BACTERIAL GENETICS All the properties of the cell, including its virulence, pathogenicity and antibiotic resistance, are

determined by it genome. This is encoded by the specific sequences of nucleotide bases in the DNA. Recall that the bacterial DNA is a single, circular dsDNA, which does NOT have a nuclear membrane and has NO histones. E.coli is an exception since its DNA is an irregular coiled bundle lying freely in the cytoplasm. In addition to the main ds-DNA, some bacterial cells may also carry one or more extra-chromosomal elements, which appear circular, and are termed PLAMIDS. Plasmids replicate INDEPENDENTLY of the main chromosome in the cell. They carry supplemental genetic information coding for certain properties such as antibiotic resistance or the ability to survive harsh environments. A third source of genetic information in the bacterial cells is provided by BACTERIOPHAGES. These are also termed bacterial viruses. They consists of a viral genome enclosed by a protein coat/ They need a bacterial host in order to multiply, and when they do so, they cause death of the bacterial cell. In some instances though, they can undergo LYSOGENY-they unergo controlled, long term replication within the bacterial cell without causing the bacterial cell’s lysis. In this way, it may become a temporary part of the cell, and change the bacterial cell’s properties. Table 1. Comparison between the 3 types of genetic information sources in the bacterial cell Genetic element Bacterial chromosome Plasmids Bacteriophages DNA TYPE DNA Configuration ds circular ds circular RNA/DNA ss/ds Size in kilobases 2.0 to 4.0 x10 9 to 60 3 to 40

Linear/circular

3 Principal mechanisms of genetic material transfer into a HOST organism: 1. TRANSFORMATION

2. TRANSDUCTION 3. CONJUGATION

TRANSFORMATION Transformation is the uptake and integration of naked DNA from the environment into the host cell. Once inside the cell, there must be homologous recombination with the chromosome of the host. Most species of bacteria are unable to take up exogenous DNA from their environment; in fact, they produce nucleases to recognize and breakdown these foreign DNA. Haemophilus and certain Bacillus however, can take up DNA through transformation. They are able to do this only in LOG PHASE or in the period of exponential growth. The Bacillus species will take it up in their SPORE phase. Transformation can be induced artificially in the lab. Incubation of E.coli at 4C, in the presence of calcium ions, then a short exposure to 42C will allow structural alterations in the cell wall, allowing passage of the DNA into the E.coli cell. In general, ANY gene may be transferred by transformation, however, this fragment is usually small and will contain a limited number of genes. Once inside the bacterial cell, the DNa has to be incorporated into the existing chromosome through RECOMBINATION in order to survive. The pieces of DNA can only recombine with the chromosome if there is a high degree of nucleic acid similarity, also known as HOMOLOGY . Recall that the bases in the DNA are paired in such a way: adenine always pairs with thymine, and guanine always pairs with cytosine. An example of this was a study conducted by Dr Frederick Griffith in the 1920’s using S.pneumoniae. After injecting heat-killed S colonies with R colonies into mice, he was able to isolate live S colonies. He concluded that the heat released the gene encoding for the capsule, and was taken up into the R colonies, thus transforming them into S colonies.

TRANSDUCTION This is the PHAGE-MEDIATED transfer of bacterial DNA. PHAGES are bacterial viruses with a DNA molecule enclosed in a protein shell. The structure of the protein shell imposes a limitation on the amount of DNA that can be enclosed by the virus, usually about 50 kilobases. Most phages carry ds-DNA coiled up inside a protein coat. There may be species of phages which have a ss-DNA or ss-RNA as well.

There are two kinds: 1. Generalized transductionWhen phages enter a bacterial cell and replicate, each phage head usually has a copy of the replicated phage genome. Sometimes, an empty phage head is produced. Bacterial DNA may be mistakenly packaged into that empty phage head, then transferred to another bacterial cell. Thus, the DNA that enters the second bacterial cell is a short segment of the chromosome of the first bacterial cell. As with transformation, recombination must occur for transduction to be successful. It is termed generalized since the phage will pick up any portion of the bacterial chromosome at random (is is not specific for a particular chromosomal segment). Phages usually attack a limited range of bacteria, so there is a close relation between these specias, so the genes transferred are within a certain species only. Phages can also pick up and transfer plasmid DNA. For example, the penicillinase gene of Staphlococci is located in a plasmid, and the phages can pick up that plasmid and through transduction, pass it on to another cell, thus transferring the penicillanase gene to other staphylococci species.

