Soil Microbial Diversity; Methods Used & Future Consideration

Contents

• Soil
• Soil microbial diversity
• Molecular methods:
 Gene probes
 Southern& Nouthernhybridization
 Microarray
 Phyloarray
 T-RFLP
 Metagenomics
 Sequence analysis
 Comparative genome

• Future consideration


Soil
Weathered end product of the action of climate and living organisms on soil parent material.

Soil microbial diversity
Diverse population of microorganisms inhabiting various horizons of soil.
Studied under following heads:
• Microorganisms in surface soil
• Microorganisms in Subsurface soil
• Microorganisms in Shallow subsurface soil
• Microorganisms in Deep subsurface soil

1. Microorganisms in surface soil

Surface soil is inhabited by Indigenous population of:
• Archaea
• Bacteria including actinomycetes
• Fungi
• Algae
• Protozoa
• Viruses.
In general, as Size of organism increases from bacteria to protozoa, the no. decreases.

(i) Bacteria – are almost always abundant in surface soil.
• Culturable bacteria- 107 to 108 / gm of soil
• Total population exceed 1010 / gm of soil.
• In Unsaturated soil aerobes outnumber anaerobes by 2 or 3 order.
• Anaerobic population increases with soil depth but rarely predominate unless soils are saturated and/ or clogged.

Based on DNA sequencing in combination with statistical approaches, diversity indicated by operational taxonomic units (OTU) where each OTU represents a different bacterial population in community. These provides estimates of diversity range upto 6300 OTU/ gm of soil.

Dominant culturable bacteria-

Mainly belong to the genus Arthrobacter, Streptomyces, Pseudomonas And Bacillus. All of them play role in nutrient cycling and biodegradation.




Examples of Important autotrophic soil bacteria

Organism Characteristics Function
Nitrosomonas Gram -, aerobe Nitrification
NH4+ to NO2-
Nitrobacter Gram -, aerobe NO2- to NO3-
Acidothiobacillus Gram -, aerobe S to SO4 2-
A. Denitrificans Gram -, fac. Anaerobe S to SO42-
A. ferrooxidans Gram -, aerobe Fe2+ to Fe3+

Important heterotrophic soil bacteria

Organism Characteristics Function
Bacillus Gram +, aerobe, spore forming C cycle, antibiotic production
Clostridium Gram +, anaerobe spore forming C cycle, toxin production
Methanotrophs Gram -, aerobe Cometabolize TCE
Ralstonia eutrophus Gram -, aerobe 2,4-D degradation
Via pJP4
Rhizobium Gram -, aerobe N2 fixation
Agrobacterium Gram -, aerobe Plant pathogen


(ii) Actinomycetes -
Characteristics-
• Structure – procaryotic
• Size- 1-2 micrometer in diameter
• Morphology – filamentous length of Cocci
• Gram stain- positive
• Abundance in soil- 106 to 108/ gm
Functions :
• Antibiotic source e.g. streptomycin from Streptomyces
• Produce goesmin, which gives soil, a characteristic earthy odour.
• Degrading complex molecules like chitin, cellulose. hemicellulose
• N2 fixation with non legume Frankia

(iii) Fungi
• Except yeast most are aerobic
• Their no. usually range from 105 to 106/gm of soil

Common genera of fungi found in soil are-

Organism Function
Penicillium & Aspergillus Nutrient cycling
White rot fungus Degrade lignin, DDT, TNT
Fusarium, Pythium, Rhizoctonia Plant pathogen
Coccidiodes immitis Human pathogen
Mycorrhiza Protection,P uptake

(iv) Algae – are typically phototrophic so found in areas where sunlight can penetrate. But actually algae are found at a depth of 1m because some algae can grow heterotrophically as well as photoautotrophically. Algal population are highest in surface 10 cm of soil.

Group of algae Examples
Chlorophyta Chlamydomonas. Found in acidic soil
Chrsophycophyta Navicula. Found in neutral & alkaline soil
Xanthophyta Botrydiopsis
Rhodophyta Porphyridium
Cyanobacteria Blue green algae

Functions:
• Carbon input by photosynthesis
• Weathered mineral particles so play role in soil formation.
• Produce extracellular polysaccharides which causes aggregation of soil particles.
• Cyanobacteria fix nitrogen, a nutrient that is usually limiting in a barren environment.

