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Chapter I Introduction & Review o(Literature

1.1. Introduction

Microorganisms constitute the oldest, vast and almost unexplored reservoir of natural

resources and are likely to provide innovative applications in challenging areas like food, energy and

climate change. They are vital for sustaining life on the biosphere directly or indirectly. The earth is

estimated to be nearly 4.6 billion years old (1 billion =109). The age of the oldest living systems has

been more difficult to establish, although many lines of evidence point to the occurrence of abundant

microbial life at least 3.5 billion years ago (Fenchel, 2002; Lazcano & Miller, 1994). There is a

general agreement that 80% of the natural history of life on Earth is exclusively a history of

microbial communities (Colwell et ai., 1996). The first line of evidence regarding the antiquity of

microbial life forms consists of ancient microbial fossils, which have been found at almost all places

where prehistoric environmental conditions support their preservation (Corsetti et ai., 2003). Flint

like siliceous rocks (cherts) may contain fossils of bacteria, particularly of large cyanobacterial cells

with specialized secondary structures embedded in silica (Kasting, 2001).

The microfossil research of Schopf (1996) and molecular chronometer studies of Doolittle et

aI., (1996) have established a framework for the divergent times of the major microbial groups: 3.5

Gy (1 Gy=1 billion year=109 years) for the cyanobacterial-like lineages, 2.1 Gy for the last ancestral

type (Progenote) common to all three domains, Archaea- Eukarya separation at 1.96 Gy, Gram

positive and Gram negative bacterial separation at 1.5 Gy, and the divergence of protists at 1.23 Gy.

The history of life on earth is unquestionably dominated by unicellular microorganisms and in terms

of relative biomass and physiological diversity, the contributions of microbes to global

environmental change far outweigh the contributions of complex multicellular macroorganisms.

During the first half of earth's evolutionary history, a set of metabolic processes that evolved

exclusively in microbes would come to alter the environments of the planet. They could adapt

themselves to live in diverse habitat because they were capable of exploiting a vast range of energy

sources (Hurst, 2001). When earth was turning to its modem shape for the initial period, prokaryotes

were the only life forms present (Mojzsis et aI., 1996) and they became the core biological machines

responsible for biogeochemical cycles that sustain other life forms and health of the planet.

A significant development in microbial ecology and evolution has been the realization that

microbial life, primarily prokaryotic life, is extremely hard, and can survive and indeed thrive in

environments previously thought uninhabitable on earth (Steven et aI., 2007). Both bacteria and

archaea (constituting prokaryotes) can live in extremes of conditions. All present day organisms in

biosphere depend on microbial activities (Pace, 1997). Their activities created environment

conducible for evolution of other organisms. Life continued to evolve giving rise to various life

forms with tremendous diversity and range of complexity as being observed on the present day earth.

Chapter 1 Introduction & Review o(Literature

Diversity of macroscopic life forms has been reasonably well documented over the centuries.

This was possible because of their large size and visibly distinguishing morphological features.

Prokaryotic diversity, on the other hand, did not draw as much attention because their existence came

to be known only with the discovery of microscope by Leeuwenhoek in 1673. Now it is known that

prokaryotes are the majority on this planet (4-6 x 103°) and their diversity is enormous and constitute

350-550 Pg of carbon (lPg = 1015g) contributing nearly as much carbon as plant (Whitman et aI.,

1998). They are incredible due to the fact that they communicate and interact among themselves as

well as with members of other domains which makes the diverse array of life possible on earth.

Bacteria provide foundation of our biosphere in the sense that they possess an unusual high

physiological and biochemical versatility. They can use reduced inorganic compounds as energy

source (electron donors) for chemolithotrophic metabolism or oxidized inorganic compounds as

electron acceptors for anaerobic respiration. Moreover, certain metabolic pathways, such as

fermentations, nitrogen fixation, methane formation and anoxic photosynthesis are only found among

prokaryotic microbes. This is also true for the biosynthesis of secondary metabolites such as certain

life saving antibiotics, various enzymes and toxins. The metabolic, physiological and genetic

diversity of prokaryotic microorganisms is far greater than that found in higher organisms. Earth gets

rid of accumulation of naturally occurring toxic substances, waste and xenobiotics by degradative

capabilities of microorganisms. Some of them obtain energy for growth by transferring electrons to a

wide range of harmful metals, such as uranium, chromium, arsenic and plutonium (Schleifer, 2004)

and other organisms in tum utilize the products of such degradative cycles making the cycle very

intricate and interdependent. Only a handful of microorganisms cause disease. On the other hand,

associations with microbes, particularly bacterial endosymbionts, are fundamental to the survival of

higher organisms. Symbionts carry out essential biochemical reactions for their eukaryotic hosts, e.g.

the biosynthesis of essential amino acids, vitamins, or degradation of certain macromolecules like

cellulose. Some marine worms use their sulfide oxidizing bacterial endo or ecto symbionts even as

sole feed source.

The benefits of improving the understanding of microbial diversity and ecology are economic

as well as social. The knowledge might provide new tools for bioremediation, biorestoration and

improved management of ecosystem as well as it offers the potential to identify new genes of

bacterial origin to be utilized for bioprospecting, biotransformations and in synthetic biology.

Although understanding the roles of bacteria in shaping the ecology and environment on earth are far

from complete, study of their diversity has become an important part in this direction.

As the existence of the microbial life was recognized only relatively recently in history (about

300 years ago), the knowledge gained is still rudimentary. Microorganisms still represent the largest

reservoir of undescribed biodiversity. The recent techniques such as ribosomal RNA gene

sequencing have helped to survey the biodiversity sufficiently faster and comprehensively. Till date

2


53 bacterial phyla are known in bacterial domain (27 phyla have culturable representatives and 26

phyla have no cultured representatives). About 13,700 bacterial species only have been formally

described and majority of them (~ 90%) lie within 4 of the 53 known bacterial phyla (Hugenholtz,

2002) and 23 out of the 53 bacterial phyla have only a few culturable representative (Keller &

Zenglar, 2004). Rest of the 26 bacterial phyla that do not have cultured representatives, their

presence is known from gene sequences only. Many lines of evidence indicate that less than 1 % of

the bacteria can be cultured so far. The term "the great plate count anomaly" was coined by Staley &

Konopka (1985) to describe the difference in orders of magnitude between the numbers of cells that

grow on nutrient media and the number countable by microscopic examination. There are different

explanations for this anomaly; growth state of cells in nature, dormancy, exceptionally high

concentration of nutrients, complex organic carbon in laboratory media and failure to provide

suitable conditions. The physiology and ecological role of these unknown "uncultured" bacterial

species in most cases can only be assessed after their isolation in pure culture or partly from

sequences of genes in culture independent approach. Thus one of the biggest challenges in

microbiology today is how to increase the percentage of cultivability or in other words how to culture

these so-called "unculturables".

Ecogenomics, direct environment shotgun sequencmg, genomics of cultured microbes and

functional genomics, together with different microbial ecological methods such as microarrays, real

time peR, FISH (Flourescent In Situ Hybridization), microautoradiography and new cultivation

techniques will contribute significantly to the understanding of hitherto uncultivated microbes of

various environment. However, attempts to develop and refine culturing techniques should continue,

as this is the only way to have a better knowledge about the biology of the microorganisms.

The biological diversity of the Indian subcontinent is one of the richest in the world owing to

its vast geographic area, varied topography and climate, and the juxtaposition of several bio

geographical regions. Because of its richness in overall plant and animal species diversity, India is

recognized as one of the 12-mega diversity regions of the world. By virtue of supporting and

sustaining rich biological, ethnic and landscape diversity, it manifests biodiversity at all levels and

spatial scales. It represents an example of conglomeration of diverse bio-climates influenced by

neighboring areas (particularly Mediterranean), the unique location, peninsular land mass, Gangetic

plains and the crown of complex chain of mountain systems - the Himalaya and Western Ghats.

India ranks seventh among the centres of diversity and origin of crop plants (Khoshoo, 1995). In

India, there are 148 endemic genera and 5,725 endemic species (Nair, 1986) that are mostly

distributed in 2 "hotspots" (Himalayas and Western Ghats along with Sri lanka) out of 25 recognized

in the world (Myers et aI., 2000).

As mentioned above, the Western Ghats is one of the two biodiversity hotspot present in India.

The Western Ghats are known to be tectonically active and an uplifted region. The high biodiversity

3


of this region may be due to large nutrients that volcanism brought in, the relatively higher thermal

gradients along this belt and widely varying elevations.

Some documentation of diversity of macroscopic life forms of India is available (Khoshoo,

1995; Krishnakumar et ai, 1998; Rajagopal & Bhat, 1998), and a few studies on fungal diversity in

the Western Ghats region have been reported (Natarajan et aI., 2005; Raviraja, 2005; Naik et aI.,

2008) unfortunately, except for a few fragmentary reports (Ghosh et ai., 2003; Bhatnagar &

Bhatnagar, 2005; Thajuddin & Subramanian, 2005, Chaudhuri & Thakur, 2006), a systematic study

of diversity of prokaryote from these and other regions of India is lacking. Most of the natural

habitats, especially aquatic ecosystems are nutritionally poor but rich in microbial flora. It is known

that majority of bacteria do not grow in standard laboratory conditions, bacteria from aquatic and

sediment habitats are notoriously difficult to obtain in culture (Kemp & Aller, 2004; Schleifer, 2004).

Study of bacterial diversity from such a habitat and their function is an interesting and challenging

area of research. In the present study, an attempt has been made to study total bacterial diversity of

aquatic sample, sediment sample and mangrove sediment sample of Western Ghats by both culture

dependent and culture independent approaches and their biotechnological potential was assessed by

screening them for various enzymes. A comparative study of the bacterial diversity in different

niches will help in understanding the niche specific occurrence, their interaction and pattern of

ubiquity and will also provide a rich bacterial resource for utilization of their biotechnological

potentials.

1.1.1. Objectives of the thesis:

India ranks tenth in the world for richness in flowering plants (17,000 species) and mammals

(372 species) (Kaveriappa & Shetty, 2001). Unfortunately, hardly any data is available for

prokaryotic diversity of India. A polyphasic approach to study culturable microorganisms and a

molecular approach to study total microbial diversity in selected niches of Western Ghats are

expected to throw some light on the richness of prokaryotes in this region. The present work is

proposed with the following objectives: -

• To study culturable bacterial diversity of selected soil/water sample(s) collected from

various niches of Western Ghats using a polyphasic approach .

• To study the total diversity of microbial community of selected sample(s) using molecular

phylogenetic approach .

• Screening of culturable microbes for important biomolecules.

The study is aimed at having some knowledge about niche specific bacterial diversity of

aquatic habitat (water as well as sediment sample) and ofa mangrove sediment in the Western Ghats.

To the best of my knowledge, the study is the first of its kind on bacterial diversity from this Western

4


Ghats region using both culture dependent and culture independent methods. Considering the impact

of bacteria on global processes that keeps earth properly functioning, the work is the beginning of

efforts to understand the bacterial diversity of various ecological niches of this region.

5


1.2. Review of literature

1.2.1. Biodiversity: what the term defines?

"By which one sees an un perishable entity in all beings and undivided among the

divided then the knowledge is pure. But if one merely sees the diversity of things with their

division and limitations, without the truth then that knowledge is merely ignorance" The

Bhagavad Gita, chapter XVIII (Hunter-Cevera, 1998).

The tenn "biological diversity" was introduced by Elliot Norse and colleagues (Harper &

Hawksworth, 1995) to define diversity at three levels of complexity: (i) genetic (intraspecies

diversity), (ii) species (numbers of species), and (iii) ecological (community diversity), but

subsequently the contracted expression" biodiversity" has become the common parlance (Wilson &

Frances, 1988). Many revisions have been proposed afterwards time to time. Each of the components

in the definition of biodiversity has a hierarchical structure spanning biomes to niches (ecological),

domains to populations (species and organismal), and population to nucleotide sequence (genetic).

The tenn biodiversity, according to Erwin (1991), is related to the number of species (species

richness), along with 'the richness of activity each species undergoes during its existence through

events in the life of its members, plus the nonphenotypic expression of its genome. Microbial

diversity includes the genetic composition of microorganisms, the environment or habitat where they

are found, and there ecological or functional role within the ecosystem. Biodiversity according to

Hunter- Cevera, (1998) is defined as "all hereditarily based variation at all levels of organization,

from the genes within a single local population or species, to the species composing all or part of a

local community and finally to the communities themselves that compose the living parts of the

multifarious ecosystem of the world". As the living world is mostly considered in tenns of species,

biological diversity commonly used as a synonym of species diversity, in particular of 'species

richness', which is the number of species in a site or habitat (Groombridge, 1992), as defined by

Erwin (1991).

Biodiversity explains the variety of life in all its fonns, levels and combinations. The 1992

United Nations Earth Summit in Rio de Janeiro defined biodiversity as "the variability among living

organisms from all sources, including 'interalia', terrestrial, and other aquatic ecosystems and the

ecological complexes of which they are part". This definition is adopted by United Nations

Convention on Biological Diversity (UNCB). Other definitions can also be found which are as

follows-

l. The range of significantly different types of organisms and their relative abundance in

an assemblage or community (Torsvik et al., 1998)

6


n. As per information theory, amount and distribution of information in an assemblage or

community (Atlas, 1984).

1.2.2. Estimating the scale:

Earth provides a large variety of habitats that supports huge array of life forms full extent of

which stilI remain unknown; especially of microorganisms. Twelve mega biodiversity regions have

been identified on earth which is divided into 25 hot-spot centers based on diversity of mainly plants

and vertebrate animal (Myers et ai., 2000). We have quit a fair idea of diversity of macroscopic life

forms but the extent of prokaryotic diversity is yet to be fully explored and so far we know only very

little of it. Bacteria, archaea (together constituting the prokaryotes), fungi, microscopic algae,

protozoa and viruses constitute the microbial world as already stated. Prokaryote cells outnumber

that of the eukaryotic cells on earth by several orders of magnitude. The number of microbial species

in nature is estimated to be in millions (Hong et ai., 2006). Even for samples obtained from

environments like soils, such estimates vary widely; from a few dozen (Hughes et ai., 2001) and

hundreds to tens of thousands (Kemp & Aller, 2004) to half a million (Dykhuizen, 1998). The

estimated amount of bound carbon, nitrogen and phosphorus in globally occurring prokaryotes are

shown in Table 1.1. An approximate estimation of diversity of biological world is summarized in

Table 1.2. Prokaryotes are very important component of biodiversity which is evident from the fact

that the recent estimate indicated that prokaryotes outdo eukaryotes both by number as well as by

biomass. According to Whitman et ai., (1998) about 500 billion tons of carbon are bound in

prokaryotes which constitute about half of the total carbon found in global biomass and in case of

nitrogen and phosphorus even 90% is estimated to be of prokaryotic origin.

Table 1.1. Estimated amounts of bound carbon, nitrogen and phosphorus in globally occurring prokaryotes. (Adopted from Schleifer, 2004)

Compound Amount of compounds Incorporation to amounts of

bound in tons compounds bound in plants

Carbon 3.5-5.5 x 1011 60-100%

Nitrogen 0.9-1.4 x 10" 10 times more

Phosphorus 0.9-1.4 x IO IU 10 times more

7


Table 1.2. Estimated numbers of species occurring on earth. (Adopted mainly from Schleifer, 2004)

= ... '" ~ Q,} Q,}

..Q .0:; Q e " = '"

Q,} .. ..:c " = Q,} Q. »

'" = .0:; '" OJ Group ... Q,} 0:

Q • 0:; "1:1 Q,} = ... ... Q,} Q,} Q. ~ = Q,} Q. - '" Q OJ

..Q '" 0: ... = OJ

e e Q ~ < = '= ~ ;Z; '" ~ 0

Microorganisms

Prokaryotes 5 >1000 <0.5 Very poor

Fungi 72 1500 4.8 Very poor

Protozoa 40 200 20 Moderate

Algae 40 400 10 Very poor

Viruses* 50 130 4 Very poor

Plants 270 320 84 Good

Animals

Nematodes 25 400 6 Poor

Crustaceans 40 150 26 Moderate

Insects 950 800 12 Moderate

Vertebrates 40 45 90 Good

Molluscs# 70 200 35 Moderate

Others# 115 250 46 Moderate

a In thousands,' adapted from Bull e/ al. 1992; # adapted from Bull & Stach, 2004

1.2.3. Microbial diversity extent- known:

The total number of prokaryotes on the earth is estimated to be 4 to 6 x 1030 and the amount of

carbon 3 to 5 X 1017 g contributed by prokaryotes corresponds to almost half of the total carbon found

in the global biomass (Whitman et at., 1998).

Prokaryotes are ubiquitous and even live in extreme habitats where a major part of other life

forms can not survive. Analysis of a range of representative habitats revealed that a major part of

prokaryotes are believed to live in oceanic sub surface followed by terrestrial, subsurface, soil and

aquatic habitat (Table 1.3). They are integral part of most of the animals including human and

performing functions vital to the survival of the host (Whitman, 1998). Several caveats must be

placed on these estimations including large variations resulting from sampling effort and

extrapolations and integrations from representative data. Nevertheless, these estimates carry some

striking inferences. The large popUlations imply that events that are rare in the laboratory may occur

frequently in nature, and they point to an enormous potential to accumulate mutations and thereby

acquire genetic diversity.

8


Table 1.3. Number and biomass of prokaryotes in the world. (Adopted from Whitman et al., 1998)

Environment Number of prokaryotic Biomass in prokaryotes cells xlO28

in Pg (lOISg)

Aquatic habitat 12 2.2

Oceanic subsurface 355 303

Soil 26 26

Terrestrial sub-surface 25-250 22-215

Total 415-640 353-546

Considering such a huge number and ubiquitousness, it is expected that prokaryotic species

diversity will be enormous. But in the laboratory conditions it has been observed frequently that only

small fractions of microbial community appear on variety of nutrient media (Staley & Konopka,

1985). One of the most important insights of microbial diversity came from reassociation kinetic

analyses of environmental DNA (Torsvik et al., 1990a). These authors also found that the numbers of

bacterial genomes present in soil and marine sediment was as great as 6,500 and 11,400,

respectively, representing an estimated 20,000 to 37,000 bacterial species (Torsvik, et ai., 2000).

