microbial genetics

 Microbial genetics(plasmid,conjugation,transduction,transformation)


Concepts
1. Recombination is a one-way process in procaryotes:a piece of genetic material (the exogenote) is donated to the chromosome of a recipient cell (the endogenote) and integrated into it.

2. The actual transfer of genetic material between bacteria usually takes place in one of three ways: direct transfer between two bacteria temporarily in physical contact (conjugation), transfer of a naked DNA fragment (transformation), or transport of bacterial DNA by bacteriophages (transduction).

3. Plasmids and transposable elements can move genetic material between bacterial chromosomes and within chromosomes to cause rapid changes in genomes and drastically alter phenotypes.

4. The bacterial chromosome can be mapped with great precision, using Hfr conjugation in combination with transformational and transductional mapping techniques.

5. Recombination of virus genomes occurs when two viruses with homologous chromosomes infect a host cell at the same time.

General recombination
 The most common form, usually involves a reciprocal exchange between a pair of homologous DNA sequences.
 It can occur anyplace on the chromosome, and it results from DNA strand breakage and reunion leading to crossing-over.

 The Holliday Model for Reciprocal General Recombination.


General recombination is carried out by the products of rec genes such as the recA protein so important for DNA repair

 In general, a piece of donor DNA, the exogenote, must enter the recipient cell and become a stable part of the recipient cell’s genome, the endogenote.
 During replacement of host genetic material, the recipient cell becomes temporarily diploid for a portion of the genome and called a merozygote.



 Movement of DNA from a donor bacterium to the recipient can take place in three ways:
1. Direct transfer between two bacteria temporarily in physical contact (conjugation)
2. Transfer of a naked DNA fragment (transformation)
3. Transport of bacterial DNA by bacteriophages (transduction).


 Whatever the mode of transfer, the exogenote has only four possible fates in the recipient (figure 13.4). First, when the exogenote has a sequence homologous to that of the endogenote, integration may occur; that is, it may pair with the recipient DNA and be incorporated to yield a recombinant genome. Second, the foreign DNA sometimes persists outside the endogenote and replicates to produce a clone of partially diploid cells. Third, the exogenote may survive, but not replicate, so that only one cell is a partial diploid. Finally, host cell nucleases may degrade the exogenote, a process called host restriction.






I. Bacterial plasmid:
 Plasmids are small double-stranded DNA molecules, usually circular, that can exist independently of host chromosomes and are present in many bacteria (they are also present in some yeasts and other fungi).

 They have their own replication origins and are autonomously replicating and stably inherited.

 Plasmids have relatively few genes, generally less than 30.

 Their genetic information is not essential to the host, and bacteria that lack them usually function normally.

 Single-copy plasmids produce only one copy per host cell. Multicopy plasmids may be present at concentrations of 40 or more per cell.

 Plasmids can be eliminated from host cells in a process known as curing.
 Some commonly used curing treatments are acridine mutagens, UV and ionizing radiation, thymine starvation, and growth above optimal temperatures.

Classification of plasmid:

 An episome is a plasmid that can exist either with or without being integrated into the host’s chromosome.

 Conjugative plasmids, have genes for pili and can transfer copies of themselves to other bacteria during conjugation.

 Fertility Factors
 A plasmid called the fertility or F factor plays a major role in conjugation in E. coli and was the first to be described.

 Most of the information required for plasmid transfer is located in the tra operon, which contains at least 28 genes.

 Many of these direct the formation of sex pili that attach the F+ cell (the donor cell containing an F plasmid) to an F- cell (figure 13.6). Other gene products aid DNA transfer.

 The F factor also has several segments called insertion sequences that assist plasmid integration into the host cell chromosome. Thus the F factor is an episome that can exist outside the bacterial chromosome or be integrated into it.

 The plasmid contains several transposable elements. IS2 and IS3 are insertion sequences; γ∂ is also called transposon Tn1000.

 The tra genes code for proteins needed in pilus synthesis and conjugation.

