Genetic recombination in Bacillus subtilis 168: effect of DeltahelD on DNA repair and homologous recombination.

The B. subtilis DeltahelD allele rendered cells proficient in transformational recombination and moderately sensitive to methyl methanesulfonate when present in an otherwise Rec(+) strain. The DeltahelD allele was introduced into rec-deficient strains representative of the alpha (recF strain), beta (addA addB), gamma (recH), epsilon (DeltarecU), and zeta (DeltarecS) epistatic groups. The DeltahelD mutation increased the sensitivity to DNA-damaging agents of addAB, DeltarecU, and DeltarecS cells, did not affect the survival of recH cells, and decreased the sensitivity of recF cells. DeltahelD also partially suppressed the DNA repair phenotype of other mutations classified within the alpha epistatic group, namely the recL, DeltarecO, and recR mutations. The DeltahelD allele marginally reduced plasmid transformation (three- to sevenfold) of mutations classified within the alpha, beta, and gamma epistatic groups. Altogether, these data indicate that the loss of helicase IV might stabilize recombination repair intermediates formed in the absence of recFLOR and render recFLOR, addAB, and recH cells impaired in plasmid transformation.     

Characterization of Bacillus subtilis recombinational pathways

Recombination in Bacillus subtilis requires the products of numerous rec loci. To dissect the various mechanisms which may be involved in genetic recombination, we constructed a series of isogenic strains containing more than one mutant rec allele. On the basis of their impairment in genetic exchange, the various loci (represented by specific rec alleles) were classified into different epistatic groups. Group alpha consists of rec genes represented by recB, recD, recF, recG, recL, and recR mutations, while group beta comprises the addA and addB mutations. Group gamma consists of the recH and recP mutations. These results suggest that B. subtilis has multiple pathways for genetic recombination and that the products of the genes within the alpha, beta, and gamma epistatic groups are involved in these alternative recombination pathways. The RecA protein is required in all three pathways of intermolecular recombination.     

Bacillus subtilis RecG branch migration translocase is required for DNA repair and chromosomal segregation

The absence of Bacillus subtilis RecG branch migration translocase causes a defect in cell proliferation, renders cells very sensitive to DNA-damaging agents and increases approximately 150-fold the amount of non-partitioned chromosomes. Inactivation of recF, addA, recH, recV or recU increases both the sensitivity to DNA-damaging agents and the chromosomal segregation defect of recG mutants. Deletion of recS or recN gene partially suppresses cell proliferation, DNA repair and segregation defects of DeltarecG cells, whereas deletion of recA only partially suppresses the segregation defect of DeltarecG cells. Deletion of recG and ripX render cells with very poor viability, extremely sensitive to DNA-damaging agents, and with a drastic segregation defect. After exposure to mitomycin C recG or ripX cells show a drastic defect in chromosome partitioning (approximately 40% of the cells), and this defect is even larger (approximately 60% of the cells) in recG ripX cells. Taken together, these data indicate that: (i) RecG defines a new epistatic group (eta), (ii) RecG is required for proper chromosomal segregation even in the presence of other proteins that process and resolve Holliday junctions, and (iii) different avenues could process Holliday junctions.     

Genetic Recombination in Bacillus subtilis 168: Effects of recU and recS Mutations on DNA Repair and Homologous Recombination

Bacillus subtilis recombination-deficient mutants were constructed by inserting a selectable marker (cat gene) into the yppB and ypbC coding regions. The yppB:cat and ypbC:cat null alleles rendered cells sensitive to DNA-damaging agents, impaired plasmid transformation (25- and 100-fold), and moderately affected chromosomal transformation when present in an otherwise Rec⁺ B. subtilis strain. The yppB gene complemented the defect of the recG40 strain. yppB and ypbC and their respective null alleles were termed “recU” and “recU1” (recU:cat) and “recS” and “recS1” (recS:cat), respectively. The recU and recS mutations were introduced into rec-deficient strains representative of the α (recF), β (addA5 addB72), γ (recH342), and (recG40) epistatic groups. The recU mutation did not modify the sensitivity of recH cells to DNA-damaging agents, but it did affect inter- and intramolecular recombination in recH cells. The recS mutation did not modify the sensitivity of addAB cells to DNA-damaging agents, and it marginally affected recF, recH, and recU cells. The recS mutation markedly reduced (about 250-fold) intermolecular recombination in recH cells, and there were reductions of 10- to 20-fold in recF, addAB, and recU cells. Intramolecular recombination was blocked in recS recF, recS addAB, and recS recU cells. RecU and RecS have no functional counterparts in Escherichia coli. Altogether, these data indicate that the recU and recS proteins are required for DNA repair and intramolecular recombination and that the recF (α epistatic group), addAB (β), recH (γ), recU (), and recS genes provide overlapping activities that compensate for the effects of single mutation. We tentatively placed recS within a new group, termed “ζ.”     

