Genome Dynamics: Part 1

Paper No. : 16: Molecular Genetics Module : 9a: Structural organization of genome: Genome dynamics: Part 1

Development Team

Principal Investigator: Prof. Neeta Sehgal Head, Department of Zoology, University of Delhi

Co-Principal Investigator: Prof. D.K. Singh Department of Zoology, University of Delhi

Paper Coordinator: Prof. Namita Agrawal Department of Zoology, University of Delhi

Content Writer: Dr. Neelam Gandhi, Hansraj College, University of Delhi

Content Reviewer: Dr. Surajit Sarkar, Department of Genetics South Campus, Delhi University

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

Description of Module

Subject Name ZOOLOGY

Paper Name Molecular Genetics; Zool 016

Module Name/Title Structural organization of genome

Module Id 9a: Genome Dynamics: Part 1

Keywords Horizontal gene transfer; conjugation: transformation; transduction; integrons; cassettes

Contents

1. Learning Outcomes 2. Introduction 3. Genome Changes in Bacteria 3.1. Causative Agents 3.2. Biological Consequences 4. Applications 4.1. Formulation of Strategies 4.2. Bioremediation 4.3. Genetic Research 5. Summary

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

1. Learning Outcomes

After studying this module, you shall be able to understand

 that genomes are not fixed entities but continue to change exhibiting new properties  the concept and mechanism of horizontal gene transfer in prokaryotes  that DNA can be transferred to members not only of the same species but also to other species, genera, or even across different domains  the evolutionary role played by horizontal gene transfer in prokaryotes  that evolutionary progress is greatly speeded by installing new genes from outside which get domesticated in the genome of the recipient  that characters conferred by these processes can have their long term implications.  the possible applications in the field of public health care, animal husbandry, agriculture and bioremediation as well as use of vectors for gene therapy and research 2. Introduction

Bacteria have been around for four billion years despite extensive changes in the environment, including introduction of a large number of antibiotics. Several bacteria adapt to multiple environments and can occupy very diverse ecological niches, e.g., Vibrio cholerae residing normally in aquatic ecosystems, can cause severe diarrhea in human and Bacillus anthracis, found in soil as dormant spores, is the causative agent of anthrax disease in humans and animals. What makes them so very successful? Research in the field of genetics is providing an insight into their success story. For a long time, the common belief was that genomes of organisms are fixed and passed only vertically from parental generation to offspring. A large number of studies have shown that genomes are not static, but exist in a state of incessant flux. This property enables them to survive in a variety of habitats and tolerate chemical onslaughts of all kinds. They are capable of expanding their genomes through horizontal gene transfer and gene duplication. Transposable elements, plasmids and viruses, all play key roles in genome dynamics. As bacteria, by employing these strategies, become resistant to most of the available antibiotics and some become more virulent, providing adequate healthcare becomes a major challenge. Hence, a clear understanding of bacterial evolution at the genetic level becomes very important. This will enable public health officials to formulate strategies to deal with infectious diseases more effectively. In addition, these studies may assist in bioremediation and genetic research.

3. Genome Changes in Bacteria

Bacteria trade genes more frantically than a pit full of traders on the floor of the Chicago Mercantile Exchange - Lynn Margulis and Dorion Sagan

In 1956 in Japan, six years after the clinical use of antibiotics, the dysentery- causing pathogen Shigella dysenteriae was found to be resistant to up to four antibiotics simultaneously (Tc, Cm, Sm, Su). This emergence of multiple resistant strains could not be attributed to co-appearance of multiple mutations in bacteria. It was soon established that bacteria were acquiring genes that confer resistance, rather than mutations arising in resident genes as rate of spontaneous mutation is very low and change 3

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

is acquired for single character. Agents such as plasmids, exogenous DNA and bacteriophages can mediate transfer of a number of genes in a single event, achieving a much faster rate of evolution of bacteria.