2. Specialized transductionBacteriophages that lyse the host cell are known as VIRULENT PHAGES, and cause a LYTIC cycle of infection. In contrast, phages that can infect a bacterial cell without causing its death are called TEMPERATE PHAGES. The bacterial cell which survives after phage infection is called a LYSOGEN, and the integrated temperate phage inside of it is termed a PROPHAGE.

The phage DNA is then inserted in to the bacterial cell DNA,and is also replicated as part of the host cell chromosome. Can more that 1 phage infect a bacterial cell?

No. Once the phage is integrated into the bacterial cell DNA, it imparts immunity against superinfection by other phages.

The lysogen phase is stable but not permanent. If the prophage is excised during the process of replication, the bacterial cell may eventually be lysed. This can be replicated in the lab by exposure of bacteria to UV light. (e.g. hospital sterilization of rooms with UV).

In contrast to generalized transduction, temperate phages normally have a specific insertion site on the chromosome and can pick up only a short length of DNA containing a few genes on either side of this site. Thus, it is termed SPECIALIZED OR RESTRICTIVE TRANSDUCTION.

Defective phages can occur. As it attaches to the bacterial DNA chromosome and picks up DNA adjacent to the phage integration site, the amount of DNA may be too much, thus the phage will lack a few phage genes. When that defective phage is transduced to a second bacterial cell, the phage can still integrates into its phage specific site, but it cannot replicate normally nor can it lyse the bacterial cell. The final product is the integration of the 1 st bacterial cell’s DNA into the 2nd bacteria’s DNA.

How does this influence the virulence of the bacteria for humans?

Since the prophage carries genes, its presence in a bacterial cell may cause the expression of certain proteins. This is called LYSOGENIC CONVERSION. For example, the diphtheria toxin of Corynebacterium diptheriae will only be produced if the Beta Phage infects the bacteria. Another common use for the phages are the LAMBDA PHAGES of E.coli. They have a genome of 50 kilobases, but one the nonessential genes are removed, they are a reduced to 30 kilobases, making room for the insertion ofr foreign DNA. The phage then infects E.coli, which in turn replicates along with the phage (phagesynthesizing factory), then the E.coli cell is lyzed and the phages infect other cells. Why E.coli? it is the ideal host since it replicates every 20 minutes. Also, E.coli has all the distinct phases in the bacterial growth curve, so its growth can be monitored in the lab.

CONJUGATION

This is the most important mechanism for widespread transfer of genetic information between bacteria. It is the direct transfer of bacterial DNA between organisms and requires CELL-TO-CELL CONTACT. Most conjugation is PLASMID- MEDIATED. PLASMIDS are small, circular, ds-DNA molecules, measuring about 2-10 kilobases. They all have the ability to replicate in bacteria. Multiple copies of plasmids can be found in a host cell, however, not all plasmids can transfer themselves. A CONJUGATIVE plasmid has the codes for the genes allowing transfer between cells. A NONCONJUGATIVE plasmid requires the help of a conjugative plasmid to transmit itself to another bacteria. NARROW-HOST RANGE PLASMIDS exist only within a single species. BROAD-HOST RANGE PLASMIDS exist in different genera of organisms. Plasmids contain the following: 1. And ORI or origin of replication that allows autonomous replication 2. One or more genes that confer specific antibiotic resistance 3. Sites for restriction endonucleases which are used for inserting specific DNA fragments. 4. Encode for virulence factors such as toxins or adhesins Plasmids capable of mediating conjugation carry genes coding for the production of a PILUS, a protein appendage which is found on the surface of the donor cells. Bacteria which carry these plasmids are called F(+) cells, making them the donor cells The tip of the pilus attaches to the surface of the recipient cell, and DNA transfer occurs. It is unsure whether the DNA is transferred through the pilus, or if the pilus serves as the mechanism for holding the two cells together. In either way, this is the reason why it has also been termed BACTERIAL SEX. Once the recipient cell acquires the F plasmid, it changes from a F(-) to a F (+) cell, thus allowing it to become a donor cell now. In a very small proportion of cells, the F plasmid is incorporated into the bacterial chromosome. Once inserted, the entire bacterial chromosome acts like a F plasmid. These are then called high frequency recombination strains (Hfr strains). This leads to 2 processes: 1. The F plasmid and the entire bacterial DNA undergoes conjugation with an F(-) cell.

