diversity of phylogenetic and lipolytic genes from soil...
TRANSCRIPT
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Diversity of phylogenetic and lipolytic genes from
soil metagenome of Drass (Ladakh)
THESIS
Submitted to the University of Jammu
For the Award of the Degree of
DOCTOR OF PHILOSOPHY
IN
BIOTECHNOLOGY
BY
PUJA GUPTA
UNDER THE SUPERVISION OF
DR. JYOTI VAKHLU
SCHOOL OF BIOTECHNOLOGY
UNIVERSITY OF JAMMU
JAMMU – 180 006
2015
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CERTIFICATE
This is to certify that:
1. the thesis entitled “Diversity of phylogenetic and lipolytic genes from soil
metagenome of Drass (Ladakh)” embodies the work done by Ms. Puja
Gupta under my supervision for the period required under statutes;
2. the candidate has put in the attendance in the School of Biotechnology for the
period required;
3. the thesis being submitted for the Degree of Doctor of Philosophy in
Biotechnology by Ms. Puja Gupta has not been submitted for any other
degree and is worthy of consideration for the award of Ph.D degree of the
University of Jammu;
4. the conduct of research scholar remained good during the period of research,
and
5. the candidate has fulfilled the statutory condition as laid down in the Ph.D
statutes.
(Dr. Jyoti Vakhlu) (Prof. M.K. Dhar)
Supervisor Director
Associate Professor School of Biotechnology
School of Biotechnology University of Jammu
University of Jammu
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Acknowledgements
I take this opportunity to sincerely thank all the people involved either directly or indirectly in
my research work.
It is my pleasure and privilege to put on record my profound gratitude to my esteemed
teacher, Dr. Jyoti Vakhlu, for initiating my interest in the field of metagenomics and for her
guidance, valuable suggestions, constructive criticism, remarkable patience and
understanding throughout the course of this work.
It is my pleasure to express deep sense of gratitude and thankfulness to Prof. Manoj Kumar
Dhar, Director, School of Biotechnology, University of Jammu, for his support and providing
all the necessary facilities for the research work.
I express my heartiest gratitude to all faculty member of the department, Dr. B.K. Bajaj, Dr.
Sanjana Kaul, Dr. Madhulika Bhagat, Dr. Ritu Mahajan and Dr. Nisha Kapoor for their
invaluable help.
I would like to thanks all the research scholars of school and my lab mates, Dr. Avneet, Ms.
Deepika Trakroo, Mr. Puneet Gupta, Ms Indu, Ms. Sheetal Ambardar, Ms. Simmi Grewal,
Ms. Ranjeet Kour, Ms. Rikita Gupta, Ms. Sakshi Sharma, Sneha Ganjoo, Shanu Magotra for
their intense support throughout my course of study.
I also want to acknowledge my friends Mr Abhimanyu Jha, Ms Meenu Sharma, Ms Preeti
Choudary, Mr. Sahil Gupta, Ms Shaina Rajput, Ms Shilpi Gupta for their continual support
and affection.
I acknowledge, with utmost humility my indebtness to my parents, parents- in –law, my
beloved husband, kids and brothers for their support and encouragement.
I also would like to convey my sincere thanks to all non-teaching staff members for their
cooperation and timely help.
(Puja Gupta)
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ABBREVIATIONS
U Units
TAE Tris, Glacial acid, EDTA
Sm/m Siemens per meter
SDS Sodium Dodecyl Sulphate
RNA Ribose nucleic acid
Ppm Parts per million
Pm Picomoles
PCR Polymerase Chain Reaction
PAGE Poly acryl amide gel electrophoresis
Nm Nanometer
mM Millimolar
mm Millimeter
ml Millilitre
MHz Mega Hertz
mg Milligram
kDa Kilo Dalton
Kb Kilobase
gm Gram
EDTA Ethylene diamine tetra acetate
DNA Deoxyribose nucleic acid
CFU Colony Forming Unit
bp Base pair
µl Microlitre
µg Microgram
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Contents
Chapter Section Page No.
Chapter 1 Introduction 1-5
Chapter 2 Review of Literature 6-16
Chapter 3 Material and Methods 17-33
Chapter 4
Chapter 5
Results and Discussion
4.1 Sampling Site
4.2 Physicochemical analysis of soil
4.3 Microbial Diversity
4.3.1 Cultivation dependent approach
4.3.1.1 Cultivable bacterial microflora
4.3.2 Cultivation independent (metagenomic) approach
4.3.2.1 Assessment of bacterial diversity
by 454 pyrosequencing
4.3.2.2 Assessment of fungal diversity
by 454 pyrosequencing
4.4 Diversity of Lipolytic genes
4.4.1 Cultivation dependent approach
4.4.1.1 Screening cultivable bacteria for production
of lipolytic enzymes
4.4.2 Cultivation independent (metagenomic) approach
4.4.1.2 Diversity of lipolytic genes by cultivation
independent approach.
Summary and Conclusion
Bibliography
Appendix
apter
34-52
34
34-35
35-47
35-37
37-47
37-42
43-47
47-52
47-48
48-52
53-56
57-88
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Chapter – 1
Introduction
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Chapter 1 Introduction
1
1. INTRODUCTION
Psychrophilic and psychrotolerant microbes inhabiting ice covered regions of
cold desert have received increasing attention during the past decade, as these play a
major role in food chains and biogeochemical cycles of these environments (Margesin
and Miteva 2011; Gesheva et al. 2012). Psychrotolerant microbes are interesting, as
besides being able to grow at temperatures close to or below freezing, they have
withheld their ability to withstand mild temperatures and are more ubiquitous and
numerous (both quantitatively and in terms of number of species) in permanently cold
environments (Ahmad et al. 2010). Various cold environments like Antarctic (Tytgat
et al. 2014), Arctic (Frank-Fahle et al. 2014), Siberian tundra (Schnecker et al. 2014),
Finland (Heino et al. 2014) have been explored for diversity of such cold adapted
microbes. The Himalayan cold deserts are characterized by a fragile ecosystem and a
complex climate due to dramatic seasonal shifts in physical and biochemical
properties. The hostile climatic conditions prevailing in these Himalayan cold deserts,
makes it an interesting habitat to study phylogenetic and functional diversity that can
be explored by the cultivation dependent and independent approach. In cultivation
dependent approach, though isolation of microbe is possible, that can be subsequently
exploited, but these methods have biases manifested by the media used and growth
conditions selected (Vartoukian et al. 2010; Delmont et al. 2011). Due to this a vast
majority of microbes resists cultivation using conventional methods (Delmont et al.
2011; Rastogi and Sani 2011) and very limited phylogenetic and functional diversity
has been catalogued so far using these traditional cultivation based methods
(Schleinitz 2011; Narihiro et al. 2014). Thus, a cultivation based cataloguing alone is
not enough for assessing the phylogenetic and functional diversity, but it needs to be
complimented with cultivation independent metagenomic techniques. Cultivation
independent metagenomic approach is based on direct isolation and analysis of
nucleic acids from environmental samples followed either by cloning & sequencing or
direct sequencing (Shivaji et al. 2011; Srinivas et al. 2011a; Serkebaeva et al. 2013;
Kim et al. 2014).
Cloning dependent metagenomic approach was the widely used cultivation
independent metagenomic method till 2011, where in the PCR products were
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Chapter 1 Introduction
2
amplified from an environmental sample, cloned and sequenced (DeSantis et al.
2007; Liu et al. 2009; Rajendhran and Gunasekaran 2011; Shivaji et al. 2011). The
sequences retrieved were compared with the reference sequences in a database such
as GenBank (www.ncbi.nlm.nih.gov/genbank), Greengenes for bacterial
identification (DeSantis et al. 2006); Unite for fungal identification (Koljalg et al.
2005; Abarenkov et al. 2010; Op De Beeck et al. 2014). Ribosomal Database Project
(RDP) database used for both bacterial and fungal identification (Cole et al. 2009).
Cloning based culture-independent metagenomic approach overcomes the limitations
of cultivation based methods (Vaz-Moreira et al. 2011) and gives a larger view of the
total microbiome than cultivation based methods. This approach revealed an
abundant array of previously unknown and uncultured microbes, including entirely
new bacterial divisions with no cultured representatives (Liu et al. 2009; Shivaji et
al. 2011; Taib et al. 2013). However owing to the cloning bias, limitation of number
of clones selected and less sequencing depth, it was realized that complete
phylogenetic diversity cannot be explored by cloning based cultivation independent
metagenomic method (Shokralla et al. 2012; Fakruddin et al. 2013; McCormack et
al. 2013). Moreover, sequencing clones after metagenomic library preparation
captures only the dominant components of microbial communities that mask the
detection of low abundance microorganisms. These low-abundance microorganisms
constitute a highly diverse “rare biosphere” in almost every environmental sample
including soil (Lauber et al. 2009; van den Bogert et al. 2011; Delmont et al. 2011;
Delmont et al. 2014). These hidden rare biosphere microbial populations offer a
potentially inexhaustible genetic reservoir and could be explored only by using high
throughput direct sequencing techniques (Cloning independent). Cloning
independent direct sequencing using next generation sequencencing technology, has
not only reduced the cost of sequencing but the time consumed is also comparatively
less. Unlike Sanger sequencing that requires in vivo amplification of DNA fragments
to be sequenced (achieved by cloning into bacterial hosts first) next-generation
sequencing technology circumvents the cloning requirement by taking advantage of
a highly efficient in vitro clonal DNA amplification.
http://www.sciencedirect.com/science/article/pii/S0944501310000261http://www.sciencedirect.com/science/article/pii/S0944501310000261
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Chapter 1 Introduction
3
In addition to look into what kind of microbes are present in the cold habitates,
cold active enzymes including α-amylases, cellulase, lipases and proteases are another
attraction to study these habitats. Cold active enzymes are valuable for
biotechnological applications and industrial processes (Aygan and Arikan 2008;
Pulicherla et al. 2011; Zheng et al 2011; Joshi and Satyanarayana 2013; Sarvanan et
al. 2013; Maiangwa et al. 2014). Cold adapted enzymes have high catalytic efficiency
and specificity at low and moderate temperatures, offering economic benefits through
energy savings (Gerday et al. 1997). Running processes at low temperatures reduces
the risk of contamination by mesophiles and saves energy. In addition,
thermosensitive biocatalysts can be easily inactivated by mild heat treatment.