This dramatic finding represents the first limitation for those wishing to compare microbial diversity

in different environments and samples, i.e. that tens of thousands of samples would be needed to

ensure complete coverage (not including the likelihood of catching the same species twice). Detailed

analysis of different soil samples by Torsvik et ai., (2002) using direct microscopic count and studies

based on community genomic complexities as determined by reassociation kinetics of total

community DNA (Table 1.4) revealed the fact that enormous diversity of bacterial species exists in a

sample which is not reflected by culture dependent approach. Extremes of habitats such as salt

crystallizing pond are expected to have less species diversity. Major taxonomic diversity also occurs

in the domain archaea, and culture independent surveys have revealed novel types in a wide variety

of habitats. Karner et ai., (2001) reported that one group of archaea, the pelagic Crenarchaeota, are

extremely abundant and the ocean contain over 1028 archaeal cells (Karner et ai., 2001).

Subsurface is a major habitat for prokaryotes, and the number of subsurface prokaryotes

probably exceeds the number found in other components of the biosphere (Table 1.3) out of the 3.8 x

1030 prokaryotes estimated to be in the oceanic and terrestrial subsurface, 97% or 3.7x 1030 occur at

depths shallower than 600 m.

9


Table 1.4. Prokaryote abundance as determined by fluorescence microscopy and total genomic diversity in prokaryotic communities calculated from the reassociation rate of DNA isolated from the community. Community genome complexity is described as numbers of base pairs (bp). Genome equivalents are given relative to the E. coli genome (4.1 x 109 bp) (Torsvik et al., 2002).

DNA source Abundance Community genome Genome

(cell/cm3) complexity (bp)

Equivalents

Forest soil 4.8x 1O~ 2.5xl01V 6000

Forest soil, cultivated prokaryotes 1.4 x 107 1.4 x 1O~ 35

Pasture soil 1.8 x lOw 1.5-3.5 X lOw 3500-3800

Arable soil 2.1 x lOw 5.7-14.0 x 10· 140-350

Pristine marine sediment 3.1 x 10~ 4.8 X 101V 11,400

Marine fish-farm sediment 7.7 x 10~ 2.0 X 10M 50

Salt crystallizing pond 6.0 x 10' 2.9 X 10' 7

1.2.4. Are microbes too diverse to count? The ability to measure bacterial diversity is a prerequisite for the systematic study of bacterial

biogeography and community assembly. It is therefore central to the ecology of surface waters, the

oceans and soils, waste treatment, agriculture, and global elemental cycles. However, the

experimental definition of bacterial diversity has never been undertaken for any naturally occurring

bacterial community anywhere, and the extent of prokaryotic diversity is widely held to be beyond

practical calculation (Wilson, 1994). Our understanding of bacterial biogeography and community

assembly is correspondingly vague, anecdotal, and controversial. For example, the global distribution

of some aquatic protozoa has been used to assert that the entire microbial world is composed of a

small number of ubiquitous organisms (Fenchel, et ai., 1997; Finlay & Clarke 1999) whereas the

apparently endemic distribution of some bacteria has been used to suggest the opposite (Cho &

Tiedje, 2000; Fulthorpe et ai., 1998). However, according to Curtis et ai., (2002) in estimating the

extent of microbial diversity, it is not necessary to count every single species or taxa in a sample. It is

sufficient to simply estimate the area under the bacterial species abundance curve for that

environment. They speculated that they could estimate the bacterial diversity on a small scale

(oceans 160 per ml; soil 6,400- 38,000 per g; sewage 70 per ml) and also diversity at a larger scale

(the entire bacterial diversity of the sea may be unlikely to exceed 2 x 106, while a ton of soil could

contain 4 x 106 different taxa).

In 9.ny community, the number of types of organisms observed increases with sampling effort

until all types are observed. The relationship between the number of types observed and sampling

effort gives information about the total diversity of the sampled community. The ideas that microbial

diversity cannot be estimated comes from the fact that many microbial accumulation curves are

10


linear or close to linear because of high diversity, small sample sizes, or both. At least for some

communities, microbiologists may be able to co-opt techniques that ecologists use to estimate and

compare the richness of macroorganisms. Plotting an accumulation (An accumulation curve is a plot

of the cumulative number of types observed versus sampling effort) or a rank-abundance curve helps

in this direction. Comparisons of accumulation curves and rank-abundance plots demonstrate that

some bacterial communities have been sampled equally well as some macroorganism communities

(Hughes et aI., 2001). Therefore, evaluating microbial diversity with statistical approaches available

for macroorganisms seems feasible. Figure 1.1 shows the accumulation curves for samples from five

communities: bacteria from a human mouth (Kroes, 1999), soil bacteria (Borneman & Triplett,

1997), tropical moths (Ricketts et at., 2001), tropical birds (Hughes, 2002), and temperate forests

(Hellmann, 1999). Differences in the richness and relative abundances of species in the sampled

communities underlie the differences in the shape of the curves. Because all communities contain a

finite number of species, if the surveyors continued to sample, the curves would eventually reach an

asymptote at the actual community richness (number of types). Thus, the curves contain information

about how well the communities have been sampled (i.e., what fraction of the species in the

community have been detected). The more concave-downward the curve, the better sampled the

community. Ultimately, microbes are too diverse to count exhaustively. While it would be useful to

know the actual diversity of different microbial communities, most diversity questions address how

diversity changes across biotic and abiotic gradients, such as disturbance, productivity, area, latitude,

and resource heterogeneity. The answers to these questions require knowing only relative diversities

among sites, over time, and under different treatment regimens.

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Proportion of individuals sampled

1

Fig. 1.1. Accumulation curves for Michigan plants (x), Costa Rican birds (.), human oral bacteria (0), Costa Rican moths (_), and East Amazonian soil bacteria (e) (Hughes, 2001)

11


Some of the possible reasons which possibly explain the huge diversity found among

prokaryotes are as follows:

a) As they have evolved 3.5 billion years ago they have experience maximum changes in

earth's climate, geography and chemistry. These changes forced them to evolve differently

giving rise to such a huge diversity.

b) This huge population size implies that prokaryotes have an enormous potential to

accumulate genetic variability due to events such as mutations.

c) The rapid growth rate of prokaryotes implies that mutations and other rare genetic events

are more likely to occur in prokaryotes. The capacity for a large number of simultaneous

mutations distinguishes prokaryotic from eukaryotic evolution.

d) For essentially asexual, haploid organisms such as prokaryotes, mutations are a major

source of genetic diversity and one of the essential factors in the formation of novel

species.

1.2.5. Global pattern in microbial diversity:

Although microorganisms are perhaps the most diverse (Torsvik et aI., 2002; Venter et aI.,

2004) and abundant type of organism on earth, the distribution of microbial diversity at continental

scales is not well understood. Ecologists describing microbial biogeography typically invoke

Beijerinck (Beijerinck, 1913) from a century ago; "every thing is every where, the environment

selects". However, few studies have attempted to verify this statement or specify which

environmental factor exerts the strongest influences on microbial communities in nature (Papke &

Ward, 2004; Horner- Devine et aI., 2004). For centuries, biologists have studied patterns of plant and

animal diversity at continental scales. Until recently, similar studies were impossible for

microorganism, arguably the most diverse and abundant group of organisms on earth. Fierer &

Jackson, (2006) presented a continental scale description of soil bacterial communities and the

environmental factors influencing their biodiversity. They collected 98 soil samples from across

North America and South America and used an rDNA finger printing method to compare bacterial

community composition and diversity quantitatively across sites. Bacterial diversity was unrelated to

site temperature, latitude, and other variables that typically predict plant and animal diversity and

community composition was largely independent of geographic distance. The diversity and richness

of soil bacterial communities deferred by ecosystem type and these differences could largely be

explained by soil pH. Bacterial diversity was highest in neutral soils and lower in acidic soils, with

soils from the Peruvian Amazon the most acidic and least diverse in their study. A global picture of

microbial diversity has remained elusive, yet it is critical to understand microbial adaptation to

different environments and their function in those environments. Determining physical and chemical

factors, such as temperature, pH, or geography, that correlate with differences between diverse 12


microbial communities will reveal how easily microbes tolerate different kinds of environmental

change and will increase our understanding of microbial ecology and evolution. In addition,

determining the environment types that contain the most phylogenetic diversity will reveal where

new sequencing efforts to catalogue global bacterial diversity will be most efficient at uncovering

deep branching lineages. Lozupone & Knight, (2007) have reported the most comprehensive analysis

of the environmental distribution of bacteria to date, based on nearly twenty two thousand 16S rRNA

sequences compiled from III studies of diverse physical environments. They clustered the samples

based on similarities in the phylogenetic lineages that they contain and found that, surprisingly, the

major environmental determinant of microbial community composition is salinity rather than

extremes of temperature, pH, or other physical and chemical factors represented in the samples. They

also found that sediments are more phylogenetically diverse than any other environment type. Many

diversity studies so far done targeting soil bacteria have clearly revealed that bacterial diversity in

soil is enormous and that the composition and diversity of soil bacterial communities can be

influenced by wide range of biotic and abiotic factors. (Dunbar et aI., 2002; Tringe et aI., 2005).

Recently with the help of a new approach, the shotgun sequencing of entire communities without

the need to construct large insert clone libraries J. Craig Venter, made a significant attempt to sequence

and achieve the inventory of Sargasso Sea (Venter et al., 2004). In February and May 2003 researchers

took sea water samples from six marine research sites in the Sargasso Sea. Using a protocol in which

the water was filtered through decreasing size filters, from plankton net, through a 3.0 micron filter, a

0.8 micron filter, and a 0.1 micron filter in order to collect different sized single cell organisms. DNA

libraries were made and sequenced. Using this whole genome shotgun sequencing, 1.045 billion base

pairs of DNA sequence were produced. Using precise mathematical algorithms previously used to

assemble sequence results from single species, the researchers were able to assemble whole genomes

and major sections of genomes from the diverse microbial community found in the ocean. They

reported the presence of a minimum of 1,800 species (180 of them were novel) and 1,214,207 new

genes. One of the most important single discoveries from the Sargasso Sea environmental shotgun

sequencing study is the 782 new rhodopsin-like photoreceptor genes (Yooseph et aI., 2007). Only a

few dozen photoreceptors have been characterized in microorganisms to date and less than 200

photoreceptors have been discovered from all species, including human where they are responsible

for our vision. Therefore, this discovery represents a substantial increase in the total number of this

family of proteins.

Sorcerer II Global Ocean Sampling Expedition (GOS) lead by Ventor (Yooseph et aI., 2007;

Rusch et at., 2007) is a landmark effort in microbial diversity study. The group amassed 6.3 billion

bases and assembled them by computational tools as they developed to handle and analyse the

massive data set. In brief they found a great degree of diversity both within and between samples and

also highly abundant ribotypes (roughly equivalent to species). The study revealed the presence of

13

Chapter 1 Introduction & Review of Literature

novel genetic information in marine microorganism and novel metabolic process. Many of the open

reading frames (roughly equivalent to genes) are unlike known gene. The GOS analysis also nearly

doubles the number of previously known proteins. This enormous amount of data allowed the

researchers to better understand the genomic structure and evolution of microorganisms, as well as

the function of important protein families such as protein kinases, which are key regulators of

cellular function in all organisms.

1.2.6. Factors controlling microbial diversity:

The major underlying principle of diversity studies is probably the assumption that interactions

between populations in a habitat lead to an organized and stable community (Atlas, 1984). Marglef

(1968) stated that diversity and stability is inversely related to productivity (Ovreas, 2000). An

explanation of this view may be that the mature community needs less energy to maintain its

structure (Atlas, 1984). In a microbial community many different organisms will perform the same

processes and probably be found in the same niche. Diversity can vary with a number of factors-

1. Stress in one part can be rapidly amplified and spread to the whole system through positive

feedback links that tie the system together (Ovreas, 2000). Bacterial communities respond

to perturbation in the same manner as do communities of higher organisms, though much

faster. This is partly due to fact that microorganism have a much higher growth rate. In

addition the bacterial community may inhabit members and are ready to take advantage of

the new situation.

2. Chemical warfare between microbes promotes biodiversity (Czaran et ai., 2002). Several

studies have been concerned with diversity in response to stress such as heavy metals

(Baath et ai., 1998; Barkay, 1987; Dahlin et ai., 1997) herbicides (Ka et ai., 1994),

antibiotics (Belliveau et ai., 1991) and toxic chemical waste (Baya et ai., 1986; Burton et ai., 1982). It is evident now that the microbial communities have a high degree of

adaptability.

3. Plasmids, bacteriophages, and transposones are genetic elements with a continuous lineage

and their own evolutionary history. The influence of these factors in generating and

maintaining gene flux and in adding to the phenotype of their host, contribute to the

evolution of bacterial genomes. Bacterial genomes contain evidences for both vertical and

horizontal gene transfers (Campbell, 1981). Bacteria are products of an evolutionary

process that has occurred over thousands and millions of years.

4. Predator-prey interaction also influences biodiversity (Lebaron et ai., 1999). The system

controlling bacterial diversity seems to be a hierarchical system where lytic viruses,

predation and system nutrient content are closely linked together (Thingstad & Lignell,

14


1997). Bacterial diversity can be regulated by a combination of size selecting grazing and

host specific viral lysis (Bratbak et al., 1994).

1.2.7. Fundamental reasons for prokaryotic diversity studies- why

so important?

In comparison to almost 1 million of insect species there are currently only about ~ 9000

validly described species ofprokaryotes (www.bacterio.cict.fr/number). Considering that each insect

carries at least millions and even billions of prokaryotes, we know only a minor fraction of

prokaryotes present on Earth (Table 1.2). Apart from such huge abundance and biomass

representation, they have many unique properties and carries out very important functions (Schleifer,

2004). These are as follows:

1. Founder of our biosphere, "the prokaryotes" was the very first kind of living organisms that

appeared on earth at least 3.5 billion years ago. Therefore, Earth has been populated by

these microbes for most of its existence. The existence and evolution of other life forms

would not have proceeded without them. Those were prokaryotes only that created an

oxygenic environment which supported the life of higher organisms.

2. Global biosphere is mainly shaped by geochemical activities of prokaryotic life as they

maintain biogeochemical cycles. They affect all geochemical processes that occur at the

earth surface, as well as deep subsurfaces and in tum get affected by these activities also.

They complete nitrogen and sulfur cycle, oxidize and reduce metals and some of them in

tum obtain energy for growth by transferring electrons to a wide range of harmful metals,

such as uranium, chromium, arsenic and plutonium.

3. Prokaryotic microbes show an unusual high physiological and biochemical versatility.

They can use reduced inorganic compounds as energy for chemolithotrophic metabolism or

oxidized inorganic compounds as electron acceptors for anaerobic respiration. Moreover,

certain metabolic pathways, such as fermentations, nitrogen fixation, methane formation

and anoxygenic photosynthesis, are only found among prokaryotic microbes. This is also

true for the biosynthesis of secondary metabolites such as certain antibiotics and toxins.

Certain microorganisms carry out "anamox" reaction that refers to the anaerobic oxidation

of ammonia where nitrite is the terminal electron acceptor. The energetics of the reaction is

much more favorable than the oxic nitrification process. The bacteria that perform this

reaction, form a deeply branching monophyletic lineage within the phylum Planctomycetes

which till recently not been obtained in pure culture and were known only from culture

independent studies oftheir phylogenies and physiologies. Completely autotrophic nitrogen

removal over nitrite (CANON) where a combination of both aerobic ammonia oxidizers

15


and anammox bacteria are utilized to remove nitrogen from wastewaters (Jetten et al.,

2001) is an important application of anamox.

4. The metabolic, physiological and genetic diversity of prokaryotic microorganism is far

greater than that found in higher organisms. Some microorganisms are highly resistant to

radiation e.g. Deinococcus radiodurance and might be used for safe management of

radioactive waste.

5. Microbes are the frontiers of life because they are also determined by prokaryotic microbes.

They can survive and even thrive under the most extreme environmental conditions. They

can be found in all habitats where the physical and chemical circumstances allow the

existence of life. Some of them can grow in' hot spring at temperatures up to 121°C

(Kashefi & Lovley, 2003), others are found in frozen water in a fresh water lake beneath 4

kilometers of ice in central east Antarctica (Karl et aI., 1999). They exist and grow at low

pH as well as in saturated salt solution. Moreover, all natural occurring and also many man

made compounds will be degraded by microbes.

6. Microbial interactions among themselves in both positive and negative manner within a

single population. One population may benefit another in a one sided comonsal way, or two

different populations may interact synergistically. Such beneficial interactions are

facilitated by close physical proximity, as in biofilms and flocs. Such co operational

interactions have been shown for populations of Myxobacteria (Dworkin, 1996; Shimkets,

1990). Cooperation in a microbial population can function as protective mechanism against

hostile environmental factors. Microbial populations within a biofilm are orders of

magnitude more resistant to antimicrobial agents than suspended cells of the same

organisms (Shapiro, 1991). Resistance to antibiotics and heavy metals and the ability to

utilize unusual organic substrates are often genetically transmitted to other members of the

population (Hardy, 1981). Mutualism which is defined as a strong, specific, beneficial

interaction essential for survival of both partners is well studied in microbial world.

According to theory of serial symbiosis, some mutualistic endosymbiotic relationship had

key roles in the evolution of higher organisms (Margulis, 1971). Endosymbiotic

methanogens have been found in anaerobic ciliate protozoa living within the rumen. It is

likely that endosymbiotic methanogens can directly use molecular hydrogen produced by

the ciliate protozoan (Heckmann & Gortz, 1992). Syntrophism term is applied to the

interaction of two or more populations that supply each other's nutritional need.

