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Journal of Pharmaceutical Research and Opinion 1: 7 (2011) 170 – 173. Contents lists available at www.innovativejournal.in JOURNAL OF PHARMACEUTICAL RESEARCH AND OPINION Journal homepage: http://www.innovativejournal.in/index.php/jpro REVIEW EFFECTIVENESS OF TRANSDUCTION AND CONJUGATION IN GENETIC TRANSFORMATION Shuaib M. Awwal*, Abalaka M. Department of Microbiology, Federal University of Technology, Minna, P.M.B 65, Niger State. Nigeria ARTICLE INFO ABSTRACT Received 23 Nov 2011 Accepted 11 Dec 2011 Bacteria have mechanisms by which they can 'obtain' extra DNA, which creates opportunities for recombination to occur. Certain species of bacteria can become competent to take up high molecular weight DNA from the surrounding. An event in which one bacterium donates DNA to another bacterium is a type of genetic transfer termed recombination leading to Genetic transformation. Transduction is DNA transfer mediated through the action of a bacteriophage while Conjugation requires the attachment of two related species through a bridge that can transport DNA. Both phenomenons provide additional genes for resistance to drugs and metabolic poisons, increased virulence and adaptation to environment effectively, thus genetic transformation. Corresponding Author: Shuaib M. Awwal Department of Microbiology, Federal University of Technology, Minna, P.M.B 65, Niger State. Nigeria [email protected] KeyWords: Transformation, Transduction, Conjugation, DNA ©2011, JPRO, All Right Reserved. INTRODUCTION Genetic transformation is a process by which free DNA is incorporated into a recipient cell and brings about change. The discovery of genetic transformation in bacteria was one of the outstanding events in biology, as it led to experiments proving without a doubt that DNA was the genetic material. This discovery became the keystone of molecular biology and modern genetics. Genes derived from unrelated species and even other kingdoms, such as bacteria, fungi, plants, animals, that would otherwise be inaccessible to an organism, can be combined in the lab usinggenetic transformation techniques.(National4-Hcouncil,1997). Bacteria can exchange or transfer DNA between other bacteria in three different ways. In every case the source cells of the DNA are called the DONORS and the cells that receive the DNA are called the RECIPIENTS. In each case the donor DNA is incorporated into the recipient’s cell's DNA by recombination exchange. If the exchange involves an allele of the recipient's gene, the recipient's genome and phenotype will have changed. The three forms of bacterial DNA exchange are (1) TRANSFORMATION, (2) CONJUGATION and (3) TRANSDUCTION. (Hurlbert, 1999). Natural genetic transformation was originally discovered in Streptococcus pneumoniae (Griffith, 1928), and ever since the pneumococcus has served as a paradigm for this important phenomenon. After (Dawson and Sia, 1931) achieved transformation in vitro in 1931, the ‘nuts and bolts’ of the transformation apparatus and its regulation has gradually been unraveled. However, much remains to be learned, especially with respect to environmental cues that promote competence development in situ, and the complex nature of the DNA uptake machinery. TRANSDUCTION Bacteriophages (bacterial viruses) have been previously described as destructive bacterial parasites. Infection by a virus does not always kill the host cell, however, and viruses can in fact serve as genetic vectors (an entity that can bring foreign DNA into a cell). The process by which a bacteriophage serves as the carrier of DNA from a donor cell to a recipient cell is transduction. Although it occurs naturally in a broad spectrum of bacteria, the participating bacteria in a single transduction event must be the same species because of the specificity of viruses for host cells. (Kathleene and Arthur, 2002). Transduction is the transfer of bacterial genes by viruses. Bacterial genes are incorporated into a phage capsid because of errors made during the virus life cycle. The virus containing these genes then injects them into another bacterium, completing the transfer. There are two different kinds of transduction: generalized and specialized. (Prescott et al., 2008) . Generalized Transduction Sometimes, during bacteriophage replication, a mistake is made, and a fragment of the host DNA gets packaged into a viral capsid. The resulting phage would be able to infect another cell, but it would not have any viral genes, so it would not be able to replicate. The cell infected by this phage would survive, and would have an extra piece of bacterial DNA present, which could undergo recombination with the host chromosome, and perhaps cause a gene conversion event. Because it is a random fragment that gets packaged into the viral capsid, any segment of the bacterial DNA can be transferred this way (hence the name 'generalized').(emunix ,2011). 