Ronald E. Yasbin

New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments

Two new shuttle vectors have been constructed by fusing the Escherichia coli plasmid pUC9 with the Staphylococcus aureus plasmids pU110 and pC194. The resulting hybrids replicate in both E. coli and Bacillus subtilis and contain seven restriction sites within a part of the lacZ gene. Insertion of foreign DNA into those sites can be easily detected in E. coli and hybrid plasmids can subsequently be transformed into B. subtilis.

The genetics and specificity of the constutive excision repair system of Bacillus subtilis

SummaryAn isogenic set of DNA repair-proficient and-deficient strains of B. subtilis, cured of all prophages, were constructed and analyzed for their sensitivities to selected mutagens. The results demonstrated that the lethal damage caused by ultraviolet (UV) radiation and by 4-nitroquinoline-1-oxide (4NQO) were repaired by the bacterial excision and/or recombination repair systems. In contrast, the lethal damages caused by ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS) were removed from the DNA by the recombination repair system of the bacteria, and not by the excision repair system.Significantly, the bacteria required both a functional recombination repair system and a functional excision repair system in order to remove the DNA damage caused by the bifunctional alkylating agent mitomycin C (MC).

Transcription-Associated Mutation in Bacillus subtilis Cells under Stress

Adaptive (stationary phase) mutagenesis is a phenomenon by which nondividing cells acquire beneficial mutations as a response to stress. Although the generation of adaptive mutations is essentially stochastic, genetic factors are involved in this phenomenon. We examined how defects in a transcriptional factor, previously reported to alter the acquisition of adaptive mutations, affected mutation levels in a gene under selection. The acquisition of mutations was directly correlated to the level of transcription of a defective leuC allele placed under selection. To further examine the correlation between transcription and adaptive mutation, we placed a point-mutated allele, leuC427, under the control of an inducible promoter and assayed the level of reversion to leucine prototrophy under conditions of leucine starvation. Our results demonstrate that the level of Leu(+) reversions increased significantly in parallel with the induced increase in transcription levels. This mutagenic response was not observed under conditions of exponential growth. Since transcription is a ubiquitous biological process, transcription-associated mutagenesis may influence evolutionary processes in all organisms.

Contribution of the Mismatch DNA Repair System to the Generation of Stationary-Phase-Induced Mutants of Bacillus subtilis

A reversion assay system previously implemented to demonstrate the existence of adaptive or stationary-phase-induced mutagenesis in Bacillus subtilis was utilized in this report to study the influence of the mismatch DNA repair (MMR) system on this type of mutagenesis. Results revealed that a strain deficient in MutSL showed a significant propensity to generate increased numbers of stationary-phase-induced revertants. These results suggest that absence or depression of MMR is an important factor in the mutagenesis of nongrowing B. subtilis cells because of the role of MMR in repairing DNA damage. In agreement with this suggestion, a significant decrease in the number of adaptive revertant colonies, for the three markers tested, occurred in B. subtilis cells which overexpressed a component of the MMR system. Interestingly, the single overexpression of mutS, but not of mutL, was sufficient to decrease the level of adaptive mutants in the reversion assay system of B. subtilis. The results presented in this work, as well as in our previous studies, appear to suggest that an MMR deficiency, putatively attributable to inactivation or saturation with DNA damage of MutS, may occur in a subset of B. subtilis cells that differentiate into the hypermutable state.

Defects in the Error Prevention Oxidized Guanine System Potentiate Stationary-Phase Mutagenesis in Bacillus subtilis

Previous studies showed that a Bacillus subtilis strain deficient in mismatch repair (MMR; encoded by the mutSL operon) promoted the production of stationary-phase-induced mutations. However, overexpression of the mutSL operon did not completely suppress this process, suggesting that additional DNA repair mechanisms are involved in the generation of stationary-phase-associated mutants in this bacterium. In agreement with this hypothesis, the results presented in this work revealed that starved B. subtilis cells lacking a functional error prevention GO (8-oxo-G) system (composed of YtkD, MutM, and YfhQ) had a dramatic propensity to increase the number of stationary-phase-induced revertants. These results strongly suggest that the occurrence of mutations is exacerbated by reactive oxygen species in nondividing cells of B. subtilis having an inactive GO system. Interestingly, overexpression of the MMR system significantly diminished the accumulation of mutations in cells deficient in the GO repair system during stationary phase. These results suggest that the MMR system plays a general role in correcting base mispairing induced by oxidative stress during stationary phase. Thus, the absence or depression of both the MMR and GO systems contributes to the production of stationary-phase mutants in B. subtilis. In conclusion, our results support the idea that oxidative stress is a mechanism that generates genetic diversity in starved cells of B. subtilis, promoting stationary-phase-induced mutagenesis in this soil microorganism.

Forespore-Specific Expression of Bacillus subtilis yqfS, Which Encodes Type IV Apurinic/Apyrimidinic Endonuclease, a Component of the Base Excision Repair Pathway

The temporal and spatial expression of the yqfS gene of Bacillus subtilis, which encodes a type IV apurinic/apyrimidinic endonuclease, was studied. A reporter gene fusion to the yqfS opening reading frame revealed that this gene is not transcribed during vegetative growth but is transcribed during the last steps of the sporulation process and is localized to the developing forespore compartment. In agreement with these results, yqfS mRNAs were mainly detected by both Northern blotting and reverse transcription-PCR, during the last steps of sporulation. The expression pattern of the yqfS-lacZ fusion suggested that yqfS may be an additional member of the Esigma(G) regulon. A primer extension product mapped the transcriptional start site of yqfS, 54 to 55 bp upstream of translation start codon of yqfS. Such an extension product was obtained from RNA samples of sporulating cells but not from those of vegetatively growing cells. Inspection of the nucleotide sequence lying upstream of the in vivo-mapped transcriptional yqfS start site revealed the presence of a sequence with good homology to promoters preceding genes of the sigma(G) regulon. Although yqfS expression was temporally regulated, neither oxidative damage (after either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment induced the transcription of this gene.

