molecular methods in rhizosphere microbiology

REVIEW ARTICLE Plant Sciences Feed 2011 - 1 (2) : 45- 64

PLANT SCIENCES FEED ISSN : 2231 - 1971 Jounal Homepage : http://psf.lifescifeed.com

MOLECULAR METHODS IN RHIZOSPHERE MICROBIOLOGY. B. DEB ROY A UT HOR A F F I LIAT ION S

ABSTRACT

Microbial Ecology Laboratory, Department of Life Science, Assam University, Silchar-788011, Assam, India.

New molecular methods that have been used in the study of rhizosphere microorganisms are based on culture-independent assays and sophisticated molecular techniques. This paper discusses a number of the new methodologies

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techniques. The first part of this paper further comprises a thorough treatise of the

developed for detection and localization of specific bacterial populations using modern electron and fluorescence microscopy combined with specific tagging recent development of reporter gene technology, i.e. using specific reporter bacteria to detect microscale distributions of rhizosphere compounds such as nutrients, metals and organic exudates or contaminants. Second half of the paper is devoted to microbial community analysis containing a thorough treatise of nucleotide- and PCR based technologies to study composition and diversity of indigenous bacteria in the natural rhizosphere. The most recent developments of functional gene and gene expression analyses in the rhizosphere based on specific

KEYWORDS Metagenome, Microarray, Microscopy, Reporter, Proteome, Transcriptome

mRNA transcript or transcriptome analysis, proteome analysis and construction of metagenomic libraries are also included. © 2011 LifeSciFeed Ventures

specific

INTRODUCTION

reporter

bacteria

to detect

microscale

distributions of rhizosphere compounds such as

methodologies that have already been or will soon be

nutrients, metals and organic exudates or contaminants.

applicable in rhizosphere microbiology were mainly

Second half of the paper is devoted to microbial

discussed with a emphasis on rhizosphere bacteria and

community analysis containing a thorough treatise of

this paper first describes a number of the new

nucleotide- and PCR based technologies to study

methodologies developed for detection and localization

composition and diversity of indigenous bacteria in the

of specific bacterial populations using modern electron

natural rhizosphere. Also included are the most recent

and fluorescence microscopy combined with specific

developments of functional gene and gene expression

tagging techniques. First half of the paper further

analyses in the rhizosphere based on specific mRNA

comprises

transcript or transcriptome analysis, proteome analysis

a

thorough

treatise

of

the

recent

development of reporter gene technology, i.e. using

and construction of metagenomic libraries.

45

The culture-independent assays and molecular

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. SINGLE-CELL STUDIES IN THE RHIZOSPHERE BY

Sample preparation for conventional SEM involves dehydration, but the so-called Environmental Scanning

MODERN MICROSCOPICAL METHODS

Electron Microscopy (ESEM) operates slightly above the

Direct microscopy

saturation vapor pressure of water in the specimen

The approaches of resin-imbedding and thin-

chamber. Under such conditions, water remains a liquid

sectioning, development of root-growth chambers

and hydrated biological specimen may be observed

(rhizosphere microcosms), and gentle root-washing

without prior preparation. The technique has been used

techniques assuring a minimum of change for microbial

to study mineral composition in the rhizosphere

activity at the root surface per se (rhizoplane), have all

(Cabala and Teper, 2007). Although the technique has

been introduced as alternatives to direct observations

not yet been applied in rhizosphere microbiology, it

in the native rhizosphere environment. Microscopy can

may soon become useful for high resolution studies of

be used in direct visualization using e.g. scanning

microorganisms in undistorted rhizosphere samples.

electron microscopy (SEM) or combined with staining of the indigenous microbial community. In addition, microscopy

can

be

used

to

monitor

specific

Molecular stains Direct detection of single cells in a complex

microorganisms, e.g. growth activity and survival of

environment

as

the

bacterial reporter strains. In this section we will dicuss

development of high-resolution technologies primarily

a broad overview of these three major approaches to

based on unique molecular staining and tagging

study rhizosphere microbiology.

systems

for bacteria

rhizosphere

combined

has

required

with advanced

fluorescence microscopy. The molecular staining

Traditional and environmental SEM

techniques combined with fluorescence microscopy are

The porous matrix of the rhizosphere soil (or bulk

often specific enough to localize complete genera or

soil) may actually be accessible by SEM, provided the

functional groups of the indigenous microorganisms in

preparation includes imbedding in a resin, followed by

rhizosphere samples, as described below.

thin-sectioning Altemuller,

of

the

1990).

specimen et

Fukui

al.,

(Postma

and

(1994)

used

conventional SEM technique to study bacterial colonization of sugar beet seeds, and observed different

for SEM, conventional epi fluorescence

microscopy (EFM) in rhizosphere samples has always been hampered by the sample destruction necessary to

Pseudomonas putida strains, respectively. Similarly,

prepare a thin specimen for staining and observation.

Dandurand et al., (1997) obtained detailed information

Hence, sample mounting in resin and subsequent thin-

on

fluorescens

sectioning to provide structurally intact specimen is

biocontrol strain on young pea root surfaces. This

also important in fluorescence microscopy (Eickhorst

approach gave evidence of a spatial pattern for root

and Tippkotter, 2008). Where mechanical distortion

colonization including cell aggregate formation along

must be avoided or kept at a minimum, an alternative is

the entire root. Finally, Fakhouri et al., (2001) studied

the use of long-distance objectives; such objectives may

the ultra-structure of root colonization, including

in some cases provide adequate depth of the working

Pseudomonas

range for “thick” rhizosphere samples (Thrane et al.,

patterns

distributions

antagonistic

of

for

a

interactions

subtilis

As

and

distribution

Bacillus

General cell stains

Pseudomonas

between

fluorescens and pathogenic Fusarium oxysporum on

2000).

46 46

tomato roots.