More emphasis has been put on cold adapted lipases in the the present study
as these were predominant in soil of Drass among other hydrolases. The commercial
use of lipases of cold origin is a billion dollar business. These are widely used for
bioremediation and degrading hydrocarbons present in contaminated soil (Aislabie et
al. 2000; Paniker et al. 2006). Psychrophilic lipases have attracted attention for
synthesis of organic substances and conversion of biomass into useful products due
to their inherent flexibility in contrast to mesophilic and thermophilic enzymes with
excess rigidity (Ramani et al. 2010; Joseph et al. 2011; Nagarajan 2012; de Abreu et
al. 2014). Most of the industrially important lipases have microbial origin (Gurung et
al. 2013; Veerapagu et al. 2013) wherein the microbes are cultivated on suitable
media, purified and then further used for the production of industrially important
compounds. Several psychrophlic and psychotrophic bacteria have been exploited
for the production of a variety of coldactive lipases across different cold habitats.
The most common ones are bacterial lipases produced from the genera Aeromonas
sp. LPB4 (Lee et al. 2003), Burkholderi (Wang et al. 2009a), Pseudomonas (Yang et
al. 2009; Madan and Mishra 2010); Photobacterium sp. (Ryu et al. 2006),
Psychrobacter sp. (Xuezheng et al. 2010), Micrococcus roseus (Joseph et al. 2011),
Rhodococcus and Serratia (Joseph et al. 2007). A novel cold-active and organic
solvent-tolerant lipase displaying remarkable stability have been reported from
Stenotrophomonas maltophilia CGMCC 4254 isolated from oil-contaminated soil
samples (Li et al. 2013). Soils from Alaskan cold habitat and other cold regions have
http://scialert.net/fulltext/?doi=jm.2011.1.24#567041_jahttp://scialert.net/fulltext/?doi=jm.2011.1.24#567100_jahttp://scialert.net/fulltext/?doi=jm.2011.1.24#567100_jahttp://scialert.net/fulltext/?doi=jm.2011.1.24#566819_ja
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Chapter 1 Introduction
4
been exploited as potential sources of novel cold-active lipase (Leonov 2010). Apart
from culture based screening for the isolation of lipase producers, culture-
independent approach of screening lipase/ lipolytic genes is sequence-based on any
of these methods such as PCR microarrays or next-generation sequencing platforms.
In PCR-based, approach and microarrays, oligonucleotide primers are designed on
the basis of conserved amino acid sequence motifs and these identify target lipolytc
genes, either directly or via polymerase chain reaction (PCR)/nucleic acid
hybridization. Gene-specific PCR yields a partial gene fragment, requiring additional
steps to obtain the up- and down-stream flanking regions. The partial gene fragment
can also be used as a probe to identify possible full-length genes in a metagenomic
library or enzyme restricted metagenomic DNA that can be excised and cloned. Bell
et al. (2002) designed degenerate primers using conserved amino acid regions within
lipase genes. Using these degenerate primers they obtain partial lipase gene
fragments from a metagenomic soil sample, and obtained the full-length lipase genes
using genome-walking PCR. High throughput random shotgun sequencing combined
with advanced assembly and primer walking strategies may identify particular genes
of interest. This method does not require heterologous gene expression and is
relatively less expensive and less laborious relative to PCR and microarray. This
approach generates vast amounts of sequence information that require advanced
computational approaches for assembly and gene function assignment (Piel 2011).
Whole-metagenome sequencing of mangroves areas using the 454 GS-FLX titanium
technology revealed profile of α/β-hydrolase fold proteins in mangrove soil
metagenomes from oil-contaminated sites (Jiménez et al. 2014). Cloning
independent direct pyrosequencing catalogue the diversity and access the novel
enzyme genes from previously uncharacterized soils (Wang et al. 2010; Mhuantong
et al. 2015). The information generated by metagenomics can also be used to develop
cultivation techniques to isolate novel microbes, hence novel gene products (Gong et
al. 2013; Narihiro et al. 2014).
The current study aimed to explore the diversity of phylogenetic and lipolytic
genes from composite soil sample of Drass by cultivation dependent and cultivation
independent metagenomic approaches. This place is unique in many respects for its
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Chapter 1 Introduction
5
geoclimatic conditions, such as high altitude, extremely cold and dry weather
characterized by hostile climatic conditions. Drass, the second coldest place in world
located at 34.428152°N, 75.75118°E is situated 60 km west of Kargil on the road to
Srinagar with an average elevation of 3,280 metres (10,764 feet) (Fig.1) It starts
from the base of the Zojila pass (the Himalayan gateway to Ladakh), a trans-
Himalayan region that separates the western Himalayan peaks from the Tibetan
plateau. The place is snowbound and inaccessible for half of the year, from October
to April giving it a cool, temperate climate. Summers are warm up to 30°C with cool
nights, while winter is long and cold with temperatures often dropping to −45°C.
Annual precipitation is almost entirely concentrated in the months from December to
May with 360 mm (14 inches) of snow. Considering the wide range of temperature
of the site and other geoclimatic conditions, it was expected that the soil sample from
this site would contain novel microbial communities encoding enzyme able to work
at wide range of temperature.
With this background, following objectives were set forth for the present study
1. Assessment of microbial diversity (both cultivable and yet to be cultivated) in
Drass soil
2. Screening and identification of lipolytic psychrozyme producing microflora
3. Isolation of lipolytic genes from the metagenomic library of Drass soil
4. Characterization of isolated lipolytic genes
http://tools.wmflabs.org/geohack/geohack.php?pagename=Dras¶ms=34.428152_N_75.75118_E_
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Chapter – 2
Review
of
Literature
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Chapter 2 Review of Literature
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2. REVIEW OF LITERATURE
Microbes have been around for billions of years and are present everywhere.
They inhabit almost all the environment, from cold (below 0°C) to hot (above 100°C),
acidic to alkaline, and saline environments etc. that might not look optimum for life to
human beings. In the last century lot of microbial secrets have been unraveled, but
there are still many unanswered questions e.g How many microbes are there? Who are
they? And what exactly are they doing? Therefore the microbial diversity associated
with various niches needs to be explored to answer these questions.
Cold habitats dominate the biosphere. About 85% of the biosphere are
permanently exposed to temperatures below 5°C. Oceans representing 70% of the
earth’s surface and 90% by volume are at or below 5°C (Margesin and Miteva 2011).
According to Morita and Moyer (2001), cold environments can be divided into two
categories: psychrophilic (permanently cold) and psychrotrophic (seasonally cold or
where temperature fluxes into mesophilic range) environments. Psychrophiles usually
grow at or below zero (0°C) and have an optimum growth temperature ≤15°C with an
upper limit of ≤20°C. In contrast, psychrotrophs can grow close to zero but have
optima temperature limits above 30°C; hence these could be considered as being cold-
tolerant mesophiles (Russell 2006).
Psychrophiles and Psychrotrophs belong to diverse genera ranging from Gram
negative bacteria (e.g. Acinetobacter, Serratia, Pantoa and Pseudomonas) to Gram-
positive bacteria (e.g. Exiguobacterium, Arthrobacter, Bacillus and Micrococcus) and
Fungi (Penicillium and Hypocrea). These organisms play a key function in food
chains, biogeochemical cycles and mineralization of pollutants (Margesin and Miteva
2011; Gesheva et al. 2012). Psychrotrophs are more interesting than psychrophiles,
besides being able to grow at temperatures close to or below freezing, they have
withheld their ability to withstand mild temperatures and are more ubiquitous and
numerous (both quantitatively and in term of number of species) even in permanently
cold environments (Kanbakan et al. 2004; Ahmad et al. 2010). Various cold
environments like Antarctic (Tytgat et al. 2014), Arctic (Frank-Fahle et al. 2014),
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Chapter 2 Review of Literature
7
Siberian tundra (Schnecker et al. 2014), Finland (Heino et al. 2014) has been explored
for microbial phylogenetic and functional diversity.
Functional diversity is the direct response of the soil microbial community to
it’s metabolic requirements and available nutrients. Knowledge of functional diversity
is equally important as taxonomic diversity, as it reflects the capacity of microbial
communities to flourish through disturbance, stress or succession that could
ultimately be more important to ecosystem productivity and their stability. Although
microbes produce various hydrolytic enzymes, the present review is focused only on
the functional diversity of cold adapted lipases owing to their wide biotechnological
and industrial applications (mentioned in detail in Table 4).
Diversity of phylogenetic and lipolytic (lipolytic) genes can be assessed by
cultivation dependent as well as independent methods. The cultivation dependent
approach has an advantage of isolation of microbe that can be subsequently
manipulated for various processes. Very limited phylogenetic and functional diversity
has been catalogued so far using traditional cultivation based methods (Schleinitz
2011; Narihiro et al. 2014). Thus, culture based cataloguing alone is not the answer
for assessing the phylogenetic and functional diversity but it needs to be
complimented with cultivation independent metagenomic techniques.