3.1. Agents Causing Genome Change

3.1.1. Plasmids

Though bacteria don’t show sexual reproduction and recombination, processes as seen in eukaryotes, they have been shown to engage in sex-like process as suggested by the work of Joshua Lederberg and Edward Tatum initially. They were working with two multiple auxotrophs of E. coli strain K12 as can be seen in the fig.1. Each, when plated independently on minimal culture medium, failed to form any colonies. However, when cells of the two auxotrophs were mixed and allowed to grow together, prototrophs were recovered at a rate of 1/107 which could grow on minimal culture medium. This showed that some kind of exchange had occurred between the two kinds of cells, though physical nature and genetic basis of this exchange became known only after further experimentation. In the Davis U tube experiment, two arms of the U tube were separated by a glass filter with pore size such that bacteria in one arm could not come in contact with bacteria present in the other arm, though they shared a common growth medium suggesting the need for a physical contact between the two kinds of cells to lead to the production of prototrophs.

Figure 1: (1): shows the recovery of prototrophs as a result of genetic recombination between two auxotrophic strains; (2): shows that genetic recombination does not occur when two auxotrophic strains are separated by a filter.

Hayes reported that if strain A cells were inactivated by exposing them to streptomycin,(inhibitor of protein synthesis), prior to the cross, number of prototrophs recovered was not affected. However, when strain B was treated with streptomycin, no prototrophs were recovered. Streptomycin, by 4

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

inhibiting growth and division of cells of B strain, stopped the process leading to the recovery of prototrophs without affecting the ability of strain A cells to transfer their genetic material. This suggested that genetic material was transferred in a non-reciprocal/unidirectional fashion.

Cells capable of working as donors of their genetic material were designated as F+ cells (F for fertility). Bacteria receiving the donor chromosome material were designated as F-. Subsequent experimentation revealed the details of the conjugation process as can be seen in Figure 2

It became established that F+ cells contain a fertility factor (F factor) that confers the ability to donate part of their chromosome during conjugation. F factor has been shown to consist of a circular, double stranded DNA molecule with about 40 genes. The transfer (tra) genes present on the F factor, establish a stable mating pair and initiate DNA transport from the donor to the recipient cell through a sex pilus. Conjugative system also includes a relaxase that nicks DNA to give a single-stranded DNA that is suitable for transfer. Pilus assembly is a function of a type IV secretion system (T4SS) in which a coupling protein links a transenvelope protein complex (a transferosome) to a nucleoprotein complex (a relaxosome), which is bound at the plasmid’s origin of transfer (ori T). Only one of the two strands of plasmid DNA passes through the fused membranes into the recipient cell. This is followed by DNA synthesis in both donor and recipient to replace the missing strand in each. The genes encoding the enzymes responsible for this part of the conjugative process are also found on the plasmid. After completion of the process now there are two donor cells each with a whole, double stranded, circular, conjugative plasmid, i.e. F- cells become F+ cells. This process is so efficient that it can quickly change an entire recipient population to donor cells. Some types of conjugative plasmids are transferred only between cells of the same species. Other types can be transferred across species, known as promiscuous plasmids.

Figure 2: Shows the process of conjugation and production of recombinant bacterium

Although these experiments showed the transfer of plasmid from donor to recipient bacterium every time conjugation occurred, they could not explain the very low rate of, as well as, the mechanism of 5

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

genetic recombination. Subsequent discoveries clarified not only the process but also enabled to draw map of E.coli chromosome.

Cavalli-Sforza treated an F+ strain of E.coli K12 with nitrogen mustard, a potent mutagen. This enabled him to recover a genetically altered strain of donor bacteria which showed a much higher rate of recombination (1/104). Later a similar strain showing a higher rate of recombination was isolated by William Hayes. Both strains were designated Hfr, for high-frequency recombination. Important differences were observed between the two types of mating such as:

 F+ X F- recipient cell becomes F+ (low rate of recombination), pattern of gene transfer random  Hfr X F- recipient cell remains F- (high rate of recombination), pattern of gene transfer non- random, and varied from strain to strain.

These differences could not be explained until the experiments conducted by Wollman and Jacob clarified the genesis of Hfr bacteria. They further postulated that integration of F factor into the chromosome determined the O site (Figure 3). When Hfr and F- strain underwent conjugation, the initial point of transfer was determined by the site of integration of F factor. Genes next to O enter the recipient first and the last to enter is F factor. As conjugation generally does not persist for too long a period, the recipient cell, though acquiring bacterial genes, remains F-. At the site O, DNA molecule of donor opens up, allowing the transfer of one strand.

