Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida.

Summary The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene. The predicted amino acid sequences of the S. typhimurium, E. carotovora and P. putida LexA proteins are 202 residues long whereas that of P. aeruginosa is 204. Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 by in S. typhimurium and E. carotovora, as in the lexA gene of E. coli, and 3 by in P. putida and P. aeruginosa. The first lexA site present in the lexA operator of all five bacteria is very well conserved. However, the second lexA box is considerably more variable. The Ala-84 — Gly-85 bond, at which the LexA repressor of E. coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S. typhimurium and E. carotovora. Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors. These residues also exist in the LexA proteins of P. putida and P. aeruginosa, but they are displaced by 4 and 6 residues, respectively. Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E. coli are highly conserved in the S. typhimurium, E. carotovora, P. aeruginosa and P. putida LexA proteins.     

Indirect and intragenic suppression of the lexA102 mutation in E. coli B/r.

Summary In Escherichia coli B/r the expression of UV inducible (SOS) functions is under the control of the recA and lexA genes. In this study we have characterized mutants which are altered in their ability to express SOS functions. These mutants were isolated as UV resistant UV nonmutable (Rnm) derivatives of the lexA102 uvrA155 mutant strain WP51. The UV resistance of these Rnm strains is a result of the suppression of lexA102 mediated UV sensitivity. Genetic mapping of rnm mutations shows that the two predominant classes, rnmA and rnmB, map in or very near the lexA and recA genes respectively. rnmA mutations differ from rnmB with respectively recA protein synthesis. rnmA mutations do not restore the ability to express high levels of recA protein after UV treatment whereas rnmB mutations result in constitutive expression of high levels of recA protein. However, both rnmA and rnmB mutant strains inhibit postirradiation DNA degradation. This shows that in rnmA strains, high levels of recA protein are not needed to inhibit postirradiation DNA degradation.The genetic map location and constitutive expression of recA protein synthesis resulting from rnmB mutations suggests that they are operator constitutive mutations of the recA gene. The result that the lexA ⁺ gene is required for the expression of UV mutagenesis in rnmB mutants shows that high levels of recA protein do not circumvent the need for the lexA ⁺ gene product in this process. Thus, while the lexA gene product is required for the induction of recA protein synthesis, lexA must have an additional role in UV induced mutagenesis.     

Identification and characterization of two <Emphasis Type="Italic">uvrA</Emphasis> genes of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar citri

Two uvrA-like genes, designated uvrA1 and uvrA2, that may be involved in nucleotide excision repair in Xanthomonas axonopodis pv. citri (X. a. pv. citri) strain XW47 were characterized. The uvrA1 gene was found to be 2,964 bp in length capable of encoding a protein of 987 amino acids. The uvrA2 gene was determined to be 2,529 bp with a coding potential of 842 amino acids. These two proteins share 71 and 39% identity, respectively, in amino acid sequence with the UvrA protein of Escherichia coli. Analyses of the deduced amino acid sequence revealed that UvrA1 and UvrA2 have structures characteristic of UvrA proteins, including the Walker A and Walker B motifs, zinc finger DNA binding domains, and helix-turn-helix motif with a polyglycine hinge region. The uvrA1 or uvrA2 mutant, constructed by gene replacement, was more sensitive to DNA-damaging agents methylmethane sulfonate (MMS), mitomycin C (MMC), or ultraviolet (UV) than the wild type. The uvrA1 mutant was four orders of magnitude more sensitive to UV irradiation and two orders of magnitude more sensitive to MMS than the uvrA2 mutant. The uvrA1uvrA2 double mutant was one order of magnitude more sensitive to MMS, MMC, or UV than the uvrA1 single mutant. These results suggest that UvrA1 plays a more important role than UvrA2 in DNA repair in X. a. pv. citri. Both uvrA1 and uvrA2 genes were found to be constitutively expressed in the wild type and lexA1 or lexA2 mutant of X. a. pv. citri, and treatment of these cells with sublethal dose of MMC did not alter the expression of these two genes. Results of electrophoresis mobility shift assays revealed that LexA1 or LexA2 does not bind to either the uvrA1 or the uvrA2 promoter. These results suggest that uvrA expression in X. a. pv. citri is not regulated by the SOS response system.     

