Genetic organization of the lexA,recA and recX genes in Xanthomonas campestris

A gene cluster containing lexA, recA and recX genes was previously identified and characterized in Xanthomonas campestris pathovar citri (X. c. pv. citri). We have now cloned and sequenced the corresponding regions in the Xanthomonas campestris pv. campestris (X. c. pv. campestris) and Xanthomonas oryzae pathovar oryzae (X. o. pv. oryzae) chromosome. Sequence analysis of these gene clusters showed significant homology to the previously reported lexA, recA and recX genes. The genetic linkage and the deduced amino acid sequences of these genes displayed very high identity in different pathovars of X. campestris as well as in X. oryzae. Immunoblot analysis revealed that the over-expressed LexA protein of X. c. pv. citri functioned as a repressor of recA expression in X. c. pv. campestris, indicating that the recombinant X. c. pv. citri LexA protein was functional in a different X. campestris pathovar. The abundance of RecA protein was markedly increased upon exposure of X. c. pv. campestris to mitomycin C, and an upstream region of this gene was shown to confer sensitivity to positive regulation by mitomycin C on a luciferase reporter gene construct. A symmetrical sequence of TTAGTAGTAATACTACTAA present within all three Xanthomonas lexA promoters and a highly conserved sequence of TTAGCCCCATACCGAA present in the three regulatory regions of recA indicate that the SOS box of Xanthomonas strains might differ from that of Escherichia coli.     

Constitutive expression of the SOS response in recA718 mutants of Escherichia coli requires amplification of RecA718 protein.

In recA718 lexA+ strains of Escherichia coli, induction of the SOS response requires DNA damage. This implies that RecA718 protein, like RecA+ protein, must be converted, by a process initiated by the damage, to an activated form (RecA) to promote cleavage of LexA, the cellular repressor of SOS genes. However, when LexA repressor activity was abolished by a lexA-defective mutation [lexA(Def)], strains carrying the recA718 gene (but not recA+) showed strong SOS mutator activity and were able to undergo stable DNA replication in the absence of DNA damage (two SOS functions known to require RecA activity even when cleavage of LexA is not necessary). lambda lysogens of recA718 lexA(Def) strains exhibited mass induction of prophage, indicative of constitutive ability to cleave lambda repressor. When the cloned recA718 allele was present in a lexA+ strain on a plasmid, SOS mutator activity and beta-galactosidase synthesis under LexA control were expressed in proportion to the plasmid copy number. We conclude that RecA718 is capable of becoming activated without DNA damage for cleavage of LexA and lambda repressor, but only if it is amplified above its base-line level in lexA+ strains. At amplified levels, RecA718 was also constitutively activated for its roles in SOS mutagenesis and stable DNA replication. The nucleotide sequence of recA718 reveals two base substitutions relative to the recA+ sequence. We propose that the first allows the protein to become activated constitutively, whereas the second partially suppresses this capability.     

Non-mutability by ultraviolet light in uvrD recB derivatives of Escherichia coli WP2 uvrA is due to inhibition of RecA protein activation

The deficiency in UV mutagenesis in uvrD3 recB21 strains of E. coli is almost completely overcome by constitutive activation of RecA protein and expression of the SOS system (by recA730 or 43 degrees C treated recA441 lexA71). When SOS was expressed but RecA protein not self-activated (recA441 lexA71 at 30 degrees C), uvrD3 recB21 still reduced UV mutagenesis at low doses. The uvrD3 recB21 combination is therefore inhibiting activation of RecA protein. It is suggested that the DNA unwinding activity of the products of the uvrD and recB genes may be involved in generating single-stranded DNA needed to activate RecA protein both for the cleavage of LexA repressor and for a further role in UV mutagenesis.     

Constitutive SOS expression and damage-inducible AddAB-mediated recombinational repair systems for Coxiella burnetii as potential adaptations for survival within macrophages

Coxiella burnetii , a Gram-negative obligate intracellular pathogen, replicates within an parasitophorous vacuole with lysosomal characteristics. To understand how C. burnetii maintains genomic integrity in this environment, a database search for genes involved in DNA repair was performed. Major components of repair, SOS response and recombination were identified, including recA and ruvABC , but lexA and recBCD were absent. Instead, C. burnetii possesses addAB orthologous genes, functional equivalents to recBCD . Survival after treatment with UV, mitomycin C (MC) or methyl methanesulfonate (MMS), as well as homologous recombination in Hfr mating was restored in Escherichia coli deletion strains by C. burnetii recA or addAB . Despite the absence of LexA, co-protease activity for C. burnetii RecA was demonstrated. Dominant-negative inhibition of C. burnetii RecA by recA mutant alleles, modelled after E. coli recA1 and recA56 , was observed and more apparent with expression of C. burnetii RecAG159D mutant protein. Expression of a subset of repair genes in C. burnetii was monitored and, in contrast to the non-inducible E. coli recBCD , addAB expression was strongly upregulated under oxidative stress. Constitutive SOS gene expression due to the lack of LexA and induction of AddAB likely reflect a unique repair adaptation of C. burnetii to its hostile niche.     