Figure 3: Shows the formation of Hfr cell by integration of F factor into the E.coli chromosome by a single crossover. The origin (O) is the region transferred first; pairing region is homologous with a region on the E.coli chromosome, a through d are representative genes in E.coli chromosome. Pairing regions are identical in plasmid and chromosome and are derived from insertion sequences.

Figure 2 c and d show how recombination occurs between the chromosome of F-, the recipient and the DNA fragment transferred from the Hfr, the donor bacterium. After recombination, which involves a double crossover, marker B becomes a permanent feature of the recipient bacterium though it still remains F-.

All these experiments and observations have helped clarify the process of recombination in F+ and F- matings. In F+ X F- matings, very low rate of recombination is due to rare integration of F factor into bacterial chromosome to convert it into Hfr cell which is then capable of transferring its DNA strand into the recipient bacterium. Therefore the recipient bacterium would be recombinant but F-.

Jacob and Adelberg discovered in one of the crosses involving Hfr lac+ strain and F- lac- strain, lac+ was transferred to lac- at a very high frequency. It was also found that the transferred lac+ was not 6

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

integrated into the recipient’s chromosome because these F+ lac+ exconjugants occasionally gave rise to F- lac- daughter cells at a frequency of 1X10-3, suggesting their genotype to be F+ lac+/F- lac-. Occasionally, the integrated F factor could excise out of the chromosome and the cell again becomes F+. This excision process happens to be faulty sometimes, (Figure 4) such that, the excised F factor carries some bacterial genes lying adjacent to the point of integration. Faulty excision occurs because there is another homologous region nearby that pairs with the original. F factors carrying bacterial genes are designated F’. Since F’ has all the genes to engage in conjugation process with an F- cell, it transfers not only a strand of F factor but also the bacterial genes that are part of it converting the recipient bacterium partially diploid for these genes, called merozygote, conferring new properties on the recipient bacterium. As F’ plasmids can confer a state of partial diploidy, they are being increasingly used in the study of genetic regulation in bacteria.

Although various experiments clarified the conjugation process, it was still not clear as to what was the mechanism of recombination in the recipient cell. Answer to this question came with the discovery of several mutants which greatly reduced recombination in bacteria. Mutant gene, recA nearly abolished recombination in bacteria; recB, recC and recD mutant genes reduced recombination by 100 times, all pointing to important roles played by wild-type products of these genes in recombination. Further investigations revealed that RecA protein has an important role in recombination involving single stranded DNA. As the double stranded DNA enters the cell, one strand is degraded, leaving a single strand which pairs up with its homologous region along the host chromosome and then RecA facilitates recombination. The recB, recC and recD are other three genes, whose product makes a complex enzyme, RecBCD protein; it is capable of unwinding double- stranded DNA, to facilitate recombination by RecA.

Figure 4 Shows the production of F’ as it exits the chromosome in which it had integrated at the insertion sequence IS1, to form Hfr. Abnormal outlooping by crossing over with a different IS2, to include the lac locus. Transfer of F’lac+ to F-lac- produces a partial diploid.

F’ factors are able to alter characteristics of even those bacteria which have mutant genes coding for RecA and RecBCD proteins.

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

Figure 4: Formation of F’ by defective excision

Transposons are DNA segments that can move around to different sites in the genome. When a transposon in the genome moves to a new location, it can sometimes carry between its ends any genes, including alleles for drug resistance, and move them along to their new locations. Sometimes, a transposon carries a drug resistance allele to a plasmid, creating an R plasmid (Figure 5). As many R plasmids are conjugative, they are effectively transmitted to a recipient cell during conjugation. Even R plasmids that are not conjugative can donate their R alleles to a conjugative plasmid by transposition.

Figure 5: Shows an R plasmid which has genes for resistance against antibiotics tetracycline (Tc), kanamycin (Kan), streptomycin (Sm), sulphanilamide (Su), ampicilin (Amp), mercury (Hg) contained in a transposon.

