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

recombination

Contents:

• Definition
• Examples
-In prokaryotic system
-In eukaryotic system
• Types
• Meselson and Wiegle exp.
• Holliday model
• Potter and Dressler’s evidence
• Meselson and Raddding
• Enzymology
• Conclusion


Recombination
Definition- Recombination is a process or set of processes by which DNA molecules interact with one another to bring about a rearrangement of the genetic information or content in an organism.

Examples of recombination in prokaryotic systems are:• Integration of the bacteriophage lambda prophage,
• Recombination of bacterial DNA following conjugation between bacteria,
• Formation of plasmid multimers.
Examples of recombination in prokaryotic systems are:• Meiosis crossing over- also called gemetic recombination.
• At the time of expression of antibody genes so as to generate great potential diversity of antibody molecules.

Types of recombination- 4 general types of recombination:
Homologous genetic recombinationAlso known as general recombination or general homologous recombination involves an exchange of genetic material between homologous chromosomes, resulting in the arrangement of genes into new combinations.
It is also called general recombination because the enzymatic machinery that mediates the exchange can use essentially any pair of homologous DNA sequences as substrates.
This is the type of recombination that is required during meiotic crossing over, for recombination following bacterial conjugation, and during the formation of plasmid multimers
Site-specific recombination
As the name implies, this type of recombination involves the exchange of genetic material at very specific sites only. Examples include the integration of bacteriophage lambda into the host chromosome to form the prophage and the rearrangement of chromosomal DNA prior to expressing antibody genes.
DNA transpositionServes as mechanism by which particular DNA sequences called transposons may be moved from one location to another on chromosomes independently.
Illegitimate recombinationA very rare form of recombination, occurs between non homologous DNA independently of any unique sequence element.

Molecular mechanism of recombination-
The Meselson - Weigle Experiment
In the simplest sense, recombination is an exchange of both strands between two DNA molecules:

This representation implies that both strands of each molecule must be broken and then rejoined. This was first demonstrated by an experiment performed by Matt Meselson and Jean Weigle in 1961.
Meselson and Weigle infected E. coli cells at the same time with phage from two different stocks of bacteriophage lambda. One stock had been prepared by growing the bacteriophage lambda c-mi- in cells grown in medium containing heavy isotopes of carbon (13C) and nitrogen (15N). The other stock had been prepared by growing bacteriophage lambda c+mi+ in medium containing light isotopes of carbon and nitrogen.


After infection, the progeny phage were isolated and banded on a CsCl gradient.
A broad band of phage particles were found on the gradient.
• Nonrecombinant phage were found, as expected, at two well-defined densities corresponding to the parental light and heavy phages.
• Recombinant phage were found - surprisingly - at all intermediate densities between these two.



They also followed the course of the infection using two genetic markers, c and mi, which were located near one end of the lambda chromosome.

When the phenotypes of the intermediate density phage particles were analyzed, recombinant phage that were c-mi+ were found near the band of "heavy" phage while recombinant phage that were c+mi- were found near the band of "light" non-recombinant phage.
These results can only be explained if recombination between the two parental phage involves breakage and rejoining of both DNA strands (as shown above).

The Holliday Model of Genetic Recombination
This model of recombination was first proposed by Robin Holliday in 1964 and re-established by David Dressler and Huntington Potter in 1976 who demonstrated that the proposed physical intermediates existed.
The basic (simple) model
Align two homologous DNA molecules.

Nick the DNA at the same place on the two molecules.
This must happen in strands with the same polarity.

Exchange strands and ligate.The intermediate that is formed is called a Holliday intermediate or Holliday structure. The shape of this intermediate in vivo is similar to that of the greek letter chi, hence this is also called a chi form.

Visualization of the next step is made easier if one molecule is now rotated through 180š with respect to the other. This also helps to emphasize the chi-shape of the intermediate:


Resolve the structure.There are two ways in which this can happen:
• If the same strands are cleaved a second time then the original two DNA molecules are generated:

• If the other strands are cleaved, then recombinant molecules are generated:


A more realistic model
The above model is too simple and does not explain a number of genetic results, including the occurence of two different recombinant bacteriophage in a single plaque in the Meselson-Weigle experiment.
These can be explained by modifying the model slightly. As before, two homologous DNA molecules must be aligned and nicked at the same place. Following strand exchange the intermediate Holliday structure is formed. At this stage a new step is introduced:
Branch migration.
Migration of the branch can occur over many nucleotides in either direction. The result is a physical transfer of part of one of the strands of one molecule with that of the other:

Once again, visualization of the next step is made easier if one molecule is now rotated through 180š with respect to the other.

Resolve the structure.
There are still two ways in which this can happen, however, the consequences are different:

• If the same strands are cleaved a second time then nonrecombinant DNA molecules are generated but they each contain a region of heteroduplex DNA that spans the region of branch migration. Such heteroduplexes are called Patch recombinants.