lexA dependent recombination in uvrD strains of Escherichia coli

Mutation of the uvrD gene of Escherichia coli is associated with an increased capacity for genetic recombination. The hyper-recombination effect is abolished by an additional mutation in lexA that limits synthesis of RecA protein and other gene products regulated by LexA repressor, and is not restored when increased synthesis of RecA protein is facilitated by a recAoc mutation. The viability of uvrD lexA strains is reduced and revertants selected on the basis of improved growth fall into three categories: those that are lexA+, or carry another mutation in lexA that directly suppresses the lexA defect; recA mutants that have lost the capacity for recombination altogether; and a third class which carry a mutation that is not in lexA or recA and which restores the hyper-rec phenotype but does not otherwise suppress the lexA defect. These results indicate that the hyper-recombination effect of a uvrD mutation is an induced response catalysed by RecA protein and at least one other lexA regulated activity.     

DNA repair mechanisms affecting cytotoxicity by streptozotocin in E. coli

Mechanisms underlying cytotoxicity by the monofunctional nitrosourea streptozotocin (STZ) were evaluated in DNA repair-deficient E. coli mutants. Strains not proficient in recombinational repair which lack either RecA protein or RecBC gene products were highly sensitive to STZ. In contrast, cells that constitutively synthesize RecA protein and cannot initiate SOS repair mechanisms because of uncleavable LexA repressor (recAo98 lexA3) were resistant to this drug compared to a lexA3 strain. Further, E. coli cells lacking both 3-methyladenine DNA glycosylases I (tag) and II (alkA) also were highly sensitive to STZ. DNA synthesis was most inhibited by STZ in recA and alkA tag E. coli mutants, but was suppressed less markedly in wild-type and recBC cells. DNA degradation was most extensive in recA E. coli after STZ treatment, while comparable in recBC, alkA tag, and wild-type cells. Although increased single-stranded DNA breaks were present after STZ treatment in recA and recBC mutants compared to the wild type, no significant increase in DNA single-stranded breaks was noted in alkA tag E. coli. Further, DNA breaks in recBC cells were repaired, while those present in recA cells were not. These findings establish the critical importance of both recombinational repair and 3-methyladenine DNA glycosylase in ameliorating cytotoxic effects and DNA damage caused by STZ in E. coli.     

Analyses of binding sequences of the two LexA proteins of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar citri

Xanthomonas axonopodis pv. citri (X. axonopodis pv. citri) possesses two lexA genes, designated lexA1 and lexA2. Electrophoretic mobility shift data show that LexA1 binds to both lexA1 and lexA2 promoters, but LexA2 does not bind to the lexA1 promoter, suggesting that LexA1 and LexA2 play different roles in regulating the expression of SOS genes. In this study, we have determined that LexA2 binds to a 14-bp dyad-spacer-dyad palindromic sequence, 5'-TGTACAAATGTACA-3', located at nucleotides -41 to -28 relative to the translation start site of lexA2 of X. axonopodis pv. citri. The two spacer nucleotides in this sequence can be changed from AA to TT without affecting LexA2 binding; all other base deletions or substitutions abolish LexA2 binding. The LexA1 binding sequence in the promoter region of lexA2 is TTAGTACTAAAGTTATAA and is located at -133 to -116, and that in the lexA1 gene is AGTAGTAATACTACT located at nucleotides -19 to -5 relative to the translation start site of lexA1. Any base change in the latter sequence abolishes LexA1 binding.     