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

The F factor contains four IS elements: two copies of IS3, one of IS2, and one of IS gamma-delta. The E. coli chromosome also has copies of these four insertion sequences at various positions. It is for this reason that F factor can integrate at a number of different sites on the chromosome by genetic recombination between the homologous sequences. Upon excision of the F factor, different chromosomal segments flanking the site of integration, can thus be picked up. Plasmids containing bacterial genes are called F’ factors.

F’ cells can show conjugation with F- cells in which F’ factor is transferred to all the F- mating cells along with the genes it happens to carry. In the F- cell no recombination is required because F’ can replicate and be maintained in the dividing F- cell population. Thus bacterial genes can be inherited by F- strains that cannot undergo normal homologous recombination.

A bacterial cell contains both conjugative and non-conjugative plasmids in addition to its genomic DNA. When a transposable element gets mobilized, all of the DNA molecules present are potential targets for insertion. With time, all of the plasmids in a bacterial lineage will acquire copies of any transposable element present in the host DNA, and the host DNA will acquire copies of any transposable elements present in the plasmids. Thus, transposable elements become distributed among all independently replicating DNA molecules. As a result, most bacteria contain different types of transposable elements in multiple copies, some in host genome, some in plasmids and some in both. Therefore, many conjugative and non-conjugative plasmids present in a bacterial cell happen to contain one or more copies of the same transposable element. Due to this homology in DNA sequences, recombination can take place between host DNA and plasmid as well as between two plasmids. When a non-conjugative plasmid undergoes recombination with a conjugative plasmid in a bacterial cell, both plasmids get transferred to the recipient bacterium during conjugation.

Bacteria possess DNA elements called integrons that code for site-specific recombinases which can bind with specific nucleotide sequence in duplex DNA. When the site is present in each of two duplex DNA molecules, the enzyme brings the sites together for reciprocal exchange.

All known integrons are composed of three essential elements for procuring exogenous genes (Figure 6)

 a gene coding for an integrase (int I),  a primary recombination site (att I), and  a strong promoter (Pc).

Integron integrases recombine, discreet units of circularized DNA known as gene cassettes, downstream of the resident Pc promoter at the proximal att I site, permitting expression of their encoded proteins. Figure 6 shows how an integron captures a cassette by site- specific recombination between att I present in integron and att C present in cassette. In general, cassettes are promoter-less genes which can be transcribed only by read-through transcription from an adjacent promoter. Once a cassette has been captured, using the same att I site, another with the att C site can be integrated immediately adjacent to attI and mRNA produced from the P ant promoter has the coding sequences for both.

More than 70 different resistance cassettes have been described, and they confer resistance to all beta- lactams, all aminoglycosides, chloramphenicol, trimethoprim, streptothricin, rifampin, erythromycin, and antiseptics of the quaternary ammonium compound family.

Integrons are found on transposons, plasmids and bacterial chromosome allowing their easy mobility and thus have played a very important role in the evolution of antibiotic resistance in bacteria.

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

Figure 6: Shows the entrapment of gene cassettes by integrons for expression

It is because of this system, bacteria can accumulate a battery of exogenous genetic loci to fight off a number of antimicrobials. Some bacteria have up to eight different resistance cassettes in a single integron.

3.1.2. Exogenous DNA via transformation

There is yet another mechanism by which bacteria can show altered characteristics. Acquisition of genes by cells from free DNA molecules in the surrounding medium, resulting in a phenotypic change in the recipient, is called transformation. In 1928, Frederick Griffith, working with Streptococcus pneumoniae, a bacterium that causes pneumonia, noticed a change in the property of the avirulent strain when injected into mice along-with the heat killed virulent strain as shown in the diagram.

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

Figure 7: Shows the transformation of avirulent strain to virulent form by heat-killed virulent bacteria

Griffith interpreted this as transformation of avirulent bacteria into virulent type by the virulent strain though the exact mechanism involved was not known at the time.

Figure 8: Shows the summary of Avery, MacLeod and McCarty’s experiment demonstrating that DNA is the transforming principle.

