DNA

Workbench 5.1 (CLCbios A/S). The contigs were analyzed using BLASTN

(http://blast.ncbi.nlm.nih.gov/Blast.cgi; Altschul, 1990) to compare the sequences of the

environmental isolates to known sequences in the database.

II.3.2. Phylogenetic Analysis

To determine the phylogenetic relationship of the eight environmental isolates

chosen for this study, the partial 16S rRNA gene sequences obtained by PCR were

aligned with their closest database matches as determined by BLASTn using SDSC

Microbiology Workbench (http://workbench.sdsc.edu/) (Subramaniam, 1998). The

sequences were aligned using CLUSTALW then assembled into a unrooted phylogenetic

21

tree using the Neighbour-Joining method, (Saitou & Nei, 1987). The phylogenetic tree

was visualized using NJ Plot (Perriere & Gouy, 1996) and Tree View software

(http://taxonomy.zoology.gla.ac.uk/rod/rod.html).

II.3.3. Liquid Culture Conditions

The eight environmental Streptomyces isolates were inoculated into 25 mL cultures

of Yeast Extract Malt Extract Dextrose (YEMED) broth, Minimal Medium (1 g K2HPO4,

1 g NaCl, 2 g (NH4)2SO4, 2 g CaCO3 and 1L dH2O) supplemented with 0.1% (w/v)

birchwood xylan or Minimal Medium supplemented with 0.1 % (w/v) glucose. Cultures

were inoculated with 1 x 104 spores per mL and incubated at 30°C with shaking 150 rpm

for 5 days.

II.3.4. Antibiotic Susceptibility of Environmental Streptomyces Isolates

The susceptibility of Streptomyces environmental isolates was assessed by plating

isolates JLS2-C7, JLS1-C4, JLS1-F12, JLS1-C12, KT1-B1, KT1-B8, JLS2-D6 and JLS1-

A6 on LB agar medium plates individually supplemented with gentamycin (50 µg/mL),

kanamycin (40 µg/mL), trimethoprim (100 µg/mL and 150 µg/mL) and streptomycin (5

µg/mL). Plates were incubated at 28°C for a growth period of 1 week and compared to

LB agar control plates without antibiotic. LB was chosen for susceptibility assays since

the streptomycete isolates were to be co-cultured with E. coli MT102 for lactone

signalling assays (see below). E. coli does not grow well on typical streptomycete

cultivation media.

II.3.5. Microscopic Analysis

Gram staining was used to confirm the eight Streptomyces isolates as gram

positive and to visualize their microscopic cell morphology. Prior to staining, the eight

22

isolates were cultured in 0.1% (w/v) birchwood xylan broth and agar medium at 28°C for

7 days. Bacterial smears of all eight environmental isolates from the 0.1% (w/v)

birchwood xylan agar media cultures were prepared by dispensing 25 µL of dH2O onto a

microscope slide and aseptically transferring the Streptomyces to the droplet using a

flamed loop. Bacterial smears of all eight environmental isolates from the 0.1% (w/v)

birchwood xylan broth cultures were prepared by dispensing 25 µL of each culture

aseptically onto a microscope slide. The Streptomyces were fixed to each slide by passing

the slide through a flame and placing the fixed slide in a crystal violet staining solution

for 20 sec to allow the crystal violet to penetrate the cell wall. The slide was rinsed with

dH2O and blotted to remove excess water. The slide was then placed in Gram’s iodine

solution for 1 min, rinsed briefly with dH2O, flooded with ethanol and rinsed again with

dH2O. Submerging the slide in Gram’s iodine allows the formation of a crystal violet -

iodine complex while decolourizing the cell with ethanol causes the cell to dehydrate

trapping the complex within the peptidoglycan layer (Davies et al., 1983; Kreig & Holt,

1984; Beveridge, 1999). The slide was placed in safarin red solution for 20 sec and rinsed

with dH2O to stain any gram negative cells. Slides were visualized under oil immersion at

1000X magnification using a compound microscope (Leica).

II.3.5.1. Cell-to-cell Communication of Environmental Isolates

II.3.5.1.1. Indole-3-Acetic Acid Production by Environmental

Isolates

Indole-3-acetic acid (IAA) is a plant-derived auxin that induces growth within the

tips of roots and shoots during plant development (Leveau & Lindow, 2005). Some

Streptomyces species secrete IAA during their growth and differentiation which has been

23

implicated as a mechanism of plant growth promotion (Manulis et al., 1994). In order to

assess whether the eight Streptomyces isolates used in this study produced indole-3-acetic

acid, each strain was subjected to a colourometric assay utilizing Salkowski’s reagent

(Glickmann & Dessaux, 1995). Minimal growth medium supplemented with 2 mg/mL of

L-tryptophan was used to cultivate the eight Streptomyces isolates for 5 days to be used

for auxin assessment. One litre of Minimal Medium contained 1 g K2HPO4, 1 g NaCl, 2 g

(NH4)2SO4, 2 g CaCO3 and 1 L dH2O. A volume of 150 µL of culture supernatant was

dispensed into replicate wells of a 96-well plate. To each well 100 μL of Salkowski’s

reagent was added and colour development was allowed to proceed for 15 min. The

presence of indole-3-acetic acid in Salkowski’s reagent causes a colour change from clear

to pink. Salkowski’s reagent is prepared as follows: 150 mL concentrated H2SO4 added to

5 mL 0.5 M FeCl·6H2O in 250 mL dH2O. The colour intensity was measured at 530 nm

using a microplate reader (Biorad XMark Microplate Spectrophotometer). A 96-well

plate was set up using 150 µL volumes of IAA standard with concentrations ranging from

0, 5, 10, 20, 30, 40, 50 µg/mL. Each standard was aliquoted in triplicate from a 5 mg/mL

IAA stock solution which was dissolved in a small volume of sterile 0.2 N NaOH and

brought up to volume with dH2O. A standard curve was used to obtain the IAA

equivalents (µg/ mL) of each isolate. YEMED and Minimal Medium were used for

comparison of IAA production both with and without L-tryptophan.

II.3.5.1.2. Lactone Assay

Lactones are signalling molecules which possess intramolecular ester bonds and are

formed from the condensation reaction of a carboxylic acid and an alcohol (Kataoka et

al., 2007). Gamma-butyrolactone molecules are secreted into the surrounding

24

environment by Streptomyces as a means of communicating population density between

strains (Horinouchi & Beppu, 1994). To validate the presence of lactones, all eight

Streptomyces isolates were streaked in the presence of E. coli MT102 harbouring the

pJBA132 plasmid which was used as an intracellular biosensor expressing a green

fluorescent protein in the presence of lactones (Anderson, 2001). All eight strains of

Streptomyces isolates were inoculated on LB medium supplemented with tetracycline to a

final concentration of 10 µg/mL in triplicate. Each strain was streaked horizontally across

the plate while the E. coli MT102 was streaked vertically across the plate, without

touching the streaked Streptomyces. Two assays were performed; the first in which

samples were inoculated simultaneously with E. coli MT102 and the second in which

mature, 48 h cultures of the Streptomyces environmental isolates were inoculated with E.

coli MT102. Streptomyces isolates were incubated for approximately seven days until the

presence or absence of fluorescence by the E. coli MT102 biosensor was confirmed in all

three replicates of each isolate. Photographs were taken on a Dark Reader (DR46B

Transilluminator) using Canon Rebel T1i camera.

To measure the concentration effect of 0 to 50% supernatant on fluorescence,

Wimpenny plating (Wimpenny, 1979) was used to prepare LB slants consisting of 6.25

mL LB and 6.25 mL of filtered culture supernatant from the eight strains of

environmentally isolated Streptomyces. The Wimpenny plates were overlaid with LB

supplemented with 10 µg/mL tetracycline. The overlay was streaked with E. coli MT102

containing plasmid pJBA132. Plates were incubated and grown for 20 days at 28°C until

fluorescence was visible in the overlay. Fluorescence was photographed on a Dark

Reader (DR46B Transilluminator) with a Cannon Rebel T1i camera.

25

To observe whether the colony size and enzymatic activity of Streptomyces

environmental isolates were affected by changes in the distance between isolates, a

distance assay was performed. Distances of 1 cm, 2 cm and 3 cm between adjacent JLS2-

C7 and JLS2-D6 strains spotted on 0.1% (w/v) birchwood xylan agar were tested.

Changes in colony size or enzymatic degradation observed by altering the distances

between colonies would indicate the exchange of proteins or molecules between isolates

and could validate gamma-butyrolactone signalling as a possible cause. Streptomyces

strains JLS2-C7 and JLS2-D6, two strains displaying antagonistic growth, were spotted

using 2.3 × 105 spores from spore stocks in triplicate onto LB supplemented with 0.1%

(w/v) birchwood xylan at distances of 1 cm, 2 cm and 3 cm and incubated for 5 days at

28ºC.

A streak test was performed to confirm the presence of a growth inhibiting

molecule by examining the growth inhibition of JLS2-C7 by JLS2-D6. Using a T

configuration for the plating of 5.0 × 104 total spores of JLS2-D6 and JLS2-C7 were

streaked without any contact being made between the two strains.

II.4. Characterization of Environmental Isolates

II.4.1. Liquid Culture

To obtain cell mass for genomic DNA extraction, single colonies of each select

environmental isolate were aseptically transferred to 5 mL of Yeast Dextrose Broth

(YDB; Per litre: 1.0 g Yeast Extract; 3.0 g dextrose) in 50 mL Erlenmeyer flasks using a

flamed loop and incubated at 30°C for 24 h on a platform shaker at 150 rpm. A 2 mL

aliquot was subcultured to 25 mL of YDB to increase culture yield and incubated at 30°C

for 24 h on a platform shaker at 150 rpm. Once cultures had sufficient growth, cells were

26

transferred aseptically to 50 mL screw-capped tubes and centrifuged at 3000 rpm for 15

minutes at 4°C. The supernatant was decanted and stored at -20°C for use in extracellular

protein analysis while the pellet was homogenized and used for the extraction of genomic

DNA.

II.4.2. Protein Purification and Precipitation

II.4.2.1. Protein Purification

To purify extracellular protein, cultures were grown as follows: 5 mL of Minimal

Medium containing 0.1% (w/v) birchwood xylan was inoculated with 1 x 104 spores per

mL of the Streptomyces strains of interest and placed on a shaker at 150 rpm and

incubated at 30°C for 5 days. Strains which did not grow in the 0.1% (w/v) xylan

Minimal Medium were inoculated into yeast dextrose broth and subsequently incubated

for the same time period and temperature. Once growth was observed of all eight

Streptomyces isolates and verified by Gram staining under a microscope (Leica) larger

cultures of 50 mL were prepared and subcultured to 500 mL cultures in order to obtain

large volumes for protein precipitation of culture supernatants.

II.4.2.1.1. Acetone Precipitation

In order to concentrate extracellular protein for further characterization, acetone

precipitation was used on 500 mL volumes of Streptomyces liquid culture supernatant by

centrifuging for 15 min at 3000 g (Sorvall RC6 Plus). One volume of acetone was added

and the mixture was inverted several times before storing overnight at -20°C. The

precipitated protein was recovered by centrifugation at 4°C for 15 min at 3,000 rpm.

Residual acetone was evaporated and the remaining pellet of precipitated protein was

frozen at -20°C. Pellets were resuspended in 50 mM Tris at pH 8.0 and a Bradford Assay

27

(Bradford, 1976) was performed to quantify the amount of extracellular protein

recovered.

II.4.2.1.2. Ammonium Sulfate Precipitation

Extracellular protein was ammonium sulphate precipitated using an 80% saturated

solution prepared in distilled H2O. The saturated solution was stored at 4°C. Liquid

cultures of the eight Streptomyces isolates were centrifuged for 20-30 min at 4,000 rpm at

4°C (Sorvall RC6 Plus) and the pellet was discarded. The saturated solution and

extracellular culture supernatant was combined before the mixture was placed on a shaker

overnight at 4°C and centrifuged for 15 min at 8,000 rpm (Sorvall RC6 Plus). The

extracellular protein pellet was resuspended in 50 mM Tris-HCl, pH 8. A 100 mL volume

of all Streptomyces liquid cultures was dialysed in a cold room at 4°C overnight in

dialysis tubing with a 12-14000 molecular weight cut off in 2 M Tris-HCl buffer at pH 8

which was replaced once after eight hours of dialysis. After samples were purified,

phenylmethylsulfonyl fluoride (PMSF) was added to inhibit protease degradation of the

proteins.

II.4.2.2. Protein Analysis

II.4.2.2.1. Quantitation by Bradford Assay

Protein quantitation was carried out as described by Bradford, 1976.

II.4.3. Non-Denaturing Gel Electrophoresis

In order to characterize the extracellular proteins precipitated from the

environmental Streptomyces isolates protein samples were analyzed by non-denaturing

discontinuous polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1973). Protein

samples were run in duplicate against a protein ladder (Spectra BR Protein ladder) and a

28

1% xylanase standard obtained from Thermomyces lanuginosus (Sigma). The resolving

gel was prepared from 3.33 mL acrylamide stock solution, (30% acrylamide, 0.8% bis-

acrylamide), 2.5 mL separating buffer (Tris-HCl, pH 8.8), 4.17 mL H2O, 10%

ammonium persulfate and 10 µL TEMED. The stacking gel was prepared from 650 µL

acrylamide stock solution (30% acrylamide, 0.8% bis-acrylamide), 1.25 mL Tris-HCl pH

8.8, 3.05 mL H2O, 10% ammonium persulfate and 5 µL TEMED.

II.4.4. Denaturing Gel Electrophoresis

To determine the molecular weight of proteins secreted from Streptomyces

environmental isolates, a sodium dodecyl sulphate (SDS) denaturing gel (Cleveland et al.,

1977) was used. Protein samples were added to loading dye containing beta-

mercaptoethanol then boiled at 100°C using a heatblock. The separating gel was prepared

from 3.33 mL acrylamide stock solution, (30% acrylamide, 0.8% bis-acrylamide), 2.5 mL

separating buffer (Tris-HCl, pH 8.8) containing SDS, 4.17 mL H2O, 10% ammonium

sulfate and 10 µL TEMED. A stacking gel was prepared using 650 µL acrylamide stock

solution, (30% acrylamide, 0.8% bis-acrylamide), 1.25 mL separating buffer containing

SDS at pH 6.8, 3.0 mL H2O, 25 µL of 10% ammonium persulfate and 5 µL TEMED. The

samples were cooled on ice immediately after boiling and loaded into the wells of the

denaturing gel. To ensure the correct pH during the run a denaturing gel electrophoresis

buffer (20 mM Tris, 1mM glycine) was used. Samples were run against a protein ladder

(Spectra BR Protein ladder). A 1% Thermomyces lanuginosus xylanase solution was used

as a positive control.

29

II.4.5. Protein Staining

To visualize protein bands on polyacrylamide gels, silver stain was used as

described by Heukeshoven, 1985. Briefly, a prefix solution of containing 100 mL

methanol, 20 mL ethanol and 80 mL dH2O was prepared to a final volume of 200 mL.

The prefix solution was added to the slab gel and the gel was heated using microwave

heating conditions at 50% power for 1.5 min. The gel was agitated at 30 sec intervals

then agitated continuously for 2 min after heating and the pre-fix solution was decanted.

A 200 mL volume of dH2O was added to the gel and the gel was heated for 2 min in a

microwave at 50% power followed by continuous agitation for 2 min to rinse the gel. A

21 µL volume of 20 mM dithiothreitol was added to 200 mL of dH2O and the solution

was added to the gel and microwaved for 2 min at 50% power while agitating at 30 sec

intervals. After heating, the gel in solution was agitated continuously for 2 min and a 1%

solution of AgNO3 was added. The mixture was microwaved for 1.5 min at 50% power

and the gel was rinsed twice using dH2O. A 100 µL volume of formaldehyde was added

to 200 mL of developer solution containing 3% (w/v) NaCO3. The developer solution

was then immediately added to the gel in two 20 mL aliquots. The gel and developer

solution was agitated for 20 sec after which the 40 mL of developer solution was

decanted and the remaining 160 mL of developer solution was added to the gel. Once the

bands had sufficiently developed the reaction was stopped by adding 2.3 M citric acid

and the gel was rinsed with dH2O. The developed gel was stored in a solution of 0.3%

(v/v) NaCO3. Protein gels were photographed on a lightbox using a Canon Rebel T1i.

30

II.4.5.1. Coomassie Blue Staining

Coomassie Blue staining was used as an alternative method to visualize

extracellular protein of the Streptomyces environmental isolates. The gels were washed

twice with 100 mM phosphate buffer at pH 7 and subjected to Coomassie Blue staining

(0.1% Coomassie brilliant blue R-250, 50% (v/v) methanol, 10% (v/v) acetic acid)

overnight. Gels were destained with 5% acetic acid and 3% glycerol. Protein was

visualized on a lightbox and photographed using a Canon Rebel T1i.

II.5. Enzymatic Activity of Environmental Isolates

II.5.1. Peroxidase Activity

The supernatants of broth-grown environmental Streptomyces isolates were

assessed for peroxidases using the decolourization of Azure B in the presence of

hydrogen peroxide which is required to activate peroxidase enzymes. A 200 µL aliquot of

supernatant from each of the eight Streptomyces isolates of interest was collected from

Streptomyces isolates cultured in both Minimal Medium supplemented with 0.1% (w/v)

glucose and Minimal Medium supplemented with 0.1% (w/v) birchwood xylan, as well

as from purified extracellular Streptomyces protein. The 200 µL aliquots were combined

with 10 µL 3.2 M Azure B stock (4.9 mg Azure B, 5 mL of 0.05 M sodium tartrate pH

4.5 and 780 µL of 0.05 M sodium tartrate buffer (1.5 g tartaric acid diluted to a final

volume of 200 mL using H2O, pH 4.5 using NaOH)) inside a cuvette. Lastly, a 10 µL

volume of 10 mM H2O2 (51 µL of 30% H2O2, 5 mL sodium tartrate at pH 4.5) was added

to initiate the reaction. The decolourization was recorded at 651 nm in a

spectrophotometer over a total time span of 5 min at 30 sec intervals in triplicate.

31

II.5.2. Xylanase Activity

II.5.2.1. Amended Growth Assay

A study in which supernatant was supplemented into the growth medium of strains

JLS2-C7 and JLS2-D6 was performed to examine the growth inhibition observed

between these isolates. A percentage of the opposing JLS2-C7 strain supernatant, 5% =

450 µL and 25% = 2.25 mL was incorporated into the approximate 9 mL total volume of

0.1% (w/v) birchwood xylan agar growth medium within a plate. Aliquots of 2.3 × 105

spores of the opposing strain JLS2-D6 were spotted on the plate to observe colony

growth and degradation in triplicate. The procedure was repeated using JLS2-D6 strain

supernatant and JLS2-C7 spore stock and cultures were incubated for 6 days at 28°C.

II.5.2.2. Assay to Determine Constitutive Versus Induced Xylanase

Activities

To determine the whether the observed xylanase activity of Streptomyces

environmental isolate JLS1-A6 was constitutive or inducible; a well diffusion assay was

performed. To perform this assay 250 mL of three liquid media types were used to

culture Streptomyces isolate JLS1-A6: Minimal Medium supplemented with 0.1 % (w/v)

glucose, Minimal Medium supplemented with 0.1% (w/v) birchwood xylan and 0.1%

(w/v) glucose and Minimal Medium supplemented with 0.1% (w/v) birchwood xylan. A

total volume of 5 mL of each Minimal Medium solution was inoculated with 5.0 x 104

JLS1-A6 spores and cultured for 48 h at 28°C in three biological replicates. A 1 mL

aliquot of culture supernatant was removed from each culture and centrifuged at 10,000

rpm for 4 min. The supernatant was filter sterilized through a 0.2 µm filter and

transferred to a clean 1.5 mL microfuge tube. A 15 µL aliquot was dispensed into 0.5 mm

32

wells in 0.1% (w/v) birchwood xylan agar plates and incubated overnight at 28°C.

Remaining supernatant was stored at 4°C. Xylanase activity was observed as clear zones

around the wells upon staining with Gram’s Iodine solution.

II.5.2.3. Induced Xylanase Assay

To determine if the extracellular xylanases of select Streptomyces isolates could be

induced, Minimal Medium agar plates containing 0.1% (w/v) birchwood xylan were

separately amended with 0.001% (w/v) xylose, 0.001% (w/v) yeast extract, and 0.02%

(w/v) glucose. Plates were inoculated with 2.3 × 105 spores and incubated for 6 days at

28°C. Clear zones were indicative of degradation and were measured for each replicate.

Results were analyzed using a one-way ANOVA at a 95% confidence interval.