• If the other strands are cleaved, then recombinant molecules are generated as before, however, each will also contain a region of heteroduplex DNA that spans the region of branch migration. Such heteroduplexes are called Splice recombinants.


Potter & Dressler's evidence for the Holliday ModelIn 1976, David Dressler and Hunt Potter published the results of a series of experiments that demonstrated the validity of the Holliday model of recombination.
They used E. coli cells containing the colicin E1 derived plasmid, pMB9. This plasmid was one of the very earliest plasmids developed for cloning in Herbert Boyer's laboratory. Normally, E. coli contain about 20 copies of this plasmid per cell. However, if the cells are exposed to chloramphenicol then, although chromosomal replication stops, plasmid replication does not and the number of plasmid molecules increases to 1000 copies per cell. With so many more copies of the plasmid in the cell, the chances of recombination increase as does the probability of observing a recombination intermediate.
When plasmid was isolated from the cells, purified by CsCl gradient centrifugation, and observed in the electron microscope, a number of candidates for intermediates were observed. These all had the appearance of "figure 8" structures. However, there are 3 possible ways such structures might arise:
• as a double-sized circular plasmid twisted over on itself.
• as two interlocking circular plasmid molecules.
• as a genuine recombination intermediate.
In order to distinguish between the three possibilities, Potter and Dressler digested their plasmid preparations with EcoRI. This enzyme will generate monomer sized linear molecules from either of the first two possible structures. However, it will generate unique chi-shaped structures from the third.
When they did this, Potter and Dressler found that between 0.5% and 3% of the molecules they observed were chi-shaped structures. The molecules were symmetrical in that the opposite arms were identical lengths and had identical denaturation patterns. Finally, they saw no such structures if they prepared their plasmids from recA- strains of E. coli.
From this evidence they concluded:
. . . the intermediates we have observed in the electron microscope provide physical evidence in support of the recombination intermediate postulated by Holliday on genetic grounds.


Matt Meselson And Charles Radding Models of Genetic RecombinationAlthough, the basic features of the Holliday model are well-established, the model does have flaws. For example, the mechanism by which two homologous regions of DNA are paired and then nicked is not well explained. In addition, the model does not explain all of the observed results in different recombination systems.
A central feature of the Holliday model as outlined above is that the heteroduplex regions in recombinant molecules will be identical in length and position. Experimental results suggest, however, that this is not necessarily so.



In order to explain such discrepancies, Matt Meselson and Charles Radding proposed a modified model. In their model, only one strand in one of the two paired homologous DNA molecules is nicked. This strand is then displaced by DNA polymerase I and invades the homologous chromosome. A Holliday intermediate is eventually formed and resolved as above. In this model, the extent of heteroduplex DNA in the resolved products, whether recombinant or nonrecombinant, will be different. Finally, gene conversion (which is a type of recombination event) in yeast follows a mechanism, proposed by Szostak, that requires double-strand breaks in one of the recombining molecules.

Enzymology of general recombination-Recombination Proteins in E. coli
The characterization of the structures and biochemical function of a number of the key proteins required for recombination is now helping us to understand details of the mechanism of recombination. The most important proteins are RecA, RecBCD, RuvA, RuvB and RuvC.

RecBCD The recB, recC & recD genes code for the three subunits of the RecBCD enzyme which has five activities: exonuclease V; a helicase activity; an endonuclease activity; an ATPase activity; and, an ssDNA exonuclease activity.
The RecBCD helicase activity can unwind DNA faster than it rewinds. Thus as it travels along a DNA molecule, it can generate ssDNA loops.
The RecBCD complex functions as a DNA exonuclease. It will bind to double-stranded breaks in DNA and degrade both strands simultaneously. However, when RecBCD encounters a Chi sequence, its activity changes. These Chi sites are recombinational Hot Spots. Characterized by 5’-GCTGGTGG-3’. Rec BCD cleaves DNA strand 4-6 nucleotides to 3’ side of Chi. The RecD subunit is released and the RecBC proteins act as a helicase to unwind the DNA in an ATP dependent reaction. This generates a ssDNA region that can serve (along with RecA) to initiate strand exchange and a recombination reaction.
the RecBCD pathway illustrates this:




RecAThe RecA protein is a multifunctional powerhouse! It has strand-exchange, ATPase and co-protease activities all packed into a compact 352 amino-acid, 38 kDa structure. It is required for all recombination pathways in E. coli.
The RecA protein will bind cooperatively to a ssDNA molecule with each monomer of RecA binding to a span of 4-6 nucleotides. Assembly of the nucleoprotein complex proceeds in a 5' -> 3' direction. The complex is both fairly stable (half-life is 30 min) and is the active species that will promote strand exchange.
The RecA protein monomer structure is shown above. Monomers associate with one another via the N-terminal alpha helix which interacts with the adjacent monomer -- forming a molecular conga line, if you will, with one helix on the shoulder of the molecule in front of it! The RecA filament that forms is helical with a pitch of 82.7 Å and it consists of 6 monomer units per turn.
RecA will promote strand exchange between DNA molecules as long as the following conditions apply:
• One of the two molecules must have a ssDNA region to which RecA can bind.
• The two molecules must share a region of homologous (i.e. nearly identical) DNA sequence - a minimum of 50 bp is required.
• There must be a free end within this region of homology which can initiate the strand exchange.
The strand exchange reaction probably involves the following steps:
• RecA binds to the ssDNA partner.
• The two molecules are aligned possible through the formation of a triple-stranded intermediate.
• Displacement of one of the old strands. This requires concurrent migration of the RecA nucleoprotein filament along the molecule - which proceeds in one direction only (5' -> 3') - and consequent winding/unwinding. ATP hydrolysis takes place during this step.
Proteins required for Resolving Holliday Junctions in E. coli