Plant Sciences Feed Vol 1 Issue 4

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. Applications of direct epifluorescence microscopy

in the following. Modern fluorescence microscopy has

of soil and rhizosphere specimen include DNA staining

thus provided an excellent insight on spatial and

with acridine orange (AO) in thin-sectioned soil

temporal colonization patterns for a large number of

samples as reported by DeLeo et al., (1997). For direct

specific bacterial inoculants, i.e. from the early binding

EFM of a root specimen, the AO may be useful to stain

of inoculant cells to a seed and to their firm

the total microbial population. One such application

establishment within roots (endophytic bacteria), in the

documented a particularly high density of bacteria near

rhizoplane, or in the rhizosphere including the mucigel

root sections presumed to release specific exudates

polymer matrix or root-adhering soil.

(sucrose or tryptophan) at the root tip or older root segments, respectively (Jaeger et al., 1999). Since the hydrophobic AO compound has a strong adsorption affinity to soil humic material, unspecific binding to the soil matrix may actually be exploited as a counter stain. AO counterstaining for observation of fungal zoospores (Pythium aphanidermatum) on cucumber roots has also been used successfully (Zhou and Paulitz, 1993). An alternative, the UV-excitable DNA stain DAPI may also stain unspecifically in some soils. Where this is a

The first applications of CLSM to study bacterial root colonization were based on strain-specific fluorescent antibody (FA) staining to follow the inoculants (Schloter et al., 1993; Hansen et al., 1997). Co-inoculation

experiments

demonstrated

that

Azospirillum brasilense strains colonizing wheat roots were mutually competitive (Kirchhof et al., 1997), while Pseudomonas fluorescens strains colonizing barley roots were not (Hansen et al., 1997).

problem, a useful DNA stain is the green fluorescent

Fluorescence probing of rhizosphere bacteria with

SYBR Green II (Weinbauer et al., 1998). Finally, a useful

rRNA-targeting oligonucleotides may result in higher

stain is also Fluorescent Brightener (FB) 28 (sometimes

hybridization signals assuming that cellular rRNA

referred to as Calcofluor White M2R), applied for wall

contents correlate with growth activity (Assmus et al.,

staining of fungi in root-soil microcosms (Thrane et al.,

1997). Hence, the rapid development of novel

1999) and imbedded thin-sections of soil. A more recent Fluorescent Brigthener Agent (FBA) 220 may actually improve staining of the soil thin-sections since crystallization problems can be avoided (Harris et al.,

fluorescence in situ hybridization (FISH) probes, staining technologies and CLSM application has resulted in numerous studies of root colonization, as exemplified here. Taxonomic probes for a selected strain or group of

2002). Today these general strains are rarely used

organisms

can

be

applied,

sometimes

even

alone, but rather to gather additional information in

physiological probes for specific cellular activity.

combination with specific cell stains.

Important early observations were the 3-D patterns of active sub-populations of both inoculant and indigenous

Specific cell stains

bacteria on root surfaces. The early works by Assmus et al., (1995, 1997) using combinations of strain-specific

introduced the possibility of 3-D reconstructions to

monoclonal antibody (Azospirillum brasilense Wa3),

obtain high resolution information on the structural and

species specific FISH probe (A. brasilense), group-

spatial composition of microbial communities in

specific FISH probe (α-Proteobacteria) and general FISH

environmental samples. CLSM applications to study

probe (domain Bacteria) on wheat roots first

microbial populations in rhizosphere samples have

demonstrated the potential of combining probes of

become numerous during the last decade or so, as seen

different specificity. More recently, Kutter et al., (2005)

laser


47

scanning microscopy (CLMS)

Confocal

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. used FISH to demonstrate different colonization

nodulation in living alfalfa roots. To specifically monitor

patterns by the pathogens Salmonella enterica and

growth-active Pseudomonas cells, Ramos et al., (2000)

Listeria spp. on barley roots. Watt et al., (2006) used

followed the colonization pattern of an unstable GFP

FISH for quantitative studies of bacteria colonizing

mutant of P. putida on young barley roots.

wheat roots; Pseudomonas and filamentous bacteria were found to comprise 10% and 4%, respectively, of the total rhizosphere community. Other Gram-negative bacteria monitored recently by FISH have been Rhizobium sp. on rape (Santaella et al., 2008) and Methylobacterium suomiense on rice and tomato (Poonguzhali et al., 2008).

Another approach for detection of single cells in the rhizosphere is the use of reporter genes in specific bacteria, also referred to as reporter bacteria, whole cell biosensors, bacterial bioreporters or monitor strains. Such cells are equipped with reporter genes that encode a product, which is easily assayed in fluorescence microscopy and related to metabolic activity or specific

Recent advances to further develop the FISH

gene expression of the host cell. Reporter bacteria

technology have been to target mRNA (rather than

respond to the bioavailable fraction of compounds in

rRNA)

their surroundings.

monitoring

expression

of

toluene

monooxygenase gene (tom) in P. putida in wheat rhizoplane samples (Wu et al., 2008a). At present, the first application of FISH to target cellular DNA monitoring occurrence of nitrite reductase gene (nirK) in indigenous denitrifier populations in environmental samples has been reported (Pratscher et al., 2009). This development is very promising for studying both occurrence (e.g. phylogeny, density, colonization pattern) and metabolic activity (gene expression) of

groups. The first group represents the non-specific reporters, which carry e.g. lux reporter genes under control of a constitutive promoter. Frequently these reporters are tagged with a complete luxCDABE cassette encoding the luciferase as well as genes involved in production of substrate for the enzyme. These bacteria will emit constant light when supplied with oxygen and energy. To track a specific bacterial

rhizosphere bacteria. REPORTER

Reporter bacteria can be classified into three

BACTERIA

AND

REPORTER

GENE

TECHNOLOGY

inoculant in the rhizosphere, the Green Fluorescent Protein

(GFP) technology in

combination with

fluorescence microscopy has had greatest importance for plant microbe interaction studies. The principle of a

the rhizosphere is insertion of fluorescence marker

non-specific reporter strain is illustrated in Figure 1.

genes in specific bacteria under study. Suitable designs

The second group of semi-specific reporters is based on

of plasmid or transposon vectors for insertion, driving

expression of reporter genes in response to stress-full

promoters and a panel of colour variants have allowed

stimuli even when the actual stress factor is unknown.

for bioluminescence

gene) or Green

Finally, the third group of specific reporters may

Fluorescence Protein (e.g. gfp gene) tagging in several

respond to presence of specific compounds or elements

bacteria and microfungi from soil and rhizosphere. The

(e.g. Jaeger et al., 1999) or to their absence (e.g. Koch et

early paper by Gage et al., (1996) described the

al., 2001). Figure 2 illustrates the principle of a specific

construction of Rhizobium meliloti GFP mutants and

reporter. Semi-specific stress reporters may be used

showed detailed CLSM images of their growth and

where bacterial physiology under adverse conditions

behaviour during the early stages of infection and

(e.g. exposure to high temperature or osmolarity,

(e.g. lux


48 48

An important approach to track single bacteria in

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. drought, or presence of reactive oxygen species) is

STUDIES OF THE RHIZOSPHERE ENVIRONMENT

controlled by global regulatory circuits (regulons) of

USING REPORTER ORGANISMS

gene expression.