Based on the above mentioned facts the layout of the review as follows:
2.1) Microbial diversity
2.1.1) Cultivation dependent approach
2.1.2) Cultivation independent approach
2.2) Functional diversity
2.2.1) Cultivation dependent approach
2.2.2) Cultivation independent approach
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Chapter 2 Review of Literature
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2.1) Microbial diversity
2.1.1) Cultivation dependent approach
Microbial diversity of various polar environments such as the Arctic and
Antarctic (Tropeano et al. 2012; Moller et al. 2013; Shivaji et al. 2013) and non-polar
alpine environments such as Italian Alps and Himalayan regions (Lipson et al. 2004;
Gangwar et al. 2009; Shivaji et al. 2011; Franzetti et al. 2013) have been assessed by
cultivation dependent methods. Diversity in polar regions differ from several high-
altitude regions such as the Himalayan ranges due to seasonal variations in
temperature that results in different physical and biochemical properties. Moreover,
polar regions are permanently frozen unlike alpine regions.
Numerous selective and nonselective media have been used for enumerating
and isolating micro-organisms. Selective media are mostly used when targeting
particular genus or species or biopropecting for specific enzymes. Nonselective
medium is preferred if the aim is to harvest diversity. Various non-selective media,
e.g Beef extract Agar medium, and YM Agar medium, PYGV agar, TSA, R2A agar,
Nutrient agar (Miteva et al. 2004; Bai et al. 2006; Zhang et al. 2008b; Sahay et al.
2013) are used for isolating microbes. The relative abundance of various taxonomic
groups of microorganisms changes with the type of the medium (Sorheim et al. 1989).
Different media giving comparable plate counts, may select for different bacterial
types thus leading to different estimates of diversity for the same soil. The choice of
the growing medium markedly affects the growth of microbe (Sorheim et al. 1989).
Small bacteria cells (dwarf cells or ultramicrobacteria) are not cultivable as they
cannot form colonies in agar. Bakken (1997) hypothesized that the culturable larger
bacteria cells have an ecological significance in soil more important than that which
appears from their small numbers as larger cells account for about 80% of the
bacterial volume.
A comprehensive work done in the last 11 years on the bacterial diversity of
cold habitats by cultivation dependent approach has been tabulated in Table-1
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Chapter 2 Review of Literature
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As cultivation dependent approach can assess limited phylogenetic diversity,
so further needs to be complemented with cultivation independent metagenomic
approach.
2.1.2) Cultivation independent metagenomic approach
Cultivation independent approach (metagenomics) involves the study of
collective genomes of microorganisms simultaneously. The term metagenomics was
coined by Jo Handelsman in 1998. The discipline of metagenomics was born when
Torsvik and Goksoyr (1978) and Pace et al. (1986) independently gave the idea that
the genomes of micro-organisms can be assessed without cultivating them. It
combines the power of Genomics, Bioinformatics and System biology.
The initial step in metagenomics is the extraction of total DNA to be used as a
template. There are two ways of extracting DNA (1) direct (in situ) extraction where
the cells are lysed within the matrix/ sample and then the DNA is recovered (Robe et
al. 2003) and (2) indirect extraction where the cells are first recovered from the
sample and then lysed for DNA recovery (Brady et al. 2007; Inceoglu O et al. 2010).
In both the techniques, the common step is to break open the cell either by shearing it
mechanically or chemically. Both methods have their own advantages and limitations.
Chemical and enzymatic treatment of the sample is gentle method and results in the
recovery of high molecular weight DNA but, can select certain species only by
exploiting biochemical properties of their cell wall. Mechanical shearing, on the other
hand, does not show such bias and is known to recover nucleic acid from more
diverse cells. However, the quality of DNA is not so good. Microbial community
(metagenomic) DNA isolation is a compromise between vigorous extractions required
for the representation of all microbial genomes and minimization of the DNA
shearing. Extracted metagenomic DNA can be used to explore both microbial
diversity and functional diversity.
Cultivation independent approach to study microbial diversity involves
analyses of selected genes such as SSU ribosomal and housekeeping genes from total
DNA extracted from an environmental sample followed by PCR amplification. The
most critical step in this approach is the primer selection used for amplification. PCR
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Chapter 2 Review of Literature
10
amplification of conserved genes such as 16S rRNA from an environmental sample
has been used extensively in bacterial ecology primarily because these genes, i) are
ubiquitous, i.e. present in all prokaryotes ii) are structurally and functionally
conserved and iii) contain variable and highly conserved regions (Rastogi and Saini
2011; Rajendhran et al. 2012). In addition, the suitable gene size (~1,500 bp) and a
growing number of 16S rRNA sequences available for comparison, in sequence
databases make it a “gold standard” in microbial ecology. 16S rRNA gene used for
bacterial identification comprises nine hypervariable regions, V1-V9, that exhibit
considerable sequence diversity among species (Elgaml et al. 2013). However, V3
hypervariable region is the target that has been the most extensively used (Kumar et
al. 2011). These hypervariable regions are generally flanked by conserved sequences
that can serve as anchors for universal or specific primer pairs (Jany and Barbier
2008). They are therefore used for species identification and allow the evaluation of
community diversity. No single region can differentiate among all bacteria therefore
different regions are amplified depending on the aim of the study (Chakravorty et al.
2007). Genomic copy number of the 16S gene varies greatly from 1 in many species
to up to 15 in some bacteria (Steven et al. 2012). Other primers used for bacterial
phylogeny are designed as per conserved genes such as RNA polymerase beta subunit
(rpoB), gyrase beta subunit (gyrB), recombinase A (recA), and heat shock protein
(hsp60) have also been used in microbial investigations (Das et al. 2014).
In fungus, internal transcribed spacers (ITS) provide a greater taxonomic
resolution than ssu rRNA genes and are generally used for fungal community surveys
in different environments (Porras-Alfaro et al. 2014). The ITS is a region located
between the 18S rRNA and 28S rRNA genes, including the 5.8S rRNA gene that
splits the ITS into two parts: ITS1 and ITS2. The ITS region undergoes a faster rate of
mutation than rRNA genes but, its sequence remains homogenous within a species.
Indeed, both ITS1 and ITS2 fulfil significant functions during rRNA maturation and
are under selective pressure (Jany and Barbier 2008).
The cultivation independent (metagenomic) community analysis can be
i) Cloning dependent
ii) Cloning independent (direct analysis)
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Chapter 2 Review of Literature
11
i) Cloning dependent method:
Till 2011, Cloning dependent was the widely used cultivation independent
method. In this method, the PCR products were amplified from an environmental
sample and then cloned and sequence the individual gene fragments (DeSantis et al.
2007). The obtained sequences are then compared to known sequences in a database
such as GenBank, Ribosomal Database Project (RDP), and Greengenes for bacterial
and UNITE fungal identification. The phylogenetic relatedness is estimated by
comparing sequence of amplicon to known microorganisms based on the homology of
16S rRNA/18S/ITS sequences and the closest affiliation of a new isolate (Taib et al.
2013). Cloning dependent culture-independent approach has revealed an abundant
array of previously unknown and uncultured microbes, including entirely new
bacterial divisions with no cultured representatives (Liu et al. 2009; Shivaji et al.
2011; Taib et al. 2013). Microbial diversity catalogued by Cloning dependent
approach has been tabulated in Table 2
Cloning dependent approach is limited in several aspects regarding
microbiome characterization and is very labor intensive. Each individual colony
represents one 16S rRNA gene, limiting most studies to relatively few sequences per
sample (sometimes in the tens or hundreds). Although it gives a larger view of the
total microbiome than cultivation based methods but complete bacterial diversity
cannot be explored, especially for rare biosphere bacteria (Carlos et al. 2012). In
addition to limited representation, restricted by the number of clones analysed, this
approach is also subjected to the bais induced by cloning.
ii) Cloning independent (direct analysis)
The alternative cloning independent metagenomic based on massively high-
throughput pyrosequencing has enabled acquisition of millions of sequences from
days worth of lab work, the same amount of data that would take years to obtain using
cloning methods, and at a fraction of the cost. It involves direct sequencing of the
phylogenitic/ functional gene amplicon without cloning them. Unlike Sanger
sequencing that requires in vivo amplification of DNA fragments to be sequenced
(achieved by cloning into bacterial hosts first) next-generation sequencing technology
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Chapter 2 Review of Literature
12
circumvents the cloning requirement by taking advantage of a highly efficient in vitro
DNA amplification. Some of the examples where microbial diversity has been
catalogued by Cloning independent (cultivation independent) approach has been
tabulated in Table 3
In addition to look into the diversity of microbial communities present in the
cold habitates, cold active hydrolytic enzymes including α-amylases, cellulase, lipases
and proteases produced by such microbes are another attraction to study these
habitats.
2) Functional diversity
Cold active enzymes have huge industrial and biotechnological significance.
The total market for industrial enzymes reached $3.3 billion in 2010 and it is
estimated to reach a value of 4.4 billion by 2015 (Sanchez et al. 2011; BBC Research
Report 2011; Adrio and Demain 2014). Among industrial enzymes, Lipases/esterases
represent a major product segment in the global industrial enzymes market with high
growth potential (Lopez-Lopez et al. 2014). The commercial use of lipases of cold
origin is a billion dollar business as in addition to other applications they are gaining
importance for the production of biodiesel (Takaya et al. 2011; Yan et al. 2014).
Global market of biodiesel is increasing rapidly (CAGR >20%). A new report
from Pike research predicts the global biofuels market shall double over the next
decade, from $82.7 billion in 2011 to $185.3 billion in 2021. Jatropha oil containing
20% saturated and 80% unsaturated fatty acids, represents a potential source for
biodiesel producton (Kumar et al. 2011b). Since feedstocks costs are about more than
85% of the total cost of biodiesel production (Fan and Burton 2009), lipase
transesterification has attracted much attention for biodiesel production as it allows
use of lower-cost feedstocks including waste cooking oil (WCO), grease, soapstocks.