170 Awwal et. al / Effectiveness Of Transduction And Conjugation In Genetic Transformation Generalized transduction in the Enterobacteriaceae is typified by the action of the lytic bacteriophages PI, P22, etc. The injected DNA is integrated into the chromosome by the host recombination mechanism or maintained by autonomous replication in cases where a replicative plasmid is transduced. (Philippe and Julian,1991) . As in transformation, once the DNA has been injected, it must be incorporated into the recipient cell’s chromosome to preserve the transferred genes. (Prescott et al., 2008). Specialized Transduction Specialized transduction occurs only with certain types of bacteriophage, such as phage lambda. Lambda has the ability to establish what is called a lysogenic infection in a bacterial cell. In a lysogenic infection, the viral DNA becomes incorporated into the host chromosome, much as the F factor did in Hfr cells. In a lysogenic infection by lambda, the DNA integrates into a very specific spot in the host chromosome. The integrated viral DNA can remain integrated for long periods of time, without disturbing the cell. Under the appropriate conditions, the viral DNA will excise itself from the chromosome, and enter the lytic phase, in which the virus replicates. The cell gets lysed, and new bacteriophage particles are released to infect other cells. As with excision of the F factor (when Hfr cells become F'), sometimes the excision of lambda is sloppy, and some bacteria DNA is excised along with it. When the resulting virus infects another cell, it will pass that bacterial DNA into the cell, along with its own DNA. If the infected cell survives, it will contain a new piece of bacterial DNA, which can undergo recombination and possibly cause gene conversion. Because the viral DNA integrates into a specific location, when it excises, the bacterial DNA removed with it will be the same in all cases. Therefore, the DNA transferred to the second cell will be the same segment of the bacterial chromosome. This is why this process is called 'specialized' transduction. (emunix, 2011). CONJUGATION In 1946 Joshua Lederberg and Tatum discovered that some bacteria can transfer genetic information to otherbacteria through a process known as conjugation. (Sridhar, 2006). Conjugation is a mode of sexual mating in which a plasmid or other genetic material is transferred by a donor to a recipient cell via a direct connection.(Kathleene and Arthur, 2002). The contact between the cells is via a protein tube called an F or sex pilus, which is also the conduit for the transfer of the genetic material. (emunix, 2011). Although mating systems have been studied in the most detail in gram-negative bacteria, conjugation systems have been identified and analyzed in streptococci, staphylococci, bacillaceae, streptomycetes, and halobacteria (Rosenshine et al., 1989). Pheromone-activated conjugation in streptococci, which has been extensively studied by Clewell and associates, is the only identified bacterial conjugation system in which a diffusible signal is required to activate cell interaction. (Clewell and Weaver, 1989) . Processes involving diffusible components might be more susceptible to environmental inhibition, such as the presence of proteases. The prototypical conjugative plasmid is the Fplasmid, or F-factor. (Holmes and Jobling, 1996) . The Fplasmid is an episome (a plasmid that can integrate itself into the bacterial chromosome by homologous recombination) with a length of about 100 kb. It carries its own origin of replication, the oriV, and an origin of transfer, or oriT. Ryan and (Ray, 2004) .There can only be one copy of the F-plasmid in a given bacterium, either free or integrated, and bacteria that possess a copy are called Fpositive or F-plus (denoted F+). Cells that lack F plasmids are called F-negative or F-minus (F-) and as such can function as recipient cells. Contact is made when a pillus grows out from the F+ cell, attaches to the surface of the Fcell, contracts, and draws the two cells together. In both gram-positive and gram-negative cells, an opening is created between the connected cells, and the replicated DNA passes across from one cell to the other. Conjugation is a very conservative process, in that the donor bacterium generally retains a copy of genetic material being transferred. (Kathleene and Arthur, 2002). When conjugation is initiated by a signal the relaxase enzyme creates a nick in one of the strands of the conjugative plasmid at the oriT. Relaxase may work alone or in a complex of over a dozen proteins known collectively as a relaxosome. In the F-plasmid system the relaxase enzyme is called TraI and the relaxosome consists of TraI, TraY, TraM and the integrated host factor IHF. The nicked strand, or T-strand, is then unwound from the unbroken strand and transferred to the recipient cell in a 5'-terminus to 3'-terminus direction. The remaining strand is replicated either independent of conjugative action (vegetative replication beginning at the oriV) or in