2024 Bacterial Genetics (PDF)

Chapter II. Bacterial genetics Many discoveries in molecular genetics are the result of work conducted on bacteria and bacteriophages (viruses called phages, that infect bacteria). 4.1 General information A bacterium is a unicellular prokaryotic microorganism, varying in size from 1 to 10 microns....

Chapter II. Bacterial genetics Many discoveries in molecular genetics are the result of work conducted on bacteria and bacteriophages (viruses called phages, that infect bacteria). 4.1 General information A bacterium is a unicellular prokaryotic microorganism, varying in size from 1 to 10 microns. It has a single chromosome, usually in circular form (e.g. Escherichia coli). Chromosomal DNA consists of a coiled double helix, supercoiled in the cytoplasm. Chemical analysis of the nuclear apparatus shows that it is composed of 80% DNA (the chromosome), 10% RNA (structural role) and 10% proteins. In addition to the chromosome, the bacterium may contain small extrachromosomal genetic elements (0.5 to 5% of the bacterial chromosome) called plasmids. Bacteria divide by scissiparity (bipartition), with one cell dividing into two every 20 minutes. Note: Exceptions are found in spirochetes (e.g., Treponema, Borrelia, Leptospira), where the chromosome is linear. Bacteria of the genus Brucella have two chromosomes. 4.1.1 Nutritional Requirements Constitutive requirements: elementary: C, H, O, N, essentially from a sugar. Specific: Growth factors for certain bacteria. energy requirements covered by oxidation-reduction reactions Bacteria can grow in either a liquid culture medium or semi-solid agar in Petri dishes. If the medium contain a carbon source (e.g., fructose or lactose) and a variety of ions (Na+, K+, Mg++, Ca++, and NH4+) in the form of inorganic salts, the medium is called a minimum medium. To grow on such a medium, a bacterium must be able to synthesise all the essential organic compounds (amino acids, vitamins, fatty acids, etc.), and is named prototrophic or wild type. These bacteria form colonies on a minimum medium: water + mineral salts + glucose (water + mineral salts = mineral medium). The bacteria requires nitrogen (N) for amino acid synthesis, which is obtained from the culture medium in its mineral form. Wild-Type Strain: A strain isolated from the natural environment that has not undergone any treatment with mutagens or genetic manipulations, often naturally prototrophic. E. coli K-12 is considered a wild strain. It is prototrophic, capable of catabolising many sugars, sensitive to most bacteriophages, thermoresistant (growth up to 45°C), non- pathogenic and sensitive to most antibiotics. If the bacterium loses the ability to synthesize one or more organic compounds due to mutation, it becomes auxotrophic. 41 4.1.2 Mutations A mutation is a change, either spontaneous or caused by a mutagenic agent, which is hereditary (stable), abrupt (discontinuous), rare (10-6 to 10-9) and independent in the characteristics of a bacterium, and is linked to a modification of the bacterial genome (DNA). The mutation may be reversible (reverse mutation). 4.1.2.1. Nutritional This type of mutation inactivates biosynthesic pathways and results in an auxotrophic mutant, which cannot grow on a minimal medium. which cannot grow on a minimal medium. Example: A prototrophic bacterial strain for histidine has the his+ gene on its chromosome. The mutated strain loses the ability to synthesise histidine and the gene becomes his-. The amino acid must be added as a supplement to the medium for the strain to grow. A supplemented medium is called a complete medium. 4.1.2.2. Carbon source Example: a bacterial strain which degrades galactose has a Gal+ gene and is supplied with this sugar in the culture medium. The mutated strain loses the ability to degrade this sugar, so it is not added to the culture medium, and glucose is used instead, which can be degraded by all bacterial strains. 4.1.2.3. Antibiotic resistance The wild strain is sensitive to antibiotics, phages and poisons. Some mutants can acquire resistance. If the antibiotic targets an enzyme, mutation in the gene coding for this enzyme can reduce or abolish the affinity of the antibiotic for the enzyme (target modification). An example is methicillin resistance in Staphylococcus aureus. Examples -A strain sensitive to streptomycin has a strs gene, After mutation its gene becomes strr ; -A strain sensitive to phage T4, has a T4s gene, After a mutation its gene becomes T4r. 4.1.3. Selection of strains 4.1.3.1. Selective media A selective medium is a culture medium that favours the growth of a single strain. A sugar, an amino acid or a growth factor can be used to make the medium selective. Phage, antibiotic and poison are also used. 4.1.3.2. Velvet replication technique (Joshua and Esther Ledberg, 1952) - Molecular origin of spontaneous mutations A bacterial population is spread out on a non-selective medium - without phage - and a colony develops from each cell. A sterile velvet piece is lightly pressed against the surface of the matrix box and the velvet attaches cells as soon as there is a colony (figure 1). 