1. There are two major path ways for homologous rrecombination in E. coli, the R
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Question
1. There are two major path ways for homologous rrecombination in E. coli, the RecBCD pathway and the RecFOR pathway. Why then, is recA- mutant completely deficient in homologous recombination?
2. In specialized transduction, only some regions of a bacterium's DNA can be transferred by a phage to another bacterium. Why is this so?
3. In a calcium-ion induced competence, what is the role of the calcium ion?
4. In both conjugation and natural transformation only one strand of DNA is taken up by cells, but recombination is necessary for maintenance of the DNA after natural transformation but not after conjugation. Why is this so?
Explanation / Answer
Answer to first part
homologous recombination, the two chromosomes do not need to be homologous over their entire length for this exchange, as a rather limited region of homology is enough. Therefore, homologous recombination is “exchange between two DNA sequences in the region of shared homology”. Homologous recombination is distinguished from other recombination events not only by the requirement for extended length of homology, but also by the enzymatic systems catalyzing the event. Homologous recombination is catalyzed by specialized and complex enzymatic machinery, at the core of which lies the still mysterious ability of the homologous recombinase protein (RecA in bacteria, RadA in archaea, Rad51 in eukaryotes) to recognize homology between two DNAs independently of their actual sequence. According to this logic, if recombination between 20 bp homologies in E. coli is completely prevented in a recA mutant, it is considered a homologous recombination event . And vice versa, even if the length of homology mediating the exchange is a significant 500 bp, but such a recombination occurs with a good frequency in a recA mutant, this RecA-independent recombination is “homology-driven illegitimate recombination” event . To provide some kind of a reference at this point, RecA-dependent recombination, although quite low, is already detectable between 12 base pair-long identical sequences in some experimental systems and becomes the predominant mode of exchange between shared homologies 100 bp or longer in most experimental systems
Answer to second part
Specialized transduction” is a phenomenon observed with a sub-class of temperate phages that, as lysogens, reside in the host chromosome. As a result of improper excision events, these phages occasionally spawn hybrid variants carrying stretches of contiguous chromosomal regions Lysogenization with such a hybrid phage via the regular site-specific recombination at the attB site generates a bacterium with a duplication of the captured region, and the majority of the specialized transduction events are of this type, which has nothing to do with homologous recombination. However, lysogenic induction of phage Lambda, for example, also generates defective phage particles, called Lambda doc, that result from attempted packaging of the Lambda copy that is still in the chromosome, and therefore approximately half of DNA in these particles is the neighboring bacterial DNA, either to the right or to the left of the prophage . Recombination of such a hybrid phage with the chromosome is by chromosomal homology and resembles generalized transduction, only it is specific to the regions contiguous with the prophage — hence the term “specialized transduction”.
The read-out in transduction is always formation of a recombinant bacterium, so the fate of the phage DNA, originally attached to the transduced chromosomal piece, is unknown. However, the situation can be turned around, when the chromosomal homology is constructed into the phage genome, and the recombination is followed by detecting recombinant phages that “picked up” markers from the chromosome, in either heteroimmune or homoimmune crosses . Probably one of the more dramatic examples of such recombination is the formation of the Lambda-reverse phage. A mutant lambda, deficient in its own phage recombination functions, cannot grow well on the wild type E. coli.
Answer to third part
Role of calcium ion in calcium induced competance
Ca2+ treatment renders the outer membrane of Escherichia coli reversibly permeable for macromolecules. whether Ca2+-induced uptake of exogenous protein into the periplasm occurs by mechanisms similar to Ca2+-induced uptake of DNA into the cytoplasm during transformation. Protein import through the outer membrane was monitored by measuring reconstitution of maltose transport after the addition of shock fluid containing maltose-binding protein. DNA import through the outer and inner membrane was measured by determining the efficiency of transformation with plasmid DNA. Both processes were stimulated by increasing Ca2+ concentrations up to 400 mM. Plasmolysis was essential for a high efficiency; reconstitution and transformation could be stimulated 5- and 40-fold, respectively, by a high concentration of sucrose (400 mM) in cells incubated with a suboptimal Ca2+ concentration (50 mM). The same divalent cations that promote import of DNA (Ca2+, Ba2+, Sr2+, Mg2+, and Ni2+) also induced import of protein. Ca2+ alone was found to be inefficient in promoting reconstitution; successive treatment with phosphate and Ca2+ ions was essential. Transformation also was observed in the absence of phosphate, but could be stimulated by pretreatment with phosphate. The optimal phosphate concentrations were 100 mM and 1 to 10 mM for reconstitution and transformation, respectively. Heat shock, in which the cells are rapidly transferred from 0 to 42 degrees C, affected the two processes differently. Incubation of cells at 0 degrees C in Ca2+ alone allows rapid entry of protein, but not of DNA.
Answer to fourth part
transformation
Many bacteria can acquire new genes by taking up DNA molecules (e.g., a plasmid) from their surroundings . The ability to deliberately transform the bacterium E. coli has made possible the cloning of many genes — including human genes — and the development of the biotechnology industry.
Conjugation
Some bacteria, E. coli is an example, can transfer a portion of their chromosome to a recipient with which they are in direct contact. As the donor replicates its chromosome, the copy is injected into the recipient. At any time that the donor and recipient become separated, the transfer of genes stops. Those genes that successfully made the trip replace their equivalents in the recipient's chromosome.
Significance of genetic recombination in bacteria.
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