A. A. Prozorov

Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology

SummaryTo determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between is pE194 derivatives carrying segments of the chromosomal β-gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for β-gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 by of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.

Toxin-antitoxin systems in bacteria: Apoptotic tools or metabolic regulators?

The results of recent (10–12 years) research in the functions of two-gene chromosomal modules are considered and generalized. One of the genes encodes a toxin protein; the product of the other gene is an antitoxin protein. In the course of balanced bacterial growth, the toxin is constantly neutralized by the antitoxin; however, certain metabolic changes (amino acid starvation, etc.) disturb the balance and then the toxin “poisons” the cell (in most cases, by destroying mRNA). As a result, bacterial growth ceases. In accordance with one group of the data, long-term inhibition of growth of most cells results in their programmed death and destruction, corresponding to apoptosis; this allows a minor part of the population to survive due to an additional nutrient source. The results of other works show that growth inhibition is mostly reversible and the functions of the relevant gene modules are restricted to the regulation of cell metabolism, i.e., transition of bacteria to the hypometabolic state. There is also a compromise point of view. The possibilities of biotechnological applications for “toxin-antitoxin” systems are discussed.

Illegitimate recombination in Bacillus subtilis: nucleotide sequences at recombinant DNA junctions.

SummaryThe illegitimate integration of plasmid pGG20 (the hybrid between Staphylococcus aureus plasmid pE194 and Escherichia coli plasmid pBR322) into the Bacillus subtilis chromosome was studied. It was found that nucleotide sequences of both parental plasmids could be involved in this process. The recombinant DNA junctions between plasmid pGG20 and the chromosome were cloned and their nucleotide sequences were determined. The site of recombination located on the pBR322 moiety carried a short region (8 bp) homologous with the site on the chromosome. The nucleotide sequences of the pE194 recombination sites did not share homology with chromosomal sequences involved in the integration process. Two different pathways of illegitimate recombination in B. subtilis are suggested.

Recombinational Rearrangements in Bacterial Genome and Bacterial Adaptation to the Environment

The rearrangement of bacterial chromosomes induced by intragenomic recombination is considered. The role of stochastic and programmed genome rearrangements in bacterial adaptation to the environment and in cell differentiation is discussed.

[The bacterial cell cycle: DNA replication, nucleoid segregation, and cell division].

Data on the bacterial cell cycle published in the last 10–15 years are considered, with a special stress on studies of nucleoid segregation between dividing cells. The degree of similarity between the eukaryotic mitotic apparatus and the apparatus performing nucleoid separation is discussed.

The virulence factors of Mycobacterium tuberculosis: Genetic control, new conceptions

The problem of Mycobacterium tuberculosis virulence, together with drug resistance, is becoming key for the design of drugs with a new mechanism of action and the production of modern concepts and tuberculosis treatment schemes. The review describes gene complexes and their products, including mycolic acids and global regulatory systems at the level of transcriptional, translational, and post-translational modification, etc. The criteria for selection of virulence/pathogenicity factors that might be used for comparative genomic analysis of strains differing in the degree of virulence were recommended. The experimental approaches and test systems for an adequate estimation of the virulence degree of different strains of M. tuberculosis were analyzed.

Mycobacterium tuberculosis mutants with multidrug resistance: History of origin, genetic and molecular mechanisms of resistance, and emerging challenges

The review summarizes the data on the Mycobacterium tuberculosis mutations that lead to multidrug resistance (MDR) to various antibiotics. MDR strains arose over the past 30 years as a variety of antituberculosis drugs were introduced in medicine, and they largely discount the results of chemotherapy for tuberculosis. The most dangerous of them are strains with extensive drug resistance (XDR), which are resistant to four or five different drugs on average. The molecular mechanisms that make a strain resistant are considered. XDR and MDR strains result from successive and usually independent resistance mutations, which arise in various regions of the mycobacterial genome. In addition, the formation of resistant strains is affected by the phenomenon of tolerance and mycobacterial latency in infected tissues.

Conjugation in Bacilli

The review considers experimental data on the conjugal transfer of plasmids in the Bacillus cereus andBacillus subtilis groups (the transfer of large self-transmissible plasmids and the mobilization of small plasmids). Conjugation in bacilli is compared with conjugation in E. coli dependent on the F factor. Conjugation of bacilli in their natural habitats is also discussed.

Additional chromosomes in bacteria: Properties and origin

The review considers papers published over the last 15 years that deal with the presence in cells of some bacterial genera and species of a second chromosome that is smaller than the main one (occasionally, of two additional chromosomes). These additional chromosomes differ from the main one in the set of genes and specific features of replication; however, they carry genes vitally important for the bacterium. The role of these chromosomes and their probable origin from megaplasmids are discussed.

Effect of the clay minerals montmorillonite and kaolinite on the genetic transformation of competent Bacillus subtilis cells.

The effect of the clay minerals montmorillonite and kaolinite on the transformation of competentBacillus subtilis cells with chromosomal DNA was studied. Clay particles were found to substantially increase the transformation frequency of competent cells, as well as the rate of their spontaneous chromosomal and plasmid transformation. The effect was ascribed to the adsorption of bacterial cells on the surface of mineral particles.