Accessing the Uncultivable: Unraveling Activities from Accessing the Uncultivable: Unraveling Activities from MetagenomicMetagenomic

Libraries Generated from Dry and Rain Libraries Generated from Dry and Rain

Forest Soils in Puerto Rico Using Functional and Forest Soils in Puerto Rico Using Functional and SequenceSequence--based Approachesbased Approaches

NCSS Conference at Las Cruces, NMNCSS Conference at Las Cruces, NM

JosJoséé

M. CruzM. Cruz--GarcGarcííaa11, Pedro J. Ondina, Pedro J. Ondina11, Manuel Ortega, Manuel Ortega11, , RosivetteRosivette

SantiagoSantiago11, , Jean C. CruzJean C. Cruz--HernHernáándezndez11, , AmarisAmaris

TorresTorres11, Francisco S, Francisco Sááncheznchez11, Orlando Soto, Orlando Soto--

CCááceresceres11, , LilliamLilliam

CasillasCasillas22, , andand

Carlos RCarlos Rííos Velos Veláázquezzquez33

University of Puerto Rico at University of Puerto Rico at MayagMayagüüezez, , 11Industrial Biotechnology Program and Industrial Biotechnology Program and 33Biology Department, Biology Department, 22University of Puerto Rico at University of Puerto Rico at HumacaoHumacao, Biology Department, Biology Department

May 11, 2009May 11, 2009

Soil: A valuable natural resourceSoil: A valuable natural resource

Why Why metagenomicsmetagenomics??

Few microorganisms can be cultivatedFew microorganisms can be cultivated

Access to uncultivable microorganismsAccess to uncultivable microorganisms

Why Why metagenomicsmetagenomics??

Bacterial Clone

Novel Activity

None

Antimicrobial agents

Biodegradation enzyme(s)

High temperature tolerance

Unraveling new activities with medical and/or Unraveling new activities with medical and/or biotechnological applicationsbiotechnological applications

Why Why metagenomicsmetagenomics??

1% 1% + 99%

Cultivating techniques Metagenomics

Why Why metagenomicsmetagenomics??

Monitoring: two approachesMonitoring: two approaches

Functional metagenomics Sequence‐based metagenomics

Expression

Transcription

Transduction

SecretionAATCGACCTAATTCTTAGCTGGATTAAG

Monitoring: two approachesMonitoring: two approaches

Functional metagenomics Sequence‐based metagenomics

•Microbial diversity

•Production of antimicrobial 

agents

•Antibiotic resistance activity

•Production of antimicrobial 

agents

•Detection of enzymatic activity

MetagenomicMetagenomic

library generationlibrary generation

MetagenomicMetagenomic

library generationlibrary generation

++

Genomic DNA extraction

Vector

Genomic DNA

Cultivable host

Ligation

Insert

Transformation

“Metagenomic Library”

Sampling SitesSampling Sites

http://www.definitivecaribbean.com/images/TemplateImages/caribbeanmap.jpghttp://www.aaa-calif.com/apps/travel/destinations/images/lonelyplanet/maps/wg-puerto-rico-2446-400x300.gifhttp://farm3.static.flickr.com/2038/1496561042_2b3b2eb84d.jpghttp://ammedia.tv/images/rain_forest3.jpghttp://www.sphaydenphotography.com/images/prrainmap.gif

Sampling SitesSampling Sites

Guánica

Dry Forest Yunque

Rainforesthttp://www.definitivecaribbean.com/images/TemplateImages/caribbeanmap.jpghttp://www.aaa-calif.com/apps/travel/destinations/images/lonelyplanet/maps/wg-puerto-rico-2446-400x300.gifhttp://farm3.static.flickr.com/2038/1496561042_2b3b2eb84d.jpghttp://ammedia.tv/images/rain_forest3.jpghttp://www.sphaydenphotography.com/images/prrainmap.gif

Sampling SitesSampling Sites

Guánica

Dry Forest Yunque

Rainforesthttp://www.definitivecaribbean.com/images/TemplateImages/caribbeanmap.jpghttp://www.aaa-calif.com/apps/travel/destinations/images/lonelyplanet/maps/wg-puerto-rico-2446-400x300.gifhttp://farm3.static.flickr.com/2038/1496561042_2b3b2eb84d.jpghttp://ammedia.tv/images/rain_forest3.jpghttp://www.sphaydenphotography.com/images/prrainmap.gif

Antimicrobial activityAntimicrobial activity

Polyketide

Synthases

amplifications Overlay assays

Library clones

Assay strains

Top agar

Overlay

Zones of inhibition

A B

Detection of Detection of ureaseurease

using the using the ureaseurease testtest

Urease

test

NH2NH2

C

O

+ OH21. urease

OO C + NH32 C

O

O-

O-

NH4+

NH4+

2. H2O

(-) (+)

Antibiotic resistanceAntibiotic resistance

Fosmid

libraries screened for antibiotic resistance

ConclusionsConclusions

A total of 8 metagenomic libraries from two forests have been generated (~850,000 clones).There are differences in microbial diversity between the two forests.Through sequence-based analysis the presence of ampliconsassociated with production of antimicrobial agents has been detected.4 examples of functional analyses have been used: production of antimicrobial agents, and epoxide hydrolase, urease, and antibiotic resistance activity.Ongoing experiments involve lipase detection, transposonmutagenesis to identify genes associated with activity, and further sequencing and screening efforts.

AcknowledgementsAcknowledgements

Dr. Jo Handelsman, Lynn Williamson and fellow researchers at University of Wisconsin-MadisonGraduate and undergraduate students of the Microbial Biotechnology and BioprospectsLaboratory at UPRMMr. Dubiezel MedinaUSDA-CSREES 2007-02386PR-LSAMP UPRM

http://http://www.cohemis.uprm.edu/gemsprwww.cohemis.uprm.edu/gemspr//

The TEAMThe TEAM

Results –

Restriction digest analysis

IKB IKB

Vector7.5 kbp

A BRestriction digest analysis of (A) Guánica

dry forest library clones and (B) Yunque

rainforest library clones digested with NotI. The line indicates the fosmid

vector. Comparing the amount of internal NotI

recognition sites between both samples, sample(A) contained DNA inserts higher in GC content than sample (B).

NotI

recognition site 5’-GCGGCCGC-3’

Diversity analyses

16S rDNA

libraries TRFLP

Genomic DNA extraction

16S rDNAPCR amplification

TOPO TA cloning Digestion

EpoxideEpoxide

hydrolasehydrolase

detection using detection using the adrenaline testthe adrenaline test

OOH

OH

O

O

OH

N

CH3

I

O

O O-

ONa

+

I

O-

OO

Na+

+ OH2

3. back - titration

adrenochrome

adrenaline

OH

OH

NHCH3

OH

1. epoxide hydrolase

2. oxidation

Epoxide

hydrolase

activity: adrenaline test

(-)

(+)

Results -

Epoxide

hydrolase

detection

Example of adrenaline test assay for Guánica

dry forest library subpool

6. Odd rows indicate actual assay, even rows represent unassayed

duplicates. Sample (A) above was induced, (B) was not. The well for the last column in row 8 for sample (B) is Escherichia coli. Under these experimental conditions, it appears induction does not modify results significantly.

12345678

12345678

A

B

Results -

Antimicrobial activity detection

(A) Example of 20 screened plates, forK. pneumoniae.

(B) Example of 40 screened plates, 20 for S. aureus

and 20 for

K. pneumoniae.

TRFLP