Paper No. : 16: Molecular Genetics Module : 09: 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 1 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 M09: 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 2 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 diarrhoea 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 3 Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1 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 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. 4 Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1 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 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 5 Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1 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 rate and the mechanism of genetic recombination. Subsequent discoveries clarified not only the process but also enabled to draw map of E.coli chromosome. Cavalli-Sforza treated 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 6 Molecular Genetics ZOOLOGY Structural organization of genome: Genome Dynamics: Part 1 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 in last F factor enters.