It also produces high purity product, enables easy separation from the byproduct,
glycerol (Takaya et al. 2011). Other applications of cold active lipases have been
tabulated Table 4 for easy comprehension.
Many microbial lipases have been commercialized in the world by various
manufacturers like Novazyme (Denmark), Amano Enzyme Inc (Japan, Biocatalysts
http://www.pikeresearch.com/
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Chapter 2 Review of Literature
13
(UK), Unilever (Netherlands) and Greenock (USA). Bacterial lipases produced from
the genera Burkholderia and Pseudomonas are commercially available. Lipase PS
isolated from Burkholderia cepacia and Lipase AK isolated from P.fluorescens are
supplied by Amano and Lipase SL and Lipase TL isolated from B.cepacia and
P.stutzeri are supplied by Meito Sangyo (Japan). Cold adapted organisms expoited for
lipase production are tabulated below in Table 5.
As a large pool of microbes and their enzymatic activities remain unexplored
by cultivation dependent approaches so complementation with the cultivation
independent metagenomic approach gives a better insight of the functional diversity
in any habitat.
Cloning dependent approach to access functional diversity includes
construction of small insert library in a standard cloning vector such as pUC (Ferrer et
al. 2009). However, for the detection of large gene clusters or operons, BAC (Kakirde
et al. 2011), Cosmid (Neufeld et al. 2011; Cheng et al. 2014) and Fosmid (Geng et al.
2012; Martínez
and Osburne 2013) may be used as vector. Metagenomic
investigations have been conducted in several environments such as soil,
phyllosphere, ocean, and acid mine drainage and have provided access to
phylogenetic and functional diversity of uncultured microorganisms (Gupta and
Vakhlu 2011). This approach (Cultivation independent) is useful in mining novel
esterases and lipolytic enzymes from environmental samples. Most of the esterases
and lipases have a size of 1-2 Kb (Nacke et al. 2011) hence the use of pUC series
vector is considered to be the best available vectors to clone these genes.
Once the metagenomic library is constructed, the most important step is
screening for the desired trait. Screening of a metagenomic library can be either
functional or/and sequence based.
Function-based screening involves assay based identification of clones that
exhibit lipolytic activity. The most popular screening method for detecting positive
clones exhibiting the desired lipolytic activity uses tributyrin agar plates, in which the
appearance of clear halos around the colonies indicates hydrolysis of the substrate.
Screenings for specifically detecting true lipases have also been used with
http://www.ncbi.nlm.nih.gov/pubmed?term=Kakirde%20KS%5BAuthor%5D&cauthor=true&cauthor_uid=21112378http://www.sciencedirect.com/science/article/pii/S016770121400027Xhttp://www.ncbi.nlm.nih.gov/pubmed?term=Mart%C3%ADnez%20A%5BAuthor%5D&cauthor=true&cauthor_uid=24060119http://www.ncbi.nlm.nih.gov/pubmed?term=Osburne%20MS%5BAuthor%5D&cauthor=true&cauthor_uid=24060119
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Chapter 2 Review of Literature
14
metagenomic libraries, using longer substrates that are not hydrolyzed by esterases
(such as emulsified triolein, tricaprylin or olive oil) in the presence of the fluorescent
dye rhodamine B. In this case, orange fluorescent halos appear around lipase-
producing colonies when irradiated with UV at 350 nm (Kouker and Jaeger 1987).
The success of such screening relies on the compatibility of the cloned genes with the
transcription and translation machinery of the heterologous host, usually Escherichia
coli. Moreover, expression of lipases can be hampered by the requirement for specific
chaperones for the correct folding of the enzyme or by its toxicity to the host cells. It
has been reported that about 40%, are recovered by functional screening when E. coli
is used as host (Craig et al. 2010; Lopez-Lopez et al. 2014).The usefulness of a broad-
host range vectors for overcoming the barrier of host compatibility has been assessed.
One of the studies proves its effectiveness using six different Proteobacteria as hosts
for the same metagenomic cosmid library, recovering different positive clones in each
host (Craig et al. 2010). Cold adapted lipases have been screened from mountain soil
(Chow et al. 2012; Ko et al. 2012), high altitude soil Taishan, China (Wei et al. 2009),
deep-sea sediments (Jeon et al. 2009), marine sediment (Chu et al. 2008; Jeon et al.
2009) and tidal flat sediment (Wu et al. 2009).
Sequence-based screenings implement PCR-based methods, microarrays or
next-generation sequencing platforms. In PCR-based, approach and microarrays,
oligonucleotide primers are designed on the basis of conserved amino acid sequence
motifs and these identify target genes either directly or via polymerase chain reaction
(PCR)/nucleic acid hybridization. Gene-specific PCR yields a partial gene fragment,
requiring additional steps to obtain the up- and down-stream flanking regions. In such
instances, the partial gene fragment can be used as a probe to identify possible full-
length genes in a metagenomic library or enzyme restricted metagenomic DNA, that
can be excised and cloned.
Bell et al. (2002) designed degenerate primers using conserved amino acid
regions within lipase genes. Using these degenerate primers they obtain partial lipase
gene fragments from a metagenomic soil sample, and obtained the full-length lipase
genes using genome-walking PCR.
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Chapter 2 Review of Literature
15
Another approach for gene isolation is microarrays technology primarily based
on DNA-DNA hybridization. It is used to monitor differential gene expression to
quantify the bacterial diversity of the environment and catalogue genes involved in
key processes (Sebat et al. 2009). Microarray technology can be used before the
shotgun sequencing for the pre-selection of genes from metagenomic libraries,
thereby reducing the sequencing burden and reducing the proportion of sequences
unassigned by the database sequence similarity searches (Sebat et al. 2009). An
advantage of such method of screening is the direct identification of gene containing
clones without expression requirements while the possible disadvantage is the high
risk of false positive clones and the detection of partial genes (Booijink et al. 2007).
High throughput random shotgun sequencing combined with advanced
assembly and primer walking strategies may identify particular genes of interest. This
method does not require heterologous gene expression and when combined with next
generation sequencing technologies may be relatively less expensive and less
laborious relative to PCR and microarray. This approach generates vast amounts of
sequence information that require advanced computational approaches for assembly
and gene function assignment (Juncker 2009). Shot gun sequencing of metagenomic
libraries of human gut microbiomes revealed clear differences between adult and
infant microbiome pool (Qin et al. 2010) while this method also revealed a higher
similarity between family relatives as compared with unrelated individuals
(Turnbaugh 2009). This approach requires that the targeted gene has sufficiently
conserved regions of appropriate distance for emulsion PCR, which is required for
pyrosequencing, so that primers or sets of primers will sufficiently cover a gene
family. This approach is likely to be most useful for genes directly responsible for
important ecosystem functions or ecological processes, such as biogeochemical
cycles, biodegradation, pathogenesis, antibiotic resistances and cell signaling.
Allgaier 2010 applied high throughput sequencing (454-titanium
pyrosequencing) for the discovery of glycoside hydrolases from a Switchgrass-
adapted Compost Community and identified 800 genes encoding glycoside hydrolase
domains that were biased toward depolymerizing grass cell wall components. Of
these, 10% were putative cellulases belonging to families GH5 and GH9. Wang and
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Chapter 2 Review of Literature
16
coworkers (2014) explored the genetic diversity of glycoside hydrolase (GH) family
10 and GH11 xylanases in Lake Dabusu, a soda lake with alkaline pH and high
salinity (10.1%). A total of 671 xylanase gene fragments were obtained, representing
78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them
having low homology with known sequences. Phylogenetic analysis revealed that the
GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria,
Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly
consisted of Actinobacteria, Firmicutes and Fungi.
High throughput sequencing has been also applied to reveal the extensive
diversity of aromatic dioxygenase genes in the environment among human and animal
fecal microbiota (Iwai et al. 2010) analysis of glucan-branching enzyme gene profiles
among human and animal fecal microbiota (Lee et al. 2014). Whole-metagenome
sequencing of mangroves areas using the 454 GS-FLX titanium technology revealed
profile of α/β-hydrolase fold proteins in mangrove soil metagenomes from oil-
contaminated sites (Jimenez et al. 2014). High throughput sequencing has not been
yet used to study the diversity of lipase genes in any cold environment.
-
Chapter – 3
Material &
Methods
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Chapter 3 Materials and Methods
17
3. MATERIALS AND METHODS
3.1. Sampling site
The soil samples were collected from different regions of Drass mountains at
34.450N, 75.77
0E located in Ladakh (J&K) during May 2010 and pooled into one
composite sample. The soil was collected 20 cm cm deep into the earth by digging
and collected in aseptic plastic bags. Hands, trowels were treated with 70% ethanol
immediately before use. The samples were transported to the laboratory in ice and
stored at -20 °C (Foght et al. 2004).
3.2. Physiochemical analysis of soil
The soil samples were sent for analysis of pH, electrical conductivity, water
holding capacity, metal ion concentration, QC research lab IIIM Jammu, India. The
pH value was measured in a 1:5 (w/w) soil water suspension using electric digital pH
meter and the salinity of a soil was determined by measuring the electrical
conductance (EC) of soil water saturation extract with the help of a conductivity
meter (Das et al. 2012) where as chemical profiling of the soil for different metallic
ion was done by following the method developed by (Raman and Sathiyanarayanan
2009). The organic carbon and organic matter were determined by rapid titration
method (Walkley and Black 1934). Sodium and Potassium were determined by flame
photometerically. Total Nitrogen and Total phosphorous was estimated by using
Kjeldahl method (Bremner and Mulvaney 1982).
3.3 Soil phylogenetic Diversity
3.3.1 Cultivation dependent approach (Bacteria)
3.3.1.1 Media used
All the chemicals used in the study were purchased from Himedia Pvt. Ltd,
India and biochemicals and enzymes were procured from Bangalore Genei India Ltd,
New England Biolabs and Fermentas (MBI).