Carbon,

nitrogen,

phosphorous

and

oxygen

availability Responses to specific nutrient limitation in soil and rhizosphere represent some of the first applications of reporter bacteria to study specific gene expression in this environment. The approach has been dominated by intensive studies of Pseudomonas sp. strains, most certainly due to the importance in degradation and nutrient cycling by this organism per se, but also due to the potential exploitation of the organism in agricultural biotechnology, e.g. for plant protection, plant growth promotion and bioremediation. Figure 1: In a non-specific bacterial reporter, the luxAB genes encoding bacterial luciferase are inserted behind a constitutive promoter located on the chromosome (or a plasmid) of e.g., Pseudomonas fluorescens. Bioluminescence depends on cellular energy, but also on oxygen and aldehyde substrate (not shown). Toxic compounds or elements such as Cu inhibit generation of cellular energy and thereby the light reaction (Sorensen and Nybroe, 2007).

Root exudates have long been considered to be the major C source supporting growth of root-colonizing bacteria in the rhizosphere of young plants. An advancement has been the use of non-specific lux tagged reporters to detect the actual C-source composition and availability in soil and rhizosphere samples through changes in metabolic activity. Hence, inoculant Pseudomonas sp. (shortly pre-starved for C) responded to both source and concentration of C; wheat root exudates gave a response comparable to that of reducing sugar monomer (glucose), rather than that of common amino acid (glutamate) or carboxylic acid (succinate) components in root exudate (Yeomans et al., 1999). The significance of N and P limitation in soil and rhizosphere has been addressed by studies including bioluminescent Pseudomonas reporter strains (Standing et al., 2003). In bulk soil neither N nor P limitation could be observed in agreement with the above observations of C limitation in this habitat. However, soil amendment


with barley straw changed the life conditions for the inoculated

Pseudomonas

strain,

encountering

N

limitation when C-rich polymers from the barley residues were degraded (Jensen and Nybroe, 1999; Koch et al., 2001).

49

Figure 2: In a specific bacterial reporter, the luxAB genes are inserted behind an environmentally induced promoter located on the chromosome (or a plasmid) of e.g., Pseudomonas fluorescens. Expression of bioluminescence, e.g. by Cu induction, occurs at concentrations below the level that inhibits the light reaction (Sorensen and Nybroe, 2007).

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. The N reporter strain used by Jensen and Nybroe

degradation

studies

is

that

concerning

the

(1999) reacted towards limitation by both NH4+ and

polychlorinated biphenyls (PCB). This group of much-

common amino acids (e.g. glutamate), and further work

attended pollutants is degraded by several soil bacteria,

should address if specific, reduced N components in

including typical degrading genera such as Ralstonia,

exudates may regulate Pseudomonas spp. growth in the

Burkholderia and Pseudomonas. The initial degradation

rhizosphere.

step is mediated by a biphenyl dioxygenase, encoded by

The study by Jaeger et al., (1999) illustrates

the bphA gene and Layton et al., (1998) constructed an

another advantage by reporter studies, namely that

R. eutropha ENV307 strain harboring the reporter

precise information on the spatial distribution of C and

plasmid pUTK60 with an insert of orf0regulatory-

N compounds can be obtained. These authors made a

bphA1 (including promoter) linked to promoterless lux.

Erwinia herbicola tryptophan-reporter strain with a

The detection limit was approx. 1μM for both

fusion between the aatl gene encoding a tryptophane

monochlorinated

aminotransferase and a inaZ ice nucleation reporter. In

polychlorinated biphenyls (Arachlor 1,242 mixture),

the rhizosphere of an annual grass (Avena barbata), the

but the linear range appeared narrow (approx. 1–

reporter showed significant induction in older root segments with lateral root formation, but not at the root

(2-CB,

3-CB

and

4CB)

and

10μM). By comparison, Cell-cell interactions in the rhizosphere

tip. Oxygen availability in the soil environment is of

Some of the most fascinating applications of

fundamental importance to expression of several

reporter bacteria have addressed the complicated plant

distinguishing traits in Pseudomonas spp., notably

bacterial, fungal-bacterial or even bacterial cell-cell

denitrification but also a number of redox-regulated

interactions in soil systems, as examplified in the

traits like fluorescent siderophore and HCN production.

following. The first well-known example is that of plant

Pollutant aromatics and their degradation

signals (flavonoid compounds) controlling early stages

containing

of the legume-Rhizobium symbiosis via activation of the

bioluminescence or gfp fusions in the functional

bacteral nod genes; an early Rhizobium reporter based

degradation genes have been presented. A first example

on a nodC-lacZ fusion was constructed by Bolanos

to illustrate the progress being made to detect

Vasquez and Warner (1997) to study activation by six

occurrence and degradation of specific chlorinated

different flavonoids from host plants (bean). Further,

aromatics,

2,4-

the identification of new, specific environmental signals

dichlorophenoxyacetic acid (2,4-D) and the degrading

regulating bacterial growth and activity in the

soil bacterium, Cupriavidus necator JMP134 (formerly R.

rhizosphere has become feasible with reporter

eutropha

techniques that can sort out activated gene promoters

Several

specific

involves

JMP134).

This

reporters

the

strain,

herbicide

harboring

the

degradation plasmid pJP4, has become the model of 2,4-

under in vivo conditions.