Syntrophism may allow microbial populations to perform activities, such as the synthesis

of a product that neither popUlation could perform alone. A classical example of such

syntrophism is exhibited by Enterococcus faecalis and Escherichia coli for conversion of

arginine to putrescine (Gale, 1940). Similarly cyclohexane is degraded by a mixed

16


population of a Nocardia species and a Pseudomonas species but not by either population

alone (Slater & Bull, 1978). Syntrophomonas sp. and Syntrophobacter sp. are hydrogen

producers and require the presence of the methanogen as hydrogen removers (Balows et

aI., 1992). In contrast to positive interaction, microorganisms produce substances toxic to

competing populations which is called amensalism. Production of ammonium by some

microbial populations is deleterious to other populations, some produce low molecular

weight alcohols, some produces antibiotics. Inhibitory substances produced by

microorganism may also act as preservatives for organic compounds in natural habitats e.g.

decomposition of cellulose in soil, organic acids are produced that prevent further

breakdown of cellulose metabolites in subsurface soil. Some examples are there for

parasitic bacteria too like Bdellovibrio that is parasitic on Gram-negative bacterial

populations (Starr & Seidler, 1971; Stolp & Starr, 1963).

7. Interactions of microbes and plants, e.g. association of nitrogen fixing bacteria and root of

leguminous plants, mutualistic relationship between Azolla and Anabaena sp. are

interesting and well studied phenomena. Rhizosphere and phyllosphere microbiota also

provide an open area for microbial diversity investigations. However, many bacteria are

known as plant pathogens too.

8. Most interactions between microbes and animals are beneficial. The mutualistic

relationship of microbial populations involves nutrient exchange and maintenance of

suitable habitat. Microbial, in particular bacterial endosymbionts are fundamental to the

survival of higher organisms. Without bacterial endosymbionts most animals would not

survive. Symbionts carryout essential biochemical reactions for their eukaryotic hosts, e.g.

the biosynthesis of essential amino acids, vitamins or the degradation of certain

macromolules. Some marine worms use their sulphide oxidizing bacterial endo or

ectosymbionts even as sole feed source. Most warm blooded animals contain extremely

complex microbial flora within their gastrointestinal tracts. In lower intestine, each gram of

feces contains approximately lOll microorganisms, belonging to up to 400 different species

(Lee, 1985). In some animals, such as pigs, and bovine animal the microbial populations of

the gastrointestinal tract contribute to the nutrition of the animals by fermenting

carbohydrates especially cellulose and animal can utilize the products of the cellulose

degradation (Kenworthy, 1973). In monogastric animals the main contribution to digestion

by intestinal microbial populations appears to be in the production of growth factors. In

some cases, microorganism supply required vitamins, to constitute an important barrier to

attack intestinal pathogen. The protozoa and bacterial population found within gut of lower

termites and wood eating cockroaches ferment cellulose anaerobically, producing CO2, H2,

and acetate. Some of the H2 and CO2 is converted to C~ by methanogenic archaea

17


(Leadbetter & Breznak, 1996). Some microbial population within termite gut fix

atmospheric nitrogen (Bonemann, 1973). The microbial populations which are maintained

within mycetomes supplement dietary deficiencies of the animal by producing growth

factors. Amphipods contain high proportions of chitinase producing Vibrio species that

partially degrade the chitin ingested by these animals. Mussels of family Mytilidae living in

deep sea thermal vent harbor methanotrophic bacteria with typical stalked internal

membrane structure. All aphids have cell clusters that are called mycetomes and the

individual cells mycetocytes. These cells harbor bacteria which upon elimination by

antibiotic treatment affect reproduction and the aphids eventually die (Baumann &

Bauman, 1994).

9. Both cultured and "uncultured" prokaryotes represent a huge genetical and

biotechnological potential and therefore an enormous source of new products and

processes. Diverse microorganisms have yielded important biological materials useful to

humans such as antibiotics (Crossley, 1986), drugs (Drews, 2000), enzymes (Cherry et ai.,

1999) and growth promoters (Parada et al., 1998).

10. Microorganisms can aid environmental restoration by oxidizing, binding, immobilizing,

volatilizing or otherwise transferring contaminants. Prokaryotic microbes are responsible

for degradation of natural products and man made harmful xenobiotic compounds. They

have capacity to remove many contaminants from environment by activity of enzymatic

processes. The most common type of bioremediation is oxidation of toxic, organic

contaminants to nontoxic products. Oxygen is the most common electron acceptor for

microbial respiration, and aerobic degradation of an extensive range of organic

contaminants, from aromatic hydrocarbons such as benzene to xenobiotics, such as

pesticides. Pseudomonas species and closely related organisms have been the most

intensively investigated for the degradation of aromatic contaminants. Microorganisms can

also anaerobically oxidize many contaminants with alternate electron acceptors such as

nitritates, sulphate and Fe (III) oxides. Geobacter species such as G. metallireduscens that

are highly enriched in subsurface environment can oxidize organic compounds with

reduction of Fe (III). Shewanella and Geothrix species release iron chelators which

solublise Fe (III) to Fe (III) oxides. Sulfate reducing bacteria such as Desulfobacula and

Desulfobacterium species, can oxidize hydrocarbons with sulfate as the electron acceptor

that are present in huge amounts in marine environments and serve as electron acceptor for

anaerobic degradation of contaminants. Microorganisms remove chlorides from

contaminant such as chlorinated solvents like tricholoroethane (TCE) is degraded by

Dehalococcoides ethanogenes. Many microbes capable of dehalogenetion are known

among which Dehalococoides species are particular important. Some can reduce inorganic

18


contaminants such as nitrate percolate and selenite. Geobacter species can remove uranium

from contaminated water by conversion of U (VI) to U (IV). Microorganisms have been

found that can accumulate heavy metals like gallium (Bull, 1991). Chemolithotrophic

bacteria, like Thiobacillus ferroxidans, and T thiooxydans are increasingly being used in

mining for controlled bioleaching of metals (Rawligs & Silver, 1995).

11. They are responsible for certain diseases in animals, plants and humans.

Thus microbial diversity is fundamental to maintenance and conservation of global genetic

resources and represent by huge diversity in terms of their metabolic activities and their interactional

ability with other life forms (Schink, 1992). Diversity analysis is therefore important (Ovreas, 2000)

in order to-

• Increase knowledge of the diversity of genetic resources in a community.

• Understand patterns in relative distribution of organisms.

• increase the knowledge of fundamental role of diversity

• identify differences in diversity associated with management disturbing

• Understand the regulation of biodiversity.

• Understand the consequence of biodiversity (to what extant does the ecosystem functioning

and sustainability depend on maintaining a species level diversity).

Many have the ability to grow anaerobically and also to adapt to diverse environmental

conditions. Due to their huge impact on numerous interconnected life processes and their metabolic

capabilities that keeps the earth properly functioning and in order to best exploit microorganisms, it

is very important to know "what is there and what can be used" (Bull, 1991).

1.2.8. Current picture of microbial diversity:

1.2.8.1. The Black Matter in Microbial Space: 'diversity black box'

The number of species in all but the simplest communities can only be estimated statistically,

typically on the basis of a small subset of species (or their small rRNA sequences) observed directly,

even for samples obtained from similar environments (soils), such estimates vary widely; number of

bacteria exceeds far than what we know presently. For example, possibly 2-13 million species in

Atlantic forest tree canopy (Lambais et aI., 2006), 2 million species in the open sea (Curtis et al.,

2002) and about 40,000 species per gram of soil (Dykhuizen, 1998), from a few dozens and hundreds

(Hughes et al., 2001; Kemp & Aller, 2004). The quality of microbial richness predictions is an

important issue as they serve as the basis of all of the paradigms of biodiversity, its role, functions

and meaning. It is therefore of principal interest to know the true extent of microbial diversity,

19


starting from that in a single environmental sample. The number of microbial species in nature may

be in millions but most have never been observed or otherwise detected.

1.2.8.2. General limitations in studying microbial diversity:

There are problems associated with studying bacterial diversity. These arise not only from

methodological limitations but also from lack of taxonomic knowledge. It is difficult to study the

diversity of a group of organism when it is not understood how to characterize the species (Kirk et

aI., 2004). Main limitations for extensive diversity analysis are given below-

i. Spatial heterogeneity:

This is major problem in soil and sediment environments as very little is known about

spatial and temporal variability of microorganisms in these environments. Bacteria are

highly aggregated in soil existing in clumps or 'hot spots. Microbial communities may have

several nested levels of organization, and that they could be dependent on different soil

properties. Microbial communities exist on such a small scale, that possibly 1-5 gram of

soil could bias results and favor detection of dominant population.

ii. Limitation of molecular-based methods:

Limitations of culture based methods have, to some extent, been overcome by usmg

molecular techniques; however, they are not without their own limitations. If method of

DNA extraction used is too gentle, Gram negative, but not Gram-positive bacterial cells

would be lysed. If the method is too harsh, both gram negative and gram-positive cells may

be lysed but their DNA may become sheared (Von Wintzingerode et ai., 1997). This

variation may lead to biases in molecular based studies. With environmental samples, it is

necessary to remove inhibitory substances such as humic acids, which can interfere with

subsequent PCR analysis. Subsequent purification steps can lead to loss of DNA or RNA,

again potentially biasing molecular diversity analysis. Differential amplification of target

genes due to different affinities of primers to templates, different copy numbers of target

genes, hybridization efficiency and primer specificity are also the causes. Although above

discussion sets forth some limitations of molecular based study which can influence the

analysis and interpretation of molecular based microbial community analysis yet these

methods provide valuable information about the microbial community as opposed to only

culture based techniques.

iii. Taxonomic ambiguity: 'species concept in prokaryotes'

Another problem associated with measuring microbial diversity in soil is the problem of

defining microbial species. There is no official definition of bacterial species (Colwell et

ai., 1995). There are many opinions and suggestions as to how to define a bacterial species.

20


For practical purposes a consensus definition was adopted. According to this view a

bacterial species is a collection of strains showing 70% or more genomic relatedness

(DNA-DNA hybridization) and ~Tm of 5°C or less of the hybrid molecules (Wayne et aI.,

1987). It has been observed that at this level of genomic relatedness the 16S rRNA gene

sequence similarity is usually 97% or more (Stackebrandt & Goebel., 1994).

iv. Inability to culture:

Many lines of evidence indicate that only a small fraction of naturally occurring

prokaryotes is culturable by standard techniques (Amann et al., 1995). As stated above

very little about their identity and possible functions is known. The total number of

described prokaryotic species known at present is about 9,000 only (www.bacterio.cict.fr)

which is less than 1 percent of the currently estimated bacterial species diversity. The

existence of these species is only known from their 16S rRNA gene sequences and

predicted from statistical analysis. Since conventional recognition of a prokaryotic species

requires its cultivation but only a minor fraction can be cultured in the laboratory. This

means that majority of the species are not known to the scientific community and remains

to be discovered as supported by many observations:

1). "Great plate count anomaly" the term, first introduced by Staley & Konopka (1985) is

based on the observation that number of organisms present in a sample seen directly

under a microscope is 10 to 100 times more than the number of colonies they form on

conventional laboratory media (Staley & Konopka, 1985). Some methods which do

not require culturing of microbes for their detection and enumeration are:

epiflourescence microscopy with stains such as acridine orange, 4', 6' -diamino 2-

phenylindol (DAPI), direct imunoflourescence, epiflourescence microscopy, and

direct viability count by nalidixic acid. Microautoradiography and stable isotope

probing have revealed that up to 36% (Zimmermann et aI., 1978) and in some cases

staining by SYTO 9 even 90 % (Janssen et ai., 2002) uncultured majority may be

metabolically active. Autoradiography combined with direct microscopic observation

(in which bacteria incubated with radiolabelled substrate, such as tritiated glucose, are

subsequently collected on a bacteriological filter placed on a glass slide, coated with

a photographic emulsion) indicated that 2.3-56.2 % of the total bacteria are

metabolically active (Meyer-Reil, 1978). Some of the reasons for their inability to

grow in the laboratory are:

• These ostensibly "uncultured" cells have permanently lost culturabilty i.e.

S-rr·S TH-17112 ~y{qJ . ~4--{ ~

21

579.3 84697 St 1111111111111111 i 11111111111111

TH17112

TH


effectively had gone to the stage of "viable but non-culturable". They are in

dormant stage from which recovery method is still unknown (Barer &

Harwood, 1999).

• They are simply unable to grow on standard isolation media.

• Cell damage caused by oxidative stress preventing growth until repair (for

instance by SOS response mechanism) has been completed.

• Inhibition by high concentrations of substrates in the media (substrate-

accelerated death).

• The induction of lysogenic phages during cultivation.

• Lack of cell-to-cell communication in laboratory media (Kaeberlein et aI.,

2002).

2). Second supporting evidence comes from measurements of the genetic diversity of

bacteria in soil or marine sediment using the reassociation kinetics of their genomic

DNA that indicated the presence of 4000 to 13000 different bacterial genomes

(Torsvik et aI., 1990a; Torsvik et al., 1994; Torsvik et al., 1993), exceeding the

number of bacterial species currently known. Such a huge number of bacterial species

were never isolated on culture media. The number of different cultured species from

the same sample was calculated to be 21 from 206 isolated morphotypes when

determined by renaturation kinetics of the cultured isolates. Table 1.5 shows ratio of

cultivable to microscopically detectable prokaryotes in different niches.

3). Further support for a high diversity of so far 'uncultured" bacterial species came from

rRNA gene based culture independent molecular phylogenetic studies. The database

consisting of these gene sequences contains many phylotypes with no close

cultivatable relatives. Some of these distantly related sequences have also been

proposed to belong to new candidate divisions consisting solely of gene sequences

whose presence has been indicated by the culture independent approach (Hugenholtz

et aI., 1998a; Rappe & Giovannoni, 2003). There are currently 53 phyla (Fig. 1.3) in

bacteria, 26 of which have got no cultured representatives (Keller & Zengler, 2004)

and continuous from the detection of novel 16S rRNA gene sequences in

environmental samples.

22


Table 1.5. Ratio of cultivable to microscopically detectable prokaryotes in percentage in various niches (data taken from Bull & Stach, 2004).

Habitat Culturability (%)

Sea water 0.001-0.1

Fresh water 0.25

Mesotrophic lake 0.1-1

Unpolluted estuarine 0.1-3 waters

Activated sludge 1-15

Sediments 0.25

Soil 0.3

0.05

~. Verrucomicrobia 7

JI

IL

~ 'l

OP3

WS3 BRC1

NKB19

OP9 wsz

vadinBE97 Chlamydiae 13

Planctomycetes 9

Firmicutes 1205

Cyanobacteria 4 Fusobacteria 25

OP10 SC4

Actinobacteria 1367 NC10

~ Bacteroidetes 220

Chiorobi8

~ -t

-t

Ir-l

t 1c!

Marine Group A Caldl!hnx 1

OS·K

C..emmatimonadetes 1 Fibrobacteres Z

Proreobacteria 1808 Oeterribacteres 7 Chrysiogenes arsenatis 1

SBR1093

SC3

Acidobacteria 3 OPB

Nitrosptae 6 Termite Group 1

TM6 SynergiSieS 1

OP5 Spirochaetes 91

B01-5 group

WS6 TM7

WS5 Guaymas 1

ABY1

Chbrofiexi 11 Deinococcus· Thef/17'Js 24

Thermodesulfobacteria 2 OP1 4 ---.l ThemlOtogae 25 - 4'-------.. -... Coprothermobacter 2

1----11 .... fJctyoglomi 2

{::~~~::~::::~~AqWftae13 ""'I Oesu!furobacterium 1

OP11

Fig 1.2. Phylogenetic tree of the domain Bacteria based on 16S rRNAgene sequences (Keller & Zengler, 2004). Bacterial phyla with cultivated representatives are shown in blue and green. Bacterial phyla with no cultured representative are shown in red Numbers in red represent the published species within a phylum.

23


1.2.8.3. Cultivating the uncultivated:

Cultivation of microorganisms has been viewed as fundamental for understanding of microbial

physiology and metabolism. It is well known that conventional cultivation approaches access only a

tiny subset of the wide diversity of microorganisms inferred to be present in any given environment.

Methods for cultivation that are based on modified traditional approaches have resulted in the

isolation of some previously uncultured, phylogenetically distinct microorganisms. Various methods

employed in improving cultivation of uncultured bacteria from environment sample by doing some

manipulations in traditional culturing methods. Some of them are as follows-

(i). Using low-nutrient media and increased incubation times, addition of pyruvate or

catalase to reduce oxidative stress, addition of carbon substrates at only low

concentrations Janssen and co-workers were able to obtain from soil, pure culture

representatives of several subdivisions of the recently recognized bacterial phyla

Verrucomicrobia and Acidobacteria (Bartscht et al., 1999; Brewer et aI., 1977;

Janssen et al., 2002; Sa it et al., 2002).

(ii). Stevenson et aI., (2004) changed various culturing conditions (oxygen concentration,

nutrient level, addition of humics and signaling molecules) to increase the

Culturability of microorganisms in soil.

(iii). Signaling compounds added to media have also been reported to aid in culturing

aquatic microorganisms (Bruns et aI., 2002; Bruns et aI., 2003).

(iv). Simulating natural environment (for example by placing microorganisms in a

diffusing chamber and incubating the chamber in an aquarium as has been done for

cultivating aquatic bacteria, simulating the organism's natural settings) (Kaeberlein et

al., 2002). Up to 40% inoculated organisms formed micro colonies, but the majority

of these did not grow after passage to Petri dishes. The one that did not continue to

grow on passage appeared to be mixed culture, and author invoked specific signaling

mechanisms to explain this behavior.

(v). Culturing under elevated CO2 concentration and/or limited O2 concentration,

incorporating detoxifying reactive oxygen species in plating media (Stevenson,

2004).

(vi). Use of gellan-gum instead of agar as solidifying agent (Tamaki et aI., 2005)

24


Some recently developed innovative culturing techniques are also employed,

(i). Single cell manipulation techniques such as optical tweezers and Laser micro

dissection, also hold promise for targeted isolation of microorganisms (Frohlich &

Konig, 2000)

(ii). Oligonucleotide probes in combination with optical tweezers to track and separate a

novel hyperthermophilic archaeon from an Obsidian Pool community (Huber et ai.,

1995).

(iii). Use of 16S rRNA-directed probes to track the progress of isolation attempts in serial

dilutions without previous knowledge of the physiology of the organisms (Rappe et

al.,2002).

(iv). High throughput methods to grow encapsulated single cells under environmentally

relevant conditions(Zengler et aI., 2002).

(v). Using the MicroDrop micro dispenser system (Bruns et aI., 2003).