42 Figure 1 The velvet replica technique. It allows the identification of mutant colonies on a mtrix plate, based on their behaviour on plates containing a selective medium. (G.S. Stent and R. Calendar, Molecular Genetics, 2nd ed. W. H. Freeman and Company, 1978.) In this way the velvet carries an ‘imprint’ of the colonies present in contact with replica dishes containing a selective medium (i.e. T1 phages). During contact between the velvet and the replica plates, the cells present on the velvet are inoculated into these replica plates in the same relative positions as their original colonies in the matrix plate. Rare mutant colonies were observed on the replicate dishes, but the multiple replicate dishes showed identical patterns of resistant colonies (Figure 2). A series of replicate dishes containing high concentrations of phage T1 and four Tons colonies. The velvet replica technique demonstrates the presence of mutants before selection. Figure 2 The same colonies appear on all three replicates, proving that the resistant colonies existed on the matrix dish, (Adapted from G. S. Stent and R. Calendar, Molecular Genetics, 2nd ed. W. H. Freeman and Company, 1978). 43 If the mutations had appeared after exposure to the selective agents, the colony patterns observed on the boxes would have been as random as the mutations. Therefore, it was concluded that the mutational events occurred prior to exposure to the selective agent. Mutation is a random process. Any allele in a cell can be affected at any time. Application exercise We consider 3 mutant strains of Escherichia coli A, B and C with the following genotypes: A: Leu+ His+ Ala- Gal+ Mal- Arg- B : Leu+ His- Ala+ Gal+ Mal+ Arg+ C: Leu+ His- Ala- Gal- Mal+ Arg+ 1- Give the composition of the culture medium for each of these strains. 2- Give the composition of the culture medium suitable for mixing the 3 strains. 3- Give the composition of a selective medium for each strain. 4.2 Genetic material transfers: Parasexuality in bacteria Bacteria can undergo genetic variation other than mutation. These variations can result from the transfer of genetic material from one bacterium to another through processes as different as conjugation, transformation, and transduction. In prokaryotes, genetic transfers are unidirectional. A fragment of exogenous genetic material enters a recipient cell. This DNA may persist in the cytoplasm (as in many plasmids) or be integrated into the bacterial chromosome through recombination. 4.2.1. Bacterial conjugation (Joshua Ledberg and Edward Tatum, 1946) The experiments were conducted with two multi-auxotrophic strains (nutritional mutants) of E. coli K12. Strain A required the presence of methionine (met) and biotin (bio) to grow, while strain B required the presence of threonine (thr), leucine (leu) and thiamine (thi). The two strains cannot grow on a minimal medium. - The strains are first placed separately on differently supplemented media, then ; - the cells are mixed and cultured for several generations, then ; - they are plated on a minimum medium (figure 3). Genetic recombination: replacement of one or more genes present in a strain by those of a genetically distinct strain. The genetic information of one chromosome is transferred to another chromosome, resulting in a change of genotype. 44 Figure 3 Genetic recombination between two auxotrophic strains. Prototrophs appear at a rate of 10-7. Ledberg and Tatum suggest the existence of genetic recombination. 4.2.1.1. Physical nature and genetic basis of recombination - The transfer of genetic material is unidirectional. When cells donate DNA, they are called F+ (F for fertility or sexual factor) or male bacteria; - Receiving bacteria receive and recombine this DNA with part of their own chromosome, are called F- or female bacteria. Bernard Davis experiment (1950) F+ and F- cells were cultured in a U-shaped tube. A glass filter with pores allowing the passage of liquid but not bacteria was inserted at the base of the tube. F+ cells were placed on one side of the filter, and F- cells on the other (figure 4). 45 Figure 4 When strains A and B are cultured in a common medium, but separated by a filter, no recombination occurs and no prototrophs appear. La conjugaison se fait grâce au pilus F ou pilus sexuel. Les pili sont des extensions tubulaires de la cellule (figure 5). Conclusion: physical contact is essential for genetic recombination. Conjugation occurs through the F pilus or sexual pilus. Pili are tubular extensions of the cell (figure 5). Figure 5: Microphotography of conjugation between F+ and F- E. coli cells. 46 DNA transfer As soon as the cytoplasmic bridge is formed, genetic transfer begins. It involves a strand of DNA, which allows the restoration of the donor bacterium's genome integrity through replication. DNA strand transfer is unidirectional, progressive and sometimes total. It takes one hundred minutes at 37ºC. Interrupting the transfer (mechanical agitation) allows the sequence of the transferred genes to be determined and the chromosome map to be established. 