D degradation; two modules of tfd genes, Rregulatory-

There has also been research using Pseudomonas

DIICIIEIIFII and Tregulatory-CIDIEIFI, are both involved

reporter bacteria to study gene-regulating signals of

in the degradation steps from ring cleavage of 2,4-

importance for interaction with plant-pathogenic

dichlorocatechol to formation of a product entering the

microfungi, including both Oomycota and “true”

TCA cycle.

microfungi. In a study, Lee and Cooksey (2000) found

assessed with reporter bacteria in soil and rhizosphere

that hyphal colonization of Phytophthora parasitica by a P. putida


50 50

A second example of pollutant compounds being

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. reporter strain led to activated promoters

adopted routinely in microbial ecology in the 1990’s

controlling ABC transporter proteins. The recent report

when the Polymerase Chain Reaction (PCR) and other

of de Werra et al., (2008) demonstrates that GFP-based

DNA-based characterization methods became available.

reporter fusions to the phlA and prnA genes essential

The rapid interest for these methods requiring the DNA

for production of antifungal compounds in P. fluorescens

(or RNA) to be directly extracted from the environment

CHA0 can be constructed to monitor biocontrol gene

was related to their capacity to overcome biases in

expression of this strain in the rhizosphere.

isolation and in vitro cultivation. The new discipline

Finally, a role of bacterial cell-cell communication

opened new possibilities in the search for knowledge

including cell density-dependent gene regulation

within the “black box” of soil microbiology. Despite the

(quorum sensing) in a large number of bacteria has also

introduction of other biases (Wintzingerode et al.,

been indicated in rhizosphere systems. Steidle et al.,

1997), the new DNA- and PCR based approaches have

(2001) constructed AHL-sensitive reporters with GFP in

provided completely new insight into the life of

P. putida and were able to demonstrate that the

microorganisms in their natural environment.

indigenous bacterial community colonizing tomato

Extraction of nucleic acids from complex systems

roots in natural soil produces N-acyl-L-homoserine

Nucleic acid

extraction

from environmental

lactone (AHL) molecules, serving as bacterial cell-cell

samples is the basis for a range of methods used in

communication

molecular

signals;

interestingly,

the

AHL

microbial

ecology.

For

complex

production appeared to be more common among plant-

environments such as bulk soil and rhizosphere two

associated than among soilborne Pseudomonas spp.

approaches have been developed for extracting nucleic

COMMUNITY

STUDIES

IN

RHIZOSPHERE

BY

acids. The first is direct extraction of the nucleic acids

NUCLEOTIDE AND PCR BASED METHODS

after in situ cell lysis which is then followed by DNA

Molecular analysis of microbial communities

purification (Ogram et al., 1987). In the second

Despite improvements in cultivation techniques to

approach the cell fraction is first separated from soil

study the microbial community composition in complex

particles before the cells are lysed and nucleic acids

environmental samples, the organisms in culture

purified (Courtois et al., 2001). Both approaches have

represent only a minor fraction of the microorganisms

advantages and disadvantages related to DNA yields,

occurring in situ as estimated by e.g. DNA re association

DNA purity for molecular purposes, and the ever

(Torsvik et al., 1990). Culturing of microorganisms is

questioned representation of the entire microbial

hampered by difficulties in reproducing natural,

diversity.

ecological niches in the laboratory media. Furthermore,

The direct DNA extraction method

symbiotic relationships might be of crucial importance

The direct DNA extraction method assumes

for community function and are often impossible to

complete in situ lysis of all microorganisms present in

maintain in the cultivation process. Hence, there is a

the sample. The disruption of the microbial cell wall

need

to

must lead to the release of all nucleic acids from all

accompany the cultivation-dependent methods to

bacteria, theoretically independently of the cell wall

obtain a deeper insight into the structure and function

sensitivity to lysis treatments, the location of bacteria in

of indigenous microbial communities.

microstructures and their interactions with soil

for

cultivation-independent

techniques

particles. Purification of the extracted DNA solution is

study the indigenous microbial community were

the second step of the direct DNA extraction methods in


51

Cultivation-independent molecular approaches to

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. which the aim is to get the best compromise between

environmental research. As mentioned previously 16S

purification efficiency and DNA loss. The direct DNA

rRNA has the advantage of being found in many copies,

extraction method generally provides the highest DNA

and has been the easiest gene transcript to detect. The

yields but some applications, such as construction of

ubiquitous presence of RNases and the very short half-

metagenomic libraries, require the extraction of large

life of RNA (especially mRNA) have made studies based

DNA frag-ments that are rarely obtained by direct

on RNA from environmental samples very difficult. For

extraction methods.

these reasons, the bacterial extraction procedure

The bacteria extraction method

described above is normally not applied when mRNA is

An alternative approach to the direct DNA

the target nucleic acid. Alternatively, quick-freezing

extraction method was developed for the recovery of

samples in liquid nitrogen, direct extraction, pre-

highly purified and large bacterial DNAs essential for

treatment of all solutions with the RNase inhibitor

preparing metagenomic DNA to detect complete gene

DEPC and baking all glassware as well as keeping all

clusters and biosynthetic pathways. The bacteria

samples on ice during the extraction procedure, is

extraction method is based on the initial separation of

advantageous.

bacteria from the soil particles prior to cell lysis and

Based on recent methods for extraction RNA,

DNA purification with gentle treatments to preserve

investigations on the diazotrophic community in the

DNA integrity. Protocols have gradually been improved

rhizosphere of different rice cultivars (Knauth et al.,

by optimizing each of the following sequential steps:

2005) and Spartina alterniflora (Brown et al., 2003) by

dispersion of soil particles, separation of the cells from

detecting nifH mRNA, as well as investigations on the

soil particles by centrifugation and/or buoyant density,

denitrifying community detecting the nirK and nirS

lysis

gene transcripts (Sharma et al., 2005), have been

of extracted

Approaches

cells

combining

and DNA purification. centrifugation-based

cell

performed.