Effect of a lexA41(Ts) mutation on DNA repair in recA(Def) derivatives of Escherichia coli K-12

Summary Derivatives of Escherichia coli K-12 carrying a deletion of the recA gene survive exposure to UV (254 nm) better if they also contain the lexA41 mutation which codes for a labile LexA protein. This effect of the lexA41 mutation is not observed in comparable strains carrying a uvr A6 mutation. Using two independent methods to detect pyrimidine dimers we found that UV irradiated RecA deficient cells removed dimers from their DNA more rapidly if they contained the lexA41 mutation than if the contained the wild-type lexA gene. Our results are consistent with the idea that a relatively high level of UvrABC incision nuclease resulting from inefficient repression of the corresponding genes by the labile LexA41 protein facilitates excision of pyrimidine dimers from the DNA of UV irradiated cells.     

Isolation and characterization of an operator-constitutive mutation in the recA gene of E. coli K-12

Summary The recA gene of E. coli is regulated by a specific repressor, the lexA protein, which binds to an operator in the recA regulatory region. We describe in this paper the isolation and characterization of a mutant thought to carry an operator-constitutive mutation in the recA gene. This mutation has the following properties: 1) It partially supresses the UV sensitivity of lexA ^– strains. 20 It maps near the recA gene. 3) It allows constitutive high-level synthesis of recA protein in both lexA ^– and lexA ⁺ backgrounds. 4) It allows constitutive synthesis of the recA messenger RNA. 5) It is cis–acting. The mutation does not restore induced cellular mutagenesis in a lexA ^– background. The expression of induced repair and mutagenesis of UV irradiated phage lambda or the regulation of the lexA gene is not affected by the presence of the mutation in either a lexA ⁺ or lexA ^– strain. These observations confirm other findings that high levels of recA protein synthesis per se is not sufficient for the expression of UV inducible functions and that the lexA protein represses other genes besides the recA gene.Abbreviations UV ultraviolet - Kd kilodalton - PAGE polyacrylamide gel electrophoresis     

Influence of plasmids carrying the lexA gene on DNA repair and related processes in Escherichia coli K-12

Summary Plasmid pLC44-14 from the Clarke and Carbon collection has been shown to carry the lexA gene. The presence of lexA was demonstrated by complementation of tsl mutants which lie close to lexA on the E. coli K-12 linkage map and are probably in the lexA gene, and by crossing the dominant lexA mutation on to pLC44-14 to produce a recombinant plasmid, pSEl, which gave the host cell the properties of a lexA mutant. The lexA gene has been cloned on to pBR322 (Little, 1980). pJL21, which carries the lexA ⁺ gene, rendered the host cell moderately sensitive to UV light, greatly reduced the extent of Weigle reactivation and mutagenesis of UV-irradiated phage , and inhibited induction of protein X by either UV light or nalidixic acid. A similar plasmid carrying a mutant lexA3 allele produced extreme sensitivity to UV light, reduced recombinant production 10 to 50-fold following Hfr x F^– conjugation crosses, and otherwise mimicked the effects of pJL21. Introduction of an amber mutation into the lexA gene carried by the plasmid greatly reduced the UV-sensitivity of the host, thereby indicating that the extreme sensitivity was due to the mutant lexA gene product. These properties of strains with lexA plasmids are thought to originate from high levels of the lexA protein in the cell due to a large plasmid copy number. This protein, which appears from other studies to regulate negatively the recA gene, may inhibit expression of recA or other DNA repair genes when present in excess amounts in the cell.     

Cloning and characterization of the recA gene of Aquaspirillum magnetotacticum

The recA gene of Aquaspirillum magnetotacticum has been isolated from a genomic library and introduced into a recA mutant strain of Escherichia coli K12. The cloned gene complemented both the recombination and DNA repair deficiency of the host and its protein product promoted the proteolytic cleavage of the LexA protein. A protein whose molecular weight is similar to that of the RecA protein of E. coli was associated with the cloned sequence.This paper is affectionately dedicated to Prof. John L. Ingraham     

Molecular cloning, sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^– mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Lysogenic induction of lambdoid phages in lexA mutants of Escherichia coli

Summary UV irradiation of lexA3 mutants of E. coli caused lysogenic induction of prophage , _i21, _i434 and 80. Maximal induction in lexA3 lysogens needed less UV than in lexA ⁺ bacteria and gave 25–100% of the normal levels of infective centres induced. Assays of gene expression arising from derepression of a defective prophage showed at least 40% of the normal levels of induction by mitomycin C in lexA3 bacteria. The need for post-irradiation protein synthesis for lysogenic induction in lexA3 lysogens was reduced by increasing the basal level of recA protein with a recA ⁺ plasmid. It is concluded that in lexA E. coli some recA protein synthesis, too small to be detected by physical means, is needed for UV induced lysogenic induction.     