 The rep genes code for proteins involved in DNA replication.

 OriV is the initiation site for circular DNA replication and oriT, the site for initiation of rolling circle replication and gene transfer during conjugation.



 Resistance Factors
 Plasmids often confer antibiotic resistance on the bacteria that contain them. R factors or plasmids typically have genes that code for enzymes capable of destroying or modifying antibiotics.

 They are not usually integrated into the host chromosome. Genes coding for resistance to antibiotics such as ampicillin, chloramphenicol, and kanamycin have been found in plasmids.



Col Plasmids

 Bacteria also harbor plasmids with genes that may give them a competitive advantage in the microbial world. Bacteriocins are bacterial proteins that destroy other bacteria.

 Bacteriocins often kill cells by forming channels in the plasma membrane, thus increasing its permeability. They also may degrade DNA and RNA or attack peptidoglycan and weaken the cell wall.

 Col plasmids contain genes for the synthesis of bacteriocins known as colicins.

I. Other Types of Plasmids

 virulence plasmids, make their hosts more pathogenic because the bacterium is better able to resist host defense or to produce toxins.
 For example, enterotoxigenic strains of E. coli cause traveler’s diarrhea because of a plasmid that codes for an enterotoxin.
 Other plasmid-borne toxins are the tetanus toxin of Clostridium tetani and the anthrax toxin of Bacillus anthracis.

 Metabolic plasmids
Carry genes for enzymes that degrade substances such as aromatic compounds (toluene), pesticides (2,4-dichlorophenoxyacetic acid), and sugars (lactose).
 Metabolic plasmids even carry the genes required for some strains of Rhizobium to induce legume nodulation and carry out nitrogen fixation.


Transposable Elements

• The chromosomes of bacteria, viruses, and eucaryotic cells contain pieces of DNA that move around the genome. Such movement is called transposition.
• DNA segments that carry the genes required for this process and consequently move about chromosomes are transposable elements or transposons.

• The simplest transposable elements are insertion sequences or IS elements

• They were first discovered in the 1940s by Barbara McClintock during her studies on maize genetics (a discovery that won her the Nobel Prize in 1983).

 A well-studied example of such a Conjugative transposon is Tn916 from Enterococcus faecalis.


 Although Tn916 cannot replicate autonomously, it will transfer itself from E. faecalis to a variety of recipients and integrate into their chromosomes. Because it carries a gene for tetracycline resistance, this conjugative transposon also spreads drug resistance

Bacterial Conjugation
 Bacterial conjugation, the transfer of genetic information by direct cell to cell contact, came from an elegant experiment performed by Joshua Lederberg and Edward L. Tatum in 1946.


 F+ * F- Mating

 In 1952 William Hayes demonstrated that the gene transfer observed by Lederberg and Tatum was polar

 That is, there were definite donor (F+) and recipient (F-) strains, and gene transfer was nonreciprocal. He also found that in F+ * F- mating the progeny were only rarely changed with regard to auxotrophy (that is, bacterial genes were not often transferred), but F- strains frequently became F+.



 The F+ strain contains an extrachromosomal F factor carrying the genes for pilus formation and plasmid transfer. During F+* F- mating or conjugation, the F factor replicates by the rolling-circle mechanism, and a copy moves to the recipient.

 The sex pilus or F pilus joins the donor and recipient and may contract to draw them together.

 Self transmissible plasmids are present in gram-positive bacterial genera such as Bacillus, Streptococcus, Enterococcus, Staphylococcus, and Streptomyces.




 Hfr Conjugation



 Because certain donor strains transfer bacterial genes with great efficiency and do not usually change recipient bacteria to donors, a second type of conjugation must exist.

 When integrated, the F plasmid’s tra operon is still functional; the plasmid can direct the synthesis of pili, carry out rolling-circle replication, and transfer genetic material to an F- recipient cell. Such a donor is called an Hfr strain (for high frequency of recombination) because it exhibits a very high efficiency of chromosomal gene transfer in comparison with F+cells.