Recently, high-resolution studies on

separation from soil particles and in-plug lysis and

transformation dynamics of the functional gene tfdA

pulsed field gel electrophoresis (PFGE) after bacteria

(Nicolaisen et al., 2008; Bælum et al., 2008) in natural

are embedded in the agarose plugs are now recognized

soil have been made. The extraction protocol used co-

as the most efficient method yielding DNA fragments

extracts of both DNA and RNA, which has the advantage

more than 300 kbp in size and providing adequate

that it offers information on both the community

purity for further molecular cloning procedures (Robe

structure in general and on the active subpopulation.

et al., 2003). Alternative methods combining gentle lysis

The protocol also offers the possibility of relating the

treatments with CsCl density purification have

transcript formation over time to the actual population

recovered very pure, 100 kbp long DNA fragments.

density thus obtaining an “activity per DNA unit”.

RNA extraction and purification

PCR BASED ANALYSIS AND COMPARATIVE STUDIES

During the last decade, focus has shifted from merely looking at diversity and community structure

OF DIVERSITY AND FUNCTION Molecular markers The basis of molecular microbial ecology is the

function. One way has been to look at gene expression

molecular markers. Molecular markers can be genes or

during different environmental fluctuations. Studies on

gene transcripts that can be identified in a complex pool

environmental RNA have mainly focused on 16S rRNA,

of nucleic acids providing information on the group of

and only slowly are studies on mRNA emerging in

organisms harbouring these genes. Information based


52 52

towards linking community structure to community

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. on DNA and RNA can answer questions related to the

Another approach is to focus on a functional group

population structure of a specific environment such as:

within the microbial community independent of the

Is a particular gene present in this population? What is

phylogenetic relationship. Functional genes encoding

the phylogenetic composition of this community? Are

enzymes central for specific metabolic processes found

particular genes expressed? How is the community

in the rhizosphere have been investigated in this

composition changed after perturbation of the

environment e.g., amoA genes from ammonia oxidizing

environment? What are the spatial or temporal

bacteria and archaea (Chen et al., 2008), nir genes from

differences in a particular habitat? Different levels of

denitrifying bacteria and nif genes from nitrogen fixing

phylogenetic resolution or different functional groups

bacteria (Babic et al., 2008).

of organisms can be detected based on the choice of a

In recent years, RNA has thus been more often

marker. Information based on the small subunit (SSU)

targeted for information on the active fraction of the

rRNA (16S rRNA of prokayotes or 18S rRNA of

population, as transcript formation is believed to follow

eukaryotes) reveals the phylogenetic relationship

metabolic activity. The SSU rRNA has the advantage of

between the organisms from where the DNA or RNA

being present in large amounts in the cells, and thereby

arose (Woese et al., 1990). Over the last decade,

being the easiest of the RNA species to detect in

sequencing of SSU rRNA from uncultured organisms

complex samples. However, microorganisms living in

has led to the development of databases (e.g. Genbank:

the soil environment are often starved, and starved cells

http://www.ncbi.nlm.nih.gov/Genbank/index.html or

have been shown to maintain their ribosomes longer.

Ribosomal Database Project: http://rdp.cme.msu.edu/)

Hence, using 16S rRNA to describe the active

and it has been verified that only a small fraction of the

populations in a soil/rhizosphere environment might

soil microorganism diversity is known. Furthermore,

be questioned. An alternative approach is to study

new genera have been proposed solely on the basis of

mRNA of functional genes, which is believed to have a

SSU rRNA sequences form environmental samples, thus

much shorter half-live than 16S rRNA.

having no representatives in culture (Fieseler et al.,

Analysing specific subpopulations based on the pool of nucleic acids directly is often not possible as

2004).

most detection systems are not sensitive enough to

marker of bacteria is the housekeeping gene rpoB,

detect the often very low amount of a specific gene in

coding for the RNA polymerase beta-subunit. This gene

the sample. By PCR, fragments of specific marker genes

is only found in one copy in all bacteria investigated,

are amplified to reach a concentration that can be

giving some advantages over the often multicopy SSU

detected by several downstream applications discussed

rRNA gene when using genetic fingerprinting for

below. The application can be divided into qualitative

diversity indexes. In addition, the rpoB gene has thus

(describing diversity overall or active), or quantitative

been

phylogenetic

(describing abundance of gene copies or gene

information for diversity studies (Case et al., 2007). The

transcripts) in the environment. To date the PCR assay

rpoB approach has been used to study Paenibacillus

has, despite its limitations, enhanced our knowledge on

spp. in the rhizosphere of sorghum under different

the soil and rhizosphere community dramatically, and

nitrogen regimes (Coelho et al., 2007); however, the

will probably do so for many years to come. For studies

advantage of 16S rRNA for phylogenetic analysis, still

on RNA, a conversion of RNA into complementary DNA

lies in the huge ribosomal database available.

(cDNA) is necessary prior to PCR amplification. This is

shown

to

contain

enough


53

An alternative to 16S rRNA as a phylogenetic

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. done in the reverse transcription (RT) assay and the

and are selected for further discussion here. The three

joint protocol for reverse transcription and subsequent

techniques are based on three different separation

PCR amplification is referred to as RT-PCR. The reverse

technologies.

transcription can be performed with specific primers

DGGE is based on different melting behaviour of

targeting a specific transcript or it can be performed

double-stranded DNA due to sequential differences in a

using random primers converting all RNA to cDNA. The

denaturing gradient during electrophoresis (Muyzer et

high sensitivity and the ability to analyse a higher

al., 1993). T-RFLP separates fragments based on length

number of samples in a shorter time makes the RT-PCR

of the terminal fragments obtained due to differences in

based detection of RNA from environmental samples

restriction endonuclease sites (as in RFLP and ARDRA)

the most promising, despite the biases introduced.