Quantification of RecA protein in Deinococcus radiodurans reveals involvement of RecA,but not LexA,in its regulation

RecA protein is essential for the very high level of resistance of Deinococcus radiodurans to DNA damage induced by ionizing radiation or other DNA-damaging agents. Since the mechanism(s) involved in the control of recA expression and the extent of RecA induction following DNA damage in this species are still unclear, we have performed a genetic analysis of the recA locus and quantified the basal and induced levels of RecA protein in wild type, recA, and lexA mutants. We found that the two genes upstream of recA in the predicted cinA ligT recA operon appear to have no role in the regulation of recA expression or function, despite the fact that the reading frames in the operon overlap. By using a translational fusion of recA to a lacZ reporter gene, we showed that induction began with no delay following exposure to gamma-radiation or treatment with mitomycin, and continued at a constant rate until it reached a plateau. The induction efficiency increased linearly with inducer dose, levelling off at a concentration fourfold above the background. The basal concentration of RecA protein measured by Western blotting corresponded to approximately 11,000 monomers per cell, and the induced concentration to around 44,000 monomers per cell. These levels remained unchanged upon disruption of the lexA gene, indicating that LexA does not plays a role in recA regulation. However, inactivation of lexA caused cells to aggregate, suggesting that LexA may control the activity or expression of as yet undefined membrane functions. Cells bearing the recA670 mutation showed an elevated constitutive expression of recA in the absence of DNA damage. This phenotype did not result from the defect in DNA repair associated with the RecA670 protein, since the increased basal level of recA expression was also found in recA670/ recA(+) diploid cells that are proficient in DNA repair. These results suggest that RecA may be involved in regulating its own expression, possibly by stimulating proteolytic modification of other regulatory proteins.     

Regulation of the SOS response in Bacillus subtilis: evidence for a LexA repressor homolog.

The inducible SOS response for DNA repair and mutagenesis in the bacterium Bacillus subtilis resembles the extensively characterized SOS system of Escherichia coli. In this report, we demonstrate that the cellular repressor of the E. coli SOS system, the LexA protein, is specifically cleaved in B. subtilis following exposure of the cells to DNA-damaging treatments that induce the SOS response. The in vivo cleavage of LexA is dependent upon the functions of the E. coli RecA protein homolog in B. subtilis (B. subtilis RecA) and results in the same two cleavage fragments as produced in E. coli cells following the induction of the SOS response. We also show that a mutant form of the E. coli RecA protein (RecA430) can partially substitute for the nonfunctional cellular RecA protein in the B. subtilis recA4 mutant, in a manner consistent with its known activities and deficiencies in E. coli. RecA430 protein, which has impaired repressor cleaving (LexA, UmuD, and bacteriophage lambda cI) functions in E.coli, partially restores genetic exchange to B. subtilis recA4 strains but, unlike wild-type E. coli RecA protein, is not capable of inducing SOS functions (expression of DNA damage-inducible [din::Tn917-lacZ] operons or RecA synthesis) in B. subtilis in response to DNA-damaging agents or those functions that normally accompany the development of physiological competence. Our results provide support for the existence of a cellular repressor in B. subtilis that is functionally homologous to the E. coli LexA repressor and suggest that the mechanism by which B. subtilis RecA protein (like RecA of E. coli) becomes activated to promote the induction of the SOS response is also conserved.     

Limited concentration of RecA delays DNA double-strand break repair in Deinococcus radiodurans R1

To evaluate the importance of RecA in DNA double-strand break (DSB) repair, we examined the effect of low and high RecA concentrations such as 2500 and 100 000 molecules per cell expressed from the inducible Pspac promoter in Deinococcus radiodurans in absence or in presence of IPTG respectively. We showed that at low concentration, RecA has a negligible effect on cell survival after gamma-irradiation when bacteria were immediately plated on TGY agar whereas it significantly decreased the survival to gamma-irradiation of DeltaddrA cells while overexpression of RecA can partially compensate the loss of DdrA protein. In contrast, when cells expressing limited concentration of RecA were allowed to recover in TGY2X liquid medium, they showed a delay in mending DSB, failed to reinitiate DNA replication and were committed to die during incubation. A deletion of irrE resulted in sensitivity to gamma-irradiation and mitomycin C treatment. Interestingly, constitutive high expression of RecA compensates partially the DeltairrE sensitization to mitomycin C. The cells with low RecA content also failed to cleave LexA after DNA damage. However, neither a deletion of the lexA gene nor the expression of a non-cleavable LexA(Ind-) mutant protein had an effect on survival or kinetics of DNA DSB repair compared with their lexA+ counterparts in recA+ as well as in bacteria expressing limiting concentration of RecA, suggesting an absence of relationship between the absence of LexA cleavage and the loss of viability or the delay in the kinetics of DSB repair. Thus, LexA protein seems to play no major role in the recovery processes after gamma-irradiation in D. radiodurans.     