(vi). Dilution to extinction, to recover oligotrophic bacteria (Button et aI., 2001) and gave

some important massages-

1. Long incubation times are likely to be required to allow maximum recovery.

2. A large proportion of the isolates were first representatives of novel lineages in the

division Acidobacteria, Actinobacteria, Proteobacteria and Verrucomicrobia.

(vii). Filter acclimatization method which employs a filtration step, which removes most of

the readily cultivable bacteria and than acclimatization procedure to from low to high

substrate concentration of standard microbial media (Hahn et aI., 2004).

(viii). Agar nodule based in situ cultivation system (Koch et aI., 2006).

(ix). Microfluidic strategy for microbe isolation and genome amplification (Marcy et aI.,

2007)

Ammonia oxidizing planctomycete so called "missing lithotroph" was isolated by density

gradient centrifugation following careful electron microscope scrutiny of multiple biofilm (Kuypers

et ai., 2003). Isolation of Sphingomonas aisakensis RB2256 (Vancanneyt et aI., 2001) ,

Cycloclasticus oligotrophus (Button, 1998), Leptospirillum Jerrodiazotrophum , a member of

Leptospirillum group III within the Nitrospira (Tyson et aI., 2005), Acidobacteria and SAR 11

phylum, that was previously described only by environmental 16S rRNA gene sequences, were

possible by using these innovative approaches and also provided unique genetic information about

oral representatives of the uncultured phylum TM7. Use of dilution culture has produced significant

increase in the culturability of planktonic lake bacteria (Bussmann et ai., 2001) and of soil bacteria

25


(Janssen et al., 2002). Although these methods contributed significantly and opened up a way to

study the "as yet" uncultured but due to these requires enough labor, time and sophisticated

fabrication work which is a bottle neck that is why still there is a need to find more practical ways to

tapping into microbial diversity.

1.2.8.4. Opening the diversity black box:

Application of molecular phylogenetic methods in the 1980s (Pace et al., 1986; Stahl et af.,

1984) based on rRNA gene sequences founded the way of accessing diversity by culture

independent approach that brought revolution as prokaryotes could now be studied without culturing

them. It also confirmed that only small proportion of total popUlations was being cultivated as

observed by direct microscopic observations and plate counts. The use of techniques e.g. analysis of

cloned 16S rRNA gene analyses, PCR-based Denaturant Gradient Gel Electrophoresis (DGGE),

Single Strand Conformation Polymorphism (SSCP) have revealed spectacular patterns of diversity

even in previously well studied habitats. As a result, the use of 16S rRNA gene based approaches

found wide acceptance in determining community structure and change in community profiles of

prokaryotes from many environmental habitats. In recent years molecular detection methods have

evolved for more extensive characterization of uncultured natural diversity. Various methods like

fluorescent in situ hybridization, metagenomics, and oligonucleotide microarray enabled

simultaneous detection of different organisms, activity in addition to the presence of organisms, in a

habitat to be determined, and biochemical pathways of uncultured organisms to be reconstructed.

However, in number of instances these methods have yet to be rigorously tested and validated with

environmental samples (Bull et af., 2004).

1.2.8.5 Reservoir of phylogenetic information: rRNA genes:

Since Woese and Fox (1977) first proposed the 16S rRNA gene as a phylogenetic tool to

describe the evolutionary relationships among organisms and Pace et al. (1986) described its use for

classifying unculturables microorganisms in the environment, a huge repertoire of 16S rRNA gene

sequences are now available in GenBank and new ones are being added at a rapid rate (Benson et af.,

2008; and RDP Release 10, Update 5; http://rdp.cme.msu.edulmisc/news. jsp#oct3008). The

development of rRNA based methods for phylogenetic analyses and bacterial identification in

combination with special databases may undoubtly be regarded as one of the milestones in the

history of microbiology. Consequently a comprehensive sequence dataset is available in generally

accessible databases in plain or processed format and number of entries is permanently increasing. A

reasonable fraction of validly published bacterial species is represented by 16S rRNA sequences. The

phylogenetic analysis of these data provides the basis for an ongoing evaluation and restructuring of

the current bacterial systematics accompanied by emendations and renaming of bacterial taxa. It is

also widely accepted to apply the rRNA technology as an integrated part of polyphasic approach for

26


description of new species and higher taxa. The rRNA genes are organized in the form of an operon

(Fig. 1.3)

16S rRNA tRNA 23SrRNA 5S rRNA tRNA

Fig. 1.3. Organization of rRNA operon in E. coli.

The copy number of this rRNA operon varies from bacteria to bacteria and starting from one to

as many as 15 has been reported (Acinas et ai., 2004). Although, protein were considered first for

these kinds of studies but later on was proved to be less efficient than to rRNAs. Different

approaches were taken into consideration. One approach in which rRNA cataloguing was done

followed by sequencing and in second approach DNA-rRNA hybridization studies were done for

phylogenetic studies (Woese, 1992) for analyzing natural population of microbes, in which unknown

diversity is anticipated, there are several reasons to focus on the rRNA molecule (Olsen et aI., 1986).

These are as follows:

1. The rRNA, is an integrated component of the protein synthesizing machinery and

functionally and evolutionary homologous in all organisms.

ii. The rRNAs are ancient molecules and are extremely conserved in overall structure.

Thus, the homologous rRNAs are readily identifiable by their sizes.

Ill. Nucleotide sequences are also conserved. Some sequence stretches are conserved

across the primary kingdoms while others vary. The highly conserved regions also

provide convenient hybridization targets for cloning the rRNA genes and for primer

directed sequencing techniques.

IV. The rRNA constitutes a significant component of cellular mass, and they are readily

recovered from all types of organisms for accumulation of a data base of reference

sequences

v. The rRNA provides sufficient sequence information to permit statistically significant

comparIsons.

VI. The rRNA genes seem to lack artifacts of lateral gene transfer between

contemporaneous organisms. Thus relationships between rRNAs reflect evolutionary

relationship of the organisms.

27


Of the three generic rRNAs, 5S rRNA, 16SrRNA, and 23S rRNA, the 5S rRNA and 16S

rRNA received most attention. The 5S rRNA, because of its relatively small size, was amenable to

sequence analyses by the late 1960s. However, 5S rRNA is still too small to provide meaningful

phylogenetic inferences.

The 16S rRNA, in contrast, is of reasonable size (~1600 bp) and contains a wealth of useful

phylogenetic information. The 23S rRNA (~3000 bp) is almost twice the size of 16S rRNA (Brosius

et ai., 1980) and is not practical for large scale sequencing.

1.2.8.6. Protein coding genes as molecular marker for phylogeny prediction:

Several characteristics of the 16S rRNA gene, such as it is present in all prokaryotes,

indispensable, mostly conserved but with some variable region have allowed it to become the most

commonly used molecular marker in microbial diversity studies. Inferences on bacterial phylogenetic

relationship based on a single molecule may have limitations. Recently attempts are being made to

use essential housekeeping genes, such as recA, RNA polymerase ~ (rpoB) , pyruvate kinase (pyk),

alanine dehydrogenase (aid), for phylogenetic analysis. Criteria of such selection were, their wide

distribution, they are unique without any paralogues, long enough for sequence information but short

enough to be economical and finally the gene(s) reflect whole genome relatedness (Zeigler, 2003).

Alternatively core house keeping gene, such as the RNA polymerase ~ (rpoB) for differentiating

closely related organisms (Case et ai., 2007), gene encoding pyruvate kinase(pyk) gene encoding

alanine dehydrogenase (aid) to distinguish Bacillus giobisporus and Bacillus psychrophilus (Palys et

ai., 2000), catalytic subunit of proton trans locating ATPase (FIF213- subunit in particular), recA

protein and RNA polymerase (13 and 13' for bacteria and B, B' and B" for archaea) (Ludwig & Klenk,

2001). Recently, as many as 30 genes have been proposed that can be used for the determination of

genome relatedness.

Some other genes that have been used for better taxonomic resolution are as follows:

i. DNA gyrase B subunit (gyrB) (Hatano et ai., 2003; Le Roux et ai., 2005; Yamamoto et ai.,

1999).

ii. DNA fragment coding for sigma factor 70 (Yamamoto & Harayama, 1998).

iii. Gene sequences coding for heat shock proteins (Griffiths & Gupta, 2001).

iv. Genes coding for elongation factor TU and F-ATPase-l3-subunit(Paradis et ai., 2005).

v. Gene encoding translation initiation factor 2 (Hedegaard et aI., 2001).

28


1.2.8.7. "Twin track approach"; combining the two approaches:

Culture-independent molecular approaches are tending to replace culture-based methods for

comparing the composition, diversity, and structure of microbial communities. Investigations based

on these approaches have led to the conclusion that traditional methods of culturing natural

populations have seriously underestimated archaeal and bacterial diversity. Culture independent

approach has shown that most of the bacteria in that grow on conventional media, are not the most

abundant in natural habitat. The molecular approaches provide a new perspective on the diversity of

prokaryotes in nature but do not provide the organisms as such in culturable form. This means that

valuable functional traits can, at best, only be inferred from phylogenetic affinities. It is assumed

with certain level of uncertainty that organisms related in phylogeny are also related to the function

in natural habitats. However, phylogenetic coherence need not correlate with physiology (Hahn et

aI., 2004; Jaspers & Overmann, 2004). This means that the pure culture approach is required more

than ever to understand the metabolic diversity of bacteria. Relatively few studies have involved a

"Twin-track" approach whereby both cultivation and direct recovery of bacterial 16S rRNA gene

sequences have been used to gain insight into the microbial diversity of natural bacterial

communities (Dunbar et aI., 1999; Hengstmann et aI., 1999; Schut et aI., 1993). Samples of DNA

extracted from seawater, soil, and cyanobacterial mats of hot springs appear to represent predominant

populations in these ecosystems, while the species that grow on culture plates are numerically

unimportant in intact natural communities. Comparative studies such as these have shown that both

plating and 16S rDNA cloning suffer from biases that can distort community composition, richness,

and structure if applied alone. Two major conclusions were drawn from these studies. (i) for the most

part, direct enrichment techniques select for populations which are more fit under the chosen

enrichment conditions and may not be numerically significant, and (ii) the growth of numerically

dominant populations may be favored by using inoculums diluted to extinction, especially in growth

medium which reflects the conditions in the habitat under study (Bull et ai., 2000). A somewhat

mixed picture emerges from comparative studies of natural microbial ecosystems. The two

approaches some times provide different assessments of relative community diversity, the

discrepancies may be because of sampling different subset of the microbial communities and to

limitations inherent in each of the two approaches for e.g. biases are involved in molecular methods

starting from nucleic acid extraction, PCR, cloning etc (Ellis et aI., 2003; Von Wintzingerode et aI.,

1997). The culturable fraction of bacteria may not be the dominant population (Hugenholtz, 2002)

and thus are less likely to be detected by sequence based molecular phylogenetic approach. In

addition, highlighting consistent relationship between environments based on dual approaches may

be highly habitat dependent- due to limited ability of a single cultural method to survey the full

extant of the bacterial communities and the influence of bacterial physiology in situ on the success of

cultivation in the laboratory.

29


It is concluded that both innovative culture dependent and culture independent methods have a

role to play in unraveling the full extent of microbial diversity in natural habitats. Such combinatorial

approaches helps in making strategies about culturing a still uncultured bacteria if the latter is in

close phylogenetic proximity of a well-defined cultured bacterial strain. It is expected that as more

attempts are made in such combinatorial approaches, our knowledge about the diversity will increase

from the point of view of both structural diversity and functional diversity.

1.2.9. Approaches to study prokaryotic diversity:

The major microbial processes of importance to global ecosystem functions and its

sustainability are the result of microbial cell metabolism, growth, death, or enzymatic function of

non-growing cells. Methods that reveal the composition of microbial communities can be applied

over time and space in response to different environmental conditions to understand the linkages

between key populations and processes. Once tools for community analysis are available they can be

applied to almost any question addressing the soil microbial state and function. Methods that are used

to open the microbial "black box" can be grouped into those that measure the members present

(structure) and those that provide some measure of functionality in natural habitat. These methods

can be positioned according to level of taxonomic hierarchy at which they resolve differences. Some

questions are adequately addressed at a coarse level of resolution, while others require a fine scale of

resolution. The coarse scale usually samples the entire community while methods for fine scale

resolution often require analysis of target populations only in order to achieve the fine -scale

resolution. A complete list of bacterial taxonomic methods and their level of taxonomic resolution

have been suggested (Vandamme et aI., 1996). It is now widely accepted that methods used to

analyze prokaryotic diversity of any niche should take into account both culture based as well as

culture independent approaches to get a maximum unbiased idea about the diversity and the

ecosystem function. Techniques used to analyze prokaryotic diversity can be divided into two types.

I. Approaches evaluating community structure

1. Culture dependent approach.

2. Culture independent approach. This includes two kinds of techniques:

a. Biochemical based approaches

b. Molecular based approaches (based mainly on 16Sr RNA gene)

II. approaches evaluating community structure and function.

1.2.9.1. Approaches evaluating community structure:

30


1.2.9.1.1. Culture dependent approach:

To gain a comprehensive understanding of microbial physiology or to access metabolic

pathways containing genes dispersed throughout the genome, cultivation of microorganisms is

required. Only 26 out of 53 bacterial phyla contain previously cultivated microorganisms, with many

phyla represented by only a few isolates and some phyla containing only one described species

(Keller & Zenglar, 2004). So far, only five phyla - Actinobacteria, Bacteroidetes, Cyanobacteria,

Firmicutes, and Proteobacteria - include species that produce bioactive molecules and represent

95% of all cultivated and published species. The rest of the phyla with cultivated members (21 phyla)

represent only 5% of all published species. Conventional cultivation of microorganisms, however, is

selective and is biased towards the growth of specific microorganisms (Eilers et ai., 2000; Ferguson

et ai., 1984). The growth of at least 105 cells in a colony on plate-count medium is required for

visualizing colonies by eye, and growth media in common use selects for microorganisms that are

fast-growing, grow to high density, are resistant to high concentrations of nutrients and are able to

grow in isolation. It could be argued that these traditional cultivation strategies use conditions that

are completely different to the natural environment of many microorganisms and are an important

contributing factor to the failure to cultivate most microorganisms in pure culture. New cultivation

methods have been developed to increase the number of culturable bacterial species. Despite the

spectacular advances in the molecular detection and circumscriptions of microorganisms and

functional genomics, organisms in culture are essential for providing an understanding of microbial

interactions, pathogenesis, phenotypic variability, and for biotechnological innovations.

1.2.9.1.1.1. Various strategies of culture dependent approach:

To study prokaryotic diversity choice of media and method of isolation of microbes depend

upon factors like the nature of target organisms intended to be cultured, the environmental niche

from where isolation needs to be made, number of samples. Choice of an appropriate medium is

crucial when culturing bacteria in nature. Some of these techniques are listed below:

1). Plating based methods

Traditionally, diversity was accessed using selective plating and direct viable counts. These

methods are fast, inexpensive and can provide information on the active, heterotrophic

component of the population. In many cases nutritionally poor media like R2A and diluted

standard media like TSBA (Tryptic Soya Agar) and NA (Nutrient Agar) gives higher viable

count than plating the environmental sample on normal standard media. The culturability of

bacteria in the bulk soil of an Australian pasture was investigated by using nutrient broth at

11100 of its normal concentration (dilute nutrient broth [DNBD as the growth

medium(Janssen et ai., 2002). Suzuki et ai., (1997) compared collection of isolates from

R2A plates with 16S rDNA sequences from clone library of sea water sample. They found

31


that most of the cultured bacteria were novel (Suzuki et al., 1997). The success of low

nutrient media at isolating some abundant bacteria has been confirmed in bulk soil from

Australian pasture, nutrient broth diluted 100 times and solidified with gellan gum resulted

in 14% of the total microscopic counts. Plating has also been used to isolate new, abundant

bacteria from some extreme environments. For example, close relatives of the recently

discovered extremely halophilic bacterium, Salinibacterium rubber Bacteroidetes phylum,

make up to 25% of the total prokaryotic community in Spanish saltier ponds (Anton et ai.,

2002). Direct plating on low-nutrient plates has been successful in isolating the most

abundant seawater bacteria in some other cases also. Gonzalez & Moran (1997) found that

up to 40 % of the colonies isolated from coastal water in the US were members of the

abundant Roseobacter clade from the Alphaproteobacteria (16% of all the marine rDNA

clones)(Gonzalez & Moran, 1997; Rappe et ai., 2000). Plating has also been used to isolate

new, abundant bacteria from some extreme environments(Anton et ai., 2002).

2). Enrichment

One way of isolating abundant natural bacteria by enrichment is to examine bacteria

growing in highest-dilution tubes during MPN enumeration experiments. This method has

been applied successfully to grow bacteria revealed as numerically abundant by 16S rDNA

cloning methods. Chin et ai. (1999) used this method to investigate anoxic rice paddy field

soil. Several isolates constituted more than 5% of the total direct count. These isolates

included members of Verrucomicrobia, Bacteroidetes and Gram-positive bacteria (Chin et

al., 1999). A similar approach has been used to culture lake water bacteria (Bartscht et ai.,

1999) in which a new synthetic medium was used to mimic natural lake water. When

compared with direct microscopy, MPN counts gave up to 7% culturability. This approach

was extended by addition of 10J.lg cAMP to the MPN tubes using artificial brackish

seawater. This gave culturability averaging about 15% (range (2-100%). From Baltic sea

(Bruns et al., 2002). Enrichment techniques have been particularly successful for isolation

of naturally abundant thermophiles in the order Aquificales from a hot-subsurface aquifer

from a gold mine Takai et ai., (2002) tested 900 enrichment media and showed that three of

them were successful for isolation of the target organisms.

3). Micromanipulation

Micromanipulation can also be a valuable aid form isolating bacteria, especially when

fluorescent in situ hybridization (FISH) with phylogenetic probes is used to visualize the

target bacteria. Huber et ai., (1995) used a strongly focused infrared laser to separate a new

hyperthermophilic archaea from a hat pool in Yellowstone National Park. After separation

by micromanipulation, the aggregates were successfully grown in pure culture (Huber et

32


aI., 1995). Morphology without FISH probes is sometimes enough to target isolation.

Kaempfer, (1997) has isolated morphologically distinct filamentous bacteria, common in

activated sludge wastewater treatment systems(Kaempfer, 1997).