(Liu et al., 1997). Finally, SSCP separates fragments

Clone libraries and fingerprinting

based on different mobility of single stranded DNA in

Sequencing of clone libraries based on PCR-

non denaturing gels (Schwieger and Tebbe, 1998). It

amplified genes obtained from environmental samples

has been shown that the results obtained based on the

offers the highest phylogenetic resolution and has led to

three methods reveal the same clustering of the

the recognition of the impressive diversity of

microbial members when used on the same soil sample,

prokaryotes. Most obtained sequences are deposited in

and the three fingerprinting methods seem equally

databases like the Ribosomal Database Project (RDP)

suited for analysing differences in community patterns

and GenBank, and can be used for comparative studies

due to physico-chemical and biological differences

on prokaryotic diversity in the environment and to infer

between the sites of investigation (Smalla et al., 2007).

phylogenetic relationships between the organisms

Advantages of the DGGE and SSCP approaches are the

detected. Cloning followed by sequencing of specific

possibility of isolating specific genetic elements for

conserved genes is of great importance in research on

subsequent sequencing, whereas this is not possible

indigenous populations.

when T-RFLP is used, omitting the opportunity to fully

Fingerprinting techniques based on PCR amplicons

identify peaks of interest. On the other hand T-RFLP is

have a high potential for screening multiple samples for

suitable for high-throughput analyses, despite the need

differences in the genetic diversity of a group of genes.

for a restriction digest step. All three methods have

Fingerprinting techniques reveal information on the

been successfully applied in rhizosphere research.

genetic diversity of the sample, but only rarely indicate

DGGE is the most extensively used fingerprinting

phylogenetic relationship of the detectedfragments.

method to study structural diversity in the rhizosphere

Applying

combining

and has been used to investigate a diverse range of

fingerprinting with sequencing (with or without a

scientific questions related to rhizosphere microbiology

cloning step in between) of selected fragments can

e.g. dynamics of methanogenic archaeal communities in

improve the data analysis and add information on

Japanese paddy soils (Watanabe et al., 2007),

community structure.

community structure of Pseudomonas spp. in relation to

a

polyphasic

approach

the antagonistic potential in the rhizosphere (Costa et

terminal restriction fragment length polymorphism

al., 2007) and effects of elevated CO2 concentrations on

(TRFLP) and single-strand conformation polymorphism

the structural diversity of microorganisms in a

(SSCP) of DNA and RNA are currently the fingerprinting

grassland (Drissner et al., 2007).

techniques most often applied in rhizosphere studies,


54 54

Denaturing gradient gel electrophoresis (DGGE),

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. Quantitative PCR

DNA- and RNA-based Stable Isotope Probing (SIP)

Most studies based on PCR are still qualitative, and

Among other applications related to the direct

only recently has quantitative PCR (and RT-PCR) based

recovery of nucleic acids from the environment are

methods been a routine in molecular microbial ecology.

those which can provide a direct access to relate

Traditional PCR rely on end-point detection of the

function of a microbial community and identification of

amplicons. Due to chemical and physical properties

the bacteria that account for it. The establishment of

included in the PCR reaction, the end-point detection of

such a relationship still remaining a great challenge in

the product is not quantitative. However, several

microbial ecology. Stable-isotope probing (SIP) was

quantitative PCR methods have been introduced and

introduced to microbial ecology by Radajewski et al.,

used in rhizosphere research including MPN-PCR

(2000) and has been used to characterize growing

(Rosado et al., 1996), competitive PCR (Mauchline et al., 2002), and most recently real-time PCR (Mavrodi et al., 2007). Real-time PCR is presented here as the most promising

method

to

quantify

genes

from

environmental samples. By real-time PCR, amplicon formation is monitored in real time using fluorescence techniques, where fluorescence detected is proportional to amplicon formation. This enables the detection of product over the full amplification curve, and inhibition of the reaction e.g., by inhibitory substances co-extracted with the nucleic acids, can easily be identified. Another major

microorganisms in environmental samples or to determine those which have the genetic potential of metabolizing a labeled substrate. For instance, the DNAstable isotope probing (DNA-SIP) technique that combines isotopic 13C tracer incorporation into the DNA or RNA and molecular approaches as described above can help to identify soil and rhizosphere bacterial populations that are actively involved in the carbon cycle. The principle of these techniques is to provide soil bacteria with

13C-labelled

material, e.g. cellulose

produced by Acetobacter xylinus as reported by El Zahar

advantage of real-time PCR is the applicability over a

Haichar et al., (2007) and photosynthates released by

wide range of initial DNA concentration of target gene

plants grown under artificial atmospheres (Ostle et al.,

extracted from the environment (Heid et al., 1996).

2003) before extracting total DNA from the soil and

Several detection formats have been developed for the

separating the 13C -labelled (heavy) and unlabelled

real-time PCR assay, SYBR green detection and TaqMan

(light) DNA fractions by ultracentrifugation.

probes being the most commonly used in molecular

METAGENOMIC DNA LIBRARIES

microbial ecology. SYBR green detection is not

Modern highthroughput technology has made

sequence specific as SYBR green binds all dsDNA in the

it possible to not only look for specific genes but look at

minor groove. Upon binding to dsDNA, fluorescence

the full genomic information present in a soil sample,

enhances about 100-fold in comparison with unbound

called the metagenome (Rondon et al., 2000). With such

SYBR green, and it is therefore well suited for detection

bacteria extraction methods clones containing soil DNA

of product formation. Being a non-specific dye, accurate

inserts ranging in size from 40 kb to 50 kb can be

quantification of nucleic acids is dependent on high

routinely obtained. Even without robot facilities

specificity of the PCR reaction per se. The TaqMan

metagenomic libraries of more than 150,000 clones can

probe format relies on the specific binding of a

be prepared and handled for molecular, biological or

hybridization probe to the target sequence and the

chemical

cleavage of the probe by the endonuclease activity of

equivalent of 1,500 E. coli genomes such libraries are


Although

representing

an

only a partial representation of the initial genetic

55

the Taq DNA polymerase.

screenings.

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. diversity of the bulk or rhizospheric soil samples tested when at least two millions of such clones would be necessary to statistically consider that all the initial genomes have been included at least once in the library. Several screening strategies of metagenomic DNA libraries were developed with direct recombinant expression as a first alternative to detect metabolic activities (Figure 3). The usual option consists in using the high transformation efficiency of E. coli for creating metagenomic libraries although this is certainly not the best heterologous host to express genes from soil bacteria (and even if expression efficiency can be artificially increased). For instance high throughput anti-infective assays can be run by spotting the E. coli recombinant clones on bacteria- or fungi-seeded agar plates subsequently analyzed to detect growth inhibition halos around the positive clones while other