Later in vitro studies conclusively proved that DNA was indeed the transforming principle. In natural settings, such as soil, free DNA can become available by spontaneous breakage of donor cells.

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

Figure 9: Transformation, wherein exogenous piece of DNA replaces a homologous part from the endogenous chromosome.

In a population of bacterial cells, only those in a particular physiological state called competence, take up DNA. The transforming DNA is incorporated into bacterial chromosome by double crossover mediated by Rec proteins as discussed earlier in the section on conjugation. Examples of bacteria undergoing transformation naturally are Haemophilus influenzae, Bacillus subtilis, Shigella paradysenteriae, Streptococcus pneumoniae. Others, like E. coli, can be induced in the laboratory to become competent. Thus following transformation, the recipient bacterium acquires new properties.

3.1.3. Bacteriophages via transduction

It was an absolutely stunning surprise to us that something as strange as viruses carrying genes from one cell to another can happen - Joshua Lederberg

Lederberg and Zinder discovered yet another method by which genes from one bacterium could be passed to another bacterium. Their experiment consisted of mixing two multiple auxotrophic strains of Salmonella, LA-22 and LA-2 and plating them on minimal culture medium; they recovered prototrophs at a rate of about 1/105. This observation suggested that this process was similar to conjugation described earlier for E.coli. To investigate further, they placed the two strains of bacteria in two arms of the Davis tube separated by a filter of pore size that allows medium to pass through but not the cells, and thus allowed to grow in common medium. When samples were removed from both sides of the filter and plated independently on minimal culture medium, prototrophs were recovered, though only from the side of the tube containing LA-22 bacteria. As the filter prevented cell contact, recovery of prototrophs could not have resulted from the process of conjugation. It was speculated that the genes phe+ and trp+ from LA-2 could have reached the other side of the tube to convert LA- 22 into prototrophs but the mechanism was not clear and it was called filterable agent (FA).

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

Figure 10: Lederberg and Zinder experiment showing the recovery of prototrophs when two auxotrophic strains share the same common medium, yet do not come in contact with each other.

Following experiments and observations helped clarify some aspects of this type of recombination.

 LA-2 cells produced FA only when grown in association with LA-22 cells but not when grown alone in culture medium and later added to LA-22 cells. This suggested some role played by LA-22 cells in the production of FA which appeared only when the two strains were grown in a common medium.  Digestion with DNase did not destroy the activity of FA, thus indicating that FA was not exogenous DNA suggesting that recombination observed was not due to transformation.  When the pore size of the filter was reduced below the size of bacteriophages, the FA could not pass across the filter.

Thus researchers proposed that LA-22 harbored a prophage P22, which upon entering lytic phase, reproduced and was released into the medium. P22 phages crossed the filter, infected and lysed some of the LA-2 cells. Sometimes, P22 phages packaged a region of the LA-2 chromosome in their heads. If this region contains phe+ and trp+ genes, and if phages pass back across the filter and infect LA-22 cells, the cells can become prototrophs upon recombination.

Another set of experiments were conducted to test the possibility of this hypothesis. Bacteria were grown for several generations in a medium containing a precursor of DNA that is heavier than normal (15N instead of 14N), then transferred to light medium (with normal precursors of DNA), containing radioactive DNA precursor (such as 32P), and coliphage P1. Transducing particles (containing bacterial DNA) were found to band at a heavier than normal position and no radioactive phage DNA (DNA synthesized after infection) was found associated with the band of transducing particles. This experiment showed that transducing particles produced by certain viruses carry only bacterial DNA.