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Chapter 3 Materials and Methods
18
3.3.1.2 Comparison of cultivable bacterial load
1gm of soil was added to 10 ml normal saline and serially diluted. The samples
were diluted upto10-3
and 10-4
dilution level and spread on respective agar plates (Table
6) and incubated at 4 °C, 15 °C, 20 °C, 30
°C for 1 week. Colony forming units
(CFU)/gm soil/sample were calculated for each of the sample using a given formula
(Devi et al. 2012).
CFU/gm = Number of colonies x dilution factor/ volume spread
3.3.1.3 Polyphasic characterization
Bacteria were characterized on the basis of colony morphology, microscopy,
pigment color, growth pattern and biochemical analysis (Hamid et al. 2003). Microscopy
of bacterial isolates was done using Gram’s staining kit (Sigma). Isolates were screened
for biochemical property using biochemical test strips (Himedia). Pure cultures were
cryopreserved in 50% glycerol at -80 °C (New Brunswick, Effendorf).
3.3.1.4 Molecular identification of bacterial isolates
Selected bacterial cultures were further identified at molecular level.
3.3.1.4 (a) Genomic DNA isolation
Genomic DNA was isolated from all the bacteria using the GES protocol
(Pitcher et al. 1989)
Reagent preparation:
GES:
Guanidium thiocyanate (5 mol/l)
EDTA (100mmol/l)
Sarkosyl (0.5% v/v)
Guanidiumthiocyanate (60 g), 0.5 mol/l EDTA at pH 8 (20 ml) and deionized
water (20 ml) were heated at 65 °C with mixing until dissolved.After cooling, 5ml of
10% v/v sarkosyl were added; the solution was made up to 100 ml with deionized water
and stored at room temperature.
Ammoniumacetate (7.5 mol/l)
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Chapter 3 Materials and Methods
19
Lysozyme(50 mg/ml)
Chloroformand isoamyl alcohol mixture (24:1 v/v)
TE buffer: TrisHcl (10mM), EDTA (1mM)
Protocol:
3ml of broth cultures were harvested at the end of the exponential growth phase by
centrifugation at l000 g for 15 min and a small (rice grain-sized) cell pellet was obtained.
The cells of Gram-positive species were resuspended in 100 µl of fresh lysozyme in TE
buffer and the suspensions were incubated at 37 °C for 30 min.
The Gram-negative species were resuspended in 100 µlof TE buffer without enzymic
treatment.
Cells were lysed with 0.5 ml of GES reagent and cell suspensions were vortexed briefly
and checked for lysis (5-10 min).
The lysates were cooled on ice and 0.25 ml of cold ammonium-acetate (7.5 mol/l) was
added with mixing gently.
It was held on ice for further 10 min and then 0.5 ml chloroform and isoamylalcohol
mixture (24:1) was added.
The phases were mixed thoroughly, transferred to a 1.5 ml eppendorf tube and
centrifuged (12000 g) for 10 min.
Supernatant fluids were transferred to eppendorf tubes and 0.54 volumes of cold 2-
propanol was added.
The tubes were inverted for 1 min to mix the solutions and the fibrous DNA precipitate
was deposited by centrifugation at 6500g for 20 s.
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Chapter 3 Materials and Methods
20
Pellet of DNA was washed in 70% ethanol and air dried; and dissolved in 50µl of milliQ
3.3.1.4 (b) Purification of genomic DNA: Genomic DNA was purified by gel elution,
phenolation and purification through column or the combination of two methods.
Gel elution: DNA was eluted from 0.7 % Low melting agarose gel by elution kit
(Macherey – Nagel, Nucleospin Extract II kit) using the manufacturer’s protocol as
under
100 mg of the gel slice mixed with 200µl binding buffer (NT) , incubated at 5-10 min
(50°C)
The contents transferred to a column, centrifuged 1 min (11000g) and flow through was
discarded
700µl of wash buffer (NT3) was added to column and centrifuged as above followed by
dry spin at for 2 minutes (11000g)
Column was kept in a fresh eppendorf tube (collection tube) and 20µl milliQ was added
to the column (incubated for 10 minutes (25°C) and centrifuged as above to collect the
DNA.
Phenolation
Crude DNA was diluted with milliQ water and an equal volume of P:C:I
(Phenol: chloroform: isoamylalcohol; 25:24:1) was added and centrifuged at 13000g for
10 minutes.
The upper aqueous layer was carefully collected in a fresh eppendorf and again
an equal volume of C: I (24:1) was added followed by centrifugation.
The upper aqueous layer was carefully collected in a fresh eppendorf and DNA
was precipitated by adding 5M NaCl(1/10 volume) and ethanol (double the volume) and
incubated at -20°C for 2 h.
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Chapter 3 Materials and Methods
21
The precipitated DNA was centrifuged at 13000g for 30 minutes and the pellet
was washed with 70% ethanol, air dried and suspended in milliQ
Purification through column
200µl of Isopropanol was added per 100µl of crude DNA
Centrifugation was done at 11000g for 1 minute and flowthrough was discarded
700µl of 70% ethanol was added and centrifuged as above
Dry spin was done for 2 minutes and flowthrough was discarded
Column was kept in a fresh eppendorf tube (collection tube) and 20µl milliQ was added
to the column (incubated at room temperature for 10 minutes) and centrifuged as above
to collect the DNA
3.3.1.4(c) PCR amplification of 16S rRNA gene from bacterial culture and
ARDRA (Amplified Ribosomal DNA RestrictionAnalysis)
Universal bacterial primers, namely Bac8f (AGTTTGATCCTGGCTCAG) &
Univ529r (ACCGCGGCKGCTGGC) based on Escherichia coli positions, were used to
amplify internal fragments of 16S rRNA gene that amplify ~ 500 bp (Fierer et al. 2007).
The sequences that showed less than 98% homology with the reported sequences in the
database were reamplified by bac8f (5-AGAGTTTGATCCTGGCTCAG-3) and 1492r
(CGG TTA CCT TGT TAC GAC TT ) corresponding to Escherichia coli positions 8–27
and 1492–1509 respectively to amplify ~1500 bp region (Yong et al. 2011b). PCR
products were analyzed by electrophoresis on 1.5% agarose gel, followed by staining
with ethidium bromide and visualization under UV light. The amplified PCR products
were purified with a PCR product purification kit (Himedia cat no. ≠ MB512).
The PCR (with Bac8f & Univ529r primers) was performed following the
protocol standardized by Fierer and coworkers (2007) with modifications: instead of
0.5µM, 100pM primer were used and instead of 25 cycles, 30 cycles PCR were run. The
template DNA concentration for PCR reaction was used as 10-50ng per 10µl of PCR
-
Chapter 3 Materials and Methods
22
reaction volume and the PCR reaction mix is tabulated in Table 7. The PCR program
was denaturation at 950C for 5 minutes followed by 30 cycles of denaturation at 95
0C
for 60 seconds, annealing at 540C for 30 seconds followed by extension at 72
0C for 90
seconds and final extension at 720C for 10 min. The PCR (with 8f & 1492r primers)
was performed following the protocol standardized by (Yong et al. 2011b). The PCR
program was denaturation at 940C for 5 minutes followed by 30 cycles of denaturation
at 940C for 1 minute, annealing at 55
0C for 40 seconds followed by extension at 72
0C
for 90 seconds and final extension at 720C for 10 min.
The amplicons were screened for duplicacy by ARDRA using Alu and Hha I
restriction enzymes. The PCR amplified 16S rDNA were purified with a purification kit
(Himedia cat no. ≠ MB512). Aliquots of purified 16S rDNA PCR products were
digested separately with two restriction endonucleases AluI, Hha I in 25 lL reaction
volumes, using the manufacturer’s recommended buffer and temperature. The
restriction was done at 37 o
C for 1 hour. Restricted DNA was analyzed by horizontal
electrophoresis in 2.5 % agarose gels.
3.3.1.4(d) Sequencing and phylogenetic analyses:
16S rDNA were custom sequenced at SciGenom Labs Private Ltd., Cochin,
Kerala, India. The resulting nucleotide sequences were assigned bacterial taxonomic
affiliations based on the closest match to sequences available at the NCBI database
(http://www.ncbi.nlm.nih.gov/) using the BLAST (ww.ncbi.nlm.nih.gov/BLAST).
Sequences of bacteria thus obtained were deposited in the GenBank nucleotide
sequence database.
3.3.2 Cultivable independent (Cloning independent)
3.3.2(a) Metagenomic DNA isolation
Manual metagenomic DNA extraction protocols developed by Zhou et al. 1996;
Wechter et al. 2003; Brady et al. 2007; Amorim et al. 2008; Pang et al. 2008; Liles et al.
2009, Inceoglu et al. 2010 were applied. The protocols are mentioned in detail below
http://www.ncbi.nlm.nih.gov/)%20using
-
Chapter 3 Materials and Methods
23
Zhou’s protocol:
a) Buffer I: (Working concentration)
Tris HCL 100mM (pH 8)
Na2HPO4 50mM
NaH2PO4 50mM
EDTA 100mM (pH 8
NaCl 1.5 M
CTAB 1%
b) Lysis buffer:
SDS 20% (9 ml)
GITC 5M (4.5 ml)
c) Purification buffer:
Choloroform 24 ml
Isoamylalcohol 1 ml
Isopropanol
5 gm of soil sample was weighed in centrifuge cups and passed through sterile
sieve (2mm mesh sieve), 75 ml of buffer I was added. Dry ice was crushed and
isopropanol was added to make slush. The cups were incubated in crushed dry ice to
freeze (~40 minutes) and shifted to water bath at 65°C for thawing (~40 minutes).