Figure 3: Schematic representation of the metagenomic approach. A sample collected in the environment including soil (1) is submitted to treatments to extract and purify bacterial DNA (2). Several possibilities exist to deal with the extracted metagenomic DNA (3). One of the most common approaches is to use PCR amplification (4) as a fist step before products are

bioassays are achieved by preparing an extract from

cloned and sequences. Bacterial diversity can thus be

each E. coli recombinant clone to analyze its potential

estimated with the help of phylogenetic trees constructed

activity versus a set of chemical reference compounds

from 16S rDNA or other gene sequences (5). Another

and of negative controls.

approach is based on the use of fingerprinting techniques such

A second alternative relies on a molecular screening of the library requiring the spotting of colonies on high density membranes or chips before hybridizing membranes with conserved domains of targeted genes, the main difficulty consisting in

as T-RFLP, DGGE and SSCP that produce fragments to characterize bacterial diversity in the analyzed sample (6). A second approach involves the direct cloning of extracted DNA (7) in a domesticated host such as Escherichia coli to produce metagenomic DNA libraries (8). These libraries can be screened according to 3 methods, including a molecular

designing the probe(s) encompassing the unknown

screening with clone DNA transferred to membranes for

genetic diversity (Demaneche et al., 2009).

hybridization (9), or a chemical screening by analyzing the

Advances like the 454 pyrosequencing technology

culture supernatant of metagenomic clones to detect

has opened new perspectives in sequencing the

compounds specifically produced by insert DNA (10). A last

metagenome of environmental DNA (Demaneche et al.,

method is to spread the clones on a selective medium to detect

2009). By pyrosequencing, adapters are added to each DNA fragment obtained from the environmental sample, and subsequently these fragments are added to a bead—one fragment per bead. This technology has

those for which insert gene expression permits clone growth (11). Screening by these two last methods is based on expression of insert genes and transfer of recombinant plasmids, fosmids or BACs in alternative bacterial hosts can increase the recovery of genes of interest. The last approach which is called to become very popular in the next future is to

sequencing, but has the disadvantage that it currently

sequence directly the metagenomic DNA with the help of new

only read 100 bases per run.

sequencing technologies such as 454.


56 56

dropped the time and costs constraints of DNA

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. DNA microarrays and whole-community studies of

hybridization signals with rhizosphere amplified DNA

diversity and function

compared to the bulk soil DNA, suggesting that this

The use of extraction-purification kits that permit

group should be considered a rhizospheric group, while

several soil samples to be processed simultaneously has

Acidobacteria, Bacteroidetes, Verrucomicrobia, and

contributed

DNA-based

Planctomycetes related probes indicated that these

approaches to study bacteria in their natural

bacteria should not be considered as rhizospheric.

environment. However, the huge biodiversity and

Microarray technologies were also developed to detect

functional capabilities of bacteria in these samples

bacterial gene families that encode key enzymes

cannot be monitored without the corresponding high-

involved in the ecosystem functioning (Wagner et al.,

throughput technologies requested to analyze DNA

2007).

solutions.

TRANSCRIPTOME ANALYSIS

to the success of the

DNA

microarrays

can

fulfil

these

requirements by hybridizing in a single step soil

In addition to study the composition and genetic

amplified and labeled DNA targets to thousands of DNA

capabilities of the bacterial community in their natural

probes targeting genes of interest immobilized on solid

environment, DNA microarrays can also be used to

surfaces, the hybridization signal of each probe being

determe the transcript level of all the genes in a given

subsequently and simultaneously recorded with a

gene pool. For this purpose the total transcript of a

detector (Wagner et al., 2007; Huyghe et al., 2008).

sample is obtained by total mRNA extraction. This been

mRNA is then converted into DNA (cDNA) by reverse

developed depending on the target genes, the so-called

transcription, labelled (usually with a fluorescent dye)

Phylo-Chips (or phylogenetic oligonucleotide arrays)

and hybridized to microarrays containing the DNA

referring to the detection of the ubiquitous ribosomal

genes whose expression is to be analyzed. The level of

genes to detect and identify theoretically any

the signal is proportional to the level of transcript

Two

main

microarray

systems have

There are two types of commercially available

and the functional gene microarrays for detection and

microarrays. One type contains the open reading frames

analysis of specific protein-encoding (functional) genes

(ORF) of the relevant genome, synthesized by PCR and

(Wagner et al., 2007). Targeting 16S or 23S rRNA genes,

spotted on a solid microarray (or macroarray) matrix.

Phylo-Chips have begun to be used for the detection and

The other type is the oligonucleotide microarrays, in

identification of microbial strains, species, genera or

which the probes are short sequences designed to

higher taxa (depending on the design of the probe)

match parts of the sequence of known or predicted

including the rhizosphere environment. For instance,

ORFs. These microarrays are produced by printing

the high-throughput analysis potential of Phylo-Chips

short oligonucleotide sequences designed to represent

detected a significant rhizosphere effect when a 170

a single gene or family of gene splice-variants by

probe microarray was used to compare the maize

synthesizing this sequence directly onto the array

rhizosphere and the bulk soil (Sanguin et al., 2006). This

surface instead of depositing intact sequences. The size

study showed that taxonomic groups such as

of the sequences varies, according to the producer (i.e.,

Sphingomonas spp., Rhizobiaceae, and Actinobacteria

60-mer probes such as the Agilent design or 25-mer

were identified in both rhizosphere and bulk soil with

probes such as produced by Affymetrix). Although the

strong hybridization signals, indicating no specific

longer probes are more specific to individual target

habitat preference for these groups. In contrast,

genes, shorter probes may be spotted in higher density

Agrobacterium spp. targeting probes yielded stronger

across the array and the cost of their production is


57

microorganism in the soil sample (Desantis et al., 2007);

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. lower. Microarrays based on oligonucleotides enable

and one with a fluorescence emission wavelength of

the quantization of transcripts from non-coding regions

670 nm (corresponding to the red part of the light

of the DNA. This is important for the analysis of small

spectrum). The two differentially labelled cDNA

RNAs (Gottesman et al., 2006) and regulatory regions of

samples are mixed and hybridized to a single

the transcripts. For specific purposes, or when

microarray that is then scanned in a microarray scanner

commercial microarrays are not available, it is possible

to visualize fluorescence of the two fluorophores

to produce microarrays “in house”. In these microarrays

(Figure 4). These microarrays provide data on the

(spotted microarrays) the probes are oligonucleotides,

transcription level of the whole genome under two

cDNA or small fragments of PCR products that

conditions, as well as a comparison of the two.

correspond to the expected transcripts. These arrays

PROTEOME ANALYSIS

may be easily customized for each experiment, and are especially

useful

for

expression

profiling

of

environmental samples.