concert with conjugation (conjugative replication similar to the rolling circle replication of lambda phage). Conjugative replication may require a second nick before successful transfer can occur. A recent report claims to have inhibited conjugation with chemicals that mimic an intermediate step of this second nicking event. (Lujan et al., 2007) If the F-plasmid that is transferred has previously been integrated into the donor’s genome some of the donor’s chromosomal DNA may also be transferred with the plasmid DNA. (Griffiths et al.,1999). The amount of chromosomal DNA that is transferred depends on how long the two conjugating bacteria remain in contact. In common laboratory strains of E. coli the transfer of the entire bacterial chromosome takes about 100 minutes. The transferred DNA can then be integrated into the recipient genome via homologous recombination. In high-frequency recombination (Hfr) donors, the fertility has been integrated into the F+ donor chromosome. The term high-frequency recombination was adopted to denote that a cell with an integrated F factor transmits its chromosomal genes at a higher frequency than other cells. (Kathleene and Arthur, 2002). Effectiveness of transduction in genetic transformation Transduction may be important for the exchange of genetic material between closely related species (identical or cross-reacting receptors) in nature. (Kokjohn, 1989). DNA carried by the transducing particle is protected from the environment and may survive for relatively long periods of time (Zeph et al., 1988). Several cases of specialized transduction have biomedical importance. The virulent strains of bacteria such as C. diphtheria, Clostridium spp., and S. pyogenes all produce toxins with profound physiological effects, whereas nonvirulent strains do not produce toxins. It turns out that toxicity arises from the expression of bacteriophage genes that have been introduced by transduction. Only those bacteria infected with a temperate phage are toxin formers. Other instances of transduction are seen in staphylococcal transfer of drug 171 Awwal et. al / Effectiveness Of Transduction And Conjugation In Genetic Transformation resistance and in the transmission of gene regulators in gram-negative rods (Escherichia, Salmonella). (Kathleene and Arthur, 2002). Effectiveness of conjugation in genetic transformation The initial studies of (Wolk et al.,1984) and (Guiney et al.,1984) demonstrated conjugative transfer between two distant groups of gram-negative bacteria, including anerobes; Subsequently, Trieu euot and collaborators (Trieu-Cuot et al.,1987) designed and constructed broad-host range conjugative plasmids having dual gram-negative and gram-positive replication origins on a mobilizable plasmid possessing a "universal" antibiotic resistance selection marker. The motivation for the study was to obtain evidence for intergeneric transfer of certain antibiotic resistance determinants in nature. Gene transfer between, distantly related organisms has also expanded outside the microbial world! The vir genes of the Agrobacterium tumefaciens Ti plasmid provide some of the tra functions for conjugal transfer from bacteria to plants. In an important experiment, BuchananWollaston and collaborators (Buchanan-Wollaston et al., 1987) showed that the mob and oriT functions of the broad-host range IncQnplasmid RSFIOIO mediate the transfer of plasmids from Agrobacterium sp. into plant cells. The broad-host range plasmid pRK2 (IncP) was unsuccessful in similar tests of transfer into plant cells. Conjugation is so successful in promoting diverse gene exchanges, an important factor is likely to be the replicative transfer of single-stranded DNA during conjugation. Single-stranded DNA is known to be refractory to most restriction systems, thus the transfer of singlestranded DNA during bacterial conjugation may escape digestion by recipient restriction systems. (Baltz and McHenney, 1989). The facile transfer of IncQ plasmids to a variety of gram-positive species is of significance in considerations of gene transfer in the environment (range and efficacy) and in aiding the manipulation of such industrially important microorganisms as Mycobacteria and Streptomycetes. CONCLUSION Transduction and specialized transduction is especially important because they explain how anti-biotic drugs become ineffective due to the transfer of resistant genes between bacteria. In addition, hopes to create medical methods of genetic modification of diseases such as Duschenne/Becker Muscular Dystrophy are based upon these methodologies. Conjugation has great biomedical importance. Special resistance (R) plasmids, or factors, that bear genes for resisting antibiotics and other drugs are commonly shared among bacteria through conjugation. Transfer of R factors can confer multiple resistances to antibiotics such as tetracycline, chloramphenicol, streptomycin, sulfonamides and penicillin. 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