Genetic and Physical Analysis of Plasmid Recombination in Recb Recc Sbcb and Recb Recc Sbca Escherichia Coli K-12 Mutants

The effect of mutations in known recombination genes (recA, recB, recC, recE, recF, recJ, recN, recO, recQ and ruv) on intramolecular recombination of plasmids was studied in recB recC sbcB and recB recC sbcA Escherichia coli mutants. The rate of recombination of circular dimer plasmids was at least 1000-fold higher in recB recC sbcB or recB recC sbcA mutants as compared to wild-type cells. The rate was decreased by mutations in recA, recF, recJ, recO, ruv or mutS in recB recC sbcB mutants, and by mutations in recE, recN, recO, recQ, ruv or mutS in recB recC sbcA mutants. In addition to measuring the recombination rate of circular dimer plasmids, the recombination-mediated transformation of linear dimer plasmids was also studied. Linear dimer plasmids transformed recB recC sbcB and recB recC sbcA mutants 20- to 40-fold more efficiently than wild-type cells. The transformation efficiency of linear dimer plasmids in recB recC sbcB mutants was decreased by mutations in recA, recF, recJ, recO, recQ or lexA (lexA3). In recB recC sbcA mutants the transformation efficiency of linear dimers was decreased only by a recE mutation. Physical analysis of linear dimer- or circular dimer-transformed recB recC sbcB mutants revealed that all transformants contained recombinant monomer genotypes. This suggests that recombination in recB recC sbcB cells is very efficient.     

Role of constitutive and inducible repair in radiation resistance of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Radiation resistance of Escherichia coli cells depends on how efficiently DNA is recovered after damage, which is determined by the function of constitutive and inducible repair branches. The effects of additional mutations of the key genes of constitutive and inducible repair (recA, lexA, recB, polA, lig, gyr, recF, recO, recR, recJ, recQ, uvrD, helD, recN, and ruv) on radiation resistance were studied in E. coli K-12 strain AB1157 and highly radiation-resistant isogenic strain Gam^r444. An optimal balance ensuring a high γ resistance of the Gam^r444 radiation-resistant E. coli mutant was due to expression of the key SOS repair genes (recA, lexA, recN, and ruv) and activation of the presynaptic functions of the RecF homologous recombination pathway as a result of a possible mutation of the uvrD gene, which codes for repair helicase II.     

Genetic recombination in Bacillus subtilis 168: contribution of Holliday junction processing functions in chromosome segregation

Bacillus subtilis mutants classified within the epsilon (ruvA, DeltaruvB, DeltarecU, and recD) and eta (DeltarecG) epistatic groups, in an otherwise rec+ background, render cells impaired in chromosomal segregation. A less-pronounced segregation defect in DeltarecA and Deltasms (DeltaradA) cells was observed. The repair deficiency of addAB, DeltarecO, DeltarecR, recH, DeltarecS, and DeltasubA cells did not correlate with a chromosomal segregation defect. The sensitivity of epsilon epistatic group mutants to DNA-damaging agents correlates with ongoing DNA replication at the time of exposure to the agents. The Deltasms (DeltaradA) and DeltasubA mutations partially suppress the DNA repair defect in ruvA and recD cells and the segregation defect in ruvA and DeltarecG cells. The Deltasms (DeltaradA) and DeltasubA mutations partially suppress the DNA repair defect of DeltarecU cells but do not suppress the segregation defect in these cells. The DeltarecA mutation suppresses the segregation defect but does not suppress the DNA repair defect in DeltarecU cells. These results result suggest that (i) the RuvAB and RecG branch migrating DNA helicases, the RecU Holliday junction (HJ) resolvase, and RecD bias HJ resolution towards noncrossovers and that (ii) Sms (RadA) and SubA proteins might play a role in the stabilization and or processing of HJ intermediates.     

The Genetic Dependence of Recombination in Recd Mutants of Escherichia Coli

RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations.     