 Because only part of the F factor is transferred at the start (the initial break is within the F plasmid), the F- recipient does not become F+ unless the whole chromosome is transferred.


 The connection usually breaks before this process is finished. Thus a complete F factor usually is not transferred, and the recipient remains F-.

 F'Conjugation
 Because the F plasmid is an episome, it can leave the bacterial chromosome. Sometimes during this process the plasmid makes an error in excision and picks up a portion of the chromosomal material to form an F'plasmid.
 It is not unusual to observe the inclusion of one or more genes in excised F plasmids. The F'cell retains all of its genes, although some of them are on the plasmid, and still mates only with an F- recipient.
 F' * F- conjugation is virtually identical with F+ * F- mating.
 Once again, the plasmid is transferred, but usually bacterial genes on the chromosome are not (figure 13.15b). Bacterial genes on the F'plasmid are transferred with it and need not be incorporated into the recipient chromosome to be expressed.


Figure 13.15 F'Conjugation. (a) Due to an error in excision, the A gene of an Hfr cell is picked up by the F factor. (b) The A gene is then transferred to a recipient during conjugation.

 The recipient becomes F'and is a partially diploid merozygote since it has two sets of the genes carried by the plasmid. In this way specific bacterial genes may spread rapidly throughout a bacterial population. Such transfer of bacterial genes is often called sexduction


 F'conjugation is very important to the microbial geneticist. A partial diploid’s behavior shows whether the allele carried by an F'plasmid is dominant or recessive to the chromosomal gene.
 The formation of F'plasmids also is useful in mapping the chromosome since if two genes are picked up by an F factor they must be neighbors.

 DNA Transformation
 Discovered by Fred Griffith in 1928.
 Transformation is the uptake by a cell of a naked DNA molecule or fragment from the medium and the incorporation of this molecule
into the recipient chromosome in a heritable form.



 When bacteria lyse, they release considerable amounts of DNA into the surrounding environment. These fragments may be relatively large and contain several genes. If a fragment contacts a competent cell, one able to take up DNA and be transformed


 Competency is a complex phenomenon and is dependent on several conditions. Bacteria need to be in a certain stage of growth; for example, S. pneumonia becomes competent during the exponential phase when the population reaches about 107 to 108 cells per ml.


 When a population becomes competent, bacteria such as pneumococci secrete a small protein called the competence factor that stimulates the production of 8 to 10 new proteins required for transformation



 The Mechanism of Transformation. (1) A long double-stranded DNA molecule binds to the surface with the aid of a DNA-binding protein (•) and is nicked by a nuclease .
(2) One strand is degraded by the nuclease. (3) The undegraded strand associates with a competence-specific protein ( ). (4) The single strand enters the cell and is integrated into the host chromosome in place of the homologous region of the host DNA


 Artificial transformation is carried out in the laboratory by a variety of techniques, including treatment of the cells with calcium chloride, which renders their membranes more permeable to DNA.


 When linear DNA fragments are to be used in transformation, E. coli usually is rendered deficient in one or more exonuclease activities to protect the transforming fragments. It is even easier to transform bacteria with plasmid DNA since plasmids are not as easily degraded as linear fragments and can replicate within the host .


 Transformation in Haemophilus influenzae, a gram-negative bacterium, differs from that in S. pneumoniae in several respects. Haemophilus does not produce a competence factor to stimulate the development of competence, and it takes up DNA from only closely related species (S. pneumoniae is less particular about the source of its DNA).
 Transduction


 Bacterial viruses or bacteriophages participate in the third mode of bacterial gene transfer


 After infecting the host cell, a bacteriophage (phage for short) often takes control and forces the host to make many copies of the virus. Eventually the host bacterium bursts or lyses and releases new phages. This reproductive cycle is called a lytic cycle because it ends in lysis of the host.