4.2.1.2. F factor It consists of a double-stranded DNA molecule, an autonomous genetic unit called a plasmid: - It replicates independently of the bacterial chromosome; - The genetic information it carries codes for the synthesis of sex pili; - The genes carried by plasmids can code for the synthesis of proteins that confer various biological properties: antibiotic resistance, bacteriophage resistance, resistance to mercurial antiseptics, etc; - They may be integrated into the bacterial chromosome or in free form (episome). Figure 6: F+ × F- cross. During conjugation, the DNA of the F factor is replicated, and a new copy enters the recipient cell, making it F+. 47 Factor F is replicated and a copy of the plasmid is transferred (single stranded) to the recipient cell. 4.2.1.3. High recombination frequency (Hfr) bacteria In 1953, William Hayes isolated a donor strain with a recombination rate of 10-4; called Hfr for high recombination frequency. Factor F contains insertion sequences that allow the integration of the plasmid into the cell's chromosome. When the F factor is integrated into the bacterial chromosome, the cell is called to be ‘Hfr’. - These strains only transfer certain genes at a high frequency (1000x). - These strains do not transfer donor status to the recipient cells F+ X F- -------------- the recipient cells become F+. Hfr X F- ------------ the recipient cells remain F-. During the Hfr x F cross, the entire copy of the Hfr cell's chromosome is rarely transferred to the F- strain because the required contact time is too long (100 minutes in Escherichia coli). The contact between cells is often broken (disruption of the cytoplasmic bridge enabling transfer) before the process is completed He crossed bacterial strains A sensitive to streptomycin (strr) and B resistant to streptomycin (strs), then spread them on : - mineral medium+ glucose+ streptomycin: development of around a hundred colonies; Crossing of strain A (strs) and B (strr) then spread on: - mineral medium+ glucose+ streptomycin: no colonies Conclusion The transfer of genetic material occurs from bacteria A (donor bacteria) to bacteria B (recipient bacteria). 48  Figure 7 Conversion of an F+ state to Hfr Integration of factor F into the bacterial chromosome (step 1). The point of integration defines the origin (O) of the transfer. During the following conjugation (steps 2-4), an enzyme cleaves the F factor, now integrated into the host chromosome, initiating chromosome transfer from this point. Conjugation is usually interrupted before transfer is complete. Only the A and B genes are transferred to the F- cells (steps 3-5), so they can recombine with the host chromosome. 4.2.1.4. Interrupted conjugation (Wollman and Jacob) Interrupted conjugation allows for determining which genes are transferred from Hfr to F- and in what order. It demonstrates that specific genes from a given Hfr strain are transferred and recombined earlier than others. 49 Hfr : thr+ leu+ aziR tonS lac+ gal+ F- : thr- leu- aziR tonS lac- gal- After mixing the two strains, samples are taken at various times and and shaken (to separate the conjugating bacteria and stop chromosome transfer). At 10 minutes, recombination of the aziR gene can be detected, but not that of the tons, lac+ ou gal+ genes. At 15 minutes, the percentage of aziR recombinants has increased, and tons recombinants begin to appear. At 20 minutes, the lac+ gene is found among the recombinants, and at 30 minutes, gal+ began to be transferred. The thr and leu genes are not shown (figure 8). 100 Fréquence relative de recombinaison Temps de conjugaison Figure 8. Progressive transfer of various genes from an Hfr strain of E.coli to an F- strain. The azi and ton genes are transferred earlier than others and recombine more frequently. 4.2.2. Bacterial transformation This process allows genetic recombination in some bacteria (Bacillus subtilis, H. influenzae, N. meningitidis, S. pneumoniae, etc). Small fragments of extracellular DNA are taken up by living bacteria in a state of competence (bacteria capable of accepting exogenous DNA), leading to a stable genetic change in the recipient cell (figure 9). Transferred DNA constitutes 1% of the genome, and the frequency of transfer is of the order of 10-4 à 10-6. 50  Figure 9 Stages in the transformation of a bacterial cell by exogenous DNA. Only one of the two strands of incoming DNA is involved in the transformation event, which is completed after the next cell division. 4.2.3. Transduction: transfer of bacterial DNA via viruses 4.2.3.1. Experiments by Norton Zinder and Joshua Ledberg (1952) Recombination in Salmonella typhimurium Using mixed cultures of two auxotrophic strains LA-22 and LA-2 ; LA-2 : phe+ trp+ met- his- LA-22 : phe- trp- met+ his+ They obtained prototrophic cells (phe+ trp+ met+ his+) at a rate of about 10-5. Analysis using a Davis U-tube showed that this recombination was not due to the presence of factor F and conjugation as in E. coli (Figure 10). 51  Figure 10 Lederberg-Zinder experiment in Salmonella. The LA-2 cells appeared to be the source of the new genetic information (phe+ and trp+). It was suggested that a filtering agent (AF) was responsible for transferring this information. Three observations led to the identification of AF: 1. AF is produced by LA-2 cells only when they grow in association with LA-22 cells. If LA-2 cells are cultured separately and their culture medium is added to LA-22 cells, recombination does not occur. Therefore, LA-22 cells play a role in the production of AF by LA-2 cells. 