13.5 ml of lysis buffer was added to the cups and incubated at 65°C and mixed
throughly by inversion for several times. Further 2 hr incubation with gentle inversion
mixing was done. Centrifugation was carried out at 15,000 g at 10C for 20 minutes.
The supernatant was transferred to fresh cups and 25 ml of choloroform:
isoamylalcohol (24:1) to each cup was added and mixed for 10 minutes.
Centrifugation at 15,000 g at 10 minutes was carried out for 20 minutes. The
supernatant was transferred in fresh cups and 0.7 % of isopropanol was added, mixed
for 5 minutes and the cups were incubated at room temperature for 20 minutes.
Centrifugation of cups at 15,000g for 40 minutes was done to collect the pellet and
supernatant was discarded. The DNA pellet was dissolved in 1 or 2 ml of T.E. Equal
-
Chapter 3 Materials and Methods
24
volume of Tris buffered with phenol-choloroform (pH 8.0) was added and then mixed
gently. The solution was centrifuged for 10 minutes. The supernatant was transferred
to a new centrifuge cup and isopropanol was added and again centrifuged for 20
minutes to retrieve the DNA pellet. The pellet was dissolved in 100μl of T.E and the
DNA was stored for few days at 4°C and -80
°C for long term storage.
Wechter’s protocol:
a) Extraction buffer
Phosphate buffer 0.2 M (pH 7.2)
PVPP 10% (w/v)
CaCl2 3M
b) Lysis buffer
SDS 20% (w/v)
Lysozyme 25 mg/ml
Proteinase K 20 mg/ml
c) Washing Solution
Ethanol 70%
d) TE (Tris Cl)
Tris-HCL 10mM (pH 8)
EDTA 1mM
One milliliter of sterile PPB (pH 7.2) was added to a 2ml microcentrifuge tube
containing 500mg of soil. After vortexing for1 min at high speed, the mixture was
centrifuged at 325x g for 30s in a micro centrifuge. The supernatant was transferred to
a 1.5-ml microcentrifuge tube, 400µl of a poly vinyl polypyrrolidone slurry (100 mg
PVPP/ml of PPB at pH 7.2) was added using a large-bore pipette tip, and the mixture
was vortexed at high speed for 30 second. To this, 2µl of 3M CaCl2 was added, and
the microcentrifuge tube was vortexed for 30s and centrifuged as stated above. The
-
Chapter 3 Materials and Methods
25
supernatant then was transferred carefully to a clean 1.5ml microcentrifuge tube, to
which 20µl of a lysozyme solution (25mg/ml) and 10µl of a Proteinase K solution
(20mg/ml) were added. The tube was inverted several times to mix the contents and
placed in a 37°C water bath for 30min and then placed in a 55°C water bath for
30min. 30µl of 20% (w/v) SDS was added to the tube, the contents were mixed by
inverting several times, and the tube was placed in an 80°C water bath for 30min.
Immediately after removal from the water bath, 400µl of the PVPP slurry was added
to the contents of the tube which was mixed by gentle inversion, placed on ice for 15
min, and then centrifuged at 8,000x g for 30min.The supernatant was transferred to a
clean 1.5ml microcentrifuge tube followed by the addition of 0.7x volumes of 100%
isopropanol, the tube was inverted several times and centrifuged at 8,000 x g for
30min. After centrifugation, the supernatant was discarded and the remaining pellet
was washed in 500µl of 70% ethanol, centrifuged for 5min at 16,000x g, and air dried
for 5min. The pellet was resuspended in 25µl of TE.
Brady’s protocol
a) Lysis buffer
Tris-HCl 100 mM
Na EDTA 100 mM
NaCl 1.5 M
Cetyl trimethyl ammonium bromide 1% (w/v)
SDS (pH 8.0) 2% (w/v)
b) Washing solution
Ethanol 70%
c) TE (Tris Cl)
Tris-HCL 10mM (pH 8)
EDTA 1mM
-
Chapter 3 Materials and Methods
26
Soil (5 g), 10 ml of preheated (70°C) lysis buffer were mixed and incubated at
70°C for 2h with constant inversions after every 30 min. The content of the bottles
was cooled to room temperature and supernatant was extracted twice by
centrifugation for 20 min. Supernatant was precipitated with two volume of ethanol
(25°C, 1hr). Following centrifugation for 30 min, DNA pellet was washed with 70%
ethanol, air dried and resuspended in TE.
Amorim’s protocol
a) Tween 80 0.1%
Sodium Phosphate buffer (pH 7)
b) TE (Tris Cl)
Tris-HCL 10mM (pH 8)
EDTA 1mM
c) Purification buffer
Choloroform 24 ml
Isoamylalcohol 1 ml
Isopropanol
Soil (5 g), 0.1% Tween 80 and 50ml Sodium Phosphate buffer (pH 7) were
mixed in a nalgene bottle (250-mL) followed by overnight incubation at room
temperature (25°C) with constant shaking. Pellet was obtained by centrifugation
(6000 g, 10 min) and washed four times with TE buffer (Tris-HCl 50 mM, EDTA 50
mM, pH 8.0) by centrifugation at 5000 g for 5 min. Cell pellet obtained was lysed
mechanically using liquid nitrogen, macerate was then transferred to a 15-mL
centrifuge tube containing 2 mL TE buffer 50/50 and an equal volume of PCI
(25:24:1). Supernatant was obtained by centrifugation at 5000g for 10min. DNA was
precipitated using 0.7 vol of chilled Isopropanol and 1/10 vol of 3M sodium acetate,
followed by overnight incubation at -20°C. DNA pellet was collected by
centrifugation at 10,000g for 15 min (4°C), washed with 70% ethanol, air dried and
resuspended in T.E
-
Chapter 3 Materials and Methods
27
Liles’s 2009
a) Lysis buffer
Sarkosyl 1%
Sodium deoxycholate 1%
Lysozyme 1 mg/ml
Tris-HCl [pH 8.0] 10 mM
EDTA [pH 8.0] 0.2 M
NaCl) 50 mM
b) ESP buffer
1% Sarkosyl,
1 mg/ml Proteinase K
0.5 M EDTA [pH 8.0])
c) 1 mM phenylmethylsulfonyl
Extracted and washed bacterial cells were pelleted by centrifugation and
embedded within low-melting-point agarose in a 1-ml syringe. The agarose plug was
then extruded from the syringe and incubated in 10 ml of lysis buffer 1 h at 37°C. The
plug was transferred into 40 ml of ESP buffer and incubated for 16 h at 55°C,
followed by inactivation of proteinase K with 1 mM phenylmethylsulfonyl fluoride
from a fresh phenylmethylsulfonyl fluoride stock in isopropanol with 1 h of
incubation at room temperature. After three 10-min washes in TE, plugs were stored
at 4°C in 10 mM Tris-HCl with 50 mM EDTA (pH 8.0).
Pang’s protocol
Reagents required
a) Extraction buffer
-
Chapter 3 Materials and Methods
28
TrisCl 100mM (pH8)
Na-EDTA 100mM (pH8)
NaCl 1.5M
b) Lysis buffer
SDS 20%
Lysozyme 10mg/ml
Proteinase K 20mg/ml
c) Polyethylene glycol (30%)
Soil (20 g) was suspended in 50 ml of DNA extraction buffer along with 1 ml
of Lysozyme (10 mg/ml) and incubated at 37ºC for 1 h. Sample was further incubated
at 65ºC with SDS (2 ml, 20%, w/v) and proteinase K (15 μl, 20 mg/ml) for 2 h and
then centrifuged at 6,000 rpm for 10 min to remove soil residue. Supernatant was
transferred into a clean tube, and then precipitated by using half-volume of PEG
(30%, w/v) and incubated at room temperature for another 2 h. DNA was pelleted and
resuspended in milliQ.
Inceoglu’s protocol (modified version of the method of Zhou et al. (1996).
Briefly, 2.5 g soil samples were mixed with 30 ml of EDTA, 50 mM Tris
buffer (pH 8.3) and centrifuged (6,000 x g, 4ºC, 30 min), after which the supernatant
was discarded. Following this initial wash, 2.5 ml of 500 mM NaCl, 50 mM Tris, 50
mM EDTA (pH 8.3) was added to the soil pellets. Lysozyme was then added at 5
mg/ml and the suspension incubated for 1 h at 37°C. Then, 140 μl of 20% SDS was
added, along with 1 mg of proteinase K, for a further 1h digestion. After incubation, 5
ml of 500 mM NaCl, 300 mM succinic acid, 10 mM EDTA (pH 5.7) were added,
followed by 700 μl of 20% SDS. The mixtures were incubated for 30 min at 65°C and
centrifuged (15,000 x g for 30 min), after which the soil pellets were discarded.
Aliquots (8 ml) of supernatant were then transferred to 50-mL Falcon tubes and
supplemented with 1 ml of 5 M NaCl followed by 1 ml of 10% cetyl trimethyl
ammonium bromide (CTAB). The suspensions were gently mixed and incubated for
30 minutes at 65ºC. Five ml of 50% polyethylene glycol - 8000 (PEG- 8000) were
then added before overnight incubation at 4ºC, centrifugation (40,000 x g, 30 min,
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Chapter 3 Materials and Methods
29
4ºC) and discarding of the supernatant. The resulting pellets were resuspended in 240
µl TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0) in Eppendorf tubes. Then, these
were extracted with one Vol. of phenol/chloroform/isoamylalcohol (25:24:1) and
subsequently with one Vol. of chloroform/isoamylalcohol (24:1). Humic acids were
further removed by adding CaCl2. 2H2O at 27 mM, incubating at 65ºC for 1 h, and
centrifuging at 14,000 x g for 20 min. The resulting supernatants, containing the soil
DNA, were precipitated with 3 M potassium acetate, centrifuged (12,000 x g, 10 min),
and the resulting pellets washed with 70% ethanol and dissolved in 20 μl TE buffer
3.3.2(b) PCR amplification
DNA extracted from multiple methods (n=3) was pooled and diluted (1/100
dilutions) with final concentration of 20 ng/µl and used as template for PCR
amplification.