(v) Protozoa
• Unicellular, eukaryotic. The common genera found in soil include-
Heteromitra cucullus, Oikomonas & cercomonas (flagellates)
Colpoda cucullus & C. sitinii (cilliates)
Naegleria gruberi, Acanthoamoeba spp., Hartmanella hyalina ( amoebae)

Testaceous & rhizopodes restrict to acid soil.
Most of the protozoans are heterotrophic and survive by consuming other microflora

(vi)Viruses
• Bacterial viruses, as well as, plant & animal viruses find their way into soil through addition of wastes.
• Soil microflora may themselves harbor viruses.

2. Microorganisms in Subsurface soil

• In subsurface environment, the same patch like distribution of microbes exists that is found in surface soil.
• Culturable count 0 to 107/ gm of soil.
• Direct count 105 to 107/ gm of soil.
• Thus the difference between culturable and direct count is large in subsurface soil than in surface soils. This is mostly due to-
VBNC- viable but non culturable
VBDC- viable but difficult to culture
• Estimate that 99% soil microbes may be VBNC or VBDC

3. Microorganisms in Shallow subsurface soil

The study of subsurface microflora is new, beginning in 1980s only.
Complicated the study of subsurface life are the facts that sterile sampling is problematic and many subsurface microbes are difficult to culture. Some of the initial studies evaluating subsurface populations were invalidated by contamination with surface microbes. Because subsurface microbiology is still a developing field, so the information is limited. Yet Information is there about zone specifically rapid water discharge have a higher no. of microorganisms. Direct count remain fairly constant, ranging from 105 to 107/ gm of soil throughout the profile of shallow subsurface systems. This is low as compared to surface soil having 109 to 1010 cells/ gm Culturable count 0 to nearly equal to direct count
This is due to-
• Nutrients are limiting in subsurface, a greater proportion of population may be in the non culturable state.
• The Physiological & nutritional requirements of subsurface organisms are not understood

4. Microorganisms in Deep subsurface soil

Earlier it was thought that the deep subsurface environment contain few microbes because of oligotrophic conditions found there. But research has shown that microbes can be found at depth of > 3 km below earth’s surface.
First evidence regarding this was given by Edward bastin, a geologist at the university of Chicago, upon examine water form deep within oil fields
He found significant high levels of H2S and HCO3-. The presence of these materials could not be explained on a chemical basis alone. So suggested occurrence of sulfate reducing bacteria.

Subsequently, Frank Greer, a microbiologist at the University of Chicago, was able to culture sulfate reducing bacteria from water extracted from oil deposit 100s m below earth’s surface.
Types of organism mainly include Aerobe and facultative anaerobe chemoheterotrophs, denitrifiers, methanogens, S reducers & S oxidizers.



MOLECULAR METHODS TO STUDY SOIL MICROFLORA

Extraction of Nucleic acid from environmental samples-

The first step in molecular analysis is the extraction of nucleic acid from environmental samples. The most common approach to extraction of community DNA from soil is to lyse the bacterial cells in siyu (direct lysis)

Community DNA- DNA concurrently extracted from populations within a sample, generating a mixture of DNA referred to as Community DNA.

1. Gene probes and probing

Gene probes consisting of single stranded DNA can be used to identify the presence of a particular nucleic and sequence within an environmental sample. Typically, probes are short sequences of DNA, known as oligonucleotides, that are complementary to the target sequence of interest. These probes are labeled in some way that facilities their detection.

In order to design a gene probes, the DNA sequence of the gene interest must be known. This gene may be unique to a particular microbial species, in which case the gene probe would allow screening of an environment sample for the presence of that MOS.

Functional gene probes-
the target gene may code for production of an enzyme unique to a metabolic pathway. In this case, the gene probe results indicate that the environment sample contain the genetic potential for that particular activity referred to as gene probe.
e.g. gene probe complementary to genes coding for enzymes involved in N2 fixation.

Phylogenetic probe-
Probes designed against specific rRNA sequences known as Phylogenetic probe. These can be specific for groups of bacteria e.g. proteobacteria or its subgroups

Universal probes-
Probes designed to detect an entire domain ( bacteria, archaea or eucarya referred to as universal probes).

Size of probe-
Range from 18 bp to as many as several 100 bp.

Labeling of probes-
1. Earlier labeling option was radioactivity, done by labeling the sequence with a radioactive material such as P32 incorporated into DNA. After hybridization probe is detected by autoradiography.