II.5.2.4. Quantitation of Xylan Degradation

Xylanase activity was determined by measuring the release of sugars from

birchwood xylan using the dinitrosalicyclic acid (DNSA) method (Miller, 1959). The

DNSA solution was prepared by dissolving 0.25 g of dinitrosalicyclic acid, 0.25 g sodium

hydroxide and 4.8 g of potassium sodium (+)- tartrate (Rochelle Salts) in a total of 25 mL

of dH2O. The assay was carried out in a microtitre plate and consisted of aliquots of 90

µL, 80 µL, 50 µL, 0 µL of 1 mg/mL xylan substrate combined with 10 µL, 20 µL, 50 µL

and 100 µL of crude enzyme and 100 µL DNSA in a microtitre plate. The microtitre plate

was heated for 15 min to 100°C to develop the colour reaction. After cooling the

microtitre plate to room temperature, the absorbance was read at 640 nm on a BioRad

XMark Microplate Spectrophotometer with xylose as a standard. One unit (U) of

xylanase activity is defined as the amount of enzyme that releases 1 µmol xylose/min/mg.

33

II.5.2.4.1. Effect of Temperature and Time on Xylanase Activity

When determining extracellular xylanase activity it was important to encompass a

range of temperatures in which xylanases function in order to determine the optimal

reaction conditions. The optimal temperature for enzyme activity was determined by

performing the standard assay procedure at temperatures of 28ºC, 37ºC and 45°C. The

optimal reaction time for xylanase activity was determined as described previously by

Ghose, 1987. An initial xylanase reaction time of 60 min was used followed by reaction

times of 30 min and 10 min using reaction volumes of 200 µL.

II.5.2.5. Xylanase Production during Growth

Crude xylanase production during growth of Streptomyces JLS1-A6 was tested at

temperatures of 28°C, 37°C, 45°C, and 60°C in buffers of pH 4, 5, 6, 7, 8, 9 for 13 days.

Streptomyces JLS1-A6 was inoculated with 2.2 x 105 spores into two separate 300 mL

culture flasks containing Minimal Medium plus 0.1% (w/v) birchwood xylan. Cultures

were sampled daily to measure protein, xylanase activity and mycelia wet weight in

triplicate starting on day 0 of the experiment. These 300 mL cultures served as biological

replicates which were sampled every day for protein quantity and xylanase-specific

activity. Streptomyces JLS1-A6 strain was tested in triplicate for protein content using a

Bradford assay (Bradford, 1976) and a Dinitrosalicylic acid (DNSA) (Miller, 1959)

assay at each temperature and pH. Protein content was used to follow growth of the

Streptomyces isolate. The DNSA assay was used to quantify the amount of reducing

sugars released by the xylanolytic activity in the culture supernatant. The xylan-specific

activity (enzymatic activity per mg of protein) was determined over the time course

34

experiment. The mycelia wet weight of JLS1-A6 was recorded for each time sampled

(Figure II.3).

Included in the xylanase reaction assay was a blank, a positive and a negative

control. The blank consisted of a range of buffers (pH 4, 5, 6, 7, 8, 9) containing crude

enzyme with no xylan substrate. The negative control consisted of uninoculated 0.1%

(w/v) birchwood xylan medium. A positive control consisting of a 1% (w/v)

Thermomyces lanuginosus xylanase solution as well as a 1% (w/v) Streptomyces

viridosporus T7A xylanase solution should have been used for comparison to

Streptomyces strains of known xylanase activity.

Xylanase activity was assayed at different pH values using the following buffer

systems aceto-acetate (pH 4, 5, 6), sodium phosphate (pH 7), and tris (pH 8, 9). To assess

the growth of Streptomyces JLS1-A6 on each of the 13 days a 600 µL aliquot was

removed from each replicate flask and dispensed into a 1.5 mL microfuge tube. The

sample was divided into three 200 µL aliquots in preweighed 1.5 mL microfuge tubes.

All three tubes were centrifuged at 10,000 g for 2 min to pellet the mycelia. The

supernatant, (~200 µL), from each sample was assayed by the Bradford method

(Bradford, 1976) by transferring the supernatant into new 1.5 mL microfuge tubes. A 600

µL volume of dH2O and a 200 µL volume of Bradford reagent were added to each of the

new tubes containing the supernatant and vortexed. A 200 µL volume from each tube was

aliquoted into a 96-well plate in triplicate and read at an absorbance of 575 nm. These

values were compared to a bovine serum albumin standard. The microfuge tubes which

contained the mycelia pellet were re-weighed and the mass was recorded.

35

The effect of temperature on the xylanolytic activity of the Streptomyces strain

JLS1-A6 was assessed at temperatures of 28°C, 37°C, 45°C, and 60°C. A 200 µL aliquot

of crude enzyme from culture supernatant of Streptomyces JLS1-A6 was added to a 1.8

mL of 0.1% (w/v) birchwood xylan substrate in sodium phosphate buffer (pH 6.5, pH 7),

aceto-acetate buffer (pH 4, 5, 6) and tris buffer (pH 8, 9) in 15 mL test tubes in triplicate.

A positive control was prepared with a 200 µL volume of 0.1% standard xylanase added

to a 1.8 mL volume of 0.1% (w/v) birchwood xylan substrate in sodium phosphate buffer

(pH 7), aceto-acetate buffer (pH 4, pH 5, pH 6) and tris buffer (pH 8, 9). A negative

control was prepared by adding 0.2 mL of uninoculated 0.1% (w/v) birchwood xylan

medium to a 1.8 mL volume of 0.1% (w/v) birchwood xylan substrate in sodium

phosphate buffer ( pH 7), aceto-acetate buffer (pH 4, pH 5, pH 6) and tris buffer (pH 8,

pH 9). A blank without 0.1% (w/v) birchwood xylan was prepared by adding 0.2 mL

crude enzyme to 1.8 mL sodium phosphate buffer (pH 7), aceto-acetate buffer (pH 4, pH

5, pH 6) and tris buffer (pH 8, pH 9). Four aliquots of 500 µL were extracted from each

pH reaction and dispensed into 1.5 mL microfuge tubes and incubated at its

corresponding temperature in a waterbath for 15 min. The reaction was stopped by the

addition of 750 µL of DNSA to each reaction tube. The tubes were boiled for 5 min,

cooled to room temperature and a 200 µL volume from each reaction tube from both

biological replicates was aliquoted in triplicate into a 96-well plate and the absorbance at

575 nm was measured with xylose as the standard. One unit (U) of xylanase activity is

defined as the amount of enzyme that releases 1 µmol xylose/min/mg.

36

Figu

re II

.3. C

rude

enz

ymat

ic ti

me

cour

se e

xper

imen

t flo

w c

hart

of S

trep

tom

yces

stra

in J

LS1-

A6

liqui

d cu

lture

0.1

% (w

/v) x

ylan

min

imal

med

ia.

37

II.6. Isolation of Nucleic Acids

II.6.1. Genomic DNA Extraction

Genomic DNA (gDNA) was extracted as described by Aljanabi & Martinez, 1997.

Briefly 50-100 mg of Streptomyces cell biomass was homogenized with a sterile pestle in

1.5 mL microfuge tubes containing 400 µL homogenizing buffer (0.4 M NaCl; 10mM

Tris-HCl, pH 8.0; 2 mM EDTA, pH 8.0). SDS was added to a final concentration of 2%,

8 µL of proteinase K (20 mg/L) was added and tubes were incubated overnight at 60°C in

a water bath. A 300 µL volume of 6 M NaCl was added to precipitate genomic DNA

which was centrifuged for 30 min to obtain a pellet. Supernatant was transferred to fresh

tubes where an equal volume of isopropanol was added and tubes were incubated at -

20°C for 1 h. Genomic DNA samples were centrifuged for 20 min, 4°C at 10,000 g.

Genomic DNA was washed with 50 µL ice cold 70% ethanol. The DNA pellet was

allowed to dry (~15 min) and resuspended in 200 µL sterile dH2O and stored at 4°C.

The quality of genomic DNA was assessed by electrophoresis on 0.85% TAE

agarose gel; with a λHind III DNA ladder. The running buffer consisted of TAE (40 mM

tris acetate, pH 8; 1mM EDTA); gels were run at 100 V for 40 minutes, stained in

ethidium bromide and visualized with UV transillumination on a gel dock (Fluorchem

SP).

Due to unsuccessful extraction of genomic DNA from a few environmental isolates

using the method described by (Aljanabi & Martinez, 1997) an alternative method

described by (Cheng & Jiang, 2006) was used. Briefly, a 1 mL volume of Streptomyces

liquid culture was centrifuged for 2 min at 8000 g. The pellet was washed twice using

400 µL of STE solution (100 mM NaCl, 10 mM Tris/HCl, 1 mM EDTA, pH 8.0) and

38

centrifuged for 2 min at 8000 g. A volume of 200 µL of Tris-EDTA buffer (TE) was

added to the pellet followed by the addition of 100 µL of a tris-saturated phenol solution.

The solution was vortexed for 2 min and centrifuged at 12,000 g for 5 min. A 160 µL

volume was extracted from the top layer and 40 µL of TE was added. Subsequently 100

µL of chloroform was added to the extracted volume and the solution was vortexed for 2

min and centrifuged for 2 min at 13,000 rpm. The chloroform extraction was performed

until no protein was present in the interface. A 40 µL volume of TE containing RNase

was added and the solution was incubated for 10 min at 37°C. Following the incubation a

100 µL volume of chloroform was added and the solution, vortexed for 2 min and

centrifuged for 5 min at 13,000 rpm. The upper layer containing the genomic DNA was

transferred to a clean 1.5 mL microfuge tube and stored at -20°C.

To successfully isolate genomic DNA from all eight environmental isolates a third

extraction method was performed (Zhou et al., 1996). Briefly 50-100 mg of Streptomyces

cell culture was pelleted by centrifugation. A 500 µL volume of extraction buffer

containing 2.5 mL of 2 M Tris-HCl, pH 8.0, 10 mL of 0.5 M EDTA, 5.0 mL of 1 M

sodium phosphate pH 8.0, 15 mL of 5 M NaCl, 10 mL of 5%

hexadecyltrimethylammonium bromide (CTAB) and 7.5 mL H2O was added to the pellet.

A 3 µL volume of proteinase K (20 mg/mL) was added and the mixture was placed on a

shaker for 30 min at 37°C. After agitation 55 µL of 20% SDS was added and the solution

was incubated for 2 h in a 65°C waterbath. The solution was mixed by inversion every 20

min. The solution was then centrifuged for 10 min at 6,000 g at room temperature.

Supernatant from the solution was transferred to a fresh 1.5 mL microfuge tube and the

pellet was extracted a second time by adding 100 µL of extraction buffer and 10 µL of

39

20% SDS and mixing the solution. The solution was incubated for 10 min at 65°C in a

waterbath and centrifuged for 10 min at 6,000 g. An equal volume of chloroform:isoamyl

alcohol (24:1 vol/vol) was added after incubation and the solution was centrifuged for 5

min at 13,000 g. The aqueous phase of the solution was transferred to a clean 1.5 mL

microfuge tube and the DNA precipitated using 0.6 vol of isopropanol for 1 h at room

temperature. The DNA was centrifuged for 20 min at 16,000 g at room temperature after

which the pellet was washed with ice cold 70% ethanol, air dried and resuspended in

dH2O. Purified DNA was stored at 4°C.

40

III. CHAPTER 3, RESULTS

III.1. Phenotypic and Taxonomic Characterization of Environmental

Streptomyces Isolates

III.1.1. Phylogenetic Determination

To determine the relatedness of the eight Streptomyces isolates used in this study,

phylogenetic analysis based on 16S rRNA gene sequence was performed (Figure III.1).

The eight isolates were observed to separate into five different clades (Figure III.1).

III.1.2. Microscopy

The cellular morphology of the eight Streptomyces isolates were observed using

light microscopy of gram-stained cultures grown in liquid and agar media containing a

0.1% (w/v) birchwood xylan. Microscopic analysis of agar-grown Streptomyces isolates

showed a filamentous mode of growth with a few of the isolates producing spores (Figure

III.3). Gram stained images of broth cultures containing birchwood xylan were

complicated by the presence of the substrate. In retrospect, a medium containing a water-

soluble carbon source would have given better results. Streptomyces isolates grown on

agar medium grew as dark-pigmented colonies (Figure III.2).

III.1.3. Antibiotic Susceptibility of the Environmental Streptomyces Isolates

Antibiotic sensitivity of the environmental Streptomyces isolates JLS2-C7, JLS1-

C4, JLS1-F12, JLS1-C12, KT1-B1, KT1-B8, JLS1-A6 and JLS2-D6 was characterized

for co-culture experiments with the lactone biosensing strain of E. coli.

41

Figu

re II

I.1. N

eigh

bour

Join

ing

tree

base

d on

par

tial 1

6S rR

NA

gen

e se

quen

ce o

f eig

ht e

nviro

nmen

tal S

trep

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isol

ates

.

Boo

tstra

p va

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are

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n at

the

node

s bas

ed o

n 10

00 re

sam

pled

dat

a se

ts. S

cale

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se c

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The

acce

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list

ed in

bra

cket

s.

42

Tabl

e II

I.1. 1

6S rD

NA

Ana

lysi

s of S

trep

tom

yces

isol

ates

use

d in

this

stud

y.

Isol

ate

ID

Acc

essi

on

Num

ber o

f cl

oses

t mat

ch

Des

crip

tion

% Id

entit

y Id

entit

y B

it Sc

ore

JLS1

-A6

FJ40

6112

St

rept

omyc

es tu

berc

idic

us

stra

in 5

US-

2

99

1491

/199

7 24

13

JLS1

-F12

FJ

1713

35

Stre

ptom

yces

chat

tano

ogen

sis s

train

L10

100

1174

/117

4 21

69

JLS2

-D6

GU

0455

39

Stre

ptom

yces

sp.

SXY

124

100

1147

/114

7 21

19

JLS2

-C7

AB

2220

71

Stre

ptom

yces

sp.

100

1423

/142

7 26

28

JLS1

-C12

FJ

2228

16

Stre

ptom

yces

sp. W

YE1

99

13

95/1

395

2567

JLS1

-C4

AB

1845

28

Stre

ptom

yces

arg

ente

olus

subs

p. T

oyon

aken

sis

99

1421

/142

7 27

81

KT1

-B1

DQ

0266

54

Stre

ptom

yces

sioy

aens

is

stra

in N

RR

L B

-540

8

99

1423

/143

2 27

67

KT1

-B8

AY

9997

94

Stre

ptom

yces

cir

ratu

s st

rain

NR

RL

B-3

250

100

878/

878

1741

43

Figu

re II

I.2. M

orph

olog

y of

eig

ht e

nviro

nmen

tal S

trep

tom

yces

isol

ates

on

0.1%

(w/v

) birc

hwoo

d xy

an a

gar m

ediu

m. I

sola

tes w

ere

grow

n fo

r 15

days

at 2

8°C

. Pho

togr

aphs

wer

e ta

ken

usin

g C

anno

n R

ebel

T1i

cam

era.

Env

ironm

enta

l iso

late

s wer

e di

stin

guis

hed

from

one

anot

her b

y th

e vi

sual

obs

erva

tion

of d

iffer

ence

s in

spor

e co

lour

, siz

e of

col

onie

s and

rate

s gro

wth

on

the

xyla

n ag

ar m

ediu

m.

44

Figu

re II

I.3. C

ellu

lar m

orph

olog

y of

the

eigh

t Str

epto

myc

es is

olat

es u

sed

in th

is st

udy.

A) S

trep

tom

yces

isol

ates

gro

wn

in M

inim

al

Med

ium

supp

lem

ente

d w

ith 0

.1%

(w/v

) birc

hwoo

d xy

lan.

B) S

trep

tom

yces

isol

ates

gro

wn

in a

gar M

inim

al M

ediu

m su

pple

men

ted

with

0.1

% (w

/v) b

irchw

ood

xyla

n. A

ll is

olat

es w

ere

grow

n fo

r 5 d

ays,

Gra

m st

aine

d an

d vi

sual

ized

und

er o

il im

mer

sion

at 1

000X

mag

nific

atio

n.

45

It was found that the eight Streptomyces isolates were susceptible to the aminoglycoside

antibiotics gentamycin, streptomycin and kanamycin, but were not susceptible to the

antimetabolite trimethoprim (Table III.2).

III.1.4. Quorum Sensing

Fluorescence produced from the pJBA132 plasmid of E. coli MT102, due to

lactone signalling, was observed from most individual Streptomyces isolates, excluding

JLS1-C12, JLS1-F12 and JLS2-C7 (Figure III.4). Fluorescence by the green fluorescent

protein (GFP) expressed from pJBA132 supports the notion that lactone communication

is occurring in isolates JLS1-A6, JLS1-C4, JLS2-D6, KT1-B1 and KT1-B8. A sequential

inoculation of the Streptomyces isolates, in which isolates were allowed to grow before

inoculation of the E. coli MT102 carrying the pJBA132, was performed initially to ensure

strong enough signalling was present to excite the GFP. Fluorescence was observed for

JLS1-A6, KT1-B8, JLS1-C4, JLS1-F12, JLS2-D6 and KT1-B1 strains producing

lactones after 168 h (Table III.4). However, when each of the eight Streptomyces strains

were inoculated on LB agar medium in combination with E. coli MT102 simultaneously,

the E. coli MT102 began to fluoresce after 48 h for JLS1-A6, KT1-B8, JLS1-C4, JLS1-

F12, JLS2-D6 and KT1-B1 strains producing lactones (Table III.3). The simultaneous

inoculation of the Streptomyces isolates and E. coli MT102 was used to examine the

initiation of lactone signalling over time.

46

Tabl

e II

I.2.A

ntib

iotic

susc

eptib

ility

ass

essm

ent o

f Str

epto

myc

es is

olat

es w

hen

plat

ed o

n LB

aga

r pla

tes s

uppl

emen

ted

with

gent

amyc

in, k

anam

ycin

, trim

etho

prim

and

stre

ptom

ycin

incu

bate

d at

28°

C fo

r a g

row

th p

erio

d of

1 w

eek

com

pare

d to

LB

aga

r

cont

rol p

late

s with

out a

ntib

iotic

.

Susc

eptib

ility

ass

ays w

ere

perf

orm

ed in

trip

licat

e us

ing

thre

e bi

olog

ical

repl

icat

es.

47

The presence of lactones in the simultaneous assay was indicated by the observation of

fluorescence near the newest growth along the edges of the petri dish rather than at the

intended E. coli streaking site indicating that diffusible lactones were being secreted by

the Streptomyces isolates at early stages of growth. Isolate JLS2-C7 inhibited the growth

of E. coli MT102 (Figure III.4). Further investigation into the type of quorum sensing

molecules secreted by the Streptomyces isolates remains to be investigated.

To determine if the concentration of extracellular signalling molecules was

influencing the fluorescence of E. coli MT102, a Wimpenny plate incorporating

Streptomyces isolate supernatant was prepared. No differences were observed in the

fluorescence emitted by E. coli MT102 with Streptomyces isolates JLS1-A6, JLS1-C12,

JLS1-C4, JLS1-F12, JLS2-D6, KT1-B1 and KT1-B8 from 0 to 50% concentration of

supernatant in the medium. Therefore, the fluorescence observed does not seem to be

correlated with the concentration gradient of extracellular signalling molecules present

within the liquid culture supernatants of Streptomyces isolates. However JLS2-C7 when

overlaid with LB containing 10 µg/ml tetracycline did allow for the growth of E. coli

MT102. In the cross-streak assay, the presence of a JLS2-C7 colony on the surface of the

agar medium inhibited the growth of the gram negative E. coli MT102. However when

the supernatant of JLS2-C7 was incorporated into the growth medium of the slant growth

of E. coli MT102 throughout the surface of the agar overlay was observed despite the

increasing concentration of JLS2-C7 supernatant (Figure III.5).

48

Figu

re II

I.4. G

amm

a-bu

tyro

lact

one

assa

y (G

BL)

of e

ight

Str

epto

myc

es is

olat

es. S

trep

tom

yces

stra

ins a

nd E

. col

i MT1

02 w

ere

sim

ulta

neou

sly

inoc

ulat

ed o

n LB

med

ium

supp

lem

ente

d w

ith 1

0 µg

/ml t

etra

cycl

ine

in tr

iplic

ate.

Sam

ples

wer

e in

cuba

ted

in th

e

pres

ence

of E

. col

i MT1

02 w

ith p

lasm

id p

JBA

132

for 5

day

s at 2

8°C

. Con

trol p

late

s of e

ach

test

stra

in w

ere

incu

bate

d w

ithou

t

E. c

oli M

T102

for 5

day

s at 2

8°C

to o

bser

ve g

row

th. A

con

trol p

late

con

tain

ing

only

E. c

oli M

T102

with

out a

test

stra

ins w

as

also

incu

bate

d fo

r 5 d

ays a

t 28°

C. P

hoto

grap

hs w

ere

take

n on

Dar

krea

der u

sing

Can

on R

ebel

T1i

cam

era.