 Bacterial viruses that reproduce using a lytic cycle often are called virulent bacteriophages because they destroy the host cell.Many DNA phages, such as the lambda phage


 The genome remains within the host cell and is reproduced along with the bacterial chromosome. A clone of infected cells arises and may grow for long periods while appearing perfectly normal. Each of these infected bacteria can produce phages and lyse under appropriate environmental conditions. This relationship between the phage and its host is called lysogeny

 Bacteria that can produce phage particles under some conditions are said to be lysogens or lysogenic, and phages able to establish this relationship are temperate phages.


 The latent form of the virus genome that remains within the host without destroying it is called the prophage. The prophage usually is integrated into the bacterial genome .

• Sometimes phage reproduction is triggered in a lysogenized culture by exposure to UV radiation or other factors. The lysogens are then destroyed and new phages released. This phenomenon is called induction.



• Generalized Transduction
Generalized transduction occurs during the lytic cycle of virulent and temperate phages and can transfer any part of the bacterial genome.

 The resulting virus particle often injects the DNA into another bacterial cell but does not initiate a lytic cycle. This phage is known as a generalized transducing particle or phage and is simply a carrier of genetic information from the original bacterium to another cell



• Abortive transductants are bacteria that contain this nonintegrated, transduced DNA and are partial diploids.

• Generalized transduction was discovered in 1951 by Joshua Lederberg and Norton Zinder(experiments with Salmonella typhimurium)


• Specialized Transduction
In specialized or restricted transduction, the transducing particle carries only specific portions of the bacterial genome. Specialized transduction is made possible by an error in the lysogenic life cycle.

 The best-studied example of specialized transduction is the lambda phage


 lysates (product of cell lysis) contain only a few transducing particles,they often are called low-frequency transduction lysates (LFT lysates).

 SUMMARY


 In recombination, genetic material from two different chromosomes is combined to form a new, hybrid chromosome. There are three types of recombination:general recombination, site-specific recombination, and replicative recombination.

 Bacterial recombination is a one-way process in which the exogenote is transferred from the donor to a recipient and integrated into the endogenote.

 Plasmids are small, circular, autonomously replicating DNA molecules that can exist independent of the host chromosome. Their genes are not required for host survival.


 Episomes are plasmids that can be reversibly integrated with the host chromosome.

 Many important types of plasmids have been discovered:F factors, R factors, Col plasmids, virulence plasmids, and metabolic plasmids.


 Transposons or transposable elements are DNA segments that move about the genome in a process known as transposition.

 There are two types of transposable elements: insertion sequences and composite transposons.


 Transposable elements cause mutations, block translation and transcription, turn genes on and off, aid F plasmid insertion, and carry antibiotic resistance genes.

 Conjugation is the transfer of genes between bacteria that depends upon direct cell-cell contact mediated by the F pilus.


 In F+ * F- mating the F factor remains independent of the chromosome and a copy is transferred to the F- recipient; donor genes are not usually transferred .

 Hfr strains transfer bacterial genes to recipients because the F factor is integrated into the host chromosome. A complete copy of the F factor is not often transferred .


 When the F factor leaves an Hfr chromosome, it occasionally picks up some bacterial genes to become an F'plasmid, which readily transfers these genes to other bacteria .


 Transformation is the uptake of a naked DNA molecule by a competent cell and its incorporation into the genome .

 Bacterial viruses or bacteriophages can reproduce and destroy the host cell (lytic cycle) or become a latent prophage that remains within the host (lysogenic cycle).


 Transduction is the transfer of bacterial genes by viruses.

 In generalized transduction any host DNA fragment can be packaged in a virus capsid and transferred to a recipient.


 Temperate phages carry out specialized transduction by incorporating bacterial genes during prophage induction and then donating those genes to another bacterium.


 The bacterial genome can be mapped by following the order of gene transfer during Hfr conjugation; transformational and transductional mapping techniques also may be used.
 When two viruses simultaneously enter a host cell, their chromosomes can undergo recombination.
 Virus genomes are mapped by recombination and hetero (duplex mapping techniques.








JSR