4). Extinction culture

Button et al. (1993) described this method (also called as dilution culture/dilution to

extinction) in which the total bacteria present in the sample is calculated microscopically

and then diluted with filter-sterilized water until only a few bacteria remain and then

growing the cells in either the unamended water or 'by adding small amounts of organic

substrates to culture them. In the original trials of the method with seawater from

Resurrections Bay, Gulf of Alaska, it was observed that almost all the marine bacteria

retained their viability (about 60 % of the direct counts)(Button et aI., 1993). Schut et al.

(1993) isolated 37 strains from Resurrection Bay and the North Sea using this approach.

Both seawater salts agar and Zobell 2214E agar gave counts that were 80 % of the total

counts. Seven of these isolates have been assigned to the new species Sphingomonas

alaskensis with isolate RB2256 as type strain. The SARI clade of Alphaproteobacteria is

one of the most abundant bacterial groups in the oceans, accounting for 26% of all rRNA

sequences isolated from sea water (Geovannoni & Rappe, 2002). Rappe et al. (2002)

reported the isolation of 11 cultures of SAR-l1 clade using extinction culture. These are

one of the smallest bacteria in culture and have been named "Candidatus Pelagibacter

ubique"(Rappe et aI., 2002).

1.2.9.1.2. Culture independent study of prokaryotic diversity:

Many culture independent approaches in which molecular biology has found their

application were developed to characterize microbial communities that are able to generate a

fingerprint of diversity as well as methods that use the conventional techniques can be used to

decipher microbial diversity such as community level physiological profiling (CLPP) and

Phospholipid Fatty Acids profiling (PFLA). The advantages of applying both of these approaches do

not require culturing the microbes thus eliminating the bias associated culture dependent method.

The application of these tools by microbial ecologists has rapidly enhanced our knowledge of

prokaryote abundance, diversity and their function in their habitat. Each of these methods measures a

different aspect of the community (diversity, in situ detection, and community dynamics). Molecular

detection methods involve (i) direct lysis of bacterial cell and (ii) The analysis the extraction of the

nucleic acids from the matrix and (iii) the analysis of targeted sequences or the whole body of

genetic information. Two main types of molecular technique are available to study bacterial

communities using DNA directly extracted from natural environments (Ranjard et aI., 2000).

33

(


1. Molecular approaches which usually investigate parts of this information by focusing on

genome sequences which are targeted and amplified by PCR that are called "partial

community DNA analysis".

2. Molecular approaches which try to investigate all the genetic information in the extracted

DNA and are called "whole community DNA analysis". Each of these methods are

described below-

1.2.9.1.2.1. Biochemical based approaches:

1). Community level physiological profiling (CLPP)

This technique was developed by Garland & Mills (1991) for a rapid and community level

physiological profiling (CLPP). This technique is widely used to characterize microbial

communities (Garland & Mills, 1991; Lehman et al., 1995). The response or CLPP

involves (1) the overall rate of color development by tetrazolium dye which gets reduced

when a substrate gets oxidized producing a visible color development. (2) The richness and

evenness of the response among well (or diversity), and the pattern, or relative rate of

substrate oxidation among well. The major strength of this approach are its (1) low man

power requirements, which enables intensive sampling across temporal and special scales

and (2) reliance on metabolic traits that could lead to functionally relevant characterization

of change in microbial communities (Garland, 1997). The technique uses a commercially

available 96-well microtitre plate to assess the potential functional diversity of the bacterial

population through sole carbon source utilization (SSCU) patterns. Gram-negative (GN)

and gram-positive (GP) plates are available from Biolog (Hayward CA, USA,

www.biolog.com) and each contains 95 different carbon sources and one control well

without a substrate. Subsequently, Biolog introduced an Eco-plate containing 3 replicates

of 31 different environmentally relevant carbon sources and one control well per replicate

(Choi & Dobbs, 1999). Inoculated popUlations are monitored over time for their ability to

utilize substrates and the speed at which these substrates are utilized. Multivariate analysis

is applied to the data and relative differences between soil functional diversity can be

assessed. This method has been used in arctic soils (Derry et al., 1999), soil treated with

herbicides (EIFantroussi et al., 1999) or inoculation of microorganisms (Bej et al., 1991).

The method has also been used successfully to assess potential metabolic diversity of

microbial communities in contaminated sites(Derry et al., 1998; Konopka et al., 1998),

plant rhizospheres (Ellis et aI., 1995) (Garrity, 1996; Grayston & Campbell, 1996), Similar

in principal to the Biolog system is the API system (Merieux, France). There are a number

of API strips available with various carbon sources that can be used to measure functional

diversity (Torsvik et al., 1990b).

34


2). Fatty acid methyl ester (FAME) analysis

A biochemical method that does not rely on culturing of microorganisms is fatty acid

methyl ester (FAME analysis). This method provides information on the microbial

community composition based on the groupings of fatty acids(Ibekwe & Kennedy, 1998;

Zelles, 1999). Fatty acids make up a relatively constant proportion of the cell biomass.

Signature fatty acids that are integral part of cell membrane can differentiate major

taxonomic groups within a community. It has been used to study microbial community

composition and population changes due to chemical contaminants (Kelly et ai., 1999;

Siciliano et ai., 2003) and agricultural practices (Bossio et ai., 1998; Ibekwe & Kennedy,

1998). Therefore, a change in fatty acid profile would represent a change in the microbial

community. For FAME analysis, fatty acids are extracted directly from soil, methylated

and analyzed by gas chromatography (Ibekwe & Kennedy, 1998). FAME profiles of

different soils can be compared using multivariate analysis. Ibekwe & Kennedy, (1998)

used phospholipids fatty acid analysis (PLFA) and CLPP to study microbial communities

in the rhizosphere of plants from the field and from green house pots and was able to

demonstrate a clear difference between microbial communities of these ecosystems.

Cellular fatty acid composition can be influenced by factors such as temperature and

nutrition(Graham et ai., 1995).

1.2.9.1.2.2. Molecular approaches:

1). Nucleic acid reassociation

DNA reassociation based on the principle that two strands of DNA which have a minimum

level of similarity will reanneal appropriate conditions are provided. It gives a measure of

genomic complexity (types of DNA molecules) of the microbial community and has been

used to estimate diversity (Torsvik et ai., 1990a; Torsvik et aI., 1996). In this method total

DNA is extracted from environmental samples, purified, denatured and allowed to

reanneal. The rate of reassociation will depend on the similarity and concentration of

sequences present. As the complexity or diversity of DNA sequences increases, the rate at

which DNA reassociates will decrease (Theron & Cloete, 2000). Time taken needed for

half of the DNA to reassociate (the half association value Cot1/2, where Co is the molar

concentration of nucleotides in single stranded DNA at the beginning of the reassociation,

and tl/2 the time in seconds for 50 % reassociation) can be used as a diversity index. It

predicts both the amount and distribution of DNA (Torsvik et ai., 1998).

2). Guanine plus cytosine (G+C) content

35


Analysis of the guanine plus cytocine (G+C) content of DNA is useful when a coarse level

of resolution is meaningful. This technique is utilizes the variation of amount of nucleotide

residues. prokaryotic DNA varies in G+C content from 24%-76% G+C versus A+T, and

that particular taxonomic groups only include organisms that vary in G+C content by no

more than 3-5% (Goodfellow & O'donnell, 1993); Vandamme et al., 1996). Hence G+C

can be related to taxonomy and can be used to detect changes in community structure

(Nusslein & Tiedje, 1999). It requires an ultracentrifuge to separate the G+C fraction. Base

composition separation is based on the principle that bisbenzimidazole binds to adenine

and thymine which altogether changes the buoyant density of DNA in proportion to its T

(hence G+ C) content. A gradient of DNA fragments of different G+C contents is then

established using CsCI density gradient centrifugation. The different fractions are then

collected using a fraction collector. The DNA in each fraction is quantified by

spectrophotometry and its G+C content is established by using a standard curve. G+C

content together with ARDRA and rDNA sequence analysis was used by Nusslein and

Teidje (1999) to study the differences in microbial diversity between a vegetative cover of

forest and a pasture in Hawaiian soil and concluded that all the three methods worked well

to detected the changes in microbial community revealing that plants have a strong

influence on microbial community composition.

3). Nucleic acid hybridization and Fluorescent In Situ Hybridization (FISH)

In the field of molecular ecology hybridization techniques has been proved to be one ofthe

most important qualitative and quantitative tool in molecular bacterial ecology (Clegg et

aI., 2000; Griffiths et aI., 1999; Guo et aI., 1997; Schramm et aI., 1996; Theron & Cloete,

2000). This technique takes in account the use of specific probes that binds to Nucleic acid

at a very specific region. Various studies it has been shown that DNA as well as RNA can

be targeted by using oligonucleotide or polynucleotide probes that has been designed from

known sequences and ranging in specificity from domain to species. These probes are

generally labeled with markers at the 5' -end (Theron & Cloete, 2000). Among many

fluorescent markers commonly used, derivatives of fluorescein and rhodamine have been

used extensively and have become most popular. To measure spatial distribution and

relative abundance of certain groups of microorganisms in environmental samples,

quantitative dot-blot hybridization in which the sample is lysed to release all nucleic acids

than ribosomal DNA sequences of interest are quantified relative to total rDNA by dot-blot

hybridization with specific and universal oligonucleotide probes. At cellular levels this

method can be applied in situ in which samples are fixed cells are permeabilized and

fluorescently labeled probes are added to the sample after addition of the probe it is

allowed to hybridize. After incubation is over the cells are visualized (Head et aI., 1998). 36


Schramm and coworkers applied this method to study spatial distribution of bacteria in

biofilms (Schramm et aI., 1996).

4). CARD-FISH

Combining FISH with catalyzed reporter deposition (CARD-FISH) has been demonstrated

to substantially enhance bacterial cell detection in situ (Schonhuber et aI., 1999). CARD

FISH has recently been applied for the identification of pelagic marine Bacteria (Pernthaler

et aI., 2002), Cyanobacteria (Schonhuber et al., 1999), Actinobacteria (Sekar et aI., 2003),

and sedimentary, marine Archaea and Bacteria (Ishii et aI., 2004), with all studies reporting

superior cell detection in comparison to conventional FISH. Tujula et al., (2006)

demonstrated the first application of CARD-FISH to study the bacterial community on the

surfaces of marine macroalgae Ulva lactuCG, Delisea pulchra, Corallina officinalis,

Amphiroa anceps, Porphyra sp. and Sargassum linearifolium (Tujula et aI., 2006). CARD

FISH involves a number of pre-treatment steps including embedding, permeabilisation and

inactivation of endogenous peroxidase followed by hybridizations with HRP probes

(Thermo Electron, Germany) targeting Bacteria (EUB338 i-iii) (Daims et aI., 1999» and

then visualized Cells were visualized with a Leica TCS-SP confocal laser scanning

microscope (CLSM).

5). PCR-based approaches

The 16S rONA has been targeted extensively to study prokaryote diversity. It allows

identification of prokaryotes and predicts phylogenetic relationships as well. Although this

technique mainly involves cloning of target genes and their sequencing of the clones, they

may sometimes becomes thousands in number to reach any conclusion making this

technique time consuming as well as expensive (Muyzer & Small a, 1998; Tiedje et al.,

1999). Due to this reason many other techniques discussed below have been developed to

assess prokaryotic diversity of natural environments.

i). Denaturing gradient gel electrophoresis DGGE)ffemperature gradient gel

electrophoresis (TGGE)

PCR -amplified 16S rRNA gene were analyzed by applying DGGE. Primers

annealing to conserved region of the rONA were used to amplify the variable V3

region flanked by primers. Different base composition in this variable region of

rDNA from the community, gave PCR products with different melting points.

The PCR products were separated according to their melting point in

polyacrylamide gels with 15-55% denaturing gradient (100% denaturant

comprised 7M urea and 40% formamide). The gels were run at 60 C. by applying

group specific probes the affiliation of the DGGE bands to main phylogenetic 37


subclasses of bacteria, was determined .furthermore, this approach allowed

identification of bands from the putative numerically dominant bacteria. The

strongest band were punched out from the gel, reanalyzed by PCR-DGGE to

ensure that they are consisted of a single sequence, and subjected to sequencing

the sequence were aligned to those obtained from various databases such as

NCBI by BLAST program in order to assign them to bacterial subclass and

probe designing. TGGE uses the same principle as DGGE except that here the

denaturing conditions are maintained using a temperature gradient. Advantages

of this technique are that it is reliable, reproducible, and rapid, mUltiple samples

can be compared simultaneously for their fingerprints and it is possible to

monitor changes in microbial populations over a spatial and temporal gradient.

Limitations include PCR biases, laborious sample handling, and variable DNA

extraction efficiency. Moreover, DNA fragments of different sequences can have

same mobilities and a single species can give mUltiple bands due to heterogeneity

in its 16S rRNA gene. Holben et al. (2004) used DGGE to assess community

diversity and to detect minority populations of bacteria in the digestive tracts of

chickens. Some researcher's have also used DGGE analyses to target catabolic

genes such as methane monooxygenase (Fjeilbirkeland et al., 2001; Knief et al.,

2003). This provides information regarding specific group of microbes.

ii). Single strand conformation polymorphism (SSCP)

Another technique that relies on electrophoretic separation based on differences

in DNA sequences is single stranded conformation polymorphism (SSCP) was

also developed to detect known or novel polymorphism or point mutations in

DNA (Orita et aI., 1989). On a polyacrylamide gel, differences in mobilities in

single stranded DNA of same sizes are caused by their folded secondary structure

(Lee et aI., 1996) resulting in a pattern that reflects number of phylotypes present

in the sample. Folding and therefore mobility of DNA fragments will be

dependent on the DNA sequences when DNA fragments are of equal size and no

denaturant is present. SSCP has all the limitations of DGGE. Also, some single

stranded DNA can form more than one stable conformation. Therefore, one

sequence may be represented by more than one band on gel. It does not require a

GC clamp or the construction of gradient gels and has been used to study

bacterial and fungal community diversity (Peters et al., 1999; Stach et aI., 2001).

This method has been also been applied to study succession of bacterial

communities (Peters et aI., 2000), rhizosphere communities (Schweiger and

Tebbe, 1998), anaerobic bioreactor (Zumstein et aI., 2000). 38


iii). Restriction fragment length polymorphism (RFLP)/Amplified ribosomal DNA restriction analysis (ARDRA)

The method is based on DNA polymorphism and involves digestion of genomic

DNA/ PCR amplified 16S rRNA gene sequences using restriction enzymes

(usually a four-cutter). Fragments are then separated on agarose or

polyacrylamide to generate a profile of microbial community. This method has

been used most frequently to screen clones (Pace, 1996) or to measure bacterial

community structure (Massol-Deya et ai., 1995; Smit et ai., 1997). Although this

method cannot be used for quantifying diversity or following specific ribotypes

but it has found its application for detecting structural changes in the microbial

community. A single species can give many restriction fragments thus sometimes

complicating study in complex communities when analysed by RFLP.

iv). Terminal restriction fragment length polymorphism (T -RFLP)

With the help of this method a community profile is generated for complex

communities as well as it provides information on diversity as each visible band

in t-RFLP represents a single operational taxonomic unit or ribotypes (Tiedje et

ai., 1999). In this method amplified product is digested with appropriate

restriction enzyme as done in ARDRA but the PCR primer is labeled with a

fluorescent dye, such as CY3 or 6-FAM (phosphoramidite fluorochrome 5-

carboxyfluorescein). Because this allows detection of only the labeled terminal

restriction fragment, it simplifies the banding pattern (Liu et ai., 1997). By doing

a profile to profile comparison a large number of bacterial species can be

identified in a profile as a data bank has been produced on the sizes of the 16S

rDNA terminal restriction fragment for a given enzyme on a large set of bacterial

species. This procedure has now been automated by use of automated sequencers

to allow sampling and analysis of large number of samples (Osborn et ai., 2000).

By using sequencing systems for T-RFLP (for separation of T-RFLP fragments)

resolution and sensitivity has been remarkably improved. Also interpretation of

peak height and area can be made, which can be further interpreted in terms of

number, and abundance of OTU's. Functional genes such as the mercury resistant

determinants (mer genes) have been studied by T-RFLP.

v). Ribosomal intergenic spacer analysis (RISA)/Automated ribosomal intergenic spacer analysis (ARISA)

The region between 16S rRNA and 23S rRNA is termed as intergenic spacer

region (lOS) and has been targeted to interpret microbial diversity in soil and

rhizosphere of plants (Borneman & Triplett, 1997), in contaminated soil (Ranjard

39


et al., 2000) and in response to inoculation (Yu & Mohn, 2001). The method

involves the analysis of the length polymorphism of the intergenic spacer region

(IGS) between the small (16S) and large (23S) rRNA genes whose size varies

from 50 bp to more than 1.5 kb depending on the species. The primers target

regions within the adjacent genes and can be defined so that part of the 16S

rRNA gene is co-amplified and directly separated on polyacrylamide gels on the

basis of size. Further sequencing of the 16S rRNA gene can provide a finer

taxonomic identification of the bands. In RISA the sequence polymorphisms are

detected using silver stain while in ARISA the forward primer is fluorescently

labeled and is automatically detected (Fisher & Triplett, 1999). ARISA increases

the sensitivity and reduces time.

vi). 16S rRNA library construction and sequencing

In this approach target genes are amplified from community DNA and then

cloning is done to separate sequences. These sequences can be characterized

individually using ARDRA and/or by sequencing. Sequencing allows a fine

identification of uncultured bacteria as well as an estimation of their relatedness

to known culturable species. There are some limitations and drawbacks of the

PeR-cloning approach as there exists bias due to the peR (i.e. choice of primers,

annealing temperature, inhibition of the enzyme by humic substances, formation

of chimeric peR products), in addition it is also time-consuming and

cumbersome since it is necessary to sample a large number of clones in order to

obtain a good diversity estimation of the amplified sequences, requires expensive

equipment (sequencer) and the cloning strategy used. Moreover, the substantial

richness found within the soil bacterial community and the number of clones per

library (usually about 100 clones) precludes the application of diversity

measurements in terms of species evenness. However, such a sampling size can

be informative for estimating diversity, both richness and evenness, by

considering higher level phylogenetic groups (i.e., subdivisions within

Proteobacteria, Firmicutes, Actinobacteria etc.). This approach has been used to

decipher prokaryotic diversity of soil, marine, freshwater and subsurface

environments.