Analysis of the Bacillus subtilis recO gene: RecO forms part of the RecFLOR function

The deduced protein product of the Bacillus subtilis gene yqfI, which is 255 residues long, shares homology (25% identity) with the Escherichia coli RecO protein. A null allele of yqfI, when present in an otherwise Rec⁺ B. subtilis strain, causes cells to become highly sensitive to DNA-damaging agents, and plasmid transformation (intramolecular recombination) is reduced by 25-fold while chromosomal transformation (intermolecular recombination) is only moderately affected (2.5-fold reduction). Therefore, the yqfI gene was renamed recO and its null allele is referred to as recO1. The recO1 mutation was introduced into recombination-deficient strains representative of the epistatic groups α (recF, recR and recL strains), β (addA5 addB72), γ (recH342) and ? (recU40). The recO mutation did not affect the sensitivity of recF, recR or recL cells to DNA-damaging agents, increased the sensitivity of recU and addAB cells and abolished the DNA repair capacity of recH cells. The recO mutation did not affect intermolecular recombination in recF, recL, recH or recU cells, but reduced (by about 9-fold) the incidence of intermolecular recombination in addAB cells. The recO mutation did not affect intramolecular recombination in the addAB, recU, recF or recL cells, but reduced it by about 75-fold in recH cells. The defects caused by the recO1 mutation can be partially suppressed by a common suppressor of the recF, recL and recR phenotypes. We therefore assigned recO to epistatic group α and predict that the RecO protein acts at the same stage of recombination as the RecF, RecL and RecR proteins, in a RecFLOR complex.     

Modulation of DNA repair and recombination by the bacteriophage lambda Orf function in Escherichia coli K-12

The orf gene of bacteriophage lambda, fused to a promoter, was placed in the galK locus of Escherichia coli K-12. Orf was found to suppress the recombination deficiency and sensitivity to UV radiation of mutants, in a Delta(recC ptr recB recD)::P(tac) gam bet exo pae cI DeltarecG background, lacking recF, recO, recR, ruvAB, and ruvC functions. It also suppressed defects of these mutants in establishing replication of a pSC101-related plasmid. Compared to orf, the recA803 allele had only small effects on recF, recO, and recR mutant phenotypes and no effect on a ruvAB mutant. In a fully wild-type background with respect to known recombination and repair functions, orf partially suppressed the UV sensitivity of ruvAB and ruvC mutants.     

Recombination is essential for viability of an Escherichia coli dam (DNA adenine methyltransferase) mutant

Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, or ruvC could not be constructed, whereas dam derivatives with recD, recF, recJ, and recR were viable. The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates. A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a dam lexA (Ind(-)) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recA and ruvAB need to be derepressed to allow for dam mutant survival. The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind(-)) derivatives. The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents.     

<Emphasis Type="Italic">Bacillus anthracis</Emphasis>, <Emphasis Type="Italic">Francisella tularensis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis>. The most important bacterial warfare agents — <Emphasis Type="Italic">review</Emphasis>

There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.     

Overexpression of the Chitosanase Gene in <Emphasis Type="Italic">Fusarium solani</Emphasis> via <Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-Mediated Transformation

To overexpress the chitosanase gene (csn) in F. solani, a vector based on pCAMBIA 1300 was constructed. The csn gene, which is under control of the Aspergillus nidulans gpdA promoter and A. nidulans trpC terminator, was introduced back into the F. solani genome by Agrobacterium tumefaciens-mediated transformation, and the herbicide-resistance gene bar from Streptomyces hygroscopicus was used as the selection marker. Transformants which showed a significant increase in chitosanase production (~2.1-fold than control) were obtained. Southern blot analysis indicated that most transformants had a single-copy T-DNA integration.     

W-reactivation and W-mutagenesis in UV-irradiated phage phil05 of Bacillus subtilis]

The survival of UV-irradiated phage ?105 on the lawns of several strains of Bacillus subtilis: wild type (strain 168) and 11 recombination-defficient mutants (recA1, recB2, recB3, recB19, recD27, recF15, recF18, recK4, recM13, recL16 and recO61) was investigated. All rec mutants have the phenotype Hcr+, i.e. normally host-cell reactivate UV-damaged phage. Small doses of UV-irradiation given to the wild type (rec+) cells increase the probability of survival of UV-irradiated ?105 phage (W-reactivation) and significantly enhance the frequency of c-mutants (W-mutagenesis). Maximal frequency of clear mutations in conditions of W-mutagenesis is 3-10(-3), i.e. is 100 times higher than the spontaneous background. Various rec mutations of host cells only diminish the level of W-reactivation but do not eliminate it completely. The most deficient in W-reactivation is recD27 mutant. Mutations recB2, B3, B19 and O61 have no effect on W-mutagenesis of UV-irradiated phage ?105 and on UV-induction of ?105, F15,F18 and L16 mutants. UV-irradiation of lysogenic cells of these mutants does not induce ?105 prophage.     