Gene expression can also be analysed at the level of translation—the final level of gene expression. Most of the experiments to analyze the composition of proteomes use

high

resolution

two-dimensional

polyacrylamide gel electrophoresis (2D gels) coupled to identification of proteins by mass spectrometry. Twodimensional gel electrophoresis makes it possible to resolve complex mixtures of cellular proteins. In this method proteins are separated by their isoelectric point and molecular mass. Proteins are extracted from microorganisms, tissues or other samples and are separated by their isoelectric point, on a gradient of pH (1st dimension). They

are then

separated by

electrophoresis on SDS-polyacrylamide (SDS-PAGE) according to their molecular mass (Neidhardt and van Bogelen, 2000). The result of this procedure is a gel with proteins spread out on its surface. These proteins are then detected by staining (usually silver or Coomassie stains). The staining provides data on Figure 4: Two-color microarray approach comparing transcriptomes in cultures grown at 32°C and 42°C, respectively.

approximate protein amounts, which is adequate for most purposes. Two-dimensional gel electrophoresis has become a very powerful tool to resolve proteins, which are the final stage of gene expression (Volker and Hecker,

prepared from two samples to be compared (e.g.,

2005). It should be noted that there exist alternative

bacteria at two temperatures) that are labeled with two

technologies, such as multi-dimensional-HPLC coupled

different fluorophores (Shalon, 1998), often one which

mass spectrometry and protein-chip techniques, for

has a fluorescence emission wavelength of 570 nm

proteomic

(corresponding to the green part of the light spectrum),

analysis

without

the

use

of

gel

electrophoresis. However, these methods have not yet


58 58

Two-Color microarrays are hybridized with cDNA

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. been extensively used in the study of gene expression in

data indicate that post-translational modifications

environmental samples (Wolff et al., 2007). Comparison

appear to be more common than expected. As an

of two samples, and analysis of proteome expression

example, when a theoretical proteome was compared to

induced by environmental and physiological changes, is

the actual proteome of A. tumefaciens, there were many

performed by overlaying two gels and quantifying the

proteins that deviated from the expected (theoretical)

difference between them. The analysis can be

molecular weight (MW) or pI as measured by their

performed by one of several image analysis programs.

vertical and horizontal migration distances, respectively

More accurate results are obtained by running the two

(Rosen et al., 2004). These proteins were clearly

samples on one gel. This sort of experiment is often

subjected to post-translational modifications, which

used to determine the effect of an environmental

changed their pI and/or molecular weight. Proteomic

change on the composition of the proteome. For this

studies can be carried out and provide solid data even

type of experiment, the culture is metabolically labelled

on organisms whose genomes are not yet sequenced.

(with L-[35S]methionine or [14C]amino acid mixture)

Future challenges for proteomics thus include the

when exposed to the stress. The protein synthesis

development of efficient technologies for the study of

pattern is obtained by autoradiography can be directly

membrane proteins and the improvement of automatic

compared with the protein level pattern. Because the

software based analysis to overcome problems with

total proteins and the newly synthesized proteins (i.e.,

incompletely separated (overlapping) spots and weak

stress-induced proteins) are on the same gel there is no

spots.

need for matching several gels and proteins that belong to different stimulons or regulons can be identified (Gottesman et al., 2006). The radioactive proteome and the total proteome (obtained by staining) can be differentially stained in the computer, and the relative gene expression is obtained by analysis of the colors. Figure 5 shows the vegetative (computer stained in green color) and heat shock (computer stained in red color) proteomes of Agrobacterium tumefaciens, as compared to the control in which the label was added to unstressed cultures. Using this method it is possible to define the protein signatures of microorganisms in response to a variety of environmental conditions and to identify environmentally induced regulons. Proteomic analysis provides information about the final levels of cellular proteins, thus measuring the level of the end product of gene expression. In addition, it provides information

Figure 5: Proteomic approach analyzing a temperature upshift. A bacterial culture growing at 32°C was incubated 20 min in the presence of 35S methionine at 32°C (left side) or 42°C (right side). The proteomes were analyzed by two dimensional electrophoresis. Each gel was autoradiographed and the image was computer-stained in red. The gel was then silverstained and the image was computer-stained in green. The overlays of radioactive and silver-stained images are shown.

Another

newly

developed

approach

is

“proteogenomics” - which combines metagenomics

previously assumed that post-translational modification

data with mass spectrometry based proteomics. It

plays only a minor role in prokaryotes. However, recent

uses proteomics approaches to verify coding regions


59

about posttranslational modifications. It has been

B. Deb Ro. (2011) Molecular Methods In Rhizosphere Microbiology…. of metagenomic sequences and quantify their activity.

REFERENCES

This technology is based on the ability to directly

[1]

measure expressed

peptides arising proteins

by

from

proteolysis of

high-throughput

liquid

chromatography tandem mass spectrometry-based proteomics. This new technology has not yet been extensively used in rhizosphere studies, but is expected to provide powerful tools in this area of research (Wilmes et al., 2008a, 2008b). CONCLUSION The new discipline of molecular microbial ecology was often seen as the “panacea”, without considering that other limitations than those related to cultivation, could strongly bias results on the actual extent of bacterial diversity and functions. However, when researchers are aware of these biases and limitations, molecular microbial ecology still provides new insight not previously obtained by the cultivation dependent approaches. In addition, better cultivation dependent approaches are developing fast, and today research moves towards a polyphasic methods approach including both lines of research in order to gain most knowledge with the least bias. Furthermore, in reaching the goal of understanding the complex microbial communities in the rhizosphere, the systematic identification and quantitation of all metabolites in the rhizosphere, termed metabolomics, can be of major importance as an additional approach to include in the studies. The present availability of a large number of whole genome sequences constitutes a leap forward in the understanding of microbial communities in the rhizosphere, as well as well as the genetic composition of their individual constituents. The

availability

of

sequences

together

with

bioinformatics tools will enable us to translate the sequences into functions.


60 60

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