1.2.9.2. Approaches evaluating community structure with function:

1) Microautoradiography-FISH (FISH -MAR)

40


This technique has been designed to detect metabolically active microbes. A metabolically

active microbe takes up specific radiolabelled substrates and can be further detected by

using pre-designed; specific 16S rRNA targeted FISH probes. Environmental samples are

first incubated for a short-term incubation with specific radio labelled substrates like 3H_

acetate, 14C-pyruvate, 14C-butyrate then thin sections of these samples are fixed on to the

glass slides and subsequent analysis by FISH and inverse confocal laser scanning

microscopy is done. This technique allows the detection of bacteria that are biologically

active in taking up a specific radiolabelled substrate. Daims and coworkers (2001) analyzed

nitrifiers and denitrifiers complex microbial communities using this technique.

2) Radioisotope array

In this method, an environmental sample is incubated with a 14C-Iabelled substrate first,

than environmental sample is subjected to RNA extraction followed by labeling with a

fluorophore further analysis with an oligonucleotide array containing DNA probe

sequences specific for the 16S rRNA genes of the bacteria of interest from the sample.

Community members that have incorporated the 14C isotope into their RNA are determined

by scanning for fluorescence and incorporation of the radioactive isotope. By using this

technique incorporation of 14C-bicarbonate by autotrophic ammonia oxidizers in activated

sludge was determined (Adamczyk et ai., 2003).

3) Stable isotope probing (SIP)

Radajewski et aI., (2000) used this technique for studying methylotrophs in a forest soil

and discovered that Acidobacterium and Beijerinckia species previously not known to be

methylotrophic were involved in the process. This technique involves analysis of the

labelled biomarker upon exposure of environmental sample to a stable -isotope (such as

i3C and 15N) enriched substrate for which utilization and assimilation has to be determined

for specific group of bacteria. In DNA-SIP identity of microorganism is linked to its

function by analyzing DNA which gets labeled upon exposure to -isotope enriched

substrate. Methodology of this technique is as follows:

a). Sample is incubated with labeled substrate ( i3CH30H or J3CH4),

b). DNA is isolated from the sample and subjected to CsCI density gradient

centrifugation. Separated heavier DNA (labelled) from lighter DNA (unlabeled)

represents the combined genomes of those bacteria, which are actively metabolizing

the labeled substrate.

c). The labeled DNA can then used as a template in PCR with primers targeting the l6S

rRNA genes or any other functional gene.

41


4) DNA microarrays

More recently DNA-DNA hybridization has been used together with DNA microarrays to

depict the microbial diversity. In DNA microarray denatured sequence fragments (single

genes or whole genomes) are attached to a solid support (in the form of an array, known as

a chip). Further steps involve random labeling of the total community DNA, hybridization

to the array and then detection and analysis of the individual dot hybridization data to

interpret the diversity of the sample. The microarray can contain specific target genes such

as nitrate reductase, nitrogenase or naphthalene dioxygenase to provide functional diversity

information or can contain a sample of environmental standards (DNA samples with less

than 70 % hybridization) representing different species found in the environmental sample

(Greene & Voordouw, 2003). The latter approach called as Reverse Sample Genome

Probing (RSGP) is used to analyze the most dominant culturable species. An internal

standard should be included both in the labelled probe and in the spotted array. This

method has been used to analyze microbial communities in oil fields (Voordouw et al.,

1991, 1992, 1993) and in contaminated soils (Shen et al., 1998; Hubert et al., 1999; Greene

et al., 2000). It is a useful technique when diversity is low; this method has got advantages

over PCR based diversity studies when diversity is comparatively low. The only limitation

associated to this technique is the problem of cross hybridization and possible detection of

only most abundant species (Greene & Voordouw, 2003).

5) Metagenomics approach

New tools like 'Metagenomics' (Environmental genomics, Community genomics) are

accessing microbial diversity to provide novel genes and biosynthetic pathways of those

bacteria, which are uncultured till date. Metagenomics is the culture-independent genomic

analysis of microbial communities. Each organism in an environment has a unique set of

genes in its genome; the combined genomes of all the community members make up the

"metagenome". Metagenome technology (metagenomics) has led to the accumulation of

DNA sequences and these sequences are exploited for novel biotechnological applications

(Ferrer et aI., 2005). Due to the overwhelming majority of non-culturable microbes in soil,

metagenome searches will always result in identification of hitherto unknown genes and

proteins. Thus, the probability of uncovering hitherto unknown sequence makes this

approach more favorable than searches in already cultured microbes. The term is derived

from the statistical concept of meta-analysis (the process of statistically combining separate

analyses) and genomics (the comprehensive analysis of an organism's genetic material)

(Rondon et aI., 2000). The method involves-

42

Chapter 1

I).

Introduction & Review o(Literature

Isolation of high quality environmental or total community DNA. The DNA isolation

methods can be divided into two categories: Direct method that involves lyses of cells

contained in the sample matrix followed by separation of the DNA from the matrix

and cell debris and indirect method wherein cells are first separated from the soil

matrix followed by cell lyses. The crude DNA recovered by both the methods is

purified by standard procedures.

II). Restriction digestion of the extracted DNA is done to cut DNA into desired size by

using either restriction endonucleases or by mechanical means such as nebulization,

French press and air nozzles. In a classical approach, small inserts libraries «lOKb)

were constructed in a standard vector [pBluescript SK (1)] and transformed in

Escherichia coli DH5a as a host strain (Henne et ai., 1999). However, small insert

libraries do not allow detection of large gene clusters or operons. To circumvent this

limitation researchers have been employing large insert libraries, such as cosmid

DNA libraries (mostly in the pWE15 vector) with insert sizes ranging from 25-35kb

(Entcheva et ai., 2001) and bacterial artificial chromosome (BAC) libraries with insert

sizes up to almost 200 kb (Beja et ai., 2000; Rondon et ai., 2000). Construction of

fosmid libraries with inserts of 40 kb of foreign DNA has been reported (Beja et ai.,

2002). Genes for antibiotic synthesis have been cloned successfully from soil in the

BAC vector, pBeloBAC 11, and the cosmid superCos 1 (Brady et ai., 2001; MacNeil et

ai., 2001; Gillespie et ai., 2002). The host for the initial construction and maintenance

of almost all the published libraries is E. coli (Daniel, 2005). Shuttle cosmids or BAC

vectors can be used to transfer libraries that are produced in E. coli to other hosts such

as Streptomyces or Pseudomonas species. The choice of the vector system depends on

the quality of isolated DNA, the desired average insert size of the library, the copy

number required, the host and the screening strategy that will be used, all of which

depend on the aim of the study. Small-insert libraries are useful for isolation of single

genes or small operons encoding new metabolic functions. Large-insert libraries are

more appropriate to recover complex pathways that are encoded by large gene

clusters or large DNA fragments for the characterization of genomes of uncultured

microorganisms.

Ill). The final step IS the screening of metagenomic libraries. For screening of

metagenomic libraries three methods are normally employed-

1. Functional screening: based either on metabolic activity (function-driven

approach). Interestingly, searches in metagenome-derived DNA libraries have

mainly focused on a rather small group of enzymes. Among these are lipases

and esterases (Voget et ai., 2003; Henne et ai., 2000). They usually display

43


exquisite chemo-, regio-, and stereoslectivities and they do not require

cofactors (Jaeger et aI., 1999). Oxidoreductases are another example of useful

catalysts with a high enantioselectivity that have been identified in

metagenome searches (Knietsch et aI., 2003). Of particular interest,

nicotinamide-dependent alcohol reductases are employed for the preparation of

deuterium or tritium labeled compounds, production of dihydroxyacetone and

as tools for enzymatic analysis of serum lipids (Hummel, 1999). Similarly,

polysaccharide-modifying enzymes such as the starch modifying enzymes are

of considerable interest to industry. Therefore, a significant number of

metagenome searches have identified polysaccharide-modifying genes (Healy

et aI., 1995; Richardson et ai., 2002). Further, the isolation of enzymes useful

for the production of bulk chemicals (Knietsch et aI., 2003), proteases (Santosa,

2001) and nitrilases (DeSantis et aI., 2002) has been reported. Metagenome

searches have also focused on the isolation of genes involved in vitamin

biosynthesis. Interest in 2,5-diketo-D-gluconic acid synthesis is related to a

new biotechnological process for the production of vitamin C (ascorbic acid)

using glucose as a substrate (Eschenfeldt et aI., 2001); and interest in biotin

biosynthesis genes is linked to the construction of biotin overproducing

bacteria for large-scale fermentation of this vitamin (Streit et aI., 2003).

Finally, the isolation of genes encoding for novel therapeutic molecules is a

very valuable area of research (Gillespie et aI., 2002).

II. Sequence based approach: Sequence analysis of large insert libraries with

environmental DNA combined with genetic and functional analysis has the

potential to provide significant insight into the genomic potential and

ecological roles of cultured and uncultured microbes. The importance of this

potential for understanding complex environments can be estimated by the

following examples.

a. Recent analysis of genome fragments recovered directly from marine

bacterioplankton suggested the presence of a new bacterial rhodopsin,

proteorhodopsin. Adaptation of proteorhodopsin proteins to different

habitats combined with the genetic and biophysical data indicate that

proteorhodopsin-based bacterial phototrophy is a globally significant

oceanic microbial process (Beja et ai, 2000).

b. Culture-independent partial or nearly complete recovery of microbial

genomes from an environmental sample by an extended random

shotgun-sequencing approach offers a highly intriguing approach to

44

Chapter 1 Introduction & Review o(Liferafure

study natural microbial communities. A recent example gave significant

insights into the community structure and the metabolism of a natural

acidophilic biofilm growing on the surface of a flowing acid mine

drainage. This was mainly possible through reconstruction of the

microbial genomes present in this niche. For this purpose the near

complete genomes of Leptospirillum group II and Ferropiasma type II

were reconstructed.

c. Analysis of the complex metagenome of model biofilms growing on

rubber-coated valves within drinking water networks using the

cultivation independent approach. This analysis revealed significant

insights into phylogenetic, catabolic and metabolic abilities of the

analyzed microbial community. Large-scale sequencing projects such as

the one initiated by Craig Venter for the metagenome of the Sargasso

Sea (Venter et ai., 2004) resulted not only in the identification of

numerous novel genes but also 180 novel bacterial species. This is a very

significant achievement of sequence based metagenome analyses.

Similar approaches have been initiated by European laboratories to

sequence complete or partial metagenomes of a phylogenetically highly

diverse biofilm (Schmeisser et ai., 2003). Complementary to this, Tyson

et ai., (2004) described the nearly complete sequence analyses of the

metagenome of an acidophilic biofilm.

d. Analysis of metagenomic DNA has been proposed as a strategy for

evaluating numbers of soil microorganisms (Miller et ai., 1999).

Aoshima et ai. (2006) reported a slow-stirring method for the isolation of

metagenomic DNA from various kinds of soil with minimal shearing.

They have obtained a linear proportional relationship between soil

bacterial biomass and the amount of DNA isolated by this method.

Therefore, the bacterial biomass could be evaluated by quantifying levels

of environmental DNA. However, co extraction of extracellular DNA

should be considered, which may lead to the overestimation of number

of living bacteria.

e. Deutschbauer et ai., 2006 have demonstrated tracking of recombinant

microorganisms by using metagenomic approach. The possibility of

genetically engineered microorganisms to be used in environmental

applications requires an understanding of the fate of recombinant DNA

introduced into the environment. To study the fate of a geneticallY

45


engineered orgamsm introduced into a soil environment, direct

extraction of DNA followed by a specific quantitative detection based on

PCR or hybridization are used to determine the persistence of the

recombinant gene.

f. An exciting extension of the metagenomics is the high-throughput

analysis of the "mobilome" or mobile metagenome, the genomics of

mobile elements from uncultured organisms (au et at., 2007). Genes

present on mobile genetic elements (MGE) that populate soil ecosystems

constitutes the mobilome of the environment. MGE include plasmids,

transposons, insertion sequences and integrons, which may move

between bacterial cells in a population or mobilize into a new host

species and introd~ce new genetic material (Jones & Marchesi, 2007).

g. As described earlier in section 1.2.5, Venter and his colleagues reported

the results of a Global Ocean sampling expedition (Rusch et at., 2007),

an environmental metagenomics project that aimed to shed light on the

role of marine microbes by sequencing their DNA without the need to

cultivate them. The analysis has been the biggest application of

metagenomics in lieu of the number of samples collected and analyzed.

A total of 41 different surface water samples (mostly marine) were

collected over a distance of 8000 km from the North Atlantic through the

Panama Canal and ended in South Pacific. Total DNA was extracted

from each sample and libraries were constructed and analysed by

shotgun sequencing approach. An extensive dataset consisting of 7.7

million reads (6.3 billion bp) of sequence data that amounted to a total of

~ 5.9 Gbp of nonredundant sequence was obtained. A total of 4, 125

distinct full length or partial 16S rRNA genes were identified. Out of

these 811 were identified as distinct ribotypes and more than half of the

ribotypes were found to be novel « 97 % similarity). They found that

relatives of SAR 11 and SAR 86 appear to be ubiquitously abundant in

all the samples.

1.2.9.3. Other molecular tools for taxonomy that require cultivation:

(i). rep-peR

Probably the most popular fine scale method to resolve differences in populations between

different sites is the use ofrep-PCR (repetitive extragenic palindromic-PCR) (Versalovik et

aI., 1994). Many organisms, both prokaryotic and eukaryotic, contain highly repetitive

46


short DNA sequences that are 1-10 base pair long repeated throughout their genomes (Zeze

et al., 1996;Longato & Bonfante, 1997) depending upon the rate of evolution, these

sequences may be diagnostic and allow differentiation down to the species or strain level

and has been used for the identification of bacteria since it provides a genomic fingerprints

of chromosome structure, and chromosomal structure is considered to be variable between

strains (Tiedje, 1999). These highly repetitive sequences are also referred as microsatellite

regions. However this method requires an isolate. It is often used as the first level screen to

indicate how closely related the isolates are. This method can be used directly on small

amounts of cells, e.g. a colony, without prior DNA extraction, thus making it possible to

analyze 60-100 strains overnight. The method is rapid, reproducible, the data were suitable

for storage in searchable database, and provides the highest level of taxonomic resolution

of any current peR based method. There are three primer sets that have been found to work

in most bacteria; these are known as REP, BOX and ERIC. It has been successfully used to

differentiate organisms like Rhizobium (Vinuesa, et al., 1998), plant pathogenic

Xanthomonas (Louws et al., 1994). Teidje et aI., (1995) evaluated the degree of endemism

in soil populations.

(ii). Rihotyping

More accurate method for genotype determination is that of the molecular biological

approach of ribotyping by comparing similarities in the rRNA gene sequences. A more

recent ribotyping technique is the patented method called riboprinting. The Qual icon

(DuPont) RiboPrinter is an automated system that takes a purified bacterial suspension,

lyses the cells, extracts the DNA, restriction endonuclease digests the DNA, separates the

digest on a gel, transfers the DNA bands to a membrane, probes the bands with non

radioisotope-labelled, 5S-16S-23S rRNA-specific probes (Southern hybridization),

photographs the membrane, and finally compares the bar code-like pattern to databases in

order to identify the genus and species(Bruce et aI., 1995). The entire process takes

approximately 8 h and requires only a small amount of growth sample from a Petri dish.

Although this method and instrument were originally developed to identify the food-borne

pathogens, Listeria, Salmonella, and Staphylococcus spp. and E. coli, it has since been used

for many applications, including the identification of spoilage bacteria in the brewing

industry (Funuahashi et aI., 1998; Leisner et al., 1999). The manufacturer has demonstrated

with food-borne pathogens that the method is close to 100% accurate at identifying genus

and species and often has the ability to differentiate at the subspecies level. This, in tum,

has utility in epidemiological studies for tracking isolates. Based on a historical database of

isolates encountered in manufacturing facilities, riboprinting may also be useful for

predicting the pathogenicity, spoilage capability, or other phenotypic expressions of the

47


organisms (Barney et ai., 2001) 16S ribosomal DNA (rDNA) sequencing of a number of

isolates showed that Pediococus damnosus isolates with distinctively different RiboPrinter

patterns had identical sequences, except for 1 bp in one strain. The sequence analysis

method was very good at identifying the organisms by genus and species but did not

differentiate at the subspecies level. The riboprinting method, on the other hand, gave the

correct genus and species and also allowed the sub speciation of many strains. It was

particularly useful in identifying an acid-resistant isolate (Barney et aI., 2001).

(iii).MALDI TOF

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF

MS) fingerprinting is a fast and reliable method for the classification and identification of

microorganisms, with applications in clinical diagnostics, environmental and taxonomical

research, or food-processing quality control. The general use of MALDI for the

characterization of large biomolecules led directly to obvious applications involving the

analysis of isolated bacterial proteins. More surprising was the observation that MALDI

TOF mass spectrometry could be applied directly to crude cellular fractions or cellular

suspensions and that the resulting data from such complex mixtures could provide evidence

for chemotaxonomic classification. Most simple analysis of a sample starts by applying a

small amount of biological material directly onto the MALDI target plate. The starting

material can be a single colony or a centrifuged portion of a liquid culture. The thin

microbial film is overlaid with matrix (oc.-cyano-4-hydroxycinnamic acid; HCCA; Jackson,

2001).

1.2.10. Bacterial Systematics: 'a roadmap to diversity'

The profounding complexities in microbial world are:

1. Large Microbial diversity

2. Heterogeneously distribute diversity over environment, space and time

3. Diversity of viable but nonculturable

4. Diversity of gene expression

5. Lateral gene transfer

Taxonomy, the principles and practice of classification of organisms that make up scattered

and unparallel diversity enables their detection and identification. The extant to which a general,

phylogenetically based taxonomy can be predictive depends on the relative contributions of

horizontally and vertically transmitted genes. Taxonomy is key to this understanding (Bull et al., 2004).

48


Bacterial systematics began in much the same way as the systematics of animals and plants.