2.

The F plasmid is excised along with a portion of the bacterial DNA, hence the F plasmid is termed F prime (F’)

For instance, the F-lac plasmid is an F’ plasmid carrying carrying the genes for E.coli lactose operon. When this plasmid is transferred to non-lactose formers, they are converted to lactose fermenters. Note that the F plasmid is confined to the Escherichia genus and other closely related enteric bacteria.

THE GENETIC BASIS OF ANTIBIOTIC RESISTANCE Thus, all of the properties of a micro-organism are determined by the genes located either on the chromosome, on the plasmid or on the bacteriophage. There are 2 types of resistance: 1. Intrinsic resistance Organisms that are a naturally insensitive to a particular drug will always exit. The most obvious determinant of bacterial response to an antibiotic is the presence or absence of the target for the action of the drug. For example: Antifungals like amphotericin B bind to the sterols on the fungal cell wall, altering it permeability, thus killing it. However, bacterial cells do not have sterols, hence they have an intrinsic resistance to the drug. 2. Acquired resistance. Three main factors affect the frequency of acquired resistance: A. The amount of antibiotic being used B. The frequency with which bacteria can undergo spontaneous mutations to reistance C. The prevalence of plasmids able to transfer resistance from one bacterium to another. How does it happen? 1. Chromosomal mutations Random spontaneous mutations occur continuously at a low frequency in all bacterial populations. These mutations may allow the resistant mutant to survive, grow and eventually become the predominant or only member of the population.

Mutation can occur by base substitution, that is, one base is inserted in place of another. It takes place at the time of DNA replication. When the base substitution results in a codon that simply causes a different amino acid to be inserted, the mutation is called a MISSENSE mutation. When the base substitution generates a termination codon that stops protein synthesis prematurely, this is called a NONSENSE mutation. Mutation may also occur by FRAMESHIFT; this occurs when one or more base pairs are added or deleted, which in turn shifts the reading frame on the ribosome, resulting in a wrong amino acid, and the production of a n inactive protein. In SINGLE LARGE –STEP MUTATIONS, the cell is able to carry out its biological functions but the drug target site is altered. If some affinity is still present to the drug, increasing the minimum inhibitory concentration may cause an effect on the bacteria. In MULTISTEP PATTERN OF RESISTANCE, the development of resistance starts slightly, then with each new mutant formed , the resistance increase, eventually leading to an organism which is highly resistant. This is the principle behind the multidrug therapy for pulmonary tuberculosis. Each drug kills the few mutants that are resistant to the other. Another example of chromosomal mutation leading to antibiotic resistance are those involving Beta-lactamases. These are enzymes produced by Staphylococcus organisms, act on the Beta-lactam ring in penicillins and cephalosporins. Mutations in the genes that encode for these enzymes can cause an overproduction, leading to drug resistance. Another is the resistance gene mecA which coded for a unique penicillinbinding protein found in methicillin-resistant S.aureus. It renders the bacteria resistant to B-lactam drugs, as well as aminoglycoside and quinolones. Thus, vancomycin is used, acting on cell wall synthesis. 2. Plasmid- mediated conjugation Of the 3 modes of gene transfer, this is of greatest importance with regards to drug resistance. R PLASMIDS are plasmids which confer resistance to one or more unrelated groups of antibiotics. They were first demonstrated in Japan in the 1950’s, where resistance to several antibiotics could be transferred by conjugation between strains of Shigella and E.coli.

This is further accelerated by the presence of TRANSPOSONS. A transposon consists of two insertion sequences flanking another gene or set of genes. The Insertion sequences are small pieces of DNA that code for the enzyme TRANSPOSASE, which allow them to jmpin and out of DNA. Meaning, the transposons can insert themselves into a donor chromosome WITHOUT having DNA homology. They do not replicate independently, but are copied as their host’s DNA is transcribed. The ability of the transposon to confer resistance is by the presence of INTEGRON, the building block of all transposons, and it allows the rapid formation and expression of new combinations of antibiotic resistance genes. The ability to move between chromosomes, plasmids or bacteriophages is an efficient method for moving genes in a bacterial population. Thus, they are frequently associated with the formation of multiple-drug resistant plasmids. This is the most common cause of drug resistance in Gram(-) bacteria.

Self study: LANDMARKS IN GENETICS, DNA STRUCTURE AND CHROMOSOME FUNCTION chapter 11, pages 75 -78