(

) In

dica

te th

e

activ

atio

n of

the

gree

n flu

ores

cenc

e pr

otei

n du

e to

the

pres

ence

of l

acto

ne si

gnal

ling.

49

Table III.3. Putative quorum sensing assessment of simultaneously inoculated

Streptomyces isolates in close proximity to E. coli MT102 containing plasmid pJBA132

on LB agar observed over a 72 h period at an incubation temperature of 28°C.

Streptomyces

Isolate

Time of observation until fluorescence (h)

24 48 72 Visual Observations

JLS1-A6 - - + + + + + + + None

KT1-B8 - - - + + + + + + None

JLS1-C4 - - - + + + + + + None

JLS2-C7 - - - - - - - - - E. coli MT102

inhibition

JLS1-F12 - - - + + + + + + None

JLS1-C12 - - - - - - - - - None

JLS2-D6 - - - + + + + + + Diffuse brown

pigment

KT1-B1 - - - + + + + + + None

1(+) indicates fluorescence 2(-) indicates no fluorescence 3(+++/---) indictates triplicate biological replicates

50

Figu

re II

I.5.W

impe

nny

plat

e as

say

for g

amm

a-bu

tyro

lact

one

sign

allin

g. L

B sl

ants

wer

e su

pple

men

ted

with

50

% (v

/v) f

ilter

ed c

ultu

re

supe

rnat

ant f

rom

Str

epto

myc

es is

olat

es o

verla

id w

ith L

B a

nd st

reak

ed w

ith E

. col

i MT1

02 c

onta

inin

g pl

asm

id p

JBA

132.

All

med

ia

cont

aine

d 10

µg/

ml t

etra

cycl

ine.

A c

ontro

l pla

te c

onta

inin

g no

cul

ture

supe

rnat

ant w

as p

repa

red

to o

bser

ve a

ny si

gnal

ling

due

to

cont

amin

ants

.

JLS2

-D6

JLS1

-C12

JLS1

-F12

JL

S2-C

7

CON

TRO

L

E. co

li M

T102

stra

in

0% S

uper

nata

nt

50%

Sup

erna

tant

51

Table III.4. Putative quorum sensing of 48 h cultures of Streptomyces isolates inoculated

with E. coli MT102 containing plasmid pJBA132 on LB agar over an observation period

of 1 week at an incubation temperature of 28°C.

1(+) indicates flourescence

2(-) indicates no fluorescence

3(+++/---) indictates triplicate biological replicates

52

Table III.5. Indole-3-acetic acid (IAA) production by Streptomyces isolates grown on

minimal media supplemented with 0.1% (w/v) birchwood xylan and 0.1% (w/v)

glucose, or grown on yeast extract-malt extract-dextrose (YEMED) medium and

YEMED supplemented with 2 mg/ml tryptophan.

1(+/-) Standard deviation of three biological replicates.

53

The uninhibited growth of E. coli MT102 on the surface of the agar overlay in the

presence of increasing JLS2-C7 supernatant concentration in the growth medium

suggests that the inhibitory molecule may not be present in the extracellular supernatant

of isolate JLS2-C7. This also suggests that the inhibitory molecule of JLS2-C7 may be an

antibiotic secreted in the presence of the E. coli MT102. Further investigation is required

to confirm whether the inhibition was indeed the result of antibiotic production by JLS2-

C7.

The time for fluorescence induction to be observed for the eight Streptomyces

isolates when inoculated simultaneously on LB agar with E. coli MT102 was compared

to the time required for fluorescence to be observed in the sequential assay where mature

Streptomyces colonies were exposed to inoculated E. coli MT102. It was observed that

fluorescence in triplicate of Streptomyces strains was achieved in 48 h in the

simultaneous study and 168 h in the sequential assay (Table III.3, Table III.4). It must be

noted that the growth rate of E. coli is much faster than that of the Streptomyces isolates

tested. More pronounced fluorescence was observed in a shorter time by Streptomyces

isolates JLS1-A6, KT1-B8, JLS1-C4, JLS1-F12, JLS2-D6 and KT1-B1 during the

simultaneous assay, suggesting that lactone signalling varies depending on whether the

cultures are actively growing or in stationary phase. The luxR receptor on the pJBA132

plasmid would be available to receive signal as soon as lactone molecules diffused into

the media from the germinated Streptomycete when inoculated simultaneously on the LB

agar. In the sequential study the production of lactone signalling from a mature stationary

phase streak took a longer period of time to reach the concentrations necessary to express

GFP from E. coli MT102 and fluorescence from Streptomyces isolates JLS1-A6, KT1-

54

B8, JLS1-C4, JLS1-F12, JLS2-D6 and KT1-B1 took much longer to be observed in

triplicate.

III.1.4.1. Cross Hatch Assessment for Enhanced Growth of Isolates

A cross-hatch assay was performed to investigate the compatibility of

Streptomyces isolates when grown in combination, and to determine whether the

combinations affected degradation of a substrate. For this assay, 48 broad-spectrum

degrading Streptomyces isolates that had been identified in a previous study were used

(Thompson-undergraduate thesis, 2009). Cross-streaking of each isolate is a method

which allows the visual observation of microbial interactions, such as sporulation or

enhanced degradation and growth. Cross streaking can be used in order to characterize

the isolates’ morphology or the interaction occurring between each isolate in all

combinations (Shirling, 1966). Visual observation of the resulting plates allowed the

selection of eight Streptomyces isolates which possessed the greatest amount of growth

and degradation on 0.1 % (w/v) oat-spelt xylan, 0.1 % (w/v) CMC and 0.1% (w/v)

cellulose agar medium (Table III.7). Growth and degradation by these eight Streptomyces

isolates was compared (Figure III.6). Varying degrees of activity were observed

depending on the types of substrates used to cultivate the isolates. When examining

growth and degradation of Streptomyces strains on birchwood xylan it appears that the

birchwood xylan substrate was more easily degraded and allowed for better growth of the

strains tested (Figure III.6). When isolates were grown on CMC medium, enhanced

growth and degradation were observed especially where isolates JLS1-E1, JLS1-E7 and

JLS1-F1 made contact with isolate KT1-B1; an increase in degradation was observed.

55

Fi

gure

III.6

. Int

erac

tion

assa

y of

Str

epto

myc

es is

olat

es. I

sola

tes w

ere

grow

n in

var

ious

com

bina

tions

on

Min

imal

Med

ium

con

tain

ing

0.1%

(w/v

) birc

hwoo

d xy

lan,

0.1

% (w

/v) C

MC

or 0

.1%

(w/v

) cel

lulo

se su

bstra

tes.

A) P

late

con

tain

s 0.1

% (w

/v) b

irchw

ood

xyla

n. B

)

Plat

e co

ntai

ns 0

.1%

(w/v

) car

boxy

met

hyl c

ellu

lose

. C) P

late

con

tain

s 0.1

% (w

/v) c

ellu

lose

. Cul

ture

s wer

e in

cuba

ted

for 1

0 da

ys a

t

28°C

and

pho

togr

aphe

d w

ith a

Can

on R

ebel

T1i

cam

era.

(

) Ind

icat

es e

nhan

ced

grow

th o

r spo

rula

tion

of is

olat

e; (

)

indi

cate

s are

a of

gro

wth

inhi

bitio

n.

56

Figu

re II

I.7. S

trep

tom

yces

isol

ates

in c

ross

hat

ch st

reak

ing

assa

y. S

train

s wer

e st

reak

ed in

diff

erin

g co

mbi

natio

ns o

n om

ni p

late

s

cont

aini

ng M

inim

al M

ediu

m p

lus 0

.1%

(w/v

) birc

hwoo

d xy

lan,

0.1

% (w

/v) C

MC

or 0

.1%

(w/v

) cel

lulo

se su

bstra

tes.

A) U

nsta

ined

omni

pla

te, a

s see

n in

Fig

ure

III.6

pan

el B

con

tain

ing

0.1%

(w/v

) car

boxy

met

hyl c

ellu

lose

. B) T

he sa

me

omni

pla

te st

aine

d w

ith 0

.1%

(w/v

) Con

go re

d. P

late

s wer

e in

cuba

ted

for a

per

iod

of 1

0 da

ys a

t 28°

C a

nd p

hoto

grap

hed

on a

ligh

tbox

usi

ng a

Can

on R

ebel

T1i

cam

era.

(

)

Indi

cate

s JLS

2-C

7 is

olat

e; (

) in

dica

tes i

sola

tes i

nhib

ited

by J

LS2-

C7;

(

) ind

icat

es K

T1-B

1 is

olat

e an

d (

)

area

of e

nhan

ced

degr

adat

ion

by is

olat

e K

T1-B

1.

57

Table III.6 Inhibition and degradation by Streptomyces isolates on 0.1% (w/v) birchwood xylan agar medium for a growth period of 10 days at 28°C in a cross-hatch assay.

Streptomyces Isolate Combination

Inhibition / Xyan Degradation 0.1% (w/v) birchwood xylan agar

Streptomyces Isolate Combination

Inhibition / Xylan Degradation 0.1% (w/v) birchwood xylan agar

JLS2-F9 vs KT1-E7 No effect JLS2-F1 vs KT1-E7 - - -/No effect JLS2-F9 vs KT1-E8 - -/+ + JLS2-F1 vs KT1-E8 - - - / No effect JLS2-F9 vs KT1-B1 No effect JLS2-F1 vs KT1-B1 No effect JLS2-F9 vs KT1-D1 No effect JLS2-F1 vs KT1-D1 No effect JLS2-F9 vs JLS2-B2 - - -/ No effect JLS2-F1 vs JLS2-B2 - - - /+ JLS2-F9 vs JLS2-B8 No effect JLS2-F1 vs JLS2-B8 No effect JLS2-F9 vs JLS2-C7 No effect JLS2-F1 vs JLS2-C7 No effect/ - - - JLS2-F9 vs JLS2-D6 - - -/- - - JLS2-F1 vs JLS2-D6 No effect/ - - - JLS2-F9 vs JLS2-D7 - -/+ + JLS2-F1 vs JLS2-D7 - - / No effect JLS2-F9 vs JLS2-D12

No effect JLS2-F1 vs JLS2-D12 - - - / No effect

JLS2-F9 vs KT1-E1 No effect JLS2-F1 vs KT1-E1 No effect/- - JLS2-G1 vs KT1-E7 No effect JLS2-A1 vs KT1-E7 - - - /- - JLS2-G1 vs KT1-E8 - -/+ + JLS2-A1 vs KT1-E8 - - - /+ + JLS2-G1 vs KT1-B1 No effect JLS2-A1 vs KT1-B1 No effect JLS2-G1 vs KT1-D1 No effect/ - JLS2-A1 vs KT1-D1 No effect JLS2-G1 vs JLS2-B2

- - - /+ + + JLS2-A1 vs JLS2-B2 - -/+

JLS2-G1 vs JLS2-B8

No effect JLS2-A1 vs JLS2-B8 No effect

JLS2-G1 vs JLS2-C7

No effect JLS2-A1 vs JLS2-C7 No effect/- - -

JLS2-G1 vs JLS2-D6

- - - / - - - JLS2-A1 vs JLS2-D6 No effect/- - -

JLS2-G1 vs JLS2-D7

- -/+ + JLS2-A1 vs JLS2-D7 - -/ No effect

JLS2-G1 vs JLS2-D12

No effect JLS2-A1 vs JLS2-D12 No effect

JLS2-G1 vs KT1-E1 No effect JLS2-A1 vs KT1-E1 No effect JLS2-H4 vs KT1-E7 - - - / No effect JLS2-A4 vs KT1-E7 - - -/- - JLS2-H4 vs KT1-E8 - - /+ + JLS2-A4 vs KT1-E8 -/ - - JLS2-H4 vs KT1-B1 No effect JLS2-A4 vs KT1-B1 Contamination JLS2-H4 vs KT1-D1 No effect/ - JLS2-A4 vs KT1-D1 Contamination JLS2-H4 vs JLS2-B2

- - - /+ JLS2-A4 vs JLS2-B2 - - - /No effect

JLS2-H4 vs JLS2-B8

No effect JLS2-A4 vs JLS2-B8 No effect

JLS2-H4 vs JLS2-C7

No effect/ - - - JLS2-A4 vs JLS2-C7 No effect

JLS2-H4 vs JLS2-D6

No effect/ - - - JLS2-A4 vs JLS2-D6 - - - / - - -

JLS2-H4 vs JLS2- - - - /+ JLS2-A4 vs JLS2-D7 - - /No effect

58

D7 JLS2-H4 vs JLS2-D12

- - / - - - JLS2-A4 vs JLS2-D12 No effect

JLS2-H4 vs KT1-E1 No effect/- JLS2-A4 vs KT1-E1 No effect JLS1-D10 vs JLS1-A6

No effect/+ + JLS1-D11 vs JLS1-A6 No effect/+ +

JLS1-D10 vs JLS1-A9

- -/No effect JLS1-D11 vs JLS1-A9 - - -/-

JLS1-D10 vs JLS1-A12

- - - / - - JLS1-D11 vs JLS1-A12 - - - /- - -

JLS1-D10 vs JLS1-B8

- - - /- - JLS1-D11 vs JLS1-B8 - - -/- -

JLS1-D10 vs JLS1-B10

No effect JLS1-D11 vs JLS1-B10 No effect

JLS1-D10 vs JLS1-B12

No effect/+ + + JLS1-D11 vs JLS1-B12 No effect/+ +

JLS1-D10 vs JLS1-C4

- - -/+ + + JLS1-D11 vs JLS1-C4 - - -/+ +

JLS1-D10 vs JLS1-C5

- - / - - JLS1-D11 vs JLS1-C5 - - / - - -

JLS1-D10 vs JLS1-C7

- - /- - - JLS1-D11 vs JLS1-C7 - - - /- - -

JLS1-D10 vs JLS1-C8

- - - /No effect JLS1-D11 vs JLS1-C8 - - - /No effect

JLS1-D10 vs JLS1-C12

No effect/+ JLS1-D11 vs JLS1-C12 No effect/- - -

JLS1-D12 vs JLS1-A6

No effect JLS1-E7 vs JLS1-A6 No effect

JLS1-D12 vs JLS1-A9

No effect JLS1-E7 vs JLS1-A9 - - - /-

JLS1-D12 vs JLS1-A12

- - - /- JLS1-E7 vs JLS1-A12 - - - /- -

JLS1-D12 vs JLS1-B8

- - - / - - - JLS1-E7 vs JLS1-B8 - - -/-

JLS1-D12 vs JLS1-B10

- - - /- - JLS1-E7 vs JLS1-B10 No effect

JLS1-D12 vs JLS1-B12

No effect JLS1-E7 vs JLS1-B12 No effect/+

JLS1-D12 vs JLS1-C4

No effect/+ JLS1-E7 vs JLS1-C4 - - - /-

JLS1-D12 vs JLS1-C5

- -/- - - JLS1-E7 vs JLS1-C5 No effect

JLS1-D12 vs JLS1-C7

- - - /- - - JLS1-E7 vs JLS1-C7 - - / - -

JLS1-D12 vs JLS1-C8

- - - /No effect JLS1-E7 vs JLS1-C8 - - - /No effect

JLS1-D12 vs JLS1-C12

No effect/- - - JLS1-E7 vs JLS1-C12 No effect/- - -

JLS1-E1 vs JLS1-A6 + +/- - - JLS1-F1 vs JLS1-A6 No effect JLS1-E1 vs JLS1-A9 - - - /- JLS1-F1 vs JLS1-A9 - - - /-

59

JLS1-E1 vs JLS1-A12

- - - /- - JLS1-F1 vs JLS1-A12 - - - /- -

JLS1-E1 vs JLS1-B8 - - - /- JLS1-F1 vs JLS1-B8 - - - /- JLS1-E1 vs JLS1-B10

No effect JLS1-F1 vs JLS1-B10 No effect

JLS1-E1 vs JLS1-B12

No effect JLS1-F1 vs JLS1-B12 No effect/++

JLS1-E1 vs JLS1-C4 - - /+ JLS1-F1 vs JLS1-C4 - - - /No effect JLS1-E1 vs JLS1-C5 -/- - JLS1-F1 vs JLS1-C5 - -/- - JLS1-E1 vs JLS1-C7 - - - /- - - JLS1-F1 vs JLS1-C7 - -/- - JLS1-E1 vs JLS1-C8 - - - /No effect JLS1-F1 vs JLS1-C8 - - - /No effect JLS1-E1 vs JLS1-C12

No effect/- - - JLS1-F1 vs JLS1-C12 No effect/+

JLS1-H8 vs JLS1-F3 No effect JLS1-H10 vs JLS1-F3 No effect/- - JLS1-H8 vs JLS1-F4 No effect JLS1-H10 vs JLS1-F4 - - - /- - - JLS1-H8 vs JLS1-F6 No effect JLS1-H10 vs JLS1-F6 No effect JLS1-H8 vs JLS1-F7 No effect JLS1-H10 vs JLS1-F7 - -/No effect JLS1-H8 vs JLS1-F12

No effect/+++ JLS1-H10 vs JLS1-F12 No effect/+ + +

JLS1-H8 vs JLS1-H1

- - - /No effect JLS1-H10 vs JLS1-H1 - - - /No effect

JLS1-H8 vs JLS1-H2

- - - /- - - JLS1-H10 vs JLS1-H2 No effect

JLS1-H8 vs JLS1-H3

- - / - - JLS1-H10 vs JLS1-H3 No effect

JLS1-H8 vs JLS1-H5

- - - /- - - JLS1-H10 vs JLS1-H5 No effect

JLS1-H8 vs JLS1-H6

No effect/- - - JLS1-H10 vs JLS1-H6 No effect/- -

JLS1-H8 vs JLS1-H7

No effect JLS1-H10 vs JLS1-H7 No effect

JLS1-H9 vs JLS1-F3 No effect JLS1-H12 vs JLS1-F3 No effect JLS1-H9 vs JLS1-F4 - - - / - - - JLS1-H12 vs JLS1-F4 No effect JLS1-H9 vs JLS1-F6 - - -/- - - JLS1-H12 vs JLS1-F6 No effect JLS1-H9 vs JLS1-F7 -/No effect JLS1-H12 vs JLS1-F7 No effect/- - JLS1-H9 vs JLS1-F12

No effect/+ + + JLS1-H12 vs JLS1-F12 No effect/+

JLS1-H9 vs JLS1-H1

- - - /No effect JLS1-H12 vs JLS1-H1 - - - /No effect

JLS1-H9 vs JLS1-H2

- - /No effect JLS1-H12 vs JLS1-H2 No effect

JLS1-H9 vs JLS1-H3

- - / - - JLS1-H12 vs JLS1-H3 No effect

JLS1-H9 vs JLS1-H5

- - - /- - - JLS1-H12 vs JLS1-H5 No effect

JLS1-H9 vs JLS1-H6

No effect/- - - JLS1-H12 vs JLS1-H6 No effect

JLS1-H9 vs KT1-H7 No effect JLS1-H12 vs JLS1-H7 No effect JLS1-D10 vs KT1- No effect JLS1-D12 vs KT1-E7 - - - /No effect