The ytkD (mutTA) Gene of Bacillus subtilis Encodes a Functional Antimutator 8-Oxo-(dGTP/GTP)ase and Is under Dual Control of Sigma A and Sigma F RNA Polymerases

The regulation of expression of ytkD, a gene that encodes the first functional antimutator 8-oxo-dGTPase activity of B. subtilis, was studied here. A ytkD-lacZ fusion integrated into the ytkD locus of wild-type B. subtilis 168 revealed that this gene is expressed during both vegetative growth and early stages of sporulation. In agreement with this result, ytkD mRNAs were detected by both Northern blotting and reverse transcription-PCR during both developmental stages. These results suggested that ytkD is transcribed by the sequential action of RNA polymerases containing the sigma factors sigma(A) and sigma(F), respectively. In agreement with this suggestion, the spore-associated expression was almost completely abolished in a sigF genetic background but not in a B. subtilis strain lacking a functional sigG gene. Primer extension analysis mapped transcriptional start sites on mRNA samples isolated from vegetative and early sporulating cells of B. subtilis. Inspection of the sequences lying upstream of the transcription start sites revealed the existence of typical sigma(A)- and sigma(F)-type promoters. These results support the conclusion that ytkD expression is subjected to dual regulation and suggest that the antimutator activity of YtkD is required not only during vegetative growth but also during the early sporulation stages and/or germination of B. subtilis. While ytkD expression obeyed a dual pattern of temporal expression, specific stress induction of the transcription of this gene does not appear to occur, since neither oxidative damage (following either treatment with paraquat or hydrogen peroxide) nor mitomycin C treatment or sigma(B) general stress inducers (sodium chloride, ethanol, or heat) affected the levels of the gene product produced.

Tight Genetic Linkage of a Glucosyltransferase and Dextranase of Streptococcus mutans GS-5:

A genetic library consisting of over 5000 clones with an average insert size of 6.9 kilobasepairs (kbp) of Streptococcus mutans GS-5 has been constructed in a bivalent plasmid vector pMK3, which is capable of replicating in Escherichia coli and Bacillus subtilis. The recombinant plasmid pSUCRI, containing a 6.0 kbp fragment of S. mutans GS-5 DNA, was the focus of this study. Using Southern hybridization, in vitro and in vivo gene expression techniques, and biochemical analysis, this clone was shown to encode the 55 kiloDalton (kDal) GS-5 gtfA gene product, as well as a 38 and a 66 kDal polypeptide. In addition to the gtfA gene, pSUCRI encodes a dextranase activity with specificity for α(1→6)-linked glucans, and with no detectable activity on mutan. The dextranase enzyme had an apparent molecular weight of 66 kDal as demonstrated by SDS-PAGE analysis of the proteins produced by a dextranase-negative deletion derivative. The pH optimum of the enzyme was approximately 6.0, and there was no detectable activity below pH 5.0. By subcloning various combinations of DNA fragments from pSUCRI, it was demonstrated that the dextranase gene (designated dexB) can be separated from the gtfA gene and still be efficiently expressed in both E. coli and B. subtilis. The dexB gene contained its own promoter and ribosome-binding site. The genetic linkage of the gtfA and dexB genes in the S. mutans GS-5 chromosome was confirmed by Southern hybridization and by the independent isolation of four distinct clones containing the gtfA gene and common flanking sequences. In addition to a glucosyltransferase and dextranase, an invertase-like activity is also encoded on pSUCRI, indicating that there is a cluster of genes on the S. mutans GS-5 chromosome which is devoted to the dissimilation of sucrose and concomitant synthesis or modification of glucans into a water-insoluble form, perhaps constituting an operon for glucan modification which can be coordinately regulated in response to environmental alterations.

Linear-programmed thermal degradation methane chemical-ionization mass spectrometry: I. Peptidoglycan, cell walls, and related compounds from Bacillus

Abstract Linear-programmed thermal degradation—mass spectrometry was used to study cell wall preparations from gram-positive bacteria of the genus Bacillus and related compounds. Also, a group of strains of Bacillus subtilis grown under various culturing conditions were compared. The temperature-resolved pyrolysis—mass spectra collected for each sample were analyzed. Many ions present in the pyrolysis—mass spectra of large complex biopolymers were found to have the same nominal masses to those found in spectra of related simpler compounds. Results indicate the importance of culture conditions if pyrolysis—mass spectrometry is used to study bacteria. Also, the pyrolysis—mass spectra of cell wall preparations from Bacillus are dominated by aminosugars.

DNA repair in B. subtilis: An inducible dimer specific W-reactivation system

SummaryThe W-reactivation system of Bacillus subtilis repairs pyrimidine dimers in bacteriophage DNA. This inducible repair system can be activated by treatment of the bacteria with UV, alkylating agents, cross-linking agents and gamma radiation. However, bacteriophage treated with agents other than those that cause pyrimidine dimers were not repaired by this unique form of W-reactivation. In contrast, the W-reactivation system of Escherichia coli repairs a variety of damages in the bacteriophage DNA.