2. The addition of DNase, which digests naked DNA, does not inactivate AF, ruling out transformation. 3. AF does not pass through the filter when the pore size is smaller than that of bacteriophages. The AF is the bacteriophage P22, present as a prophage on the chromosome of LA-22 cells. The P22 enters the lytic phase and is released by LA-22 cells, passes through the filter, and infects some LA-2 cells. During the lysis of LA-2, the phages can package a piece of the LA-2 chromosome. The phage then passes through the filter again to infect LA-22 cells. The new lysogenised cells can behave as prototrophs. 52  Figure 11 Generalised transduction 4.2.3.2. The nature of transduction Other studies have revealed the existence of transducing phages in other bacterial species (E. coli, Bacillus subtilis and Pseudomonas aeruginosa). - Specialised transduction A small fragment of bacterial DNA is packaged with the viral chromosome, and only a few genes present in the transducing phage. This is an abnormal excision of the prophage when a productive cycle follows a lysogenic phase. Instead of the phage DNA being excised in its entirety. An "hybrid" DNA molecule composed of a fragment of phage DNA and a fragment of bacterial DNA is excised. This bacterial DNA fragment is adjacent to the prophage's integration site. The phages involved are temperate phages whose genome has the ability to integrate (prophage) and excise at specific points on the bacterial genome (strict integration site). 53 This is the case with bacteriohage , whose genome integrates into the chromosome of E. coli K12 between the Gal and Bio genes. By excision of the bacterial chromosome, two types of genes will be transduced:  gal et  bio. - Generalised transduction The phage DNA is completely excluded and only the bacterial DNA is packaged (erroneously and randomly). It is the bacterial DNA that is injected instead of the viral DNA; it may remain in the cytoplasm, or recombine with the homologous region of the host chromosome. Transducing phages are DNA viruses (T4 and P1 in E. coli, P22 in Salmonella typhimurium). If the bacterial DNA remains in the cytoplasm, it does not replicate and is transmitted to one of the daughter cells during each division. This phenomenon is known as abortive transduction. If the bacterial DNA recombines with the region of the host chromosome that is homologous to it, the transduced genes are replicated as an integral part of the chromosome and are transmitted on to all the daughter cells. This process is called complete transduction. 4.3 Mixed infection in viruses 4.3.1. Mapping in bacteriophages Genetic recombination in bacteriophages was discovered during mixed infection experiments, in which two different mutant strains can simultaneously infect the same bacterial culture (Hershey experiments). These experiments are performed so that the number of viral particles exceeds the number of bacteria, so that most bacteria are simultaneously infected by both types of virus. In a study using the T2/E. coli system, the parental viruses are either of the h+ r genotype (wild-type host spectrum, rapid lysis) or of the h r+ genotype (extended host spectrum, wild-type lysis). Note. Wild-type plaques are small, mutant plaques are large. In the progeny h+ r+ and h r recombinants were detected in addition to the parental genotypes. Recombination frequency = (h+ r+) + h r/ total number of plaques X 100 Two loci are involved, the recombination is qualified as intergenic. 4.3.2. Intragenic recombination in the T4 phage: the rII locus A detailed study of the rII locus of phage T4 was carried out (Seymour Benzer experiments). Mutants of the rII locus produce detectable plaques of lysis after spreading E. coli B. In addition to lysis of E. coli B, rII mutants are unable to grow in E. coli K12 (λ). Wild-type phages can lyse both B and K12 strains. 54 If phages from two mutant strains simultaneously infected E coli B, exchanges between mutant sites in the locus would produce rare wild-type recombinants, which would replicate and produce wild-type phages (figure 12). Figure 12. Intragenic recombination between two mutants of the rII locus of phage T4 complémentation. K12 bacteria infected simultaneously with pairs of different rII mutant strains show that some pairs lyse the K12 bacteria. This is complementation. The fact that two mutants can complement each other suggests that the rII region contains two functional units, both of which are necessary for the wild-type phenotype. Cistrons Cistrons Figure 13.1. Two mutations in different cistrons: Complementation 55  Cistrons Cistrons Figure 13.2. Two mutations in the same cistron: No complementatio The phages carrying two mutations are complemented by a wild-type genome; cis- complementation. However, the result is unpredictable when the two mutations are carried by two different phages: a trans situation. These observations led to the term cistron; the smallest functional genetic unit used to describe a complementation group. In modern terminology, a cistron represents a gene. Cistron. A portion of a DNA molecule coding for a single polypeptide chain; defined by a genetic test as a region within which two mutations cannot complement each other. 56