60

E7 JLS1-D10 vs KT1-E8

-/+ + JLS1-D12 vs KT1-E8 No effect/+ + +

JLS1-D10 vs KT1-B1

No effect JLS1-D12 vs KT1-B1 No effect

JLS1-D10 vs KT1-D1

No effect/+ + JLS1-D12 vs KT1-D1 No effect/+

JLS1-D10 vs JLS2-B2

- - - /+ + + JLS1-D12 vs JLS2-B2 - - - /+ + +

JLS1-D10 vs JLS2-B8

- - - /+ + + JLS1-D12 vs JLS2-B8 - - - /+ + +

JLS1-D10 vs JLS2-C7

No effect JLS1-D12 vs JLS2-C7 No effect

JLS1-D10 vs JLS2-D6

- - - /- - - JLS1-D12 vs JLS2-D6 - - - /- -

JLS1-D10 vs JLS2-D7

- - - /+ + + JLS1-D12 vs JLS2-D7 - - - /+ + +

JLS1-D10 vs JLS2-D12

No effect JLS1-D12 vs JLS2-D12 No effect

JLS1-D10 vs JLS2-E1

No effect/- - JLS1-D12 vs JLS2-E1 No effect

JLS1-D11 vs KT1-E7

- - - /- - JLS1-E1 vs KT1-E7 - - - /No effect

JLS1-D11 vs KT1-E8

-/+ JLS1-E1 vs KT1-E8 No effect/+ +

JLS1-D11 vs KT1-B1

No effect JLS1-E1 vs KT1-B1 No effect

JLS1-D11 vs KT1-D1

No effect JLS1-E1 vs KT1-D1 No effect/+ + +

JLS1-D11 vs JLS2-B2

- - - /+ + + JLS1-E1 vs JLS2-B2 - - - /+ + +

JLS1-D11 vs JLS2-B8

- - - /+ + + JLS1-E1 vs JLS2-B8 - - - /+ + +

JLS1-D11 vs JLS2-C7

No effect JLS1-E1 vs JLS2-C7 No effect

JLS1-D11 vs JLS2-D6

- - - / - - - JLS1-E1 vs JLS2-D6 - - - /- -

JLS1-D11 vs JLS2-D7

- - - /+ + + JLS1-E1 vs JLS2-D7 - - - /+ + +

JLS1-D11 vs JLS2-D12

No effect/- - JLS1-E1 vs JLS2-D12 - - - /No effect

JLS1-D11 vs JLS2-E1

No effect/- - JLS1-E1 vs JLS2-E1 - - /No effect

JLS1-E7 vs KT1-E7 - - -/No effect JLS1-F1 vs KT1-E7 - - - /No effect JLS1-E7 vs KT1-E8 - -/+ + + JLS1-F1 vs KT1-E8 No effect/+ + JLS1-E7 vs KT1-B1 No effect JLS1-F1 vs KT1-B1 No effect JLS1-E7 vs KT1-D1 No effect/+ JLS1-F1 vs KT1-D1 No effect/+ JLS1-E7 vs JLS2-B2 - - -/+ + + JLS1-F1 vs JLS2-B2 - - - /+ + + JLS1-E7 vs JLS2-B8 - - - /+ + + JLS1-F1 vs JLS2-B8 - - - /+ + + JLS1-E7 vs JLS2-C7 No effect JLS1-F1 vs JLS2-C7 No effect

61

JLS1-E7 vs JLS2-D6 -/- JLS1-F1 vs JLS2-D6 - - - / - - - JLS1-E7 vs JLS2-D7 - - - /+ + + JLS1-F1 vs JLS2-D7 - - - /+ + + JLS1-E7 vs JLS2-D12

- - - /No effect JLS1-F1 vs JLS2-D12 - - - /No effect

JLS1-E7 vs JLS2-E1 - -/No effect JLS1-F1 vs JLS2-E1 No effect/- - - JLS1-H8 vs KT1-E7 No effect JLS1-H9 vs KT1-E7 - - - /No effect JLS1-H8 vs KT1-E8 - - - /- JLS1-H9 vs KT1-E8 - - - /No effect JLS1-H8 vs KT1-B1 - - - /No effect JLS1-H9 vs KT1-B1 - - - /No effect JLS1-H8 vs KT1-D1 - - - /No effect JLS1-H9 vs KT1-D1 - - - /- - - JLS1-H8 vs JLS2-B2

No effect/+ + + JLS1-H9 vs JLS2-B2 No effect/- - -

JLS1-H8 vs JLS2-B8

- - - /- - - JLS1-H9 vs JLS2-B8 - - - /- - -

JLS1-H8 vs JLS2-C7

- - - /No effect JLS1-H9 vs JLS2-C7 - - - /No effect

JLS1-H8 vs JLS2-D6

- - - /No effect JLS1-H9 vs JLS2-D6 - - - /No effect

JLS1-H8 vs JLS2-D7

- - - /No effect JLS1-H9 vs JLS2-D7 No effect

JLS1-H8 vs JLS2-D12

- - - /No effect JLS1-H9 vs JLS2-D12 - - -/No effect

JLS1-H8 vs JLS2-E1 - - - /No effect JLS1-H9 vs JLS2-E1 - - - /No effect JLS1-H10 vs KT1-E7

No effect JLS1-H11 vs KT1-E7 - - - /No effect

JLS1-H10 vs KT1-E8

- - - /No effect JLS1-H11 vs KT1-E8 - - - /No effect

JLS1-H10 vs KT1-B1

- - - /No effect JLS1-H11 vs KT1-B1 - - /No effect

JLS1-H10 vs KT1-D1

- - -/No effect JLS1-H11 vs KT1-D1 - - /No effect

JLS1-H10 vs JLS2-B2

No effect/+ + + JLS1-H11 vs JLS2-B2 No effect/+ + +

JLS1-H10 vs JLS2-B8

No effect JLS1-H11 vs JLS2-B8 - - - /- - -

JLS1-H10 vs JLS2-C7

- - - /No effect JLS1-H11 vs JLS2-C7 - - - /No effect

JLS1-H10 vs JLS2-D6

- - -/No effect JLS1-H11 vs JLS2-D6 - - -/No effect

JLS1-H10 vs JLS2-D7

- - - /- - - JLS1-H11 vs JLS2-D7 - - - /- - -

JLS1-H10 vs JLS2-D12

- - - /No effect JLS1-H11 vs JLS2-D12 - - - /No effect

JLS1-H10 vs JLS2-E1

- - - /No effect JLS1-H11 vs JLS2-E1 - - - /No effect

JLS1-H12 vs KT1-E7

- - -/No effect JLS1-H9 vs JLS1-A6 No effect

JLS1-H12 vs KT1-E8

- - -/No effect JLS1-H9 vs JLS1-A9 No effect

JLS1-H12 vs KT1-B1

- - /No effect JLS1-H9 vs JLS1-A12 - - / - -

62

JLS1-H12 vs KT1-D1

- - - /- - - JLS1-H9 vs JLS1-B8 No effect/- - -

JLS1-H12 vs JLS2-B2

- - /No effect JLS1-H9 vs JLS1-B10 No effect/- - -

JLS1-H12 vs JLS2-B8

No effect JLS1-H9 vs JLS1-B12 -/- - -

JLS1-H12 vs JLS2-C7

- - - /No effect JLS1-H9 vs JLS1-C4 No effect/- - -

JLS1-H12 vs JLS2-D6

- - -/No effect JLS1-H9 vs JLS1-C5 No effect/- - -

JLS1-H12 vs JLS2-D7

No effect JLS1-H9 vs JLS1-C7 - - - /- - -

JLS1-H12 vs JLS2-D12

- - - /No effect JLS1-H9 vs JLS1-C8 No effect/- - -

JLS1-H12 vs JLS2-E1

- - - /No effect JLS1-H9 vs JLS1-C12 No effect/- - -

JLS1-H8 vs JLS1-A6

- - - /+ + JLS1-H10 vs JLS1-A6 No effect/- - -

JLS1-H8 vs JLS1-A9

No effect JLS1-H10 vs JLS1-A9 No effect/- - -

JLS1-H8 vs JLS1-A12

No effect JLS1-H10 vs JLS1-A12 No effect/- - -

JLS1-H8 vs JLS1-B8

No effect JLS1-H10 vs JLS1-B8 No effect/- - -

JLS1-H8 vs JLS1-B10

- - /- - JLS1-H10 vs JLS1-B10 No effect/- - -

JLS1-H8 vs JLS1-B12

No effect/- JLS1-H10 vs JLS1-B12 No effect/- - -

JLS1-H8 vs JLS1-C4

No effect/- - - JLS1-H10 vs JLS1-C4 No effect/- - -

JLS1-H8 vs JLS1-C5

No effect/- - - JLS1-H10 vs JLS1-C5 No effect/- - -

JLS1-H8 vs JLS1-C7

- - - / - - - JLS1-H10 vs JLS1-C7 - - - /- - -

JLS1-H8 vs JLS1-C8

No effect/ - - JLS1-H10 vs JLS1-C8 No effect/- - -

JLS1-H8 vs JLS1-C12

No effect/- JLS1-H10 vs JLS1-C12 No effect/- - -

JLS1-H11 vs JLS1-A6

+ +/No effect JLS1-H12 vs JLS1-A6 + +/No effect

JLS1-H11 vs JLS1-A9

No effect JLS1-H12 vs JLS1-A9 No effect/- - -

JLS1-H11 vs JLS1-A12

No effect JLS1-H12 vs JLS1-A12 No effect

JLS1-H11 vs JLS1-B8

No effect JLS1-H12 vs JLS1-B8 No effect/- -

JLS1-H11 vs JLS1-B10

No effect/- - JLS1-H12 vs JLS1-B10 No effect/- -

JLS1-H11 vs JLS1-B12

No effect JLS1-H12 vs JLS1-B12 No effect

63

JLS1-H11 vs JLS1-C4

No effect/- - - JLS1-H12 vs JLS1-C4 No effect/- - -

JLS1-H11 vs JLS1-C5

No effect/- - - JLS1-H12 vs JLS1-C5 No effect/- - -

JLS1-H11 vs JLS1-C7

- -/No effect JLS1-H12 vs JLS1-C7 -/No effect

JLS1-H11 vs JLS1-C8

No effect/- - JLS1-H12 vs JLS1-C8 No effect/- -

JLS1-H11 vs JLS1-C12

No effect/- - - JLS1-H12 vs JLS1-C12 No effect

1(-) Isolate growth hindered.

2 (- -) Isolate growth partially inhibited.

3 (- - -) Isolate growth completely inhibited.

4 (+) Isolate xylan degradation unaffected.

5 (+ +) Isolate xylan degradation increased (1-2 mm).

6 (+ + +) Isolate xylan degradation increased (>2 mm).

7 (No effect) Neither growth nor xylan degradation was affected.

64

Tabl

e II

I.7. S

trep

tom

yces

isol

ates

exh

ibiti

ng e

nhan

ced

grow

th o

r deg

rada

tion

on a

cro

ss-h

atch

ass

ay. C

ultu

res w

ere

grow

n fo

r 10

days

at

28°

C o

n 0.

1% (w

/v) x

ylan

aga

r med

ium

.

1 A

ll is

olat

es te

sted

with

in th

e cr

oss h

atch

ass

ay m

ust i

nter

act w

ith g

reat

er th

an fo

ur is

olat

es to

qua

lify

as a

n en

hanc

ed is

olat

e.

2 (*

) Ind

icat

es th

e di

ffus

ion

of m

ycel

ia a

ssoc

iate

d pi

gmen

ted

mol

ecul

es in

surr

ound

ing

agar

med

ium

dur

ing

subs

trate

deg

rada

tion.

3 (+

) Ind

icat

es x

ylan

deg

rada

tion

obse

rved

in th

e m

ediu

m su

rrou

ndin

g th

e is

olat

es.

4 (N

/A) N

o is

olat

es a

pplic

able

65

Figu

re II

I.8.

Deg

rada

tion

prod

uced

by

the

eigh

t iso

late

s on

min

imal

med

ium

con

tain

ing

0.1%

(w/v

) birc

hwoo

d an

d 0.

1% (w

/v) o

at-

spel

t xyl

an a

gar m

edia

. A o

ne w

ay A

NO

VA

was

use

d to

cal

cula

te si

gnifi

canc

e ba

sed

on a

95%

con

fiden

ce in

terv

al. (

*) In

dica

tes t

he

varia

nce

of th

e m

eans

is st

atis

tical

ly si

gnifi

cant

bet

wee

n oa

t-spe

lt xy

lan

and

birc

hwoo

d xy

lan

degr

adat

ion.

024681012141618

JLS1

-F12

JLS2

-D6

JLS1

-A6

JLS1

-C4

JLS1

-C12

JLS2

-C7

KT1-

B1KT

1-B8

Average Clearing Zone (mm)

Stre

ptom

yces

Str

ain

0.1

% (w

/v) O

at-s

pelt

Xyla

n0.

1% (w

/v) B

irchw

ood

Xyla

n

* *

* *

* *

* *

66

Figu

re II

I.9. D

egra

datio

n of

0.1

% (w

/v) b

irchw

ood

xyla

n by

Str

epto

myc

es is

olat

es. I

sola

tes w

ere

obse

rved

on

Min

imal

Med

ium

cont

aini

ng 0

.1%

(w/v

) whe

n gr

own

indi

vidu

ally

for 5

day

s at 2

8°C

in d

uplic

ate.

(

)

Indi

cate

s Str

epto

myc

es c

olon

y; (

)

indi

cate

s deg

rada

tion

zone

of 0

.1%

(w/v

) birc

hwoo

d xy

lan

agar

med

ium

.

67

This increase in degradation is continued until those isolates contact strain JLS2-C7

(Figure III.7). The results from the cross-hatch assay of the eight Streptomyces isolates

which had the greatest degradative capability or enhanced growth on 0.1% (w/v)

birchwood xylan are summarized in Table III.7. All eight environmental isolates were

able to degrade 0.1 % (w/v) oat-spelt xylan better than 0.1 % (w/v) birchwood xylan

(Figure III.8).

Streptomyces JLS2-C7 was inhibited by JLS2-D6 (Figure III.10). Streptomyces

isolate JLS2-D6 was streaked across the plate past Streptomyces isolate JLS2-C7, but no

inhibition of surface growth by isolate JLS2-C7 was observed (Figure III.10 A).

Streptomyces isolate JLS2-C7 grew slowly (Figure III.10 A). When Streptomyces isolates

were streaked in the opposing positions the results indicate Streptomyces isolate JLS2-C7

was inhibited by isolate JLS2-D6 (Figure III.10 B) while JLS2-D6 was not inhibited by

isolate JLS2-C7. Enzymatic clearing by Streptomyces isolates JLS2-C7 and JLS2-D6

(Figure III.10 C and D) suggests there is no decrease in xylan breakdown of either strain

below the surface of the agar. However, there appears to be subtle inhibition of the

growth of isolate JLS2-C7 by isolate JLS2-D6 above the surface of the agar.

Each Streptomyces isolate displayed varying amounts of degradation or growth

according to their combination in the pair assay (Figure III.11). Growing the different

combinations of these streptomycete pairs on large plates containing 0.1% (w/v)

birchwood or 0.1% (w/v) oat-spelt xylan agar medium allowed for the observation of

different morphological behaviours between the isolates. In the pair assay, a lack of

growth was observed between isolates JLS2-D6 and JLS2-C7 when compared to all other

combinations of isolates in the individual growth assays (Figure III.11). As seen in Figure

68

III.11 A, the colony size of isolate JLS1-C4 was larger in size when grown in close

proximity to neighbouring isolates JLS2-C7 and KT1-B1 than when grown individually

on the same 0.1% (w/v) xylan medium despite the same number of spores being used to

inoculate the medium (Figure III.11). There were differences observed for the growth of

JLS2-C7 despite the use of equivalent inoculum of 1.0 x 104 spores. Although enhanced

degradation and growth were the most common themes observed amongst the strain

combinations, cell signalling and antibiotic production could both positively or

negatively affect the rate of xylan degradation of some neighbouring strains. All clearing

zones produced by each individual strain showed no significant difference (p > 0.05) in

the clearing zone size at 95% confidence interval when compared to their respective

duplicate (Figure III.11 B).

III.1.4.2. The Effects of Streptomyces Extracellular Supernatant on

Neighbouring Strains

Streptomyces survive in the presence of other bacteria by acquiring nutrients

through their substrate mycelia and secreting antibiotics to ward off competitors during

nutrient depletion. Several strains were able to degrade xylan effectively and inhibit the

growth of neighbouring strains. Two strains in particular, JLS2-C7 and JLS2-D6,

displayed antagonistic behaviour in which the growth size of each was negatively

affected.

69

Figure III.10. Streptomyces isolate growth inhibition. A and B, inhibition of Streptomyces

isolate JLS2-C7 by JLS2-D6 on 0.1% (w/v) birchwood xylan agar media. Plates were

inoculated with 1.5 x 105 spores and incubated at 28°C for 5 days. C and D, visualization

of enzymatic xylan degradation in all replicate plates stained with Gram’s iodine solution

to observe degradation. Degradation can be observed as clear zones of 0.1% (w/v)

birchwood xylan agar medium surrounding the inoculated JLS2-C7 and JLS2-D6

isolates. Arrows indicate the direction of inoculation.

70

Figu

re II

I.11.

Pai

r mor

phol

ogy

stud

y of

Str

epto

myc

es is

olat

es. A

) Deg

rada

tive

clea

ring

of 0

.1%

(w/v

) oat

-spe

lt xy

lan

med

ia su

bstra

te

by e

ight

sele

cted

Str

epto

myc

es is

olat

es in

all

com

bina

tions

vis

ualiz

ed u

sing

Gra

m’s

iodi

ne st

aini

ng a

nd p

hoto

grap

hed

on a

ligh

tbox

.

(

) In

dica

tes J

LS2-

D6

and

JLS2

-C7

inte

ract

ion

and

(

) i

ndic

ates

JLS2

-C4

inte

ract

ion

with

JLS2

-C7

and

KT1

-B1.

B) A

liquo

ts

of 5

.0 x

104

spor

es fr

om q

uant

ified

spor

e st

ocks

from

eac

h st

rain

wer

e sp

otte

d in

dup

licat

e on

0.1

% b

irchw

ood

xyla

n ag

ar fo

r cle

arin

g

zone

size

com

paris

on.

71

Figu

re II

I.12.

The

eff

ects

of S

trep

tom

yces

ext

race

llula

r sup

erna

tant

on

neig

hbou

ring

isol

ates

. Str

epto

myc

es e

xtra

cellu

lar s

uper

nata

nt

was

aut

ocla

ved,

filte

red,

or u

nalte

red

as a

con

trol t

o te

st fo

r ant

agon

istic

eff

ects

bet

wee

n st

rain

s JLS

2-C

7, a

nd JL

S2-D

6. A

men

ded

auto

clav

ed su

pern

atan

t or f

ilter

ed su

pern

atan

t was

supp

lem

ente

d in

to th

e op

posi

ng st

rain

gro

wth

med

ium

to a

sses

s inh

ibiti

on o

f

Stre

ptom

yces

isol

ates

JLS

2-C

7 an

d JL

S2-D

6 on

bot

h 0.

1 %

(w/v

) birc

hwoo

d an

d 0.

1% (w

/v) o

at-s

pelt

xyla

n su

bstra

te. A

) 2.3

× 1

05

spor

es o

f Str

epto

myc

es st

rain

JLS2

-C7

wer

e sp

otte

d on

aga

r Min

imal

Med

ium

am

ende

d w

ith d

iffer

ing

conc

entra

tions

of 0

.005

% (v

/v)

and

0.02

5 %

(v/v

) of S

trep

tom

yces

stra

in J

LS2-

D6

auto

clav

ed su

pern

atan

t, fil

tere

d su

pern

atan

t or u

nalte

red

supe

rnat

ant.

B) O

ppos

ing

isol

ates

use

d fo

r am

endm

ent a

nd in

ocul

atio

ns. T

otal

vol

ume

of a

gar m

inim

al m

edia

was

9 m

L. G

row

th o

f eac

h St

rept

omyc

es is

olat

e

was

mea

sure

d in

trip

licat

e af

ter 5

day

s and

foun

d to

be

stat

istic

ally

sign

ifica

nt u

sing

a st

uden

t T-te

st (p

< 0

.05)

at a

95%

con

fiden

ce

inte

rval

. Cul

ture

s wer

e in

cuba

ted

for 6

day

s at 2

8°C

. (*)

Indi

cate

s no

grow

th o

bser

ved.

72

Based on this negative interaction an amended growth study was performed in which a

percentage of the opposing strain supernatant, 0.005 % (v/v) and 0.025 % (v/v), was

incorporated into a 0.1% (w/v) birchwood and oat-spelt xylan agar growth media and 2.3

x 105 spores of the opposite strain were spotted on the media to observe growth and

clearing. The resulting growth of each isolate on the supplemented agar growth medium

was quantitated and compared (Figure III.12). Streptomyces JLS2-C7 does not grow on

0.1% (w/v) birchwood xylan agar supplemented with JLS2-D6 supernatant, but seems to

grow efficiently on 0.1% (w/v) birchwood and oat-spelt xylan when the supernatant of

JLS2-D6 was autoclaved or filtered (Figure III.12). Isolate JLS2-D6 grew well on 0.1%

(w/v) birchwood xylan agar medium containing JLS2-C7 supernatant, but grew on 0.1%

(w/v) oat-spelt xylan only when JLS2-C7 supernatant was autoclaved (Figure III.12).

Reduced growth by strain JLS2-D6 was observed when the agar medium contained the

filtered supernatant of JLS2-C7. This one-sided inhibition suggests the production of a

growth inhibiting compound, such as an antibiotic.

III.1.4.3. Extracellular Signalling and the Effect on Xylan Degradation

by Streptomyces Isolates

The presence and type of extracellular molecules being exchanged in Streptomyces

liquid culture supernatant of JLS2-D6 and JLS2-C7 were investigated. The differences in

the clearing zone sizes produced by JLS2-C7 and JLS2-D6 on medium containing filtered

culture supernatant indicates that the molecule responsible for the activity between the

strains is a small molecule (Figure III.13). Most proteins and small molecules would pass

through a 0.2 µm filter.