When genes are transferred from one bacterium to another by the agency of bacteriophages, the process is termed transduction. Bacteriophages show either a lytic or a lysogenic cycle. Figure 11 below compares the two types of cycles and their consequences. In lytic cycle, bacteriophage, adsorbs to a specific receptor on the bacterial cell surface injecting its DNA into the bacterial cytoplasm. This 13

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

is followed by transcription of phage genes, replication of phage genome, synthesis of viral proteins and packaging of viral genomes into capsids to form virions. At the end of the lytic cycle, phage produces two proteins, holin, to create holes in the cytoplasmic membrane and endolysin to hydrolyze peptidoglycan layer and thus hundreds to thousands of virus particles are released into the surrounding medium. Phage, upon entering the bacterium, causes breakage of bacterial chromosome and during packaging stage of the virus, any fragment of bacterial chromosome which can fit in viral head can get packaged. As the infective property resides with the protein coat, this aberrant phage can infect another bacterium, transferring the bacterial genes carried by it. Since any random sample of genes can thus get transferred, the process is termed generalized transduction. All virulent bacteriophages do not cause transduction such as T-even phages which degrade the host DNA and reutilize the mononucleotides thus produced for phage-DNA synthesis.

However, if the bacteriophage DNA gets integrated into bacterial DNA to become a prophage, it does not cause lysis of the cell and is replicated along with the bacterial chromosome. Transcription of most of the phage genes, including those needed for lytic cycle are repressed by a specific repressor. Prophages have been linked to a phenomenon of lysogenic conversion, i.e., non-virulent bacterial strain becoming virulent. Prophages have contributed to virulence of a number of bacterial pathogens such as E.coli, Streptococcus pyogenes, Salmonella enterica and Staphylococcus aureus. While most of the genes carried by prophages are repressed, virulence genes are organized as “morons”, representing discrete, autonomous genetic elements, flanked on one side by sigma70-like promoter and factor-independent transcriptional terminator on the opposite side, allowing their expression.

Temperate phages have also been implicated in the formation of biofilms as well as sporulation in some bacterial species such as Bacillus subtilis and Clostridium difficile, both properties greatly enhancing survival and dispersal.

Once in a while, under any stressful condition, there may be a proteolytic cleavage and displacement of the phage repressor bound to the early promoter , the prophage loses its integrated state and enters lytic cycle when its chromosome loops out of the bacterial chromosome. For example, phage lambda has att region in its DNA which is homologous to att region of E.coli chromosome and it is in this region phage DNA integrates into bacterial DNA. Markers flanking att region in E.coli chromosome are gal and bio (for galactose and biotin). If excision is imprecise, then either of the markers can go along with the phage chromosome, leaving a little portion of the phage chromosome behind. Transduction achieved by such phages is called specialized transduction. This is of considerable significance, as virulence factors encoded by pathogenicity is l and SaPI1 of Staphylococcus aureus are transduced between bacterial cells by bacteriophages, greatly speeding up the evolution of this species.

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

Figure 11: Shows the difference in the process of generalized and specialized transduction

Phages, which have shown the production of generalized transducing particles, include E. coli phage P1, Salmonella phage P22, and Bacillus subtilis phages PBS1 and SP10.

Transduction by bacteriophages can include any bacterial DNA such as linear chromosome fragments, plasmids, transposons, insertion elements. An important feature of transduction is that “donor” and “recipient” cells need not occur at the same place or at the same time.

In most of the natural environments such as, oceans, lakes, soil, even with-in the bodies of humans and animals as well as managed environments such as sewage treatment plants, bacteria as well as bacteriophages occur most abundantly. This relative high concentration of bacteria and bacteriophages allows frequent infections and consequent transductions to proceed. Commensal bacteria have been shown to contain a number of antibiotic resistance genes which can be acquired by pathogenic members of the gut microbial community. Another reason for transduction process to occur rather frequently is the basic structural attribute of phages which allows them to persist for prolonged periods of time in their extracellular phase quite resistant to various environmental stresses such as temperature and radiation. This contrasts with the process of transformation mediated by naked DNA, which is quite susceptible to environmental stresses. 15

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

3.2. Biological Consequences of Genome Change in Bacteria

3.2.1. Development of antibiotic resistance & spread

Bacterial-genome sequencing is revealing the important role played by phages and plasmids in bacterial evolution leading to emergence of new strains and species. Through plasmids, antibiotic resistance alleles can spread rapidly throughout a population of bacteria, a very effective strategy for the survival of bacteria. Research has shown there is frequent horizontal gene transfer among bacteria in human body, which contains over 1,000 species of such micro-organisms amounting to over 100 billion in number. Bacteria have been discovered which are able to make enzyme called NDM-1, or New Delhi metallo-beta lactamase 1. This enzyme enables bacteria to grow in the presence of nearly all known antibiotics. The resistant strains have been found in 180 people in India, Pakistan, U.K., Australia, Canada, Netherlands, U.S. and Sweden.