Systematists of both microbes and macrobes began with the observation that organisms fall into

clusters of very similar organisms, and they demarcated and named these clusters. These species

demarcations were originally based entirely on phenotype, principally on morphology in the case of

animals and plants, in the case of bacteria it was solely based on and on metabolism. However, the

practices of microbial and macrobial systematics diverged in the 1940s and 1950s when a theory

based concept for species was brought into the systematics of animals (Mayr 1942) and plants

(Stebbins 1957). First, with Mayr's biological species concept (Mayr 1942), and later with many

alternative concepts of species (de Queiroz 1998). Systematists of animals and plants sought to make

their species more than just clusters and aimed to identify species that represented the fundamental

units of ecology and evolution. Despite the plethora of modem species concepts used today, nearly

all these concepts share certain standard attributes, which are as follows: species are cohesive i.e.

some force acts to constrain divergence within species (Meglitsch, 1954) they are irreversibly

separate because there is no force of cohesion that constrains divergence between species (Wiley,

1978); they are ecologically distinct and thus able to coexist within a community (Valen, 1976) and

they are monophyletic i.e. each species is invented only once. It has long been understood that

diversity within the highly sexual animal and plant species is constrained by a powerful force of

genetic exchange. It has also been observed that bacterial species, which recombine sexually at a low

rate, can also be defined so as to meet the criterion of genetic exchange, as well as the other attributes

of species (Cohan et aI., 2001).

The taxonomy of prokaryotesarrived much later than other kingdom. Carl von Linne classified

microscopic organisms in the genus "chaos" and in 1874 Theodor Billroth (1829-1894) believed that

there was only one bacterial species, Coccobacteria septica, which could occur in any form

depending on growth conditions. Initial studies on the taxonomy of prokaryotes and protozoa were

based on higher eukaryote systematics, as exemplified by Ehrenberg (1795-1876) who classified

them into several genera, and Ferdinad Cohn, a trained botanist an adherent of Ehrenberg, who was

one of the first to hold that bacteria could be arranged into genera and species that had a high degree

of constancy although his views did not begin to be accepted until the isolation of pure cultures. The

most widely accepted classification, strictly based on morphological criteria and summarizing all the

species described by the end ofthe 19th century, was that ofMigula (1897).

Classification of prokaryoted which is data dependent (Goodfellow & O'Donnell, 1993)

changes its shape with each influx of new technology and new data. Technological developments

such as the development and application of chemotaxonomy (Goodfellow & O'Donnell, 1994),

Numerical taxonomy (Sneath & Sokal, 1973), small subunit (SSU) rRNA (SSU rRNA=16S rRNA in

prokaryotes) sequencing (Woese, 1987), DNA: DNA pairing (Grimomt, 1988), and molecular

fingerprinting techniques (Stackebrandt & Goodfellow, 1991; Mougel et ai, 2002) have led to

49


fundamental establishment in systematics. For the past three decades, whole-genome DNA-DNA

hybridization has allowed quantification of the fraction of genome that is not shared across individual

organisms. Early on, systematists determined a criterion of DNA-DNA hybridization that frequently

corresponded to the established, phenotype-based species demarcations. Annealing of 70% or less

genome became a 'gold standard' for demarcating organisms into different species (Johnson 1973;

Wayne et al. 1987).

The integrated application of many techniques (polyphasic taxonomy) and as described by

Wayne et al., (1987), has its profound impact in prokaryotic systematics for the past 20 years

(Vandamme et al., 1996; Priest & Goodfellow, 2000). Use of the 16S rRNA as a universal tool has

revealed fundamental taxonomic relationships from domains (Woese, 1987) to the diversity of

prokaryotes in ecological niches (HugenhoItz et al., 1998a; Ward, 1998). The 16S rRNA sequencing,

in prokaryotes is an essential component of modern polyphasic taxonomy (Stackebrandt et ai, 2002).

Systematics and taxonomy, like the rest of biology are currently in the throes of new

technological revolution. Researches in this direction have all resulted in ~9000 validly described

prokaryotic species (J.P. Euzeby, http://www.bacterio.cict.fr) and 7,06,103 prokaryotic 16SrRNA

sequences in the ribosomal database project, the RDPII data base (Maidak et al., 2001,

http://rdp.cme.msu.edu/misc/html). Whole genome sequencing and developments in high

throughput sequencing technology like pyrosequencing are providing unprecedented amounts of

data. Prokaryotic systematics is undoubtedly facing the imbalance between high throughput

sequencing and the concept of polyphasic taxonomy (Stackebrandt et. al., 2002). Currently,

taxonomy is reliable only at the level of broad phylogenetic groups (well delineated by even partial

16S sequences) and at the species level within certain well studied taxa such as the genus

Mycobacterium (Goodfellow & Magee, 1998). For many genera, identification of species remains a

major problem, as exemplified by the genera Nocardia (Goodfellow et al., 1999) and Rhodococcus

(Goodfellow et al., 1998).

1.2.10.1. Polyphasic taxonomy versus genomic taxonomy:

With the development in chemistry, molecular biology, and computer science prokaryotic

systematics has changed drastically .a relatively large set of techniques are being used routinely for

prokaryote classification. However it is of primary importance to understand at which level these

methods carry information. The kind of information that each technique retrieves is directly related

to its resolving power. Currently prokaryote taxonomists agree that a reliable classification can be

achieved only by applying wide range of techniques and the approach is termed as 'polyphasic

approach'. Polyphasictaxonomy, the term first coined by Colwell (1970), is now considered as a

standard approach for characterization of a prokaryotic taxon. This approach implies that the all

genotypic, phenotypic and phylogenetic information must be simultaneously investigated as

50


extensively as possible genomic parameters are gained from all data that can be retrieved from

nucleic acids either directly through sequencing or indirectly through parameters such as G+C mol%

DNA-DNA similarity, 16S rRNA gene sequence analysis, or DNA-based typing methods such as

restriction fragment length polymorphism, low frequency restriction pattern analysis, randomly

amplified polymorphic DNA, Amplified fragment length polymorphism, rep -PCR , and amplified

ribosomal DNA restriction analysis.

Phenotypic refers to the way in which the genotype is expressed, the visible or otherwise

measurable physical and biochemical characteristics of an organism. Phenotype information is

retrieved by the use of classical phenotype analysis including chemotaxonomic studies incorporated

in polyphasic taxonomy. Phenotypic information is derived from a wide range of morphological

observations of cells and colonies (shape, endospore, flagella, inclusion bodies) and physiological

and biochemical features (temperature growth range, salt concentrations, resistances to antimicrobial

agents, enzyme activities, metabolism of compounds) among others. Chemotaxonomy markers refers

to those chemical constituents of the cells that are useful to characterize prokaryotes, including

components such as cell wall composition, cellular fatty acids, isoprenoide quinines and polyamines.

Techniques such as serotyping, electrophoretic profiles (whole cell protein profile,

lipopolysaccharide profiles, multilocus enzyme electrophoresis), and spectroscopy (Fourier transform

infrared spectroscopy, UV resonance Raman spectroscopy) provide strain- unique patterns that might

be useful for identification and discrimination purposes.

Each of these methods has varying levels of capacity to resolve of different hierarchical levels

in bacterial classification (Vandamme et ai., 1996). The polyphasic approach has been considered a

consensus approach in describing novel taxa and drawing relationships among various taxa in

prokaryote taxonomy (Abraham et ai., 1999; Anderson & Wellington, 2001).

In the last few years due to significant development in sequencing techniques and

bioinformatic analysis in prokaryotic systematics has resulted in sequencing of a large number of

prokaryotic genomes over the last few years. At the time of writing, genomes of 720 bacteria and 53

archaea have been completely sequenced and those of 1253 bacteria and 37 archaea are in different

stages of completion (www.ncbi.nlm,nih.gov/genomesllproks.cgi and www.genomes.org). These

data are increasingly being used to compare the entire genomes from organisms to deduce their

relationships and phylogeny. Few novel approaches for assessing taxonomic relationship based on

whole genomes are as follows (reviewed by Coenye et ai., 2005).

1. Comparison of overall gene content and order.

2. Comparative sequence analysis of conserved macromolecules (Santos & Ochman, 2004;

Zeigler, 2003).

51


3. Genome Blast Distant phylogeny (GBDP) that includes phylogenetic inference on the basis

of whole genome sequence information.

4. Comparison of dinucleotide relative abundance between organism and its relation with 16S

rRNA gene sequence and DNA-DNA-hybridization (Karlin et ai., 1997).

5. Comparison of presence or absence of certain specific molecular features in the genome

(Fitz-Gibbon & House, 1999; Wolf et ai., 1999).

6. Comparison of overall metabolic reactions or pathways (Podani et ai., 2001; Hong et ai.,

2004).

In addition to these methods, DNA microarray and subtractive hybridization based methods are

emerging as techniques to study the overall genome difference of related microorganisms.

Microarray technique for example are now available for identification of specific bacteria (Hamels et

ai., 2001; Volokhov et ai., 2003) especially those which are of clinical significance. Ribosomal RNA

based phylogenetic DNA microarrays (called 'phylochips') that consists of collection of

oligonucleotide probes that detect the target microorganisms at multiple taxonomic levels of

specificity are now increasingly being developed and applied for rapid identification purposes in

diagnostic and environmental microbiology (Guschin et ai., 1997; Wilson et ai., 2002). Multilocus

sequence typing is also becoming a strong tool to differentiate closely related microorganisms

(Maiden et ai., 1998; Helgason et ai., 2004). Although it seems too early to speculate on how the

different new genomic data will be used in the developing genomic taxonomy but at least the road

map towards a genomic taxonomy of prokaryotes is now under construction" (Coenye et ai., 2005).

1.2.10.2. Bacterial species concept:

The ultimate unit of classification is the species. The foundation of species concept was laid

down after the establishment of Linnaean system of classification in the 18th century on the basis of

metazoan diversity. In the beginning of 20th century, the biological species concept (BSC) was

developed by Ernst Mayr, which defines species as "groups of actually or potentially interbreeding

natural populations which are reproductively isolated from other such groups". BSC has been shown

to be successful for animal world, particularly for insects but for animals reproducing

parthenogenetically, algae, plants, fungi and prokaryotes there was difficulty in applying this

concept. Early definitions of bacterial species were often based on monothetic groups described by

subjectively selected sets of phenotypic properties (Goodfellow, 1997) had severe limitations as, for

example, strains which varied in key characters could not be identified as a member of an already

classified taxon. Additionally, these original classifications were produced simultaneously by

different microbiologists that applied different criteria to the classification of the same group of

organisms. The number of species in a genus was influenced by the aims of the taxonomist, the

52


extent to which the taxon had been studied, the criteria adopted to define the species and the ease by

which the strains could be brought into pure culture. Some classifications were defined unevenly, for

example when members of environmentally and medically important genera had been under

classified and those in industrially significant taxa over classified (Goodfellow, 1997). Moreover,

this practice often leads to nomenclatural confusions, where a single species could be simultaneously

classified under several different names (Van Niel, 1952). Until the discovery of DNA as an

information-containing molecule, prokaryote classification was based solely on phenotypic

characteristics. The development of numerical taxonomy (Sneath, 200 1), in which the individuals are

treated as operational taxonomic units that are polythetic (they can be defined only in terms of

statistically co varying characteristics), resulted in a more objective circumscription of prokaryotic

units. The discovery of genetic information gave a new dimension to the species concept for

microorganisms. Parameters like G+C content and overall DNA-DNA similarity have additionally

given insight into phylogenetic relationships. Thus, the species concept for prokaryotes evolved into

a mostly phenetic or polythetic. This means that species are defined by a combination of

independent, covarying characters, each of which may occur also outside the given class thus not

being exclusive of the class (Van Regenmortel, 1997). There is no official definition of a species in

microbiology. However, from a microbiologist's point of view "a microbial species is a concept

represented by a group of strains, that contains freshly isolated strains, maintained in vitro for

varying periods of time, and their variants (strains not identical with their parents in all

characteristics), which have in common a set or pattern of correlating stable properties that separates

the group from other groups of strains" (Gordon, 1978). This definition only applies to prokaryotes

which have been isolated in pure culture (essential for the classification of new prokaryotic species),

and excludes uncultured organisms which constitute the largest proportion of living prokaryotes.

However, a prokaryote species is generally considered to be "a group of strains that show a high

degree of overall similarity and differ considerably from related strain groups with respect to many

independent characteristics", or a collection of strains showing a high degree of overall similarity,

compared to other, related groups of strains" (Colwell, 1995). There are, in the literature, at least

three different species definitions that, to date, tend to disappear due to the unification of criteria: (i)

taxospecies, defined as a group of organisms (strains, isolates) with mutually high phenotypic

similarity that form an independent phenotypic cluster, (ii) genomic species as a group showing high

DNA- DNA hybridization values, and (iii) nomenspecies as a group that bears a binomial name

(Colwell, 1995).

The species concept in prokaryotes is not theory based rather it is more arbitrary,

anthropocentric and is made for practical purposes. There are two main problems in species concept

of prokaryotes. First, it is difficult to compare species unit of prokaryote with its counterpart in

eukaryote and secondly, the use of the devised unit may not be satisfactory to a given scientist who is

53


working in the very same field due to reductionistic, monistic or plurastic use of taxonomy

(RossellO-Mora,2003).

By 1970, the concept of polyphasic taxonomy was developed and was subsequently applied in

description as well as delineation of prokaryotic species. According to this, the genotypic and

phenotypic characters of the strains should be evaluated thoroughly so as come to a conclusion

(Colwell, 1970; Vandamme et ai, 1996). By the end of 1980s following this concept, the species

definition was based on overall comparison of the genomes of the strains. The over all genome

relatedness was measured by DNA-DNA hybridization (DDH) methods and difference in melting

temperature (~Tm) of the heteroduplexes versus homoduplex of the DNA strands. This led to a

definition of bacterial species in 1987 as described earlier "species generally would include strains

with approximately 70 % or greater DNA-DNA relatedness and with 5 °C or less ~Tm" (Wayne et

ai., 1987). The DDH data is very much consistent with recently available complete genome sequence

data. However, DDH is time consuming, cannot be applied for uncultivated bacteria and cannot be

compared with greater ease like a gene sequence in a database (Gevers et ai., 2005). By the early

1990s the availability of 16S rRNA gene sequence data was increasing very rapidly. Around middle

of 1990 a correlation between the 16S rRNA gene sequence similarity and overall genome

relatedness was made. It was observed that, for strains that showed less than 97% 16S rRNA gene

sequence similarity among them, overall genomic relatedness was less than 70% (Stackebrandt &

Goebel, 1994). Although, 16S rRNA gene loses its resolving power beyond 97% sequence similarity

it proved very useful for delineating bacterial taxa or candidate taxa in both culture dependent study

as well as culture independent study. But, such overdependence of organismal evolution and

demarcating point using only one-gene sequences has several drawbacks. Therefore to have better

resolution a combination of different innovative methods like multi locus sequence typing (MLST),

analysis of bacterial cell with FTIR or MALDI-TOF, application of typing methods (AFLP, RAPD,

ARDRA etc) in addition to 16S rRNA gene sequence data and DNA-DNA relatedness data (if

required) has been suggested for taxonomic conclusion (Stackebrandt et ai., 2002). It is worth

mentioning that the delineation at species level (or any hierarchy) should also be supported by

phenotypic, chemotaxonomic and other as many evidence as possible (Stackebrandt et ai., 2002).

1.2.11. Prokaryotic phyla: known so far:

1.2.11.1. Current picture in domain bacteria:

Molecular techniques involved in surveying the uncultured majority in natural environment

have uncovered profound knowledge of microbial diversity over the past decade. The number of

estimated microbial phyla over the past two decades, expanding from 11 in 1987 to 36 in 1998

(Hugenholtz, 2002) to 53 phyla (Keller & Zengler, 2004) proposed at present (Raymond, 2008),

54


1. Phyla with cultured representatives

Carl Woese (1987) published a benchmark paper in microbial biology, the first comprehensive

synthesis of bacterial evolution placed in the context of all life forms. From the 16S rRNA sequences

and catalogs available through 1987, Woese and colleagues were able to delineate 11 major groups

or lineages, which since have variously been referred to at the taxonomic rank of kingdom, phylum,

class, order, and division. The 11 original phyla recognized in 1987 has been divided into 12 phyla as

the Gram-positive bacteria are now recognized as two separate phyla, the Firmicutes (low G+C) and

Actinobacteria (high G+C). The 12 major bacterial divisions identified still represent most of the

taxa that can be readily cultivated and characterized by using cultivation methods, these are

Firmicutes (low G+C) Actinobacteria (high G+C), Proteobacteria (classical Gram-negative bacteria)

which on the basis of cultivation-dependent and -independent approaches, are generally recognized

as one of the most successful microbial groups on the planet which includes two of the most studied

genera of microorganisms, Escherichia and Pseudomonas. Other phyla included in original phyla are

Cyanobacteria, Thermotogae, Chlorojlexi, and Bacteroidetes. Cyanobacteria are oxygenic

photosynthetic bacteria, Thermotogae that are sheathed, obligatory anaerobic, fermentative

heterotrophs. Chlorojlexi (green non-sulfur), as originally defined, included the thermophilic

phototroph Chlorojlexus, the mesophilic, gliding chemoheterotroph Herpetosiphon, and the

hyperthermophilic chemoheterotroph Thermomicrobium. These microbes exhibit divergent metabolic

strategies. Cytophaga, Bacteroidetes, and Flavobacterium form a major lineage, known now as the

Bacteroidetes phylum commonly known as (CFB group; Cytophaga, Flavobacterium and

Bacteroidetes). Five other bacterial phyla were also included namely Chlamydiae, Planctomycetes,

Spirochaetes, Chlorobi and Deinococcus-Thermus. The only phyla of bacteria known to have cell

walls that are not composed of peptidoglycan are Planctomycetes and Chlamydia. They have a

number of other odd features as well, particularly the presence of an intracellular compartment in

some species that contains the cell's DNA. The first members of the phylum Planctomycetes were

isolated in the 1970s as heterotrophs growing in dilute media. With the discovery that the

Planctomycetes responsible for anaerobic ammonia oxidation (the anamox reaction), physiological

diversity within the phylum expanded further. Four genera have been described: Pirellula,

Planctomyces, /sosphaera, and Gemmata under the phylum Planctomycetes. At a relatively early

stage in the application of environmental gene cloning methods, it became apparent that the

Planctomycetes were far more prevalent in the environment than would have been suspected from

their scant presence in culture collections (DeLong et al., 1993; Ehrich et al., 1995). It is now

apparent that they are common in soils and sediments, as well as the fresh, marine and hot spring

environments with which they were originally associated.