73

Figu

re II

I.13.

Ass

essm

ent o

f put

ativ

e ex

trace

llula

r sig

nalli

ng m

olec

ules

from

Str

epto

myc

es is

olat

es. A

) A 5

µL

aliq

uot o

f

Stre

ptom

yces

isol

ate

JLS2

-D6

0.1%

(w/v

) liq

uid

cultu

re su

pern

atan

t was

filte

red

(filt

er si

ze =

0.2

µm

) and

ass

esse

d fo

r act

ivity

aga

inst

JLS2

-C7

unfil

tere

d 0.

1% (w

/v) b

irchw

ood

xyla

n liq

uid

cultu

re su

pern

atan

t usi

ng w

ell d

iffus

ion

assa

y in

0.1

% (

w/v

) birc

hwoo

d xy

lan

agar

pla

tes.

Plat

es w

ere

stai

ned

with

Gra

m’s

iodi

ne. B

) A 5

µL

aliq

uot o

f Str

epto

myc

es st

rain

JLS2

-D6

cultu

re su

pern

atan

t was

filte

red

(filt

er si

ze =

0.2

µm

), au

tocl

aved

and

ass

esse

d fo

r act

ivity

aga

inst

a 5

µL

of J

LS2-

C7

unfil

tere

d cu

lture

supe

rnat

ant u

sing

wel

l

diff

usio

n as

say

in 0

.1%

(w/v

) birc

hwoo

d xy

lan

agar

stai

ned

with

Gra

m’s

iodi

ne. A

n un

inoc

ulat

ed 0

.1%

(w/v

) birc

hwoo

d xy

lan

med

ia

cont

rol w

as u

sed

for c

ompa

rison

.

74

Figu

re II

I.14.

Dis

tanc

e as

sess

men

t of S

trep

tom

yces

isol

ate

inte

ract

ions

aff

ectin

g de

grad

atio

n an

d co

lony

gro

wth

. Str

epto

myc

es

envi

ronm

enta

l iso

late

s JLS

2-D

6 an

d JL

S2-C

7wer

e ob

serv

ed fo

r diff

eren

ces i

n gr

owth

and

deg

rada

tion

capa

bilit

y in

rela

tion

to th

e

dist

ance

bet

wee

n th

e co

loni

es. A

liquo

ts o

f 2.

3 x

105 sp

ores

wer

e sp

otte

d on

0.1

% (w

/v) b

irchw

ood

xyla

n ag

ar m

edia

at d

ista

nces

of 1

cm, 2

cm

and

3 c

m. P

late

s wer

e in

cuba

ted

at 2

8° C

for 5

day

s the

n st

aine

d w

ith G

ram

’s Io

dine

to o

bser

ve c

lear

ing

zone

s. C

lear

zon

es

are

obse

rved

as u

nsta

ined

aga

r med

ium

surr

ound

ing

the

inoc

ulat

ed c

olon

y co

mpa

red

to th

e JL

S2-C

7 is

olat

e gr

own

indi

vidu

ally

on

the

sam

e 0.

1% (w

/v) b

irchw

ood

xyla

n m

ediu

m.

[B]

75

To examine if the distances between the isolates JLS2-C7 and JLS2-D6 affected

degradation and growth of the isolates, a proximity assay was performed. An aliquot of

2.3 x 105 spores of the each opposing isolate was spotted at distances of 1 cm, 2 cm and 3

cm from one another and compared to the clearing zone size of isolates cultured in

isolation (Figure III.14). A decrease in the degradative clearing zone produced by isolate

JLS2-C7 was observed when compared to the JLS2-C7 isolate grown individually on the

same 0.1% (w/v) birchwood xylan medium (Figure III.9).

III.1.5. Extracellular Protein Analysis of Streptomyces Isolates using PAGE

To examine the types of extracellular xylanases produced by the eight Streptomyces

isolates, extracellular protein profiles were examined (Figure III.15). Streptomyces isolate

JLS2-D6 shows two protein bands of approximately 50 kDa and 70 kDa. These proteins

correspond with the proteins of similar molecular weight observed in the Thermomyces

lanuginosus standard of 99.9% purity. KT1-B1 has one protein band with a molecular

weight of 50 kDa which also corresponds with similar sized proteins in the xylanase

standard. It must be noted that the xylanase standard used was fungal in origin. Isolates

JLS1-C4 and JLS1-A6 produce low molecular weight proteins of 15 kDa. Seven proteins

of 17 kDa, 35 kDa, 39 kDa, 42 kDa, 48 kDa, 70 kDa, and 115 kDa were observed in the

extracellular supernatant of JLS1-F12. Without purifying the proteins to homogeneity or

performing zymography, it cannot be determined that any of these proteins are xylanases.

Silver staining, a more sensitive method, was also used to examine the protein

profiles of liquid culture supernatants of the eight Streptomyces isolates. The detection

limit of Coomassie blue is 50-100 ng of protein while that of silver staining is 0.5-5 ng of

protein (Williams, 2001; Nishihara & Champion, 2002).

76

Figu

re II

I.15.

Ext

race

llula

r pro

tein

pro

files

of S

trep

tom

yces

isol

ates

gro

wn

in M

inim

al M

ediu

m c

onta

inin

g 0.

1% (w

/v) b

irchw

ood

xyla

n. A

) 20%

SD

S-PA

GE

of a

mm

oniu

m su

lfate

pre

cipi

tate

d ex

trace

llula

r pro

tein

s of S

trep

tom

yces

isol

ates

ran

in d

enat

urin

g bu

ffer

stai

ned

with

Coo

mas

sie

Blu

e. X

ylan

ase

from

The

rmom

yces

lanu

gino

sus (

7 µg

pro

tein

); St

rept

omyc

es JL

S2-D

6 pr

otei

n; p

rote

in la

dder

;

Stre

ptom

yces

KT1

-B1

prot

ein;

Str

epto

myc

es J

LS1-

C4

prot

ein;

Str

epto

myc

es JL

S1-A

6 pr

otei

n; S

trep

tom

yces

JLS

2-F1

2 pr

otei

n;

Stre

ptom

yces

JLS

1-C

12 p

rote

in. A

ll sa

mpl

es w

ere

load

ed in

to e

ach

wel

l usi

ng 4

0 µg

of p

rote

in. B

) 20%

SD

S PA

GE

of a

mm

oniu

m

sulp

hate

pre

cipi

tate

d ex

trace

llula

r Str

epto

myc

es p

rote

in st

aine

d w

ith si

lver

nitr

ate.

Fer

men

tas P

ageR

uler

pro

tein

ladd

er w

as u

sed

as a

size

stan

dard

. All

sam

ples

wer

e lo

aded

into

eac

h w

ell u

sing

40

µg o

f pro

tein

.

77

Protein from JLS1-A6, JLS2-D6, JLS1-F12, and 1% xylanase standard were

electrophoresed on a 20% SDS-PAGE gel in duplicate on a non-denaturing gel (Figure

III.15B). Streptomyces isolate JLS2-D6 shows several protein bands of 5 kDa, 10 kDa, 13

kDa, 15 kDa, 20 kDa, 22 kDa, 30 kDa, 34 kDa, 36 kDa, 40 kDa, 45 kDa, 50 kDa, 60

kDa, 65 kDa, 70 kDa of which 20 kDa, 22 kDa, 40 kDa, and 45 kDa are the most intense.

JLS1-A6 and JLS1-F12 show four proteins of 5 kDa, 10 kDa, 20 kDa and 22 kDa

compared to the Coomassie-stained gel in which none of the previously mentioned

proteins sizes of isolate JLS1-F12 were observed. Proteins of 20 kDa and 22 kDa were

observed in the extracellular supernatant of all three isolates when visualized using silver

nitrate.

III.1.6. Assessment of Lignin Peroxidase using Azure B-dye Decolourization

Assay

Extracellular peroxidases degrade complex organic compounds and are vital for

increased degradation of lignin and conversion of low value biomass such as agricultural

wastes into fuel, as well as turnover of plant material in the environment. In order to

assess extracellular peroxidases present in the supernatant of the eight environmental

isolates, an Azure B dye-decolourization assay was carried out (Table III.8).

Streptomyces isolates JLS1-C4, JLS2-D6, JLS1-C12, and JLS1-A6 exhibited higher

amounts of lignin peroxidase activity from partially purified protein than from crude

extracts grown in Minimal Medium supplemented with 0.1% (w/v) glucose or birchwood

xylan. JLS1-C4 showed the most lignin peroxidase activity at 0.3 Abs/min/mg.

78

Table III.8. Extracellular lignin peroxidase activities of eight environmental Streptomyces

isolates as determined by the Azure B dye decolourization assay.

1(+/-) Standard deviation of triplicate measurements.

79

Figu

re II

I.16.

Xyl

anas

e ac

tivity

of p

artia

lly p

urifi

ed e

xtra

cellu

lar S

trep

tom

yces

pro

tein

. A) S

peci

fic e

nzym

atic

act

ivity

of x

ylan

ase

enzy

me

from

Str

epto

myc

es is

olat

es p

artia

lly p

urifi

ed p

rote

in fr

om c

ultu

res g

row

n in

Min

imal

Med

ium

with

0.1

% (w

/v) b

irchw

ood

xyla

n fo

r 5 d

ays a

t 30°

C. B

) Ave

rage

xyl

an su

bstra

te d

egra

datio

n of

Str

epto

myc

es is

olat

es o

bser

ved

whe

n cu

lture

s wer

e gr

own

on

Min

imal

Med

ium

aga

r con

tain

ing

0.1%

(w/v

) birc

hwoo

d xy

lan.

Err

or b

ars a

re th

e st

anda

rd d

evia

tion

of th

ree

biol

ogic

al re

plic

ates

.

Ave

rage

deg

rada

tion

was

mea

sure

d us

ing

the

radi

us o

f the

col

ony;

from

the

cent

er to

the

edge

of g

row

th o

n th

e ag

ar o

f thr

ee

biol

ogic

al re

plic

ates

. (*)

Indi

cate

s no

xyla

nase

-spe

cific

act

ivity

det

ecte

d.

80

Tabl

e II

I.9. C

ompa

rativ

e an

alys

is o

f ass

ay d

ata

for c

hara

cter

istic

s of e

ight

Str

epto

myc

es is

olat

es o

f int

eres

t.

(-) n

o si

gnal

(+) p

rese

nce

of si

gnal

(+/-)

stan

dard

dev

iatio

n

81

III.1.6.1. Enzymatic Activity

The eight environmental Streptomyces isolates were compared to examine

whether IAA production, lactone signalling, lignin peroxidase and xylanase activity were

linked. Activity of the partially purified protein samples of the Streptomyces isolates

JLS1-C4, JLS2-D6, KT1-B1, JLS1-F12, JLS1-C12 seem to correspond positively with

the amount of xylanase activity observed in the 0.1% (w/v) birchwood xylan agar plate

assay (Figure III.16). The four most enzymatically active strains JLS1-C4, JLS1-F12,

JLS2-D6 and KT1-B1 produce the largest clearing zones. Streptomyces isolate JLS1-A6

did not exhibit a high degree of xylanase-specific activity in liquid culture, yet produced a

large degradation zone on solid medium (Table III.9). A statistical difference in the

amount of IAA produced in the absence and presence of tryptophan was found and was

expected (p < 0.5; student T test at 0.95% confidence interval). IAA production in the

presence of tryptophan was higher for JLS1-F12, JLS1-C4, KT1-B1, producing 41.23,

13.48, 44.78 IAA equivalents respectively. Isolates JLS1-F12, JLS1-C4 and KT1-B1 had

slightly higher xylanase-specific activities of 0.125 µmol/min/mg, 0. 622 µmol/min/mg

and 0.706 µmol/min/mg compared to isolates JLS1-A6 and JLS1-C12 which had lower

xylanase-specific activities of 0.0243 µmol/min/mg and 0.0688 µmol/min/mg and IAA

production of 8.043 and 4.492 IAA equivalents in the presence of tryptophan. Isolate

JLS2-C7 produced 59.13 IAA equivalents in the presence of tryptophan, but did not

exhibit xylanase-specific activity while isolate JLS2-D6 did not produce any IAA

equivalents in the presence of tryptophan, but possessed xylanase-specific activity of

0.721 µmol/min/mg. The results suggest that higher IAA production in the presence of

82

tryptophan may correspond to higher amounts of xylanase-specific activity in specific

Streptomyces strains.

III.1.6.2. Constitutive or Inducible Nature of the Xylanase Enzymes of

Streptomyces Isolate JLS1-A6

In nature, the degradation of hemicellulose and its xylan constituent requires the

biosynthesis of cellulolytic and xylanolytic enzymes. Streptomyces isolate JLS1-A6 was

chosen to investigate whether xylanase activity was inducible or consititutive.The JLS1-

A6 isolate produced xylanase activity of 2.43 x 10-2 µmol/min/mg, an average

degradative clearing zone size measuring 12 mm on a 0.1% (w/v) birchwood xylan agar,

and lignin peroxidase activity of 0.1 Abs/min/mg. Streptomyces isolate JLS1-A6 crude

extracellular enzyme preparations did not exhibit xylanase activity when grown in

Minimal Medium containing 0.1% (w/v) glucose or when tested by well diffusion.

However, xylanase activity was observed from extracellular supernatants of JLS1-A6

cultivated in either Minimal Medium containing 0.1% (w/v) birchwood xylan or 0.1%

(w/v) birchwood xylan plus 0.1% (w/v) glucose (Figure III.17). To examine whether

nutritional amendments to the growth medium could influence xylanase activity of JLS1-

A6, 0.1 % (w/v) birchwood xylan, 0.001 % (w/v) xylose, 0.001 % (w/v) yeast extract,

and 0.02 % (w/v) glucose were separately added to Minimal Medium agar. Streptomyces

colony size as well as xylan degradation were measured and compared. Statistical

analysis showed that the carbon source used does not alter the degradation of the xylan

(Figure III.18).

83

Figure III.17. Assessment of the inducible nature of the xylanase enzyme of Streptomyces

isolate JLS1-A6 using well diffusion. Xyl = 0.1% (w/v) birchwood xylan liquid culture;

Glu = 0.1% (w/v) glucose liquid culture; Xyl + Glu = 0.1% (w/v) birchwood xylan and

0.1% (w/v) glucose liquid culture. A and B display replicates of 0.1% (w/v) birchwood

xylan agar media inoculated with filtered (filter size = 0.2 µm) extracellular crude

enzyme from a 0.1% (w/v) glucose Minimal Medium culture of Streptomyces JLS1-A6.

A lack of activity was observed in A and B and the control plate from three biological

replicates of Streptomyces environmental isolate JLS1-A6 liquid cultures containing

0.1% (w/v) glucose. Xylanase activity was observed in the wells in which the liquid

culture medium from three biological replicates of isolate JLS1-A6 contained only 0.1%

(w/v) birchwood xylan or both 0.1% (w/v) birchwood xylan and 0.1% (w/v) glucose. C)

Represents a control plate containing 0.1% (w/v) birchwood xylan agar medium.

84

Figure III.18. Induction of xylan degradation by eight Streptomyces isolates with varying

carbon sources. Clearing zones which represent xylan degradation were measured in

triplicate after growing 2.3 x 105 spores of Streptomyces isolates for 6 days at 28°C on

Minimal Medium containing 0.1% (w/v) birchwood xylan agar amended with 0.001%

(w/v) xylose, 0.001% (w/v) yeast extract, 0.02% (w/v) glucose to assess induced

degradation. The degradation zone from the edge of the colony to the edge of the clear

zone by each isolate was recorded and statistically analyzed for all amendments using a

one-way ANOVA at a 95% confidence interval. The differences in clearing were not

found to be statistically significant. Error bars are standard deviation of triplicate

technical replicates.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Birchwood (0.1%) Xylose(0.001%) Yeast (0.001%) Glucose (0.02%)

Clea

ring

Zone

(mm

)

JLS-1 A6 JLS-1 C12 JLS-1 C4 JLS-1 F12 JLS-2 D6 JLS-2C7 KT-1 B8 KT-1 B1

85

III.1.6.3. Temperature and pH Effects on Xylanase Activity

Environmental factors such as temperature and pH can affect the rate of xylan

substrate degradation by xylanases released by Streptomyces. The size of degradation

zones produced by Streptomyces isolate JLS1-A6 on 0.1% (w/v) birchwood xylan were

used as indicators of increased or decreased xylanase activity. Degradation zone sizes

observed under varying pH (4, 5, 6, 7, 8, and 9) and temperature ranges (28°C, 37°C,

45°C) correspond with the amount of crude enzyme activity observed in the DNSA assay

(Figure III.19). The greatest mean enzymatic clearing observed (3.8 mm), as determined

by measuring each colony from the edge of the colony to the edge of the clearing zone,

was found to be statistically significant from all other clearing zone sizes produced at pH

4, 5, 6, 8 and 9, using a one-way ANOVA (p = 0.03) at 95% confidence interval. The

largest clearing zone was observed in the xylan agar medium adjusted to pH 7 using

sodium phosphate buffer at 28°C (Figure III.19, III.20). When the effects of temperature

were examined, the largest degradation zone observed was 11 mm at 45°C in 0.1% (w/v)

birchwood xylan agar (pH 7) (Figure III.19) and was found to be statistically significant

using a one-way ANOVA (p = 0.01). Crude xylanase activity of JLS1-A6 was

determined to be 1.0 x 10-2 µmol/min/mg at pH 5, pH 6 and pH 7 (Figure III.20).

Maximum levels of extracellular xylanase activity at all pH and temperatures tested were

observed on days 12 and 13. A maximum xylanase activity of 1.56 x 10-3 µmol/min/mg

was observed at pH 4 and 28°C on day 13. Statistical analysis of the maximum specific-

xylanase activity values indicate that xylanase activity at pH 4 was statistically different,

(p = 0.01) at a 95% confidence interval, than xylanase activities observed at all other

reaction temperatures and pH values (Figure III.23, III.24, III.25, III.26 and III.27).

86

Increasing the reaction temperature from 37°C to 60°C did not significantly alter

xylanase production, (p = 0.1) at a 95% confidence interval. These results suggest that the

crude xylanases of Streptomyces isolate JLS1-A6 are optimally active at pH 4 and 28°C.

87

Figure III.19. pH and temperature optimization for Streptomyces extracellular xylanase

degradative activity in 0.1% (w/v) birchwood xylan agar. A) Xylanase degradation of

0.1% (w/v) birchwood xylan agar at pH 4 by isolate JLS1-A6 for 24 h at 28°C. B)

Xylanase degradation of 0.1% (w/v) birchwood xylan agar at pH 7 by isolate JLS1-A6

for 24 h at 28°C. C) Xylanase degradation of 0.1% (w/v) birchwood xylan agar at pH 10

by isolate JLS1-A6 for 24 h at 28°C. D) Xylanase degradation of 0.1% (w/v) birchwood

xylan agar at pH 7 by isolate JLS1-A6 for 24 h at 28°C. E) Xylanase degradation of 0.1%

(w/v) birchwood xylan agar at pH 7 by isolate JLS1-A6 for 24 h at 37°C. F) Xylanase

degradation of 0.1% (w/v) birchwood xylan agar at pH 7 by isolate JLS1-A6 for 24 h at

45°C. G) Degradative clearing of 0.1 % (w/v) birchwood xylan agar medium of pH 4, 7,

and 10 at 28°C. H) Degradative clearing of 0.1 % (w/v) birchwood xylan agar medium at

pH 7 after 24h incubation at 28°C, 37°C and 45°C. (*) Indicates no degradation observed.

88

Figure III.20. Specific-xylanase activity of Streptomyces isolate JLS1-A6 on 0.1% (w/v)

birchwood xylan substrate. Xylanase activity was investigated using DNSA

(dinitrosalicyclic acid) a pH range of 4, 5, 6, 7, 8, 9 and temperatures of 28°C, 37°C, and

45°C. The range of 50 mM buffers used to prepare a 0.1% (w/v) birchwood xylan

solution were sodium phosphate buffer (pH 5, 6, 7), Tris buffer (pH 8 and 9) and aceto-

acetate buffer (pH 4). Temperatures for enzyme activity were assessed at 28°C, 37°C and

45°C. Absorbance was measured at 575 nm using a xylose standard (one unit = 1 µmole

xylose/min/mg). Error bars represent standard deviation of three biological replicates.

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

pH 4 pH 5 pH 6 pH 7 pH 8 pH 9

Spec

ific

Activ

ity (μ

mol

/min

/mg)

pH

28 °C 37 °C 45 °C

89

Figure III.21. Growth curve and protein content of Streptomyces strain JLS1-A6.The

growth curve was based on JLS1-A6 wet mass (mg wet wt) and extracellular protein

(µg/mL) over a period of 13 days at 28°C of two biological replicates. Culture sampling

was performed every 24 h in triplicate for wet weight and for protein content. Error bars

represent standard deviation of triplicate technical measurements.