Effect of alkylating agents on the expression of inducible genes of Escherichia coli

Increasing doses of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine, diethyl sulphate and ethylmethane sulphonate cause an inhibition of the expression of the recA and sfiA genes of wild-type Escherichia coli. This behaviour was not observed in a lexA56 mutant which has a defective LexA repressor that is unable to bind to the SOS operator. Furthermore, an ada-1 mutant showed the same behaviour as the wild-type strain indicating that the adaptive proteins are not responsible for the inhibition of recA and sfiA at high doses of alkylating agents. These results suggest that the inhibitory effect of these alkylating agents may be found in the interaction between the LexA repressor and the control regions of sfiA and recA. On the other hand, high doses of either UV light or mitomycin C produced only a slight decrease in the induction of recA and sfiA, whereas bleomycin had no effect. The fact that a repressor structurally related to LexA repressor, such as LacI protein, showed the same behaviour as the LexA repressor when a Lac+ strain was treated with alkylating agents, suggests that these compounds can modify the binding abilities of repressors to DNA, producing a limited or even abolished release of repressors, and so decreasing the expression of inducible genes.     

Effects of overproduction of single-stranded DNA-binding protein on RecA protein-dependent processes in Escherichia coli

Overproduction of single-stranded DNA-binding protein (SSB) in Escherichia coli led to a decrease in the basal level of repressor LexA. Expression of the LexA-controlled genes was increased differentially, depending on the affinity of the LexA repressor for each promoter: expression of the recA and sfiA genes was increased 5-fold and 1.5-fold, respectively. Despite only a slight effect on expression of sfiA, which codes for an inhibitor of cell division, bacteria overproducing SSB produced elongated cells. In fact, the effect on cell shape appeared to be essentially independent of the expression of the sfiA and recA genes. Bacteria overproducing SSB were therefore phenotypically similar to bacteria partially starved of thymine, in which filamentation results from both sfiA-dependent and sfiA-recA-independent pathways. These data indicate that excess SSB acts primarily by perturbing DNA replication, thereby favoring gratuitous activation of RecA protein to promote cleavage of LexA protein. When bacteria overproducing SSB were exposed to a DNA-damaging agent such as ultraviolet light or mitomycin C, the recA and sfiA genes were fully induced. Induction of the sfiA gene occurred, however, at higher doses in bacteria overproducing SSB protein than in bacteria with normal levels of SSB. Whereas the efficiency of excision repair was apparently increased by excess SSB, the efficiency of post-replication recombinational repair was reduced as judged by a decrease in the recombination proficiency between a prophage and ultraviolet-irradiated heteroimmune infecting phage. Following induction of ssb+ bacteria with mitomycin C, the cellular content of SSB was slightly increased. These results provide evidence that SSB modulates RecA protein-dependent activities in vivo. It is proposed that SSB favors the formation of short complexes of RecA protein and single-stranded DNA that mediate cleavage of the LexA and lambda repressors, while it delays the formation of long nucleoprotein filaments, thereby slowing down RecA-promoted recombinational events in uninduced as well as in induced bacteria.     

The Escherichia coli K-12 lexA2 gene encodes a hypocleavable repressor.

LexA2 repressor was partially inactivated after mitomycin C or UV light treatment in a recA+ or recA85(Prtc) (protease constitutive) host background. LexA2 protein was cleaved, but the reaction was slower than that observed for LexA+ repressor. lexA2 had a C-to-T transition at nucleotide 461 (Thr-154 to Ile).     

New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis.

To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+. With respect to other RecA functions, recA1730 was recessive to recA+. This demonstrates that RecA protein has an additional role in mutagenesis beside mediating the cleavage of LexA and UmuD proteins.     

Purification and characterization of the RecA protein from Streptococcus pneumoniae

Streptococcus pneumoniae is a naturally transformable bacterium that is able to take up single-stranded DNA from its environment and incorporate the exogenous DNA into its genome. This process, known as transformational recombination, is dependent upon the presence of the recA gene, which encodes an ATP-dependent DNA recombinase whose sequence is 60% identical to that of the RecA protein from Escherichia coli. We have developed an overexpression system for the S. pneumoniae RecA protein and have purified the protein to greater than 99% homogeneity. The S. pneumoniae RecA protein has ssDNA-dependent NTP hydrolysis and NTP-dependent DNA strand exchange activities that are generally similar to those of the E. coli RecA protein. In addition to its role as a DNA recombinase, the E. coli RecA protein also acts as a coprotease, which facilitates the cleavage and inactivation of the E. coli LexA repressor during the SOS response to DNA damage. Interestingly, the S. pneumoniae RecA protein is also able to promote the cleavage of the E. coli LexA protein, even though a protein analogous to the LexA protein does not appear to be present in S. pneumoniae.