PCR amplification of 16S rRNA for assessment of bacterial diversity
Small region (V1-V3) of the 16S rRNA gene was amplified from the total soil
DNA by PCR using bacterial Universal primers set 27F5`-
AGAGTTTGATCCTGGCTCAG-3` and 519R 5`-ATTACCGCGGCTGCTGGCA-
3`) (Kumar et al. 2011a). 0.5µl (10- 50 ng/ µl) of DNA was used per 10µl of PCR
reaction volume and the PCR reaction mix is given in Table 8. The PCR program
included initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation
at 94°C for 45s, annealing at 55
°C for 45s and extension at 72
°C for 1 min and 30s
followed by a final extension at 72°C for 10 min
PCR amplification of ITS for assessment of fungal diversity
Inter transcribed spacer region (ITS1-ITS4) was amplified using Universal
primer pair ITS1f (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4r (5’-
TCCTCCGCTTATTGATATGC-3’) (White et al. 1990). DNA was amplified
according to the PCR program as Denaturation at 95°C for 5min followed by 30
cycles of denaturation at 95°C for 45s, annealing at 55
°C for 30s, extension at 72
°C for
60s and final extension at 72°C for 10min. PCR reaction volume and the PCR reaction
mix is given in Table 8. The amplicon was further purified from agarose gel by gel
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Chapter 3 Materials and Methods
30
elution column (Qiagen). The purified amplicons thus obtained (16S and ITS) were
send for pyrosequencing at research and testing laboratory (Lubbock, TX, USA)
3.3.2(c) Analysis of data of 454 pyrosequencing
Bar-coded pyrosequencing
Bacterial and fungal tag-encoded FLX-Titanium amplicon pyrosequencing
(bTEFAP and fTEFAP) and data processing were performed at the Research and
Testing Laboratory (Lubbock, TX) (www.research and testing.com). The bacterial
primers based on V1-V3 region were used and sequencing was performed, extended
from 27F numbered in relation to Escherichia coli 16S ribosome gene (forward 27F
5`- AGAGTTTGATCCTGGCTCAG-3`). Single step PCR reaction (35 cycles) was
used and 1 U of Hot Star High fidelity Polymerase was added to each reaction
(Qiagen, Valencia, CA). The fungal primers were based on the ITS region and
sequencing was performed forward from 458F in relation to Candida albicans (Dowd
et al. 2008).
Quality filtering and Phylogenetic analysis
Uchime tool was used to remove the chimeric sequences (Edgar et al. 2011).
Quality trimmed sequences were clustered into operational taxonomic units (OTUs)
using CD-hit (Balzer et al. 2013) with a cut-off value of 99% sequence identity.
Candidate OTUs were assigned to phylogeny using RDP (Cole et al. 2009; Krober et
al. 2009) scheme set at 80% confidence value. Rarefaction curves and diversity
indices were calculated by using RDP pyrosequencing pipeline (Cole et al. 2009).
Pvclust tool was used to obtain the Bootstrap Probability (BP) value the
Approximately Unbiased (AU) (Shimodaira and Hasegawa 2001, Suzuki 2006).
Cluster dendrogram was constructed with AU/BP values (%).
3.3.2(d) Reference datasets
Bacterial data comparison
A total of four reference datasets was obtained from NCBI. Two reference
datasets were selected randomly from the Antarctic study with accession no
SRX206452, SRX206985 and remaining two reference datasets were selected
http://www.research/
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Chapter 3 Materials and Methods
31
randomly from the Arctic study with accession no SRX017110. The sources of
Antarctic and Arctic samples were from Grove Mountains, East Antarctic and
Foreland of MidreLoven glacier, respectively.
Fungal data comparison
A total of two reference datasets was obtained from NCBI with accession no
ERX253153 for Arctic dataset. The data for Antarctic were not submitted by the
authors on NCBI-SRA, so sequences were obtained through mail on request. The
source of Antarctic sample data were McMurdo Dry Valleys (Dreesens et al. 2014)
and the source of Arctic sample were Alaskan permafrost (Penton et al. 2013)
4.4 Diversity of Lipolytic genes
4.4.1 Screening cultivable bacteria for production of lipolytic enzymes
Agar medium (1.5% w/v) containing substrate 0.4% (w/v) tributyrin for
esterase and olive oil (1%) for lipases were inoculated with freshly grown cultures
and incubated at 4°C, 10°C, 20°C, 30°C for one week (Gangwar et al. 2009). For
screening lipases, syringe filtrated olive oil (1%) and a florescent dye rhodamine B
(0.001% w/v) was added to the autoclaved cooled growth medium with vigorous
stirring. The plates containing bacterial cultures were observed for an orange
fluorescence under UV light at 350nm (Ranjitha et al. 2009).
Tributyrin medium:
Tryptone 1%
Yeast extract 0.5%
NaCl 1%
Tributyrin 1%
Gumacacia 1%
pH 7.0
Agar 1.5%
Lipase screening media was stirred for 30 minutes to make tributyrin emulsion.
Autoclaving of above media was done at 15 psi for 20 minutes at 7.0 pH.
http://www.ncbi.nlm.nih.gov/pubmed/?term=Penton%20CR%5BAuthor%5D&cauthor=true&cauthor_uid=24014534
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Chapter 3 Materials and Methods
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Rhodamine agar media
Nutrient agar 2.8%
NaCl 0.4%
Rhodamine B 0.1mg
Olive oil 3.1ml
pH 7.0
Agar 1.5%
Nutrient agar media was autoclaved separately at 121°C, 15 psi for 20 minutes.
Separate autoclaved tube was used to prepare rhodamine B solution dissolved in ethanol.
Rhodamine B solution was sterilized by passing it through 0.2μm filter. After autoclaving
the nutrient agar media was cooled at room temperature and rhodamine solution and olive
oil was added respectively. The lipase positive cultures were visualised on UV
transilluminator (Bangalore Genei).
4.4.2 Diversity of lipolytic genes by cultivation independent approach.
4.4.2 (a) PCR amplification
Metagenomic DNA amplified by using degenerate primers LIPF (LIPF 5-
GACCRATYGTSCTSGTVCAYGG–3’) and LIPR2 (5’-GCCRCCSTGRCTRTGR
CC – 3’) that target oxynion hole and active site. The PCR reactions (10-20 μl)
contained between 10-50 ng genomic DNA and the PCR reaction recipe is given in
Table 9. Touch down PCR was run. Initial denaturation at 94°C for 5 min, followed
by 4 cycles of denaturation at 94°C for 30s, annealing at 65°C for 1 min, and
elongation at 72°C for 2 min. Cycling conditions were then altered: the same
denaturation and elongation conditions were used, but the annealing temperature was
reduced to 64°C with a reduction of 1°C every cycle for 14 cycles. Cycling conditions
were again adjusted: the denaturation and elongation conditions were maintained, but
the annealing temperature was set at 50°C for 20 cycles. Following the last cycle, a
final elongation step at 72°C for 3 min was performed. For Aliquots of the PCR
reaction mixtures were subsequently analyzed by 1.5 % agarose gel electrophoresis.
Primer pair lip LipF and LipR2
yielded a single band. The band excised, eluted and
send for Pyrosequencing (research and testing lab).
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Chapter 3 Materials and Methods
33
4.4.2(b) Bioinformatic analysis
OTU filtering was done with CD-hit 454 software (Balzer et al. 2013).
BLASTp was performed against Lipase Engineering Database (Widmann et al. 2010;
Jimenez et al. 2015) to assign the class to lipolytic sequences. BLASTp was also
performed (available in the National Centre for Biotechnology Information) to assign
phylogeny.
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Chapter – 4
Results &
Discussions
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Chapter 4 Results and Discussion
34
4. RESULTS AND DISCUSSION
The present study was undertaken with an aim to explore diversity of
phylogenetic and lipolytic genes from the soil metagenome of Drass located in
Ladakh region of Jammu and Kashmir. Both approaches cultivation independent and
cultivation dependent were used to achieve the aim.
4.1. Sampling site
Drass, a town in the Kargil district of Ladakh region (Jammu and
Kashmir) India at 34.45°N, 75.77°E with an average elevation of 3,280 meters
(10,764 feet) is the second coldest inhabited place in the world. Drass is characterized
by hostile climatic conditions and remoteness. Summers are warm with temperature
up to 35°C with cool nights and winters are long and cold with temperatures often
dropping to −40°C (Sagwal 1997).
4.2. Physio- Chemical analysis of soil
Drass soil is sandy, coarse, slightly alkaline and nutrient-poor with 0.72%
organic content (Table10). Low organic content of the soil is reported due to lack of
vegetation, poor microbial activities, coarse sediments and low humus (Kastovska et
al. 2005; Rawat and Adhikari 2005; Sagwal 1997). High altitude cold desert soils,
have been reported to be originated from weathered rocks with large proportion of
sand gravel and stone with low water (Dwivedi et al. 2005). Coarse alkaline sandy
soils have been reported from Ladakh (Namgail et al. 2012; Charan et al. 2013) and
other Himalayan cold environments (Pradhan et al. 2010; Shivaji et al. 2011) Cold
desert high altitude soils have poor water and nutrient holding capacity (Charan et al.