0

50

100

150

200

250

300

350

00.0010.0020.0030.0040.0050.0060.0070.008

0 2 4 6 8 10 12 14

Prot

ein

(μg/

mL)

Gro

wth

(mg

wet

wt)

Time (Days)

Growth curve of biological replicates of strain JLS1-A6

Protein content of biological replicates of strain JLS1-A6

90

Figu

re II

I.22.

Cru

de e

xtra

cellu

lar x

ylan

ase

activ

ity o

bser

ved

durin

g a

time

cour

se e

xper

imen

t of S

trep

tom

yces

isol

ate

JLS1

-A6

grow

n

in M

inim

al M

ediu

m w

ith 0

.1%

(w/v

) birc

hwoo

d xy

lan

and

asse

ssed

usi

ng a

reac

tion

carr

ied

out a

t pH

4. T

wo

biol

ogic

al re

plic

ates

of

Stre

ptom

yces

isol

ate

JLS1

-A6

wer

e as

sess

ed o

ver t

he c

ours

e of

13

days

for e

xtra

cellu

lar x

ylan

ase

activ

ity. A

ceto

-ace

tate

buf

fer w

as

used

to te

st e

xtra

cellu

lar x

ylan

ase

activ

ity a

t pH

4 w

hile

reac

tion

tem

pera

ture

s of 2

8°C

, 37°

C, 4

5°C

and

60°

C w

ere

used

to a

sses

s

tem

pera

ture

eff

ects

on

extra

cellu

lar x

ylan

ase

activ

ity. E

xtra

cellu

lar x

ylan

ase

activ

ity w

as m

easu

red

in tr

iplic

ate

usin

g a

DN

S

colo

urom

etric

ass

ay. E

rror

bar

s rep

rese

nt th

e st

anda

rd d

evia

tion

of th

ree

tech

nica

l rep

licat

es.

00.

0002

0.00

040.

0006

0.00

080.

001

0.00

120.

0014

0.00

160.

0018

0.00

2

12

34

56

78

910

1112

13

Xylanase Specific Activity (μmol/min/mg)

Tim

e (D

ays)

Reac

tion

Tem

pera

ture

28°

CRe

actio

n Te

mpe

ratu

re 3

7°C

Reac

tion

Tem

pera

ture

45°

CRe

actio

n Te

mpe

ratu

re 6

0°C

91

Figu

re II

I.23.

Cru

de e

xtra

cellu

lar x

ylan

ase

activ

ity o

bser

ved

durin

g a

time

cour

se e

xper

imen

t of S

trep

tom

yces

isol

ate

JLS1

-A6

grow

n

in M

inim

al M

ediu

m w

ith 0

.1%

(w/v

) birc

hwoo

d xy

lan

and

asse

ssed

usi

ng a

reac

tion

carr

ied

out a

t pH

5. T

wo

biol

ogic

al re

plic

ates

of

Stre

ptom

yces

isol

ate

JLS1

-A6

wer

e as

sess

ed o

ver t

he c

ours

e of

13

days

for e

xtra

cellu

lar x

ylan

ase

activ

ity. A

ceto

-ace

tate

buf

fer w

as

used

to te

st e

xtra

cellu

lar x

ylan

ase

activ

ity a

t pH

5 w

hile

reac

tion

tem

pera

ture

s of 2

8°C

, 37°

C, 4

5°C

and

60°

C w

ere

used

to a

sses

s

tem

pera

ture

eff

ects

on

extra

cellu

lar x

ylan

ase

activ

ity. E

xtra

cellu

lar x

ylan

ase

activ

ity w

as m

easu

red

in tr

iplic

ate

usin

g a

DN

S

colo

urom

etric

ass

ay. E

rror

bar

s rep

rese

nt th

e st

anda

rd d

evia

tion

of th

ree

tech

nica

l rep

licat

es.

0

0.00

05

0.00

1

0.00

15

0.00

2

12

34

56

78

910

1112

13

Xylanase Specific Activity (μmol/min/mg)

Tim

e (D

ays)

Reac

tion

Tem

pera

ture

28°

CRe

actio

n Te

mpe

ratu

re 3

7°C

Reac

tion

Tem

pera

ture

45°

CRe

actio

n Te

mpe

ratu

re 6

0°C

92

Figu

re II

I.24.

Cru

de e

xtra

cellu

lar x

ylan

ase

activ

ity o

bser

ved

durin

g a

time

cour

se e

xper

imen

t of S

trep

tom

yces

isol

ate

JLS1

-A6

grow

n

in M

inim

al M

ediu

m w

ith 0

.1%

(w/v

) birc

hwoo

d xy

lan

and

asse

ssed

usi

ng a

reac

tion

carr

ied

out a

t pH

6. T

wo

biol

ogic

al re

plic

ates

of

Stre

ptom

yces

isol

ate

JLS1

-A6

wer

e as

sess

ed o

ver t

he c

ours

e of

13

days

for e

xtra

cellu

lar x

ylan

ase

activ

ity. A

ceto

-ace

tate

buf

fer w

as

used

to te

st e

xtra

cellu

lar x

ylan

ase

activ

ity a

t pH

6 w

hile

reac

tion

tem

pera

ture

s of 2

8°C

, 37°

C, 4

5°C

and

60°

C w

ere

used

to a

sses

s

tem

pera

ture

eff

ects

on

extra

cellu

lar x

ylan

ase

activ

ity. E

xtra

cellu

lar x

ylan

ase

activ

ity w

as m

easu

red

in tr

iplic

ate

usin

g a

DN

S

colo

urom

etric

ass

ay. E

rror

bar

s rep

rese

nt th

e st

anda

rd d

evia

tion

of th

ree

tech

nica

l rep

licat

es.

0

0.00

05

0.00

1

0.00

15

0.00

2

12

34

56

78

910

1112

13

Xylanase Specific Activity (μmol/min/mg)

Tim

e (D

ays)

Reac

tion

Tem

pera

ture

28°

CRe

actio

n Te

mpe

ratu

re 3

7°C

Reac

tion

Tem

pera

ture

45°

CRe

actio

n Te

mpe

ratu

re 6

0°C

93

Figu

re II

I.25.

Cru

de e

xtra

cellu

lar x

ylan

ase

activ

ity o

bser

ved

durin

g a

time

cour

se e

xper

imen

t of S

trep

tom

yces

isol

ate

JLS1

-A6

grow

n

in M

inim

al M

ediu

m w

ith 0

.1%

(w/v

) birc

hwoo

d xy

lan

and

asse

ssed

usi

ng a

reac

tion

carr

ied

out a

t pH

7. T

wo

biol

ogic

al re

plic

ates

of

Stre

ptom

yces

isol

ate

JLS1

-A6

wer

e as

sess

ed o

ver t

he c

ours

e of

13

days

for e

xtra

cellu

lar x

ylan

ase

activ

ity.

Sodi

um p

hosp

hate

buf

fer

was

use

d to

test

ext

race

llula

r xyl

anas

e ac

tivity

at p

H 7

whi

le re

actio

n te

mpe

ratu

res o

f 28°

C, 3

7°C

, 45°

C a

nd 6

0°C

wer

e us

ed to

ass

ess

tem

pera

ture

eff

ects

on

extra

cellu

lar x

ylan

ase

activ

ity. E

xtra

cellu

lar x

ylan

ase

activ

ity w

as m

easu

red

in tr

iplic

ate

usin

g a

DN

S

colo

urom

etric

ass

ay. E

rror

bar

s rep

rese

nt th

e st

anda

rd d

evia

tion

of th

ree

tech

nica

l rep

licat

es.

0

0.00

05

0.00

1

0.00

15

0.00

2

12

34

56

78

910

1112

13

Xylanase Specific Activity (μmol/min/mg)

Tim

e (D

ays)

Reac

tion

Tem

pera

ture

28°

CRe

actio

n Te

mpe

ratu

re 3

7°C

Reac

tion

Tem

pera

ture

45°

CRe

actio

n Te

mpe

ratu

re 6

0°C

94

Figu

re II

I.26.

Cru

de e

xtra

cellu

lar x

ylan

ase

activ

ity o

bser

ved

durin

g a

time

cour

se e

xper

imen

t of S

trep

tom

yces

isol

ate

JLS1

-A6

grow

n

in M

inim

al M

ediu

m w

ith 0

.1%

(w/v

) birc

hwoo

d xy

lan

and

asse

ssed

usi

ng a

reac

tion

carr

ied

out a

t pH

8. T

wo

biol

ogic

al re

plic

ates

of

Stre

ptom

yces

isol

ate

JLS1

-A6

wer

e as

sess

ed o

ver t

he c

ours

e of

13

days

for e

xtra

cellu

lar x

ylan

ase

activ

ity.

Tris

buf

fer w

as u

sed

to te

st

extra

cellu

lar x

ylan

ase

activ

ity a

t pH

8 w

hile

reac

tion

tem

pera

ture

s of 2

8°C

, 37°

C, 4

5°C

and

60°

C w

ere

used

to a

sses

s tem

pera

ture

effe

cts o

n ex

trace

llula

r xyl

anas

e ac

tivity

. Ext

race

llula

r xyl

anas

e ac

tivity

was

mea

sure

d in

trip

licat

e us

ing

a D

NS

colo

urom

etric

ass

ay.

Erro

r bar

s rep

rese

nt th

e st

anda

rd d

evia

tion

of th

ree

tech

nica

l rep

licat

es.

0

0.00

05

0.00

1

0.00

15

0.00

2

12

34

56

78

910

1112

13

Xylanase Specific Activity (μmol/min/mg)

Tim

e (D

ays)

Reac

tion

Tem

pera

ture

28°

CRe

actio

n Te

mpe

ratu

re 3

7°C

Reac

tion

Tem

pera

ture

45°

CRe

actio

n Te

mpe

ratu

re 6

0°C

95

Figu

re II

I.27.

Cru

de e

xtra

cellu

lar x

ylan

ase

activ

ity o

bser

ved

durin

g a

time

cour

se e

xper

imen

t of S

trep

tom

yces

isol

ate

JLS1

-A6

grow

n

in M

inim

al M

ediu

m w

ith 0

.1%

(w/v

) birc

hwoo

d xy

lan

and

asse

ssed

usi

ng a

reac

tion

carr

ied

out a

t pH

9. T

wo

biol

ogic

al re

plic

ates

of

Stre

ptom

yces

isol

ate

JLS1

-A6

wer

e as

sess

ed o

ver t

he c

ours

e of

13

days

for e

xtra

cellu

lar x

ylan

ase

activ

ity.

Tris

buf

fer w

as u

sed

to te

st

extra

cellu

lar x

ylan

ase

activ

ity a

t pH

9 w

hile

reac

tion

tem

pera

ture

s of 2

8°C

, 37°

C, 4

5°C

and

60°

C w

ere

used

to a

sses

s tem

pera

ture

effe

cts o

n ex

trace

llula

r xyl

anas

e ac

tivity

. Ext

race

llula

r xyl

anas

e ac

tivity

was

mea

sure

d in

trip

licat

e us

ing

a D

NS

colo

urom

etric

ass

ay.

Erro

r bar

s rep

rese

nt th

e st

anda

rd d

evia

tion

of th

ree

tech

nica

l rep

licat

es.

0

0.00

05

0.00

1

0.00

15

0.00

2

12

34

56

78

910

1112

13

Xylanase Specific Activity (μmol/min/mg)

Tim

e (D

ays)

Reac

tion

Tem

pera

ture

28°

CRe

actio

n Te

mpe

ratu

re 3

7°C

Reac

tion

Tem

pera

ture

45°

CRe

actio

n Te

mpe

ratu

re 6

0°C

96

IV. CHAPTER 4, DISCUSSION

Since the isolates used in the present study were previously uncharacterized

environmental isolates, a preliminary taxonomic and phylogenetic analysis was

performed. Phylogenetic analysis by Neighbour-Joining (Saitou & Nei, 1987; Altschul,

1990) showed that the eight isolates grouped into five different clades (Figure III.1). To

determine the relatedness of xylanases secreted by each of the eight isolates, xylanase

translated gene sequence rather than 16S rRNA gene sequence would need to be

compared (Torronen et al., 1993).

The effect which neighbouring Streptomyces isolates had on xylan degradation

when grown in close proximity to each other was investigated. As xylan is a complex

substrate, it is no surprise that its degradation requires multiple enzymatic activities.

Sequential degradation of xylan requires several enzymes to first de-branch the xylan

backbone and subsequently cleave the β-1,4-glycosidic linkages between adjacent

xylopyranose units. Raweesri et al. (2008) reported the enzyme kinetics of purified

extracellular enzymes and demonstrated that crude xylanase preparations, such as those

used in this thesis, function at higher temperatures than purified enzymes due to the xylan

substrate, protecting the conformational change of xylanases from thermal shock

(Ratanachomsri et al., 2006). However, our data indicates that the optimal xylanase-

specific activity for the JLS1-A6 isolate was encountered at 28°C which is the lowest

reaction temperature utilized during the time course experiment. A possible explanation

could be the difference in xylanases produced by the streptomycete isolate investigated

which clearly was acidotolerant while that of Raweesri et al. (2008) was alkitolerant.

97

A knowledge gap exists in the discovery of an extracellularly secreted enzyme

system capable of efficiently degrading natural lignocellulose and hemicellulose and its

xylan constituent effectively (Peng et al., 2010).

Streptomyces isolates KT1-B1 possessed the highest xylanase-specific activity

(7.06 x 10-1 µmol/min/mg) and also produced a large clearing zone indicative of xylan

degradation on solid media. This appeared to be a trend amongst the other most

enzymatically active isolates JLS1-C4, JLS2-D6, JLS1-F12 (Figure III.16). In retrospect,

it would have been best to choose one of these enzymatically active isolates for xylanase

characterization instead of JLS1-A6.

Obtaining adequate concentrations of partially purified protein from isolate KT1-

B1 proved to be difficult due to the slow growth of the KT1-B1 isolate in liquid culture in

comparison to all other strains and low concentration of extracellularly secreted protein

obtained during ammonium sulphate precipitation.

Isolate JLS1-A6 did not exhibit much xylanase-specific activity in liquid culture,

yet produced a zone of xylan degradation of similar size compared to KT1-B1.

Streptomyces isolate JLS1-A6 showed a limited number of extracellular proteins, but

whether or not any of these proteins is responsible for the observed xylanase activity on

agar plates is unknown. As previously mentioned the assessment of xylan degradation

was performed using partially purified enzyme obtained from liquid culture. Mycelial

growth and development differ in both liquid and solid media. In a solid medium

Streptomyces undergoes morphological differentiation (Chater & Hopwood, 1993). In

liquid medium Streptomyces mycelia grow as large aggregates. Differences in culture

medium can lead to many biochemical and morphological changes, causing differences in

98

the secretion of xylanases and uptake of nutrients by each isolate. The isolate KT1-B1,

although shown to be most efficient degrader in this study in isolation, exhibited even

more substrate degradation when grown in the presence of neighbouring strains.

A structural reason for hemicellulose and xylooligosaccharide hydrolysis resistance

is large amounts of acetylation present within hemicelluloses (Beg, 2001). Enzymes such

as acetyl esterases, which liberate acetyl groups from acetylated polysaccharides,

cooperate with xylanase and β-xylosidase during the hydrolysis of birchwood xylan

(Chungool, 2008). The combination of secreted enzymes from the neighbouring strains

may have led to an increase in the observed xylan degradation by Streptomyces isolate

KT1-B1.

The overall production of extracellular xylan-degrading enzymes by Streptomyces

will dictate their rate of xylooligosaccharide degradation and thus the production of easily

fermentable sugars, such as xylose. The use of both oat-spelt and birchwood xylan

substrate was useful in examining the spectrum of xylanase activity. The debranching and

degradation of xylan depends upon the presence of a suitable substrate for specific

binding (Beily, 1985). The overall action of xylanase enzymes depends in part on their

individual specific activities and accumulation of degradation products. Debranching

enzymes, such as α-L-arabinofuranosidases, work together with xylanases as part of the

xylanolytic enzyme system to degrade xylan. In the present study, a larger degradation

zone was observed on oat-spelt xylan-containing growth medium and reflects the results

reported in the literature in which oat-spelt xylan was shown to possess a higher degree of

branching producing xylooligosaccharides of higher degrees of polymerization

(Kormelink & Voragen, 1993; Elegir et al., 1994; Sun et al., 2002) (Figure III.8). For

99

example, one study has shown an increase in enzymatic specific activity of acetyl ester

xylanases on oat-spelt xylan indicating the presence of acetyl O-groups substituents

which need to be removed from the xylan backbone moiety (Dupont, 1996). The presence

of acetyl ester xylans in combination with the amount of acetylated polysaccharides in the

substrate is another example of how the rate of xylan degradation can be affected by the

type of xylan substrate structure (Dupont, 1996). Although the structure of the xylan,

whether it is oat-spelt or birchwood xylan influences the extent of degradation, it must

also be mentioned that the enzyme specificities also play a key role in xylan degradation.

Endoxylanase isoenzymes have been shown to possess different activities to produce

xylose, xylobiose and xylooligosaccharides in Streptomyces sp. B-12-2 (Elegir et al.,

1994).

Streptomyces use quorum sensing to assess the population density of surrounding

bacteria regulating gene expression for developmental changes, such as sporulation

(Takano, 2006; Nishida et al., 2007; Kato et al., 2007). Streptomyces synchronize gene

expression and development to their surrounding environment by secreting signalling

molecules, such as gamma-butyrolactones, into the environment (Lazazzera, 1999;

Chater, 2001). Peptide molecules accumulate within the local environment and their

presence can be measured using biosensors such as the pJBA132 plasmid as used in this

study. The pJBA132 plasmid upon contact with autoinducer molecules will express a

green fluorescence protein which becomes excited from exposure to blue light

(Williamson et al., 2005) (Figure IV.1). When the protein is struck by UV light, its

electrons rise to an excited state and upon termination of the UV light signal the electrons

return to a ground state emitting photons which can be observed (Williamson et al.,

100

2005). The ability of the green fluorescent protein to be expressed is due to a LuxR

transcriptional activator. The presence of fluorescence was observed visually in many of

the Streptomyces isolates including KT1-B1, KT1-B8, JLS1-A6, JLS1-C4, and JLS2-D6,

using the LuxI gene at low level concentration (Figure III.4). The dynamics of quorum

sensing of acyl- homoserine lactones (acyl-HSL) (Eberhard et al., 1981) or gamma-

butyrolactones (GBLs) (Takano, 2006) and relation to bioluminescence begin with the

production of basal levels of lactones which are structurally similar to that of the LuxI

type protein (Stevens & Greenburg, 1995; Williamson et al., 2005). Gamma-

butyrolactones are structurally similar to acyl-homoserine lactones, differing only by a

carbon side chain, and are secreted in working concentrations of 10-8 to 10-9 M in

Streptomyces (Teplitski et al., 2000; Takano, 2006; Yang et al., 2005). Streptomyces

secrete gamma-butyrolactones (2,3-di-substituted-gamma-butyrolactones) in response to

changes in population density (Du et al., 2011). As more gamma-butyrolactone molecules

are produced by the Streptomyces the molecules begin diffusing down the concentration

gradient within the lipid bilayer of the cell and out into the extracellular environment.

This is possible because of the hydrophilic lactone ring and the hydrophobic side chain

allowing the molecule to be amphipathic (Fuqua et al., 2001). When the threshold level of

localized lactones is exceeded by individual production of basal levels of gamma-

butyrolactones by each individual streptomycete the gamma-butyrolactone accumulation

interacts with a transcription factor controlling expression of quorum sensing genes

which can include the LuxI homolog (Hentzer & Givskov, 2003; Koch et al., 2005).

As previously mentioned the developmental stage and gene expression in

Streptomyces is influenced by the presence of lactone molecules (Teplitski et al., 2000).

101

As the population density of both the Streptomyces and surrounding bacterial population

increase, nutrients become depleted. Quorum sensing is utilized to control cell growth

and communicate with members of the same or similar species to adapt to new

environments (Ishihama, 1999). The initiation of sporulation in Streptomyces is most

effective when cells are crowded and nutrients are depleted (Lazazzera, 1999).

The possibility of a link between the efficiency of the xylan degradation of

Streptomyces in a xylan-rich medium and the secretion of lactone molecules was

investigated in order to assess if lactone signalling can mediate the degree of xylan

degradation or vice versa. When examining the eight Streptomyces isolates in the

presence of the E. coli MT102 it was observed that simultaneous inoculation of the

Streptomyces isolates with the E. coli MT102 reporter strain produced fluorescence from

all strains in a shorter period of time (48 h) than when a mature Streptomyces colony was

inoculated with E. coli MT102 (168 h) (Table III.3, III.4). This served as a preliminary

assay to determine whether or not lactone signalling was present between these strains.