2. non radioactive alternatives include probes labeled with Digoxigenin (DIG), biotin or fluorescein which can be incorporated by chemical synthesis. Different labels are detected by binding the respective Antibiotic or streptavidin- alkaline phosphate conjugate which when reacted with appropriate substrate will give a signal.

2. Southern and Northern Hybridization

Gene probes can also be used to detect target DNA or RNA on gels following electrophoreses in a process known as Southern hybridization (DNA) or Northern hybridization (RNA).
e.g. to know whether a gene is plasmid or chromosomally borne. This can be done using
Southern hybridization analysis. In this, all plasmids within microbe being studied are extracted and separated by gel electrophoresis. The plasmid DNA is then transferred on to a nylon membrane by blotting and membrane is subsequently probed, only the DNA molecules that contain target sequence hybridize with probe, thus allowing detection of these plasmids containing the target sequence.




Northern blotting is the analogous process used to analyse RNA. In this, total RNA has been extracted from environmental sample, run on gel and transferred to membrane and specific RNA is detected using appropriate probe.



Although extraction and stability of RNA are problematic, this technology can be used in gene expression studies to show induction of a specific gene.
Detection of DNA sequences give information about the presence of a gene in a population, whereas detection of RNA provides information about the expression of gene is given population.

4. Microarrays

A high throughput screening tool that is used to study gene expression. It is basically a collection of oligonucleotides or gene probes that have been “arrayed” on to a glass chip or slide. This microarray or gene array or gene chip is then hybridized with mRNA from a sample to determine which genes are being expressed.

Microarray is of two types:
• Printed or spotted-
 printed cDNA
 oligonucleotide
• Synthesized or in sillico


Printed cDNA arrays-
cDNA probes are made from mRNA transcripts of gene on interest using RT-PCR.

Printed oligonucleotide array
DNA sequences of gene of interest are used to design unique 35 – to 70- nucleotide probes which are then synthesized commercially and deposited on to the micro array slide and subsequently chemically linked to the slide.

In sillico synthesis
Some manufactures synthesize their oligonucleotides directly on the microarray chip called in sillico synthesis.
The upper range for printed microarrays is 1,60,000 probes/ array.
Agilent technologies’ high density microarrays format include 2,40,000 probes/ array Affymetrix microarrays contain over 1 million pobes/ array.

Problems
Only targets with high enough similarity or homology to the probes will bind, this can make specificity an issue.
e.g. when soil community DNA are analyzed, a reduced detection limit mat result, presumably due to target and probe sequences being diverse and consequently not highly homologous with designed probes. Inhibitors, cross reactive cDNAs and RNAses may also result in decreased sensitivity.

5.Phyloarrays

Phylochips allow to follow to follow population dynamics and community profile changes across a wide variety of species on the same array, based on 16s rRNA hybridizations.

Gary Andersen at Lawrence Berkeley national lab. Developed the idea of a phylochip
For the department of homeland securiby with DNA signatures for 9000 known species in phyla of bacteria and archaea.

A variety of less dense phyloarrays have also been developed to target a variety of microbial populations such as Enterococcus spp. Etc.

6. T-RFLP Analysis



Reffered to as terminal restriction fragment length polymorphism. In this method, DNA is extracted from microflora and digested with particular restriction endonuclease enzyme. For a T-RFLP analysis DNA is extracted from a microbial community and then amplified with a primer pair for a specific gene of interest, where one of the primers is flouresently labeled. The amplicon is subsequently digested with one or more restriction enzymes. Fragments are then separated on an automated DNA analyzer. And only fragments containing the fluorescently labeled primers are detected. Primer binding sites are located at the ends (or termini) of the amplicon, and fragments are differentiated based on sequence differences in regions extending from that binding site of the labeled primer, thus the name T-RFLP.
Microbial diversity in a community can be estimated based on the number and peaks heights of the terminal restriction fragment (T-RF) patterns. Which are easily visvualized on electropherograms. A variety of software programs are available for T-RFLP data analysis, facilitating highly sensitive and reproducible results.





7. Metagenomics

Refers to the genetic analysis of an entire microbial community. Term was coined by handelsman et.al. (1998).