<Emphasis Type="Italic">RAD18</Emphasis> gene product of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> controls mutagenesis induced by hydrogen peroxide

Within eukaryotes, tolerance to DNA damage is determined primarily by the repair pathway controlled by the members of the RAD6 epistasis group. Genetic studies on a yeast Saccharomyces cerevisiae model showed that the initial stage of postreplication repair (PRR), i.e., initiation of replication through DNA damage, is controlled by Rad6–Rad18 ubiquitin-conjugating enzyme complex. Mutants of these genes are highly sensitive to various genotoxic agents and reduce the level of induced mutagenesis. In this case, the efficiency of mutagenesis suppression depends on the type of damage. In this study we showed that DNA damage induced by hydrogen peroxide at the same mutagen doses causes significantly more mutations and lethal events in the rad18 mutant cells compared to control wild-type cells.     

Different effects of recJ and recN mutations on the postreplication repair of UV-damaged DNA in Escherichia coli K-12.

Two mutations known to affect recombination in a recB recC sbsBC strain, recJ284::Tn10 and recN262, were examined for their effects on the postreplication repair of UV-damaged DNA. The recJ mutation did not affect the UV radiation sensitivity of uvrB and uvrB recF cells, but it increased the sensitivity of uvrB recN (approximately 3-fold) and uvrB recB (approximately 8-fold) cells. On the other hand, the recN mutation did not affect the UV sensitivity of uvrB recB cells, but it increased the sensitivity of uvrB (approximately 1.5-fold) and uvrB recF (approximately 4-fold) cells. DNA repair studies indicated that the recN mutation produced a partial deficiency in the postreplication repair of DNA double-strand breaks that arise from unrepaired daughter strand gaps, while the recJ mutation produced a deficiency in the repair of daughter strand gaps in uvrB recB cells (but not in uvrB cells) and a deficiency in the repair of both daughter strand gaps and double-strand breaks in uvrA recB recC shcBC cells. Together, these results indicate that the recJ and recN genes are involved in different aspects of postreplication repair.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the plant pathogenic fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25°C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern b lot analy s is. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in R. necatrix and provide a simple and reliable method for genetic manipulation.     

Biochemical Measure of the Time and Frequency of Radiation-induced Allelic Recombination in <Emphasis Type="Italic">Ustilago</Emphasis>

Diploids with different mutations in the structural gene for nitrate reductase recombine to produce measurable enzyme activity. Radiation induces recombination with high frequency, but only in cells which will survive the treatment. The results suggest that a recombination repair mechanism is induced in irradiated cells.     

Enhancement of Escherichia Coli Plasmid and Chromosomal Recombination by the Ref Function of Bacteriophage P1

The Ref activity of phage P1 enhances recombination between two defective lacZ genes in the Escherichia coli chromosome (lac- x lac- recombination). Plasmid recombination, both lac- x lac- and tet- x tet-, was measured by transformation of recA strains, and was also assayed by measurement of beta-galactosidase. The intracellular presence of recombinant plasmids was verified directly by Southern blotting. Ref stimulated recombination of plasmids in rec+ and rec(BCD) cells by 3-6-fold, and also the low level plasmid recombination in recF cells. RecA-independent plasmid recombination, either very low level (recA cells) or high level (recB recC sbcA recA cells), was not stimulated. Ref stimulated both intramolecular and intermolecular plasmid recombination. Both normal and Ref-stimulated lac- x lac- chromosomal recombination, expected to be mostly RecBC-dependent in wild-type bacteria, were affected very little by a recF mutation. We have previously reported Ref stimulation of lac- x lac- recombination in recBC sbcB bacteria, a process known to be RecF-dependent. Chromosomal recombination processes thought to involve activated recombination substrates, e.g., Hfr conjugation, P1 transduction, were not elevated by Ref activity. We hypothesize that Ref acts by unknown mechanisms to activate plasmid and chromosomal DNA for RecA-mediated recombination, and that the structures formed are substrates for both RecF-dependent (plasmid, chromosomal) and Rec(BCD)-dependent (chromosomal) recombination pathways.