Since 1987, 14 bacterial phyla such as Verrucomicrobia, Fusobacteria, Caldithrix,

Gemmatimonadetes, Fibrobacteres, Defferibacteres, Acidobacteria, Nitrospira, Synergistes,

55


Thermodesulfobacteria, Coprothermobacter, Dictyogiomi, Aquificae and Desulfurobacteria

(Hugenholtz, 2002; Hugenholtz et ai., 1998a; Pace, 1997) were also included. Included in this group

are phyla of predominantly thermophilic and chemolithoautotrophic microorganisms. Some members

of the phylum Aquificae can oxidize hydrogen gas as an energy source for chemolithotrophic growth

(Huber, et al., 1992). The Desulfurobacteria grow by sulfur reduction anaerobically (sulfate reducing

bacteria), and Thermodesulfobacterium hydrogenophilum grows by sulfate reduction.

Phylum Acidobacteria is apparently ubiquitous and abundant in nature, as based on results

from cultivation-independent molecular ecology studies, especially in soils (Buckley & Schmidt,

2002; Barns et al., 1999). This phylum includes three species of divergent physiology

(Acidobacterium capsuiatum, Hoiophaga foetida, and 'Geothrix fermentans'), thus making it difficult

to predict many characteristics of the Acidobacteria-related microorganisms detected in

environmental samples (Barns et al., 1999). The phylum Nitrospira is also noteworthy in that it

includes obligately chemolithotrophic, nitrite-oxidizing genus Nitrospira, and the obligately

chemolithotrophic, ferrous iron-oxidizing genus Leptospirillum. Members of this phylum are also

apparently ubiquitous in the natural environment, judging by the large number of Nitrospira rRNA

gene clones that have been reported in the past few years. Under the phyla which are under

represented in culture are Verrucomicrobia, Chioroflexi, and Gemmatimonadetes including

Pianctomycetes.

Since 1995 the phylum Verrucomicrobia has been recognized as a separate phylum of

Bacteria, but currently counts only a small number of cultivated microorganisms as members. This

phylum was known from two genera and five species of prosthecate, aerobic heterotrophs isolated

from freshwater environments which are Verrucomicrobium vinosum, Prosthecobacter fusiformis, P.

debontii, P. vanneervenii, and P. dejongeii. Although an obligately anaerobic, heterotrophic genus

(Opitutus terrae) from paddy field soil has recently been isolated and characterized. In addition to the

named microorganisms, three taxonomically uncharacterized isolates of "ultramicrobacteria" from

paddy field soil with 16S rRNA gene sequences closely related to 0. terrae have also been described.

A rich diversity of microorganisms awaits exploration within this phylum, which includes major

clusters of rRNA gene clones that are ubiquitous in natural freshwater and soil microbial

communities (Buckley & Schmidt, 2002; Zwart et al., 2002).

The phylum Chioroflexi (green non-sulfur bacteria), deep-branching lineage of the domain

Bacteria, was one of the initial group of phyla recognized early in the application of small subunit

rRNA sequencing to taxonomic questions within the Bacteria. Chioroflexi rRNA genes appear

frequently in clone libraries constructed from subsurface oceanic bacterioplankton (Bano &

Hollibaugh, 2002; Giovannoni & Rappe, 2000), freshwater bacterioplankton (Urbach et al., 2001),

soils (Chandler et al., 1998), sediments (Coolen et ai., 1998) and geothermal hot springs (Hugenholtz

et al., 1998a). Thus cultivation-independent investigations of the diversity present in a wide variety

56


of microbial communities have found members of this phylum to be ubiquitous in the natural

environment. The limited number of cultivated members of this phylum is an interesting group of

diverse phenotypes, which include gliding, filamentous isolates that contain some form of

bacteriochlorophyll (Chloroflexus, Oscillochloris, Chloronema, and Heliothrix); filamentous,

mesophilic, strict aerobic chemoheterotrophs (Herpetosiphon) which do show gliding motility; a

hyperthermophilic, irregular rod-shaped, nonmotile aerobic chemoheterotroph (Thermomicrobium

roseum); and an irregular cocci-shaped isolate able to reductively dechlorinate tetrachloroethene

('Dehalococcoides ethanogenes').

Identified first as a candidate phylum in 2001, the phylum Gemmatimonadetes has been

recognized as a main line of descent within the Bacteria. It has since been proposed as a phylum,

only cultivated representative of the genus is Gemmatimonas aurantiaca (Zhang et aI., 2003). It has

become a diverse assemblage of environmental rRNA gene clone sequences. At least four subgroups

can be clearly delineated within this phylum. Zhang and coworkers hypothesized that one identifying

feature of this phylum may be that its members possess a gram-negative cell envelope lacking

diaminopimelic acid in their peptidoglycan similar to one possessed by G. aurantiaca (Zhang et aI.,

2003).

2. Candidate Phyla of Uncultured Microorganisms

Extensive use of 16S rRNA gene cloning and sequencing tools to identify microorganisms in

natural samples has revealed an enormous diversity within bacterial phyla. It is also apparent that

some of the recovered clone sequences did not appear to belong to any of the known bacterial phyla

(Fuhrman et al., 1993; Liesack & Stackebrandt, 1992). It was later discovered that a portion of these

"unaffiliated" environmental gene clone sequences were providing scientists with the first evidence

of such ubiquitous, but not yet recognized, phyla as the Verrucomicrobia and Acidobacteria. Many

artifacts (e.g., chimeric gene clones, peR errors, sequencing errors) and methodological errors (e.g.,

improper reference or outgroup taxon selection, inadequate quantity of sequence information,

improper alignment, use of an inappropriate alignment mask) can cause the misplacement of gene

clone sequences in phylogenetic trees. Unaffiliated clones from different studies frequently clustered

together in further analyses to form monophyletic groups supported the conclusion that many of

these sequences were in fact real and formed major lines of descent within the bacterial domain

which did not contain cultivated relatives. These lineages have since become known as candidate

divisions or phyla, with the term candidate implying that no cultures yet exist to represent the group

(Hugenholtz et aI., 1998a; Hugenholtz et aI., 1998b). Phrase "candidate phylum" has been given for

these deeply diverging clusters of sequences that are phylogenetically equivalent to phyla of cultured

microorganisms as delineated in Bergey's Manual of Systematic Bacteriology.

57


Before 1998, the OS-K group (named after a 16S rRNA gene clone recovered from a

microbial mat of thermal Octopus Spring); Marine Group A (named after gene clones recovered from

Pacific Ocean bacterioplankton samples); and Termite Group 1 (named after gene clones recovered

from the intestine of the termite Reticulitermes speratus), were three groups of sequences composed

solely of environmental gene clones were generally thought to form main lines of descent within the

domain Bacteria. Hugenholtz and coworkers defined a candidate phylum as "an unaffiliated lineage

in multiple analyses of datasets with varying types and number of taxa and having <85% identity to

reported sequences, indicating its potential to represent a new bacterial division [phylum]"

(Hugenholtz et at., 1998b) and "as a lineage consisting of two or more 16S rRNA sequences that are

reproducibly monophyletic and unaffiliated with all other division [phylum] level relatedness groups

that constitute the bacterial domain" (Hugenholtz et aI., 1998a; Dalevi et aI., 2001).

Hugenholtz and coworkers (Hugenholtz et aI., 1998b) described 12 lineages that potentially

represented candidate phyla from a Yellowstone hot spring (designated as OPI-12). Recently, a

novel thermophilic chemoheterotrophic filamentous bacterium was obtained from a hot spring in

Japan that was enriched through various isolation procedures that belongs to the phylogenetic group

termed OP5 (Mori et at., 2008). In that same year, Dojka and coworkers (Dojka et aI., 1998)

described 6 lineages four of them formed candidate phyla from a hydrocarbon- and chlorinated

solvent-contaminated aquifer (WS 2, 3, 5 and 6). Afterwards eight candidate phyla have emerged

from the expanding public databases of 16S rRNA gene sequences these are SC3 and SC4 identified

from arid soil, NC 10, BRCl, identified from bulk soil and rice roots of flooded rice microcosms,

Guaymasl, identified from hydrothermally active marine sediments, NKB 19, identified from deep

sea sediments and activated sludge, and SBRI093, identified from activated sludge. Poribacteria

were detected in 2004 in marine demosponges and, to date, remain unknown from any other

environmental niche (Fieseler et aI., 2004). No pure culture of Poribacteria is available but,

according to specific fluorescence in situ hybridization analysis, they can occur in high numbers in

these sponges. Some uncertainty exists in determining the total number of candidate phyla; however

this number currently at about 26.

Result of cultivation-independent molecular surveys has revealed that the bacterial domain

consists of many more divisions, with few or no cultured representatives. Currently there are 53

phylogenetically well-resolved bacterial divisions are present (Pace, 1997; Hugenholtz et aI., 1998a).

In the domain Bacteria, phyla with no cultivated representatives, demonstrate that the microbial

species in culture collection centres provide only a limited and incomplete picture of microbial

diversity.

58


1.2.11.2. Current picture of domain Archaea:

From 16S rRNA phylogeny two main line of decent (phyla) has been delineated within archaea

"Crenarchaeota" and "Euryarchaeota". Euryarchaeota shows the greatest phenotypic diversity among

known cultivable species comprised of halophiles, methanogens, thermoacidophiles and some

hyperthermophiles. In contrast the phenotypic diversity of cultivable Crenarchaeota is much more

limited with only the hyperthermophiles. The total number of phylum-level lineages in the archaeal

tree is 18, of which 8 (44%) have cultivated representatives and 10 (56%) have none. A higher tally of

23 phyla is arrived at if lineages not meeting the selection criteria are included in the estimate. These

include "Methanopyri" currently represented by a single sequence, and environmental group C3,

which has no full-length representatives. Most archaeal research has concentrated on the cultivated

methanogenic (such as Methanococci) and thermophilic (such as Thermoprotei and Thermococci)

lineages. As is the case with the Bacteria, most candidate Archaeal phyla are completely

uncharacterized at this point. However, some environmental Archaeal 16S rRNA sequences detected

by PCR indicate the presence of new lineages that branch between Crenarchaeota and Euryarchaeota.

Barns and coworkers have suggested a tentative third phylum, the Korarchaeota (Barns et a!., 1996),

which was further shown to be firmly inside the Crenarchaeota (Robertson et al., 2005) but

representative of this lineage have not yet been isolated in pure culture. Sequences that branch even

deeper than Korarchaeota in the archaeal16S rRNA tree were also reported (Takai et al., 2001a, b). A

new phylum Nanoarchaea was proposed by Huber et al. (2002) based on the discovery of a parasitic

archaea of reduced size (Nanoarchaeum aquitans). It possesses the smallest cellular genome known to

date (less than 0.5 Mbp) and lives in parasitic association witha hyperthermophilic Crenarchaeote of

the genus Jgnicoccus and branches deeply in the Crenarchaeote tree. More phyla are very likely have

to be defined in Archaea and this could produce an archaeal classification more similar to the bacterial.

1.2.12. An introduction to Western Ghats:

The Western Ghats, identified as one of the biodiversity hotspots of the world, is a 1600 km long

chain of mountain ranges running parallel to the western coast of the Indian peninsula (Praveen &

Nameer, 2009). It has a land area of3702 Sq. Kms and a coast line of 104 Kms. They extend from the

mount of the river Tapti (21°N) to the South of India (about 8°N), the only gap in the chain being

Palghat Gap (Radhakrishna, 1993). Based on the topography and geology the Western Ghats can be

divided into three major regions (Praveen & Nameer, 2009):

North Western Ghats (Surat to Goa): This region consists of the most homogeneous part of

the Ghats, hugging the coast for almost 600 km. It corresponds to the western edge of the vast plateau

formed by the basaltic outpourings of the Deccan trap. Its elevation is generally between 700 to 1000

m and with some peaks even higher for example Kalusbai (1646 m) and Mahabaleshwar (1438 m).

59


Central Western Ghats (Goa to Nilgiris): The basaltic outpourings cease to the north of Goa.

However, towards the south, the Ghats consists of complex pre-Cambrian rocks. In the central Western

Ghats, the rocks are predominantly of Dharwar system (among the oldest in India) and Peninsular

Gneiss. The elevation generally range between 600 m to 1000 m up to 13° 30' N (except Kodachadri:

1343m). From Kudremuch (1892 m) and up to Wynad, the edge of the plateau is very often higher

than 1000 m and with several peaks ranging between 1713 and 2339 m. Towards 11° 30' N, the

Western Ghats rise abruptly in the Nilgiris horst which is made of Charnokites rock. The Nilgiri

Mountains constitute an elevated plateau attaining a maximum height of2637 m at Dodda Betta.

Southern Western Ghats (South of the Palghat Gap): This region is mostly formed of

Charnokites rocks. The Ghats which are interrupted by a gap (Palghat Gap) of about 30 km wide

reappear abruptly as the Anamalai-Palani block whose high plateau attain a height of 2695m in the

Anaimudi peak, the highest point in South India. They end almost at the southern tip of India, about 20

km before Kanniyakumari. This last part, which is very rugged, culminates at 1869 m in the

Agastyamalai peak.

1.2.12.1. Climate and soil type:

The climate of the Western Ghats shows rainfall gradients and a temperature gradient. The

western slopes of the Ghats are subject to direct influence of rain-bearing winds of the south-west

monsoon. They receive 2000 to 7500 mm of rainfall. These totals decay rapidly to <800 mm towards

the east within a distance of 7 to 60 km. The second, north-south gradient is determined by the time of

arrival and withdrawal of the monsoon. The temperature gradient is mostly related to increase in

altitude. However, it is not uniform throughout the Ghats because of variability in the relief from south

to north. In general, the mean temperature of the coldest month varies from 23°C at sea level to 12°C

at 2300 m. Soil type is laterite and lateritic clayey-loamy soil.

As already stated, Western Ghats is one of the 25 hot spot for the diversity in the world due to its

unique position in the South Asia. Studies on exploration of animal species, plant species, and fungi

have been started. In the Western Ghats, based on the ecological factors and floristic composition, four

major forests and 23 floristic types have been distinguished. These types are closely correlated with the

temperature and rainfall regimes. Wet evergreen, dry evergreen, moist deciduous and dry deciduous

forests are clearly distinguished by the mean annual rainfall, whereas low, medium and high elevation

wet evergreen types are distinguished by the decrease in minimum temperature with increasing

altitude. In addition to forests, high altitude grasslands are another unique ecosystem in the Western

Ghats.

60


1.2.12.2. Floral diversity:

Four thousand species of flowering plants are known from the Western Ghats. The gymnosperm

flora is represented by Cycas circinalis (Cycadales), Decussocarpus wallichianus (Coniferales) and

Gnetum ula and G. contractum (Gnetales). Amongst the lower plants around 150 species of

pteridophytes, 200 species of bryophytes, 200-300 species of algae and 800 species of lichens are

known. There are 600 species of fungi known from the Western Ghats.

Fifty-six genera of flowering plants are considered endemic to the Western Ghats (Nayar, 1996).

The validity of endemism at generic and higher taxonomic levels is however subject to systematic

revisions. Study survey for endemic plants of Western Ghats and West Coast distributed in Goa was

carried out which resulted in the collection of 113 endemic species. Although the exact number

keeps varying with the author and time, what is of interest is that nearly 38% of all species of

flowering plants in the Western Ghats are endemic. Further it is to be noted that 63% of India's

evergreen woody plants are endemic to the Western Ghats. Nearly 650 species of plants in the Western

Ghats are trees. The Nilgiri Mountain is considered as the most important centre of speciation of

flowering plants in the Western Ghats. Among which 82 species are endemic to these hills. These

mountains are also unique in having a mosaic of mountain forests and savannas often referred to as the

'shola-grassland' complex.

1.2.12.3 Faunal diversity

Scientific research on the invertebrates of the Western Ghats has largely been restricted to a few

groups of organisms. As with any other tropical region, the invertebrate diversity of Western Ghats is

best known by the butterflies. Out of 300 species 37 species are endemic. The biodiversity study on

insects of Western Ghats has been done extensively. A total of 15, 260 individuals belonging to 12

order, 59 families and 94 genera were collected in 293 sampling sessions from 39 localities in the

Western Ghats (Subramanian, 2003). There are around 218 species of primary and secondary

freshwater fishes in the Western Ghats. Fifty three percent of all fish species (116 species in 51 genera)

in the Western Ghats are endemic (Talwar & Jhingran, 1991; Jayaram, 1999; Menon, 1999; Daniels,

1991). One hundred and twenty one species of amphibians are known from the Western Ghats

(Daniels, 1991). Of these, 94 species are endemic. The phytodiversity of Western Ghats is explored,

identified and documented by the Southern and Western Circles of BSI located at Coimbatore and

Pune, respectively. This documentation has been published in the form of District and State Floras

such as Flora of Karnataka: Analysis (VoU), Flora of Tamilnadu: Analysis (Vol. 1-3), Flora of

Maharashtra: Monocotyledons (Vol. I), Flora of Goa (two volumes), Flora of Kerala (Grasses), Flora

of Cannanore, Flora of Thiruvananthapuram, Flora of Palaghat, Flora of Nasik and Flora of

Mahabaleshwar. Faunal surveys of Western Ghats are being conducted by ZSI through its Regional

61


Stations in Pune, Chennai and Kozhikode. A document on faunal diversity of Nilgiri Biosphere

Reserve has been published.

Diversity of plants and animals are reasonably documented and recently two reports on the

fungal diversity were published (Natarajan et ai., 2005; Raviraja, 2005; Manoharachary et ai., 2005).

There is almost no report which can throw some light on the prokaryotic world of such a pristine area

in India i. e. The Western Ghats. This work therefore was initiated with the aim to investigate

prokaryotic diversity of a few selected ecological niches ofthis region.

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