Of the eight Streptomyces isolates KT1-B1, KT1-B8, JLS1-A6, JLS1-C4, and JLS2-D6

displayed putative lactone signalling while JLS1-C12 and JLS1-F12 did not. There

appears to be a relationship in which the presence of lactone signalling is linked to more

degradation of xylan substrate (Table III.9). Streptomyces isolate JLS2-C7 did not allow

the growth of E. coli MT102 suggesting the production of an antibiotic by this isolate.

Whether the inhibitory molecule produced by JLS2-C7 is an antibiotic which is selective

against gram negative bacteria requires further investigation. It is also possible that a

lactone molecule produced by JLS2-C7 could have inhibited the growth of the gram

102

negative E. coli MT102 as AHL molecules have the capability to inhibit other signals

(Pai & You, 2009).

The delayed fluorescence of the green fluorescent protein observed during the

sequential inoculation quorum sensing assay may have been due to an increased acidic

environment associated with extracellularly secreted enzymes surrounding the mycelia

(Susstrunk et al., 1998). Studies have presented a variety of acids produced by

Streptomyces, such as benzoic, pyruvic, α-ketoglutaric, citric, succinic, lactic and oxalic

acids when in aggregate culture or filamentous mats, and that acidic environments can

degrade AHL molecules (Leirmann et al., 2000; Rozycki & Strzelczyk, 1986). Neither

the secretion of organic acids into the medium by any of the isolates nor evidence of a

lowered pH was obtained in this study. While the preliminary data show that lactone

signalling is present in Streptomyces isolates KT1-B1, KT1-B8, JLS1-A6, JLS1-C4, and

JLS2-D6 the details as to the type of lactone or quorum sensing molecules which are

being exchanged between isolates is still unknown. In a study by Yang et al., 2005

butanolides from Streptomyces coelicolor were detected using His-tagged receptor

proteins and electrospray tandem mass spectrometry.

Many Streptomyces have the ability to secrete IAA which is a plant hormone

coordinating growth activity in the young tissues of plants (Li et al., 2009). Some

Streptomyces have the ability to produce and secrete IAA when grown in the absence or

presence of L-tryptophan. It has been hypothesized that IAA serves as an endogenous

regulator of spore germination and Streptomyces differentiation (El-Raheem & El-

Shanshoury, 1991). The link between IAA and xylanase production by Streptomyces has

not to our knowledge been previously investigated. We observed an increase in the

103

amount of IAA produced by the eight Streptomyces isolates when they were grown in the

presence of tryptophan compared to conditions in which exogenous tryptophan was not

made available (Table III.5 and Table III.9).

No relationship between the amount of IAA produced and degradation was observed.

To determine the cause of the observed change in the xylanase-specific activities

between the JLS2-C7 and JLS2-D6 Streptomyces isolates, the effect of culture

supernatant on activity was tested (Figure III.13). The observed clearing zone produced

by the JLS2-C7 supernatant in the presence of both filtered or autoclaved JLS2-D6

supernatant was unaltered, suggesting that the molecule responsible for the change in

activity was not a protein. Autoclaving would denature any proteins present in the culture

supernatant (Figure III.13). To ascertain whether a protein was responsible for the

decrease in degradation observed for JLS2-C7 on agar medium (Figure III.14), culture

supernatants could be treated with a protease. Protease-treated supernatants would be

expected to increase the size of the clearing zone produced by JLS2-C7 if a protein were

the responsible effector molecule. Protease untreated supernatants would be expected to

decrease he size of the xylanase-specific degradative clearing zone produced by isolate

JLS2-C7. If the molecule responsible for inactivation was a lactone molecule, it is

expected that inhibition would still be observed after autoclaving. The lysis of a gamma-

butyrolactone molecule occurs if the lactone ring is opened; with ring opening being

determined by both ring strain of the gamma-butyrolactone isomer and reaction

conditions (Moore et al., 2005; Rasmussen & Givskov, 2006). Gamma-butyrolactones

have been shown to withstand temperatures of 165°C with polymerization occuring at

180°C, due to ring opening (Korte & Glet, 1966; Mao et al., 2003). The heat stability of

104

gamma-butyrolactones may allow them to maintain conformation under autoclaving

temperatures of 121°C.

Xylan contains acetyl, α-arabinofuranosyl and 4-O-methyl-D-glucuronosyl residues

joined to a β-1,4-linked xylan backbone (Rahman et al., 2003). It has been demonstrated

that the degradation of xylan involves the sequential attack of xylan by debranching

enzymes acetylesterase, α-L-arabinofuranosidase, and α-glucuronidase to remove side

chains substituents of heteroxylans in a cooperative manner leaving constituent sugars.

Specific xylanases, such as endo-1,4-β-xylanase, hydrolyze β-1-4 glycosidic linkages of

the xylan backbone randomly, using an endo-type of attack, to form short

xylooligosaccharides which results in decreased polymerization and further degradation

into xylobiose and xylose end products.

The extracellular proteins must be further investigated to determine whether any of

them are indeed xylanases. Zymographic analysis is one way in which this could be

accomplished (Royer & Nakas, 1990; Nakamura et al., 1993; Rawashdesh et al., 2005).

Access to the xylan components in plants is governed by lignocellulose

degradation. Many species of actinomycetes, including Streptomyces, secrete lignin

peroxidases extracellularly to break down lignocellulose in order to access hemicellulosic

and xylan substituents (Mason et al., 2001). In vivo lignin degradation is highly

dependent on the oxidative system utilized by the Streptomyces strain with the final

structure of degraded lignin monomers being controlled by the physiochemical

constrains, such as amount of lignin (Demont-Caulet et al., 2010).

105

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106

During the degradation of lignin, a carbohydrate intermediate called acid

precipitatable polymeric lignin (APPL) is produced (Crawford et al., 1983). APPL was

shown to cause an enhanced effect on peroxidase, esterase, endoglucanase and xylanase

activity (Ramachandra et al., 1987). However, isolates in this study were not in the

presence of a lignin substrate and it is important to take that into consideration when

forming conclusions about the levels of lignin peroxidase (LiP) released. The observed

amounts of lignin peroxidase activity from the eight Streptomyces environmental isolates

were determined using an Azure B dye decolourization assay in Minimal Media

supplemented with glucose and xylan. The Streptomyces environmental isolates used in

this study produced higher levels of LiP activity when birchwood xylan was a carbon

source rather than glucose (Table III.8). Glucose has been shown to cause a repression of

LiP accumulation often leading to a delay in peak LiP activity due to glucose regulation

by Streptomyces viridosporus T7A; upon depletion of glucose in the medium LiP activity

was shown to rise (Zerbini et al., 1999). Glucose regulation by the environmental isolates

may have accounted for the low levels of LiP activity observed from all eight isolates.

Xylan has been shown to be rapidly used as a carbon source yielding more LiP activity in

Streptomyces sp. F2621 (Tuncer et al., 2004). LiP is secreted at low levels into

extracellular supernatant (Tuncer et al., 2004).

Catabolite repression of xylanase activity has been reported in a study by Bertrand

et al. (1989) where 1% (w/v) glucose was added to a 1% (w/v) xylan solution such that

xylanase activity was repressed until all glucose had been metabolized. Catabolite

repression could have accounted for the lack of activity observed from the JLS1-A6

isolate cultured in minimal media supplemented with glucose, but does not explain the

107

xylanase activity observed from JLS1-A6 isolate cultured in Minimal Media

supplemented with glucose and xylan (Figure III.17). Therefore, it would appear that the

putative xylanase activity of Streptomyces isolate JLS1-A6 is inducible (Figure III.17).

To confirm this, the JLS1-A6 isolate would need to be cultured in Minimal Medium

supplemented with glucose, to validate a lack of xylanase activity, then subsequently

have xylan, which has been washed in order to eliminate xylose sugar, added to the

culture and reassessed for xylanase activity using well diffusion. The induction of

xylanases from mycelia grown on xylan-containing medium has been reported for

Streptomyces lividans and Streptomyces flavogriseus strains. However, when these strains

were grown on cellulose medium only residual amounts of xylanase activity could be

detected (Kluepfel et al., 1986; Ishaque & Kluepfel, 1981).

During the time course assessment of extracellular xylanase activity for

Streptomyces JLS1-A6, frequent spikes in xylanase activity was observed at pH 4 (Figure

III.22). Studies have shown that many Streptomyces spp. prefer alkaline environments for

growth (Leirmann, 2000; Elegir, 1994). However, Streptomyces strains gathered from

forest soil can withstand and often proliferate in conditions as low as pH 4 depending on

the acidity of the soil environment from which the Streptomyces strain was isolated. A

study, performed by Hagedorin et al. (1984), investigated carbon source utilization of

acidotolerant Streptomyces strains and found that carbon source utilization increased

when the growth medium was adjusted from a neutral to a more acidic pH.

No extracellular protein was detectable in the culture medium of isolate JLS1-A6

until day five of sampling. Preliminary assessment of the growth cycle of Streptomyces

isolate JLS1-A6 suggested completion of the life cycle after 13 days, but that this does

108

not necessarily correlate with extracellular enzyme activity. It is recognized that the time-

course assay could have been performed better. For example, a more appropriate isolate

could have been chosen. This mistake was made by not analyzing the data in a timely

manner and choosing the isolate randomly. The xylan substrate should have been washed

thoroughly to remove any available xylose which would be used preferentially by the

organism. Also, the data for xylanase activity could have been analyzed as the data was

collected so that the peak in xylanase activity could have been determined. Another flaw

in the experiment was the lack of a positive and negative control. While Streptomyces

viridosporus T7A, a well-studied degrader and xylanase producer was available, it was

not utilized as a control. Finally, the experiment should not have been terminated until it

had been unequivocally demonstrated that xylanase activity had peaked.

Further investigation using pure enzyme would be necessary to fully characterize

the type of optimized xylanase activity produced by any of the isolates used in this

investigation. In hindsight an environmental isolate exhibiting a high xylanase-specific

activity in liquid culture, such as KT1-B1, may have been more appropriate. To

determine the functionality of the extracellular xylanase(s) of an appropriately chosen

isolate, thin layer chromatography could be used to elucidate the lengths of the resultant

xylooligosaccharides produced during degradation (Morosoli et al., 1986), and the

stereochemical course of substrate hydrolysis analyzed using proton NMR spectroscopy

both before and after the addition of xylanases (Gebler et al., 1992). Based on the lengths

of observed xylooligosaccharide end products the xylanase-specific activity could be

matched with those activities of particular glycosyl hydrolase families documented within

109

the CAZY database (Coutinho and Henrissat’s Carbohydrate-Active Enzymes server at

http://afmb.cnrs-mrs.fr/~cazy/CAZYindex.html).

110

V. CONCLUSION

Enzymes are at the forefront of many industrial processes and may reduce the cost

of production of industrially important commodities by saving on energy consumption.

Xylan degradation is important for industries such as feedstock optimization,

bioconversion of lignocellulose to fermentable products, pulp and paper bleaching, juice

clarification and many others (Bajpai, 1999; Beily, 1985; Kirk et al., 2002). Streptomyces

are the dominant xylanolytic species of Actinobacteria using enzymes, such as xylanases,

which are specific in their mode of action and active at mild temperatures to degrade

recalcitrant plant biomass.

In this study, eight Streptomyces isolates were investigated. One isolate, JLS1-A6

was chosen for detailed characterization. Well diffusion assays determined that xylanase

activity is inducible for isolate JLS1-A6. A time course experiment was utilized to

determine the timing of maximum xylanase activity. Maximum xylanase activity

produced by Streptomyces isolate JLS1-A6 was determined to be 1.56 x 10-3

µmol/min/mg and optimal conditions for extracellular xylanase activity were determined

to be pH 4 and 28°C. This is very low activity. While this isolate was an excellent

degrader on solid medium, the isolate under-performed in liquid culture.

111

VI. FUTURE DIRECTIONS

To further investigate the stimulatory effects of indole acetic acid on strain growth

and subsequent xylanase production, exogenous IAA could be applied to those isolates

proven to secrete indole acetic acid (Matsukawa, 2007; Mazzola & White, 1994). In order

to determine which proteins observed on SDS-PAGE gels were xylanases, zymography

incorporating a xylan overlay could be used (Royer & Nakas, 1990). Putative xylanases

must be purified to homogeneity for any useful characterizations to be performed. Kinetic

studies on purified protein could be used to determine the mode of action on a variety of

xylan substrates (Collins et al., 2002; Rahman et al., 2003).

112

VII. APPENDIX

VII.1. Introduction

VII.1.1. Transposon Mutagenesis

Transposon mutagenesis is a tool used to create random insertions using

transposable pieces of DNA (Chiang and Rubin, 2002). Transposon systems utilizing the

Tn5 transposon and the sequencing of rescue plasmids in order to identify the insertion

site are useful in this regard.

There is no general approach for transforming Streptomyces species (Petzke &

Luzhetskyy, 2009). In the present study, electroporation (Pigac & Schrempf, 1995), and

bacterial conjugation (Giebelhaus et al., 1996), were used to insert foreign DNA into

Streptomyces using the PBT20 mini transposon (Kulasekara et al., 2005), by a

transposase from a donor, E. coli SM10 (Simon et al., 1983), to a recipient Streptomyces.

The pBT20 plasmid utilizes a mariner transposon delivery plasmid that contains a R6K

gamma origin of replication which relies upon the presence of the pir gene for replication

(Kulasekara et al., 2005; Chiang & Rubin, 2002; Ferrieres et al., 2010) (Figure VII.2.3).

The eight isolates used in the present study were screened to ensure their sensitivity to the

appropriate antibiotics. Antibiotic selectivity was used to eliminate donor strains leaving

only knockout mutants which were detected using plate assays on 0.1% (w/v) birchwood

xylan agar medium supplemented with Gm50. A non-degradative phenotype would be

expected when compared to a xylan degrading wild type for the purpose of this study.

113

VII.2. METHODS

VII.2.1. Maintenance of Bacterial Strains

Plasmids and oligonucleotides used in this study are listed in tables VII.3.3, VII.3.4 and

VII.3.5.

VII.2.2. Assessment of Mutagenized Streptomyces Environmental

Isolate JLS1-A6

In retrospect, quality control steps and recognizing what a contaminant looks like

compared to the streptomycete isolate used would have been advantageous to ensure

putative mutants were actually derived from isolate JLS1-A6 and not from contaminants.

JLS1-A6 stocks and subsequent ‘mutants’ were assessed for contamination by plating

stocks onto a variety of media types including LB agar, potato dextrose agar (PDA; 24 g

Potato Dextrose Broth, 15 g agar per litre) and yeast dextrose agar (YDA) both with and

without 0.1% (w/v) birchwood xylan (Figure VII.2.1). The plates were visually assessed

for their morphological characteristics using a dissecting scope (Leica) (Figure VII.2.2).

Unfortunately, the putative mutants were butyrous and possessed none of the typical

morphological characteristics of a streptomycete.

VII.2.3. Growth Media and Storage Conditions

Patch plates of putative JLS1-A6 Tn mutants (which were later determined to be

contaminants) from transposon mutagenesis using the pBT20 mini transposon were

prepared by picking 196 ‘transformants’ and patch plating onto LB medium

supplemented with 50 µg/ml gentamycin. Patch plates were stored at 4°C to serve as

stock plates. To preserve the mutants for long term storage stock plates of all 196 putative

114

mutants were prepared using Hogness buffer 10X stock (40 mL glycerol; 1 M

dipotassium phosphate, 3.6 mL; 1 M monopotassium phosphate, 1.3 mL; 1 M tri-sodium

citrate, 2 mL; 1 M magnesium sulfate, 1 mL) and diluting to 1X in LB supplemented with

Gm50. Stocks were sealed with aluminum sealing tape within a 96-well plate and stored at

-20°C.

VII.2.4. Antibiotic Resistance of Environmental Isolates

In order to determine the concentrations of antibiotic which could be used during

transposon mutagenesis for selection or counter-selection of each Streptomyces strain of

interest each strain was plated on LB plates supplemented with gentamycin (50 µg/ml),

kanamycin (40 µg/ml), and trimethoprim (100 µg/ml, 150 µg/ml) and incubated at 28°C

for a growth period of 1 week. Controls were prepared in duplicate by plating the eight

environmental isolates on LB without antibiotic. LB agar was chosen because it was the

recovery medium used after electroporation of electrocompetent cells and subsequent

plating of transposon mutants.

VII.2.5. Plasmid DNA Extraction

To extract the pBT20 plasmid DNA necessary for transposon mutagenesis 1.5 mL

overnight cultures of E. coli SM10 used to mobilize the plasmid, were inoculated in

triplicate and incubated at 37°C. Overnight cultures were centrifuged for 2 min at 12,000

rpm after which the supernatant was decanted. The plasmid was isolated using the EZ-10

Spin Column Plasmid DNA Miniprep Kit as per manufacturer’s direction. The resultant

purified DNA was frozen at -20°C.

115

VII.2.6. DNA Transformation

VII.2.6.1. Culture Conditions

In order to prepare electrocompetent cells, YEME and LB were used to grow the

cells. One litre of YEME contains 3 g yeast extract, 5 g peptone, 10 g malt extract and

340 g sucrose. A litre of LB medium contains 10 g Tryptone, 1.75 g NaCl and 1.75 g

yeast.

VII.2.6.2. Electrocompetent Cell Preparation

The preparation of electrocompetent cells for use in transposon mutagenesis was

performed as per Pigac & Schrempf (1995). Briefly, Streptomyces mycelia were grown in

100 mL of YEME (see below) liquid culture for 24 h at 30°C and 150 rpm. Mycelia were

harvested using centrifugation for 10 min at 10,000 rpm at 4°C (Sorvall RC6 Plus) and

resuspended in 100 mL of ice cold 10% sucrose. Mycelia were centrifuged for 10 min at

10,000 rpm at 4°C then resuspended in 50 mL of 15% ice cold glycerol. Mycelia were

suspended in 10 mL of 15% glycerol inoculated with 100 µg/mL lysozyme and incubated

at 37°C for 30 min. The cell suspension was washed twice with ice cold 15% glycerol

and the pellet was resuspended in a solution of 1 mL 30% (w/v) PEG1000, 10% glycerol

and 6.5% sucrose dissolved in dH2O. The mycelia suspension was dispensed in 100 µL

aliquots in microfuge tubes and frozen at -80°C. Plasmid DNA was quantified by diluting

2 µL of extracted DNA in 98 µL autoclaved dH2O and reading its absorbance at 260 nm

using an Eppendorf Bio Photometer.

VII.2.6.3. Electroporation

There is no procedure which exists that is equally efficient for transformation of all

Streptomyces species (Petzke & Luzhetskyy, 2009); therefore it was important to test

116

whether the presence of PEG would possess the same enhancing effect observed in

electrotransformation as it does in classical PEG - assisted protoplast transformation. A

pulse of 2000 V was used to electroporate the mycelia followed by a 3 hour recovery at

30°C. LB supplemented with MgSO4 (250 µg/ml) was used to recover the cells after the

electroporation of the cells and the cells were recovered by agitation for 3 h at 30°C.

Recovered mycelia were serial diluted and plated on LB agar supplemented with

gentamycin (50 µg/mL) and incubated for 24 hours at 28°C to select for transformants

containing the pBT20 plasmid. Variations in the procedure for electrotransformation,

including lack of lysozyme and PEG, have been observed to allow transposition in vivo as

there is no established method of transformation from species to species (Mary-Servais,

1997). A single genome equivalent = total genome length/average insert size = 1 x 107 bp

/ 584 bp = 17123 mutants are required for coverage of the genome.

VII.2.7. Sucrose Utilization Assay of Putative Tn Mutants

To further characterize the putative Tn mutants a carbon utilization medium free

from multiple sources of carbohydrates was prepared. A 250 mL volume of basal mineral

salt agar containing 0.66 g (NH4)2SO4, 0.59 g KH2PO4, 1.41 g KH2PO4·3H2O, 0.25 g

MgSO4 and 250 µL of Pridham and Gottlieb trace salts (Pridham and Gottlieb, 1966)

containing 0.64 g CuSO4, 0.11 g FeSO4, 0.79 g MnCl2, 0.15 g Zn SO4 dissolved in 100

mL dH2O was prepared. The ingredients were dissolved and adjusted to pH 6.8-7.0 using

1 N NaOH or 1 N HCl and 15 g of agar was added. The media was autoclaved and

allowed to cool to ~ 60°C. Filter sterilized sucrose from a 50% stock solution was added

to give a final sucrose concentration of 1%. The mixture was agitated and 25 mL of the

medium was added to 10 petri dishes.

