The Role of recN in stationary phase mutagenesis in bacillus subtilis

Here, we examine mutagenic programs that are independent of growth, such aspects of the evolutionary process are novel and have been implicated in the formation of cancers in animal cells and the acquisition of antibiotic resistance in animal pathogens. Adaptive or stationary phase mutagenesis is a genetic program to in increase diversity in cells under conditions of stress whereby cells escape non-dividing conditions. Previous research has shown that recombination functions are required to generate mutations that promote growth in Escherichia coli cells starved for carbon. This project tests the hypothesis that recombination functions are required for the generation of mutations that promote growth in response to amino acid starvation stresses in Bacillus subtilis cells. In B. subtilis cells, recN, in addition to recA, mediates recombination events and may influence the formation of adaptive mutations. A RecN- strain will be generated by standard molecular techniques and compared to a RecN+ strain for its ability to accumulate mutations that affect amino acid biosynthesis. We speculate that recN does not affect stationary phase mutagenesis in B. subtilis and discussed other novel mechanisms mediating the generation of mutations in non-dividing cells

Horizontal DNA transfer mechanisms of bacteria as weapons of intragenomic conflict

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated

Optimal Strategy for Competence Differentiation in Bacteria

A phylogenetically diverse subset of bacterial species are naturally competent for transformation by DNA. Transformation entails recombination of genes between different lineages, representing a form of bacterial sex that increases standing genetic variation. We first assess whether homologous recombination by transformation is favored by evolution. Using stochastic population genetic computer simulations in which beneficial and deleterious mutations occur at many loci throughout the whole genome, we find that transformation can increase both the rate of adaptive evolution and the equilibrium level of fitness. Secondly, motivated by experimental observations of Bacillus subtilis, we assume that competence additionally entails a weak persister phenotype, i.e., the rates of birth and death are reduced for these cells. Consequently, persisters evolve more slowly than non-persisters. We show via simulation that strains which stochastically switch into and out of the competent phenotype are evolutionarily favored over strains that express only a single phenotype. Our model's simplicity enables us to derive and numerically solve a system of finite- deterministic equations that describe the evolutionary dynamics. The observed tradeoff between the benefit of recombination and the cost of persistence may explain the previously mysterious observation that only a fractional subpopulation of B. subtilis cells express competence. More generally, this work demonstrates that population genetic forces can give rise to phenotypic diversity even in an unchanging and homogeneous environment

Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks

Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis

Inducible error-prone repair in B. subtilis. Progress report, September 1, 1978-August 31, 1979. [Role in mutagenesis]

The mechanism of activation and the mode of action of the SOS system in the bacterium Bacillus subtilis is under study. Interesting aspects of the SOS system in B. subtilis are: (1) the differences between SOS functions in this bacterium and in the enteric bacteria; (2) the spontaneous activation of SOS functions in component cells; and (3) the difficulty in obtaining consistent results for mutation studies in this bacterium. In order to characterize the SOS system of B. subtilis, it was proposed to: (1) isolate bacteria mutated in genes controlling various repair function; (2) investigate inducible repair; (3) determine the role of endogeneous Bacillus prophages in SOS functions; and (4) develop a tester system for potential carcinogens from competent Bacillus subtilis cells. Research has been able to: (1) isolate strains of B. subtilis in which the endogeneous prophages have been removed or neutralized; (2) demonstrate the association of one SOS function with prophage SPB; (3) demonstrate that the survival of uv-irradiated B. subtilis is not significantly altered by the removal and neutralization of the endogeneous prophages; (4) develop competant B. subtilis into a tester system; and (5) show that DNA polymerase III is absolutely necessary for W reactivation. In addition, uv and mitomycin C resistant mutants have been isolated and inducible postreplication repair in excision-repair deficient mutants of B. subtilis has been studied. The last two results are somewaht confusing but highly exciting in regards to DNA repair mechanisms in B. subtilis

Inducible error-prone repair in B. subtilis. Final report, September 1, 1979-June 30, 1981