RuvARuvA is a small protein whose function is to recognize a Holliday junction thereby assisting the RuvB helicase to promote branch migration.
The crystal structure of the E. coli RuvA protein was solved at a resolution of 1.9 Å. The protein forms a tetramer in an unusual manner - though one that is ideally suited to its function. To quote from the summary part of the paper:
"Four monomers of RuvA are related by fourfold symmetry in a manner reminiscent of a four-petaled flower. The four DNA duplex arms of a Holliday junction can be modeled in a square planar configuration and docked into grooves on the concave surface of the protein around a central pin that may facilitate strand separation during the migration reaction."
The RuvA protein is 203 amino acids in length but only 190 of them could be assigned in the crystal structure. Most of the missing assignments represent amino acids in a flexible part of the protein.





More recently, the crystal structure of RuvA complexed with DNA has been solved first at 6.0 A and then at 3.1 A.
Finally, examine these three model structures showing how a Holliday intermediate could interact with RuvA:






The RuvA-Holliday junction complexes have the predicted structure with a four-fold symmetry. The DNA bound at the junction is not flat but slightly concave to maximize the contact between the DNA and the protein and, as a result, the axes of the four helices are inclined at about 10 degrees. In the centre of the complex, eight residues corresponding to Glu-55 and Asp-56 from each of the four subunits, form an acidic central pin which may repel the DNA backbone away from the centre of the junction thereby, opening up the centre.
These refined structures have also shown that there are only 4 unpaired bases in the centre of the complex. This is a lot fewer than found for typical helicases.

RuvBThe RuvB protein is a helicase that catalyzes branch migration of Holliday junctions. By itself it cannot bind to DNA efficiently. It functions in combination with RuvA. Like other helicases, RuvB functions as a hexamer; but, unlike other helicases, RuvB encloses double-stranded DNA not ssDNA.
Electron microscopy has shown that RuvB is a heptamer in solution and that it converts to a hexamer ring when it binds to DNA. Electron microscopy has also shown that the two hexamer rings of RuvB lie contacting RuvA on the two opposite sides of a RuvAB-Holliday junction complex.
RuvCThe RuvC protein resolves the Holliday intermediate. It functions as a dimer to cleave two of the four strands that make up the central part of the intermediate. Since binding is symmetrical, RuvC can bind to the Holliday intermediate in two equally likely ways. Hence, Holliday intermediates can be resolved in two different, but equally likely, ways.
RuvC does have some sequence specificity. It cleaves DNA at the 3'-side of thymidine, preferentially at the consensus 5'-A/TTT|C/G -3' where | indicates the site of cleavage
The interaction of RuvC with Holliday junction is shown:


A Model for Resolving Holliday Junctions in E. coliThe crystal structures of RuvA and RuvC, as well as detailed electron microscope visualization of Holliday junction protein complexes allow for a reasonable model to be drawn showing how branch migration occurs and how the junction is resolved:

It i sknown now that RuvC functions with RuvA and RuvB in a higher order complex, termed the RuvABC resolvasome. However, it is also clear that the known structure of RuvC cannot be docked with the detailed RuvA-Holliday complex structure - steric conflicts are found. It is believed that there must be some other structural changes that occur within the resolvasome; but, these have yet to be identified.
The structure of RuvB at 1.9A has been determined recently. The proposed model of the complete resolvasome fits well with the hypothetical models have have been devised based on the structures of RuvA and RuvC. It has also been possible to devise a model to explain DNA translocation through RuvB as a result of ATP hydrolysis.

Conclusion-
• Chromosomal segregation, shuffles together the genetic material carried by different members of a sexual species.
• This genetic mixing unties the evolutionary fate of alles at one locus from the fate of alleles at neighboring loci and can increase the amount of genetic variation found within a population.
• In the process, however, recombination separates advantageous gene combinations, the very gene combinations that enabled the parents to survive and reproduce.
• Whether or not the adaptation of a population to an environment is more rapid in the presence of recombination, that is, whether or not recombination speeds up the evolutionary process, depends critically on the ways in which this process is modelled. As we shall see, the effect of recombination depends on the population size, the initial population composition, and the selection regime under consideration.