117

Figure VII.2.1. Morphological assessment of Tn mutants Tn-33, Tn-62, Tn-102, Tn-70

and Tn-81 for sporulation. Streak plates were qualitatively assessed for the presence of

spores after 5 days incubation at 28°C.

Putative Mutants (#33, 62, 102, 70,

81)

LB Gm50

LB Gm50 + 0.1% xylan

Potato Dextrose Agar (PDA) Gm50

PDA Gm50 + 0.1% xylan

Yeast Dextrose Agar (YDA) Gm50

Oatmeal agar Gm50

118

Figu

re V

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119

VII.2.8. Enhanced Degradation Capability Assay

All putative mutants were streaked onto LB agar media plates supplemented with

0.1% (w/v) birchwood xylan, CMC, cellulose and 50 µg/mL gentamycin. Plates were

incubated for 48 h at 28°C and degradative activity was visualized using Gram’s Iodine

staining.

VII.2.9. Enhanced Mutant Degradation

The enhanced degradation observed by several putative mutants was assayed using

a comparison of the enzymatic degradation capabilities of the three enhanced degradation

mutants against that of the JLS1-A6 wild type strain on a variety of media types including

LB, PDA, and YDA supplemented with 0.1% (w/v) birchwood xylan and gentamycin (50

µg/mL). Transformants were streaked on LB medium containing 0.1% (w/v) birchwood

xylan, PDA containing 0.1% (w/v) birchwood xylan, YDA containing 0.1% (w/v)

birchwood xylan all of which were supplemented with Gm50. JLS1-A6 wild type was

streaked on LB containing 0.1% (w/v) birchwood xylan, PDA containing 0.1% (w/v)

birchwood xylan and YDA containing 0.1% (w/v) birchwood xylan. Plates were

incubated for 48 h at 28°C and stained with Gram’s iodine to increase the visual contrast

of resulting clearing zones.

VII.2.10. DNA Analysis

VII.2.10.1. Amplification of Gentamycin Resistance Cassette in

pBT20

In order to ensure that the plasmid had inserted into the JLS1-A6 transformants,

PCR was performed using Gm-specific primers, Gm-up (5’-

TGGAGCAGCAACGATGTTAC-3’) and Gm-down (5’-

120

TGTTAGGTGGCGGTACTTGG-3’), to amplify the 548 bp Gm resistance cassette in the

pBT20 transposon. 160 ng and 320 ng of Tn-70 DNA and 220 ng and 440 ng of Tn-81

DNA were loaded along with 2 ng of pBT20 DNA positive control and 30 pmol of each

Gm-up and Gm-down primers. The DNA was electrophoresed using a 1% agarose gel

which was used to resolve the 548 bp Gm amplicon. DNA containing different

concentrations of DMSO (0.2 mg, 0.4 mg, 0.6 mg and 0.8 mg), used to ensure self-

annealing of primers did not occur and validate the presence of the pBT20 transposon

within transformant, was subjected to PCR amplification.

VII.2.10.2. Restriction Digest

In order to determine that the plasmid was successfully extracted from overnight

cultures of E. coli SM10 a restriction digest was performed to compare the size of the

plasmid obtained during the digest to the known size of the pBT20 plasmid. To validate

the 6587 bp size of the pBT20 plasmid a restriction digest using the enzymes EcoR1, and

HindIII was performed. A 1 µL volume of 10X buffer (Fermentas), 6.5 µL of H2O, 2 µL

plasmid DNA and 0.5 µL EcoR1 (Fermentas) were combined in a 1.5 mL microfuge

tube. The microfuge tube was incubated for 1 h at 37°C in a waterbath.

VII.2.10.3. Locating the Inserted Transposon

PCR was performed to confirm transposon insertion into the JLS1-A6 genome

using two different concentrations of gDNA to determine optimal amplification

conditions. The PCR reaction was as follows 160 ng or 320 ng of gDNA, 0.2 mg DMSO,

10 µmol dNTPs, 5 µL 10X Taq buffer, 150 µmol MgCl2, 30 pmol Gm-up and Gm-down

primers, and 2.5 U of Taq Polymerase. The PCR reaction GM PCR, consisted of 1 cycle

for 5 min at 95°C followed by 30 cycles of denaturing at 95°C for 45 sec; annealing at

121

62°C, 30 sec; extension at 72°C, 30-45 sec and 1 cycle (extension at 72°C, 10 min)

followed by a hold step at 4°C. The agarose gel showed a 548 bp amplicon in the form of

a doublet within both the 160 ng and 320 ng µL gDNA reactions for Tn mutants 70 and

81 when 5 µL of PCR amplified DNA was loaded into each well, but not in any of the

other mutants.

VII.2.10.4. PCR Optimization

Due to a 548 bp doublet amplicon being present PCR was assessed for optimal

conditions. Reactions were run using only one primer (Gm-up or Gm- down) to ensure

self-annealing did not occur. Three PCR programs were designed with annealing

temperatures of 50°C, 55°C, and 62°C. To ensure sufficient annealing, at a temperature

5°C lower than the lowest Tm of the Gm-up (Tm 55.4°C) and Gm-down (Tm 57°C)

primers, 50°C was used as the temperature of PCR optimization. The 50°C reaction was

performed with differing concentrations of 25 mM to 50 mM concentrations of MgCl2

and MgSO4. Finally, gradients of dimethyl sulfoxide (0, 0.2 mg, 0.4 mg, 0.6 mg, 0.8 mg)

were used to facilitate DNA strand separation and inhibit interfering reactions. PCR

amplified DNA contained a single 548 bp amplicon from transposon mutant #70 when

zero DMSO was incorporated.

VII.2.10.5. Semi-random PCR

Semi-random (arbitrary) PCR was used to determine the location of the pBT20

insertion using primers which are specific for the ends of the pBT20 transposon and

primers of random sequence which may anneal to chromosomal DNA flanking sequences

close to the insertion site in two rounds of PCR amplification (Caetano-Anolles et al.,

1992). In the first round of amplification, random primer HIB17 and transposon-specific

122

primer GmR-RT are expected to result in a weakly amplified flanking DNA sequence of

the pBT20 transposon along with many other amplified sequences due to the random

primer (Table VII.3.5). During the second round of PCR, product from the first PCR

reaction will be enriched. This is due to the first round creating single primer

amplifications which contain palindromic termini and produce hairpin loops that cause

interference (Medina et al., 2008). Round two will incorporate nested primers 3’-Gm-

reverse, which is complementary to the pBT20 transposon, but closer the junction site

between the transposon and the chromosome, and mariner-Tn-reverse which contains a

sequence identical to the 5’ end of random primer HIB17 (Figure VII.2.4). The PCR

reactions were set up as described by Mandel (2005). Briefly round 1 of PCR reactions

contained: 10 µL Taq buffer, 10 µL dNTPs (2.5 mM), 1 µL HIB17 (30 pmol), 1 µL

GmrRT (30 pmol), 5 U Taq and 77.5 µLdH2O. Round 2 PCR reaction contained: 10 µL

Taq buffer, 10 μL dNTPs (2.5 mM), 1 μL 3’-Gm reverse (30 pmol), 1 µL mariner-Tn-

reverse (30 pmol), 5 U Taq and 67.5 µL dH2O. The PCR reaction GM PCR 1, consisted

of 1 cycle (denaturing at 95°C, 5 min), 5 cycles (denaturing at 94°C, 30 sec; annealing at

30°C, 30 sec; extension at 72°C, 1 min 30 sec), 30 cycles (denaturing at 94°C, 30 sec;

annealing at 45°C, 30 sec; extension at 72°C, 2 min), and 1 cycle of 72°C for 5 min

followed by a hold step at 4°C. GM PCR 2 reaction conditions were as follows: 30 cycles

(94°C, 30 sec, 55°C, 30 sec; 72°C, 1 min 30 sec) and 1 cycle of 72°C for 5 min followed

by a hold step at 4°C.

123

Figure VII.2.3. pBT20 plasmid. pBT20 plasmid with transposon construct containing

gentamycin resistance and the gene for Mariner C9 transposase enzyme cultured within

E. coli SM10 cells. Gentamycin (Gm), ampicillin resistance marker (Amp), beta

lactamase (bla), R6K gamma origin of replication (R6K). Adapted from Kang et al.,

2007.

124

Figure VII.2.4. Arbitrary PCR of pBT20 transposon using specific and degenerate

oligonucleotide primers. Two rounds of amplification will amplify flanking regions

surrounding the inserted transposon. In the first round Tn specific primer and random

primers weakly amplify flanking DNA sequences. In the second round 3’-Gm-reverse

and Mariner-Tn-reverse will enrich the first round products closer to the junction site.

Adapted from Caetano-Annoles, 1993.

125

VII.2.11. Bi-parental Mating (Conjugation)

Biparental mating was used as an alternative method to electroporation for

transposon mutagenesis (Giebelhaus et al., 1996) with the following modifications. 8.5 x

108 spores per mL were inoculated within separate biological replicates of LB or YEME

instead of tryptic soy broth. Plates were overlaid with 3 mL of Difco nutrient broth

containing 0.3% agar, 300 µg trimethoprim (Tp), and 150 µg gentamycin (Gm).

Trimethoprim is a tetrahydrofolate reductase inhibitor which disrupts the production of

folic acid in bacteria and was used to counter select for E. coli SM10. While gentamycin

is an aminoglycoside inhibitor which disrupts the binding of the 30S subunit of the

ribosome inhibiting protein translation and was used select of Streptomyces which

contain the Gmr cassette from the inserted transposon.

Control plates were set up in duplicate to ensure E. coli SM10 was susceptible to

Tp100 and Streptomyces JLS1-A6 was not susceptible to the Tp100 concentration. A

concentration of Gm50 was previously used to effectively prevent the growth of all

environmentally isolated Streptomyces strains of interest and was utilized to counter

select Streptomyces JLS1-A6 without the transposon. Morphology of the resulting

exconjugates was assessed for Streptomyces characteristics. Exconjugants were tested by

restreaking on Tp100 and Gm50 double control media to ensure no false positives were

present.

126

VII.3. RESULTS

VII.3.1. Quality Control of Streptomyces isolate JLS1-A6

Putative mutants Tn-33, Tn-62, Tn-102, Tn-70, and Tn-81 were streaked on PDA,

YDA, LB and oatmeal agar supplemented with Gm50 to observe mutant colony growth.

Streaking on the different media types was done as an attempt to induce sporulation and

collect transposon mutant spore stocks. None of the putative Tn mutants sporulated on

any of the media tested and exhibited a mucoid phenotype. This was the first indication

that the mutants were contaminants.

VII.3.2. PCR Amplification of the pBT20 Transposon

Genomic DNA from the putative Tn mutants Tn-33, Tn-62, Tn-102, Tn-70, Tn-81

and JLS1-A6 wild type was extracted, as per Aljanabi & Martinez (1991), and subjected

to PCR reaction using Gm-up (30 pmol) and Gm-down primers (30 pmol) with a

positive pBT20 DNA control against a 100 bp ladder. Despite multiple attempts at

amplification no amplicon was present using the Gm-up and Gm-down primers which

indicate that no transposition occured in the genome of the Streptomyces JLS1-A6 isolate.

This was a further indication that the putative mutants were contaminants.

VII.3.3. pBT20 Extraction

The pBT20 plasmid was successfully isolated from E. coli SM10 and quantified

using a spectrophotometer at 260 nm. The isolated pBT20 plasmid was subjected to

electrophoresis after digestion with EcoR1 and HindIII to confirm the correct size of

6587 bp. EcoRI created a single cut resulting in a band size of 6587 bp while HindIII

made three cuts resulting in band sizes of 5957 bp, 528 bp and 102 bp.

127

Table VII.3.1. Average clearing zone sizes created by 196 putative JLS1-A6 transposon

mutants on 0.1% (w/v) birchwood xylan, cellulose and carboxymethyl cellulose.

128

Table VII.3.2. T – test statistical analysis of clearing zones created by mutants #33, 62,

102 in LB and YDA supplemented with 0.1% (w/v) birchwood xylan compared to JLS1-

A6 wild type.

129

Tabl

e V

II.3.

3. P

lasm

ids i

nves

tigat

ed fo

r use

in th

e tra

nspo

son

mut

agen

esis

of J

LS1-

A6

envi

ronm

enta

l iso

late

.

130

Table VII.3.4. Donor strains of E. coli utilized during transposon mutagenesis of

Streptomyces environmental isolate JLS1-A6.

Donor Strains

Genotype References

E. coli SM10 E. coli SM10 allows the mobilization of plasmids containing oriT into a broad range of recipient strains. The oriT transfer origin of RP4, a broad host-range conjugative plasmid, can be transferred between mostly gram negative bacteria. It can also be transferred to gram positive bacteria if the RP4 delivery machinery is expressed in trans using inserted genes into the chromosome or a plasmid. This strain carries a RP4 derivative along with a lambda pir (π) dependent suicide vector. It was designed by Puhler in the early 1980s.

Ferrieres L., Hemery G., Nham T., Guerout A.M., Mazel D., Beloin C., Ghigo JM. (2010). Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery. J Bacteriol, 192(24),6418-6427.

131

Tabl

e V

II.3.

5. O

ligon

ucle

otid

e pr

imer

s use

d in

Gm

cas

sette

am

plifi

catio

n an

d ar

bitra

ry P

CR

of t

he fl

anki

ng

regi

ons s

urro

undi

ng th

e pB

T20

geno

mic

inse

rtion

.

132

VII.3.4. Putative Mutant Patch Plating

Differences were observed in the morphologies of the putative mutants on patch

plates. A bright yellow diffusible pigment was observed for three of the putative

transformants, Tn-33, Tn-62, and Tn-102, but was absent in all others. These three

pigmented colonies degraded 0.1% (w/v) birchwood xylan and 0.1% (w/v) cellulose more

efficiently than JLS1-A6 wild type. The other putative transformants exhibited little to no

degradation of xylan, CMC or cellulose (Table VII.3.1). The non-degrading putative

transformants were used as negative controls in degrader versus non degrader assays on

LB and yeast dextrose agar supplemented with0.1% xylan (Table VII.3.2).

Tn-113, Tn-116 produced brown diffusible pigments on LB agar; these putative

mutants did not exhibit enhanced degradation. In addition to the pigments observed, all

transformants exhibited a mucoid appearance after electroporation on LB, xylan,

cellulose and CMC media types. In retrospect, this indicated that the cultures were

contaminated.

VII.3.5. Enzymatic Activity of Enhanced Degradation Mutants

A comparision between the size of the degradation zone on YDA and PDA was

made (Table VII.3.2). Clearing zones observed for the three putative degrading mutants

Tn-33, Tn-62 and Tn-102 were significantly larger than those observed for the JLS1-A6

wild type strain on LB agar medium and YDA supplemented with 0.1% (w/v) birchwood

xylan. Little to no growth or enzymatic activity was observed from the degradation

mutants Tn-33, Tn-62, Tn-102 on PDA while the JLS1-A6 wild type strain displayed

high amounts enzymatic activity resulting in clearing zones on PDA.

133

VII.3.6. Putative Transposon Mutant Screening

One hundred ninety six putative mutants were screened on Minimal Medium

containing 0.1% (w/v) birchwood xylan, 0.1% (w/v) cellulose and 0.1% (w/v) CMC

supplemented with Gm50 and the average degradation zone was determined (Table

VII.3.1). It was observed that more mutants grew on 0.1% (w/v) CMC with an average

clearing zone size of 0.34 mm than on 0.1% (w/v) cellulose with an average clearing zone

size of 0.7 mm and even less grew on 0.1 % (w/v) birchwood xylan with an average

clearing zone size of 0.9 mm. The average clearing zone sizes observed on 0.1% (w/v)

birchwood xylan and 0.1% (w/v) cellulose were statistically larger than those observed on

the 0.1% CMC media, but not statistically different from one another. However, the

largest clearing zones produced by a small number of mutant colonies were observed on

the 0.1% (w/v) carboxymethyl cellulose. It should be emphasized that while degradation

was observed, what were initially believed to be mutants were found out to be

contaminants.

VII.3.7. Sucrose Utilization Assay of Putative Tn Mutants

To confirm putative mutants as Streptomyces Tn mutants Tn-33, Tn-62, Tn-102,

Tn-70 and Tn-81, as well as JLS1-A6 parent strain were streaked on basal medium

supplemented with sucrose to induce sporulation. Tn mutants Tn-33, Tn-62, and Tn-102

grew as mucoid colonies on the medium and did not sporulate. Tn mutants Tn-70 and Tn-

81 did not grow on the basal medium supplemented with sucrose. Parent strain JLS1-A6

successfully established sporulating colonies on the basal medium supplemented with

sucrose. This result indicates that the putative mutants were contaminants.

134

VII.3.8. Locating the Inserted Transposon

To verify the transposon in mutants of isolate JLS1-A6, the gentamycin resistance

cassette of the transposon was amplified using Gm-up and Gm-down primers (Table

VII.3.5). Genomic DNA from Tn mutants Tn-33, Tn-62, Tn-102, Tn-70, Tn-81 and

JLS1-A6 wild type was extracted, as per Aljanabi & Martinez (1991), and subjected to

PCR amplification using Gm-up (30 pmol) and Gm-down primers (30 pmol) for the Tn

mutants with pBT20 DNA as a positive control. Wild type JLS1-A6 was successfully

amplified using PCR primers 27F and 1492R, however we were unable to amplify the

Gm resistance cassette from putative JLS1-A6 mutants using Gm-up and Gm-down

primers even when various concentrations of DMSO were used to optimize PCR

conditions. The inability to amplify the Gm resistance cassette indicates that the

transposon did not insert into the genome providing further evidence that they were

contaminants.

135

VII.4. DISCUSSION

In order to identify the location of degradation genes, transposon mutagenesis of

Streptomyces isolate JLS1-A6 was attempted but failed. The transposon used, possesses a

cut and paste mode of action in which the transposon is inserted into regions of the

genome where it is flanked by indirect repeats (Reznikoff, 2003). Transposon end

recognition sequences are required for transposition without the presence of a RNA

intermediate (Reznikoff, 2003).

Streptomyces are unique because both its aerial hyphae and substrate mycelia are

multigenomic. The coenocytic substrate mycelia contain numerous copies of a linear

chromosome separated by cross walls while the aerial hyphae can contain 50 or more

chromosomes within a single compartment (Ruban-Osmialowska et al., 2006). After

several attempts of replicating transposon mutagenesis using electroporation had failed,

bacterial conjugation involving pRK2013 helper plasmid and pRBrha B out plasmid was

attempted (Table VII.3.3). However since pBT20 contains a R6K gamma origin of

replication, a Gm resistance cassette and an Amp resistance marker this plasmid was

chosen to be used in conjunction with E. coli SM10λ pir which contains RP4 conjugative

machinery, a transfer origin (oriT) which allows mobilization of plasmids to gram

positive or negative recipient strains, π protein control in trans, and a π protein dependent

suicide vector (Ferrieres et al., 2010) (Table VII.3.4).

pBT20 contains a conditionally replicating, self-ligating transposon which requires

the presence of the pir π protein (Ferrieres et al., 2010). When used in vitro the mariner

based genetic element employed by the pBT20 plasmid has low specificity and broad

host range capability to result in a large number of insertions of gene inactivating

136

elements (Chiang & Rubin, 2002). Bacterial DNA can be protected from degradation by

modifying specific sequences using methylation which are recognized by corresponding

restriction enzymes from the system or cleaving DNA which contains foreign

modifications (Blumenthal et al., 1985). The methylation of the pBT20 plasmid DNA by

the Streptomyces methyl-specific restriction system (Flett et al., 1997) is a possible cause

as to why the transposon was not being integrated into the Streptomyces genome as is the

contamination problem encountered when attempting this experiment.

In yet another attempt to produce transposon mutants in JLS1-A6 electroporation

was performed (Pigac & Schrempf, 1995) with growth on yeast extract malt extract

medium for the preparation of electrocompetent mycelia. Polyethylene glycol (PEG) was

tested to determine whether its addition facilitated electrotransformation (Mazy-Servais et

al., 1997). The pBT20 plasmid was used in failed attempts to mutagenize Streptomyces

isolate JLS1-A6 to produce mutants with inactivated degradation genes (Figure VII.2.3).

137

VII.5. CONCLUSION

Transposon mutagenesis was attempted to further characterize the degradative

potential of isolate JLS1-A6 however all attempts failed. The mucoid phenotype of the

putative mutants indicates that a contaminant was carried through the mutagenesis

experiments. Future attempts at mutagenesis in this isolate should include harvesting the

cultures at different growth stages to determine which is most amenable to genetic

manipulation.

138

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