The research performed under this contract has been concentrated on the relationship between inducible DNA repair systems, mutagenesis and the competent state in the gram positive bacterium Bacillus subtilis. The following results have been obtained from this research: (1) competent Bacillus subtilis cells have been developed into a sensitive tester system for carcinogens; (2) competent B. subtilis cells have an efficient excision-repair system, however, this system will not function on bacteriophage DNA taken into the cell via the process of transfection; (3) DNA polymerase III is essential in the mechanism of the process of W-reactivation; (4) B. subtilis strains cured of their defective prophages have been isolated and are now being developed for gene cloning systems; (5) protoplasts of B. subtilis have been shown capable of acquiring DNA repair enzymes (i.e., enzyme therapy); and (6) a plasmid was characterized which enhanced inducible error-prone repair in a gram positive organism

Inducible error-prone repair in B. subtilis. Progress report, September 1, 1979-February 28, 1981

The mechanism of activation and the mode of action of the SOS system in Bacillus subtilis are being investigated. Interesting aspects of the SOS system in B. subtilis include: (1) the differences between the SOS functions in this bacterium and in the enteric bacteria; (2) the spontaneous activation of SOS functions in competent cells; and (3) the difficulty in establishing the presence of error-prone repair in this bacterium. In order to characterize the SOS system of B. subtilis, attempts will be made to: (1) isolate bacteria mutated in genes controlling various repair functions; (2) investigate inducible repair; (3) determine the role of endogenous prophages in DNA repair phenomena; and (4) utilize competent B. subtilis as a tester system for the detection of potential carcinogens. Data obtained during the past 18 months demonstrate: (1) the ability of the B. subtilis Comptest to detect potential environmental carcinogens; (2) the importance of DNA polymerase III in W-reactivation in B. subtilis; and (3) the control the bacteriophage SP..beta.. has over the inducible DNA modification system in B. subtilis. Furthermore, the data also suggests the lack of error-prone repair in B. subtilis, and the differences which exist between the Bacilli and the enteric bacteria with regards to SOS phenomena. In order to further characterize inducible repair functions in B. subtilis, results will also be presented on attempts to mobilize error-prone repair systems of other bacterial species

Cloning of the Bacillus subtilis recE+ gene and functional expression of recE+ in B. subtilis.

By use of the Bacillus subtilis bacteriophage cloning vehicle phi 105J23, B. subtilis chromosomal MboI fragments have been cloned that alleviate the pleiotropic effects of the recE4 mutation. The recombinant bacteriophages phi 105Rec phi 1 (3.85-kilobase insert) and phi 105Rec phi 4 (3.3-kilobase insert) both conferred on the recE4 strain YB1015 resistance to ethylmethane sulfonate, methylmethane sulfonate, mitomycin C, and UV irradiation comparable with the resistance observed in recE+ strains. While strain YB1015 (recE4) and its derivatives lysogenized with bacteriophage phi 105J23 were not transformed to prototrophy by B. subtilis chromosomal DNA, strain YB1015 lysogenized with either phi 105Rec phi 1 or phi 105Rec phi 4 was susceptible to transformation with homologous B. subtilis chromosomal DNA. The heteroimmune prophages phi 105 and SPO2 were essentially uninducible in strain YB1015. Significantly, both recombinant prophages phi 105Rec phi 1 and phi 105Rec phi 4 were fully inducible and allowed the spontaneous and mitomycin C-dependent induction of a coresident SPO2 prophage in a recE4 host. The presence of the recombinant prophages also restored the ability of din genes to be induced in strains carrying the recE4 mutation. Finally, both recombinant bacteriophages elaborated a mitomycin C-inducible, 45-kilodalton protein that was immunoreactive with Escherichia coli recA+ gene product antibodies. Collectively, these data demonstrate that the recE+ gene has been cloned and that this gene elaborates the 45-kilodalton protein that is involved in SOB induction and homologous recombination