Therapeutic treatment of farm animals with antibiotics has selected for resistant bacteria that are found in food when they survive the production processes, as in the production of raw milk cheeses, e.g., plasmid pK214 in bacterium Lactococcus lactis (Figure 12).

Figure 12: Shows a plasmid pK214 harboring a number of antibiotic genes from various sources

Researchers from China have recently reported the presence of bacteria, which are resistant to colistin, in pigs, meat and a small number of patients.

Temperate phages have greatly contributed to converting non-virulent strains to virulent strains as well as caused extensive transfer of virulence genes to non-virulent strains by transduction. 3.2.1. Spread of nitrogen fixing property

Bacterium Rhizobium is very important both ecologically and agriculturally. Very large (>250 kb) conjugative plasmids in Rhizobium contain genes which allow bacteria to invade the host- plant root cells and carry out conversion of atmospheric dinitrogen to ammonia, thus fulfilling the nitrogen needs of the plant. Mobile genetic elements have also been shown to carry genes important in global carbon cycling. Phages of photosynthetic, marine blue-green bacteria carry genes involved in photosynthesis as also the genes which enable host bacteria to survive in nutrient poor conditions of the ocean. 16

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

3.2.2. Bio transformation of xenobiotics

Bacterial species have been described containing naturally occurring plasmid- encoded genes which allow biotransformation of hydrocarbons, thus indicating their potential role in bioremediation applications. Such plasmid-encoded genes have been found to be organized either in large operons or on genomic islands, some of which are conjugative.

Soil bacteria show abundance of transposons carrying genes which can degrade pollutants such as radioactive and metallic substances, and thus play important role in soil transformation. Nigel Williams in 1997 reported that Bacillus subtilis has acquired genes, through the agency of bacteriophages, to resist heavy metal in its environment.

4. Applications

4.1. Formulation of Strategies

Appearance of bacteria, resistant to multiple antibiotics makes treatment a big challenge. Hence, strategies will have to be formulated to avoid over- use and misuse of antibiotics in medical prescriptions, treatment of farm animals and proper disposal of antibiotics.

4.2. Bioremediation

Bacteria capable of degrading heavy metals and hydrocarbons have these genes either on plasmids or transposons and thus have the potential to be used for remediation of degraded soils.

4.3. Genetic Research

Plasmids, transposons and viruses have been used extensively to determine functions of genes. Engineered plasmids are being used in DNA cloning and the phenomenon of transformation allows plasmid vectors to be introduced into and expressed by E. coli cells.

5. Summary

Genetic flexibility and adaptability of their genomes under environmental stress make bacteria the ultimate survivors. Presence of a large number of gene cassettes conferring resistance against a variety of commonly used antibiotics, capture of these cassettes by integrons, association of integrons with conjugative plasmids and transposons, are all responsible for the fast spread of antibiotic resistance in microbes via HGT. Mechanisms assisting in this transfer are conjugation, transformation and transduction, which are rather very prevalent considering the enormous numbers of microbes present within the guts of all animals, in the sewage treatment plants and in the soil and water bodies. As treatment of microbial infections becomes a serious challenge, it is becoming clear that restraint must be observed with regard to antibiotic usage such as medical prescriptions, treatment of farm animals and disposal in water bodies.

Prophages have contributed to virulence of a number of bacterial pathogens such as E.coli, Streptococcus pyogenes, Salmonella enterica and Staphylococcus aureus.

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1

It is also becoming apparent that these same mechanisms can also be harnessed for the purposes of bioremediation as well as spreading nitrogen-fixing capability to a greater number of plants. The entire discussion reveals that there are a number of options for future genetic research. Study of gene function as well as gene therapy has been greatly facilitated by development of powerful gene transfer techniques using plasmids, and viral vectors.

Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1