This involve the cloning of large fragments of DNA extracted from the environment, allowing the analysis of multiple genes encoded on a continuous pieces of DNA as well as allowing screening of large environment fragments for functional activities.
Specialized vectors are required for the creation o large insert libraries.
1. Bacterial artificial chromosomes (BACs) can handle fragments of 300 kb.
2. Yeast artificial chromosomes (YACs) can incorporate upto 2MB inserts. But their transformation efficiency is 100 times less than bacs.
3. Most recent development in metagenomic sequencing is 454 sequencer.

In this technology, DNA is fractionated into small fragments that are fixed on to small beads. The small fragments then undergo a pyrosequencing step or sequencing by synthesis in which each DNA fragment is amplified by PCR to determine its sequqnce.

Its advantage is that it can be used to sequence large amount of DNA at low cost compared to BAC sequencing.

Its disadvantage that the length of 454 sequences is quite short (300 bp). Thus it becomes difficult to put these short sequences together to provide a picture of a microbial community.

Two main approaches exist for the analysis of metagenomic data-
1. Sequence based
2. Functional metagenomis

Sequence based-
Analysis can be directed e.g. clone libraries can be sequenced after PCR or probe screening for phylogenetic markers such as 16S rRNA genes present.

Functional metagenomics-
Refers to the use of a functional screen to identify clones for subsequent sequencing.

Metagenomic analysis are extremely data intensive. IMG/ M (available at http ://img.jgi.doe.gov/) is a metagenome data management and analysis system that provides tools and viewers for analyzing both metagenomes and isolate genomes individually or in a comparative manner.
The potential of metegenomics is to allow insight into how microbial communities function and also to help unlock the vast genetic potential held with in the diverse population.

8. Sequence analysis

Advance is automated DNA sequencing have made the task of sequencing very routine and economical. DNA samples are generally sent o commercial sequencing lab or core facilities. A variety of sequence database have been compiled to catalog information and make it accessible to entire scientific community.
Several software programs allow researchers to utilize these database to identify sequences, translate a DNA sequence into protein, and look for homology or relatedness based on sequence.

The most common use of databases in studying soil microflora are in identification of isolates by their 16S rRNA gene sequence. The national centre for biotechnology information (NCBI) is an excellent resource for current freeware bioinformatics tools including the basic local alignment sequence tool (BLAST). This program allows researchers to submit a query sequence that is subsequently compared to all other sequences in database and scored for similarity and identity. Other tools such as Clustalw used to perform sequence comparisons where homologous sequences or unique sequence can be identified. Some time this information is used in phylogenetic analyses to identify genetic relationships and ancestory.
e.g. 16S rRNA gene sequence from an unknown oil isolate can be compared with other sequences in databases to identify the organism.


9. Comparative genomics

Advents in recombinant DNA and Sequencing tech. have resulted in the availability of large amount of sequence information. Sequencing is no longer limited to specific gene targets or relatively short DNA fragments, but can be applied to whole genomes. The first organism whose genome was completely sequenced was Haemophilus influenzae in 1995
According to Genome online database (GOLD) as of Jan 2007 >600 genome have been sequenced & 2200 ongoing projects. The availability of vast amount of sequence information, including whole genome sequences, has led to the creation of a new field of genomics reffered to as Comparative Genomics. Data management systems and analysis platforms can be used for comparison of subsets of genes or whole genomes. The Joint Genome Institute (JGI) provides such a platform, Integrated Microbial Genomes (IMG)

Comparative genomics examines both similarity & difference of genomes to-
• Draw function of particular gene
• Identify regulatory regions
• Find evidence of evolution
• Find genetic exchange by providing insights into the mobility of chromosomal sections and lateral gene transfer.
Bacterial and archeal thermophiles share same habitats and there is abundant evidence from genome analysis that lateral gene transfer is common in group
E.g. Thermotoga maritima genome have approx. 20% of genes that have homology to hyperthermophilic archaea Pyrococcus spp. (Nelson et al 1999)


Future consideration of soil microflora-

• Studying soil microflora, it has been reported that some microbes (Geobacter) can generate electricity. So used in production of microbial fuel cells to generate electricity.
• Some microbes can be used for the conversion of toxic compounds into non toxic forms or less toxic ones.
e.g. PAHs (polycyclic aromatic hydrocarbons) like pyrene is converted into less toxic or non toxic compounds by various microbes like Bacillus, Aeromonas and Pseudomonas