2013) and so is true for Drass. Electric conductivity (EC) of the Drass soil is 12 ds/m
which suggests that the soil is extremely saline or salt rich. Organic carbon content,
availability, soil texture and salinity influences microbial community composition and
diversity (Fierer et al. 2003; Fierer et al. 2007). Physicochemical profile of the soil
shows that it is not supportive of high microbial activity as is true of other similar
soils (Pradhan et al. 2010; Shivaji et al. 2011).
http://en.wikipedia.org/wiki/Kargil_Districthttp://en.wikipedia.org/wiki/India
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Chapter 4 Results and Discussion
35
The soil contains ferrous, magnessium, aluminium, iron, potassium and
calcium in abundance whereas tracer elements such as cobalt, copper, nickel, arsenic,
lead and zinc are present in low concentration. Metals play an important role in
biological process as essential micro-elements. Heavy metals at elevated
concentrations are known to effect soil microbial population and their associated
activities (Ahmed et al. 2005; Anyanwu et al. 2011).
4.3 Microbial Diversity
4.3.1 Cultivation dependent approach (bacteria)
4.3.1.1 Bacterial isolation and characterization
Both oligotrophic and nutrient rich media were selected to obtain maximum
cultivable bacteria. About 600 isolates were randomly selected (100 each from six
different media: Nutrient agar, LB agar, King,s B agar, TSA, Minimal media, R2A agar)
used in the study. Since the average summer and winter temperature varies between 4°C-
30°C, the bacteria were isolated within this temperature range. The growth pattern of
individual bacterial culture were studied and placed into psychrophilic (4–20°C),
psychrotrophic (4–30°C), and psychrotolerant mesophilic (4–37°C), mesophilic (25–
40°C) groups (Sahay et al. 2013) (Table 13). Maximum bacterial load (including
pigmented and non-pigmented 5.0± 0.07x 106
CFU/ml at 30°C CFU/ml was obtained
using NA (Table 11) but maximum number of pigmented bacteria 2.9±0.17x106
were
obtained with R2A media (Fig. 2-3). Pigment production was intense at 4°C and
decreased with increase in incubation temperature which is in accordance with earlier
studies on bacterial diversity of Puruogangri ice core (Zhang et al. 2008b) and
Himalayas (Venkatachalam et al. 2014). R2A is a oligotrophic medium and allows
cultivation of many pigmented bacteria in particular that will not readily grow on fuller,
complex organic media. R2A has been used to isolate bacteria from various cold
environments e.g glaciers (Foght et al. 2004), marine surface waters (Agogue et al.
2005), ice cores (Zhang et al. 2008b) and Antarctic soils (Dieser et al. 2010; Peeters et
al. 2012). The pigments produced by these bacteria are reported to be carotenoids and
has been co-related with cold adaptation of microorganisms by many workers
(McDougald et al. 1998; Cho et al. 2000; Daniela et al. 2012; Mojib et al. 2013).
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Chapter 4 Results and Discussion
36
4.3.1.2 Diversity measures
Diversity indices were used to compare between the communities obtained by
using different media. More community complexity was found using R2A media (Table
12). Overall Shannon-Wiener index (H) was 3.2 that is in accordance with previous
reports from Himalayan bacterial diversity (Pradhan et al. 2010; Shivaji et al. 2011;
Yadav et al. 2014).
4.3.1.3 Molecular and phylogenetic analysis of 16S rDNA sequences of isolates
Bacterial isolates were screened for duplicacy by colony/cell morphology
analysis, pigmentation, conventional biochemical tests that narrowed the 600 isolates
into 99 isolates. These selected isolates were subjected to 16S rRNA gene amplification
followed by restriction digestion with (Alu I and Hha I). On the basis of ARDRA
profiling, representative isolate from each cluster were sequenced and the nucleotide
sequences were deposited in the NCBI GenBank database (Accession numbers:
JX978884-JX978891, JX978892-JX978896, JN088486, JN088488, JN088491,
KF555604-KF555606, KF555608- KF555624, KF682428, KF682429 and HE774268,
KM188063). The nearest phylogenetic neighbor of all the 40 representative isolates were
identified through BLAST analysis of the 16S rRNA gene sequences against nucleotide
database available in the National Centre for Biotechnology Information (NCBI) (Table
13).
Drass isolates represented both Gram-positive and Gram-negative heterotrophic
bacteria belonging to three major phylogenetic groups organized into three clusters
Proteobacteria (37.5%), Firmicutes (32.5%) and Actinobacteria (30%) (Fig. 4).
Proteobacteria dominates (37.5%) the culturable bacterial diversity of Drass with
Gammaproteobacteria (35%) as the dominant class represented by genera Pseudomonas,
Acinetobacter, Serratia, Pantoea. Pseudomonads represented the dominant genera
among Gammaproteobacterium. Alphaproteobacteria is however represented by single
genera i.e Paracoccus (Dr32) (Fig. 5). The results are in accordance to the previous
studies on Himalayan that reports Firmicutes, Actinobacteria, and Proteobacteria as the
most common phylum (Shivaji et al. 2011).
Bacterial isolates showed 99% similarity with the reference sequences in the
Genbank except for Dr 46 that showed 96% similarity with Pantoea agglomerans (Fig.
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Chapter 4 Results and Discussion
37
6). DNA-DNA hybridization will be carried with close relatives to confirm and publish
as novel species. The bacteria isolated and characterized from Drass soil have been
reported from other cold environments also. The genera Arthrobacter, Bacillus,
Sporosarcina, Rhodococcus, Pseudomonas were reported in the culturable bacterial
diversity of Pindari glacier (Shivaji et al. 2011). The genera Acinetobacter, Bacillus,
Pseudomonas were reported in the culturable bacterial diversity of Kafni glacier
(Srinivas et al. 2011b). The genus Arthrobacter is the dominant bacteria in Qinghai-
Tibet Plateau permafrost (Zhang et al. 2007a), Brevibacterium and Acinetobacter are
present in abundance in Dry Valley soils of Antarctica (Cary et al. 2010),
Planomicrobium, Mycetocola, Rhodococcus, Sporosarcina have been reported from
Himalayan soils in India and Nepal (Venkatachalam et al. 2014) and an Arctic glacier
(Reddy et al. 2009). Exiguobacterium (Gram positive and facultatively anaerobic) have
been repeatedly isolated from ancient Siberian permafrost (Rodrigues et al. 2009).
Members of genera Exiguobacterium are adapted to long- term freezing at temperatures
as low as -12oC where intracellular water is not frozen and grow at subzero temperatures,
displaying several feature of psychrophiles, such as membranes composition. Genus
Pantoea (Selvakumar et al. 2008; Venkatachalam et al. 2014), Dietzia (Mayilraj et al.
2006), Staphylococcus and Citricoccus (Yadav et al. 2015) have been reported from
Indian Himalayas. Members of the genus Paracoccus have been reported from Qinghai-
Tibet Plateau permafrost (Zhu et al. 2013).
4.3.2 Cultivation independent (metagenomic) approach
Only three metagenomic DNA extraction protocols, developed by Zhou and
coworkers (1996); Wechter and coworkers (2003), Pang and coworkers (2008) worked
efficiently on the soil of Drass (Fig. 7). DNA extracted using these three protocols were
pooled and used as a template for PCR amplification. Multiple DNA extraction
protocols were employed since no single method of metagenomic DNA isolation is
efficient enough to represent all the bacterial diversity (Delmont et al. 2011).
4.3.2.1 Assessment of bacterial diversity by 454 pyrosequencing
Metagenomic DNA was diluted 1/100 times and used as template for PCR
amplification.The hypervariable region V1-V3 of 16SrRNA genes were amplified from
http://link.springer.com/search?facet-author=%22G.+Selvakumar%22http://aem.asm.org/search?author1=Tom+O.+Delmont&sortspec=date&submit=Submit
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Chapter 4 Results and Discussion
38
the extracted metagenomic DNA using the universal bacterial primers 27F 5`-
AGAGTTTGATCCTGGCTCAG-3`and 519R 5`-ATTACCGCGGCTGCTGGCA-3
(Dowd et al. 2008). Targeting V1-V3 regions of bacterial 16S rRNA genes provide two
advantages: first, the V1–V3 regions are more divergent and thus can provide more
phylogenetic resolution than other regions. Secondly, RDP and other databases store
maximum sequences that correspond to the V1–V3 region of 16SrRNA gene
(Chakravorty et al. 2007). Hence more sequences are available for comparison and it
facilitates the correct phylogenetic analysis from the phylum to the genus level (Jeraldo
et al. 2011; Eren et al. 2014). Short pyrosequencing reads also assess the microbial
diversity reliably as near-full-length sequences if appropriate primers are chosen (Will C
et al. 2010). The 500 bp amplicon was gel eluted and sent for pyrosequencing in
research and testing laboratory (Lubbock, TX, USA) (www.researchandtesting.
com/next-generation-sequencing-service.html (Fig. 8)
Bacterial diversity in metagenome of Drass soil
The amplicons were pyrosequenced and 3000 sequence reads were generated.
Downstream quality filtering resulted in 2819 high quality sequences with average
read length of ≥ 200 bp. Phylogenetic analysis revealed Acidobacteria,
Proteobacteria, and Actinobacteria as highly abundant in the soil of Drass
representing 39% of the total bacterial sequences. In addition, Chloroflexi,
Bacteroidetes, Verrucomicrobia, Planctomycetes, Firmicutes, Nitrospira,
Armatimonadetes (former candidate division (OP10), Gemmatimonadetes,
Cyanobacteria, were also identified at the relatively low abundance (
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Chapter 4 Results and Discussion
39
However, this framework would be benefitted via deeply sequenced longitudinal time
series datasets.
Comparative account of bacterial diversity across Drass, Antarctic and Arctic
soil samples
The 16S rRNA gene sequences from the present study (Drass soil) and those
retrieved from NCBI (Arctic and Antarctic soil) were clustered at 99% sequence
identity (Egge et al. 2013). A Significant proportion (2.2%, 2.8% and 1.9%) of the
total sequences from Drass and Antarctic (ANT1 & ANT2) sa