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.     

Operator-constitutive mutation in the <Emphasis Type="Italic">recA</Emphasis> gene enhances radiation resistance of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Plasmid pUC19-recAo^c carrying a mutant allele of the recA gene, which plays the key role in the control of the SOS repair system and homologous recombinational repair, causes a 1.5-fold increase in radiation resistance of Escherichia coli ΔrecA cells, as compared to the wild-type recA ⁺ cells. The protective effect of this plasmid is drastically reduced in mutant lexA3 recAΔ21 deficient in the LexA protein and in induction of the SOS regulon. Plasmid pUC19-recAo^c effectively suppresses UV sensitivity of the ΔrecA mutant. Mutation recAo20 allows constitutive high-level synthesis of the RecA protein. This mutation impairs the SOS box in the operator site of the recA gene and enhances heterology of the dimer LexA binding site. These data confirm that high level of the RecA protein synthesis per se is not sufficient for the expression of γ-inducible functions and that the derepression of lexA-dependent genes, other than recA gene, is necessary for the complete induction of the SOS repair system.     

Construction of a fused operon consisting of the recA and kan (Kanamycin resistance) genes and regulation of its expression by the lexA gene

Summary The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the recA gene was inserted into the promoter region of the cloned kan gene to produce a fused operon, recA-kan. Plasmid pMCR685 carrying recA-kan expressed a low level of activity of the kan gene product (kanamycin phosphotransferase; KPT) in the wildtype cells of Escherichia coli, while the plasmid showed an increased level of the activity in the Spr^- mutant cells which produce the inactive lexA protein. The KPT activity in the wildtype cells harboring the plasmid increased 6-to 11-fold upon treatment of the cells with mitomycin C or nalidixic acid, both of which are known to induce synthesis of recA protein.Expression of the recA-kan operon fusion was remakably repressed by the lexA gene cloned into a plasmid carrying the operon fusion. Higher concentrations of mitomycin C were required for maximal induction of KPT activity in the cells harboring the resulting plasmid pMCR687. These results strongly suggest that the lexA gene product can by itself repress the recA gene, and that pMCR687 is a useful vector to clone genes whose expression is harmful to the host cell growth.     

Analysis of the expression of the Rhodobacter sphaeroides lexA gene

The regulation of the Rhodobacter sphaeroides lexA gene has been analyzed using both gel-mobility experiments and lacZ gene fusions. PCR-mediated mutagenesis demonstrated that the second GAAC motif in the sequence GAACN₇GAACN₇GAAC located upstream of the R. sphaeroides lexA gene is absolutely necessary for its DNA damage-mediated induction. Moreover, mutagenesis of either the first or the third GAAC motif in this sequence reduced, but did not abolish, the inducibility of the R. sphaeroides lexA gene. A R. sphaeroides lexA-defective (Def) mutant has also been constructed by replacing the active lexA gene with an inactivated gene copy constructed in vitro. Crude extracts of the R. sphaeroides lexA(Def) strain are unable to form any protein-DNA complex when added to the wild-type lexA promoter of R. sphaeroides. Likewise, the R. sphaeroides lexA(Def) cells constitutively express the recA and lexA genes. All these data clearly indicate that the lexA gene product is the negative regulator of the R. sphaeroides SOS response. Furthermore, the morphology, growth and viability of R. sphaeroides lexA(Def) cultures do not show any significant change relative to those of the wild-type strain. Hence, R. sphaeroides is so far the only bacterial species whose viability is known not to be affected by the presence of a lexA(Def) mutation. Received: 31 January 2000 / Accepted: 3 April 2000     

Molecular cloning,sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^? mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Base pair substitution and frameshift mutagenesis induced by apurinic sites and two fluorene derivatives in a recA441 lexA (Def) strain

Summary One of the consequences of the induction of the Escherichia coli SOS system is the increased ability of the cells to perform mutagenesis. Induction of the SOS system is the result of derepression of a set of genes through a regulatory mechanism controlled by LexA and RecA. In response to an inducing signal, RecA is activated in a form that facilitates the proteolytic cleavage of LexA repressor. Previous works have shown that activated RecA plays a second role, i.e. it is required for the establishment of base pair substitution mutations promoted by UV irradiation. Using a forward mutatonal assay and recA441 lexA(Def) host bacteria, we show that the result can be extended not only to other mutagens promoting base pair substitution mutations (Apurinic sites, Ap sites and N-hydroxy-N-2-aminofluorene, N-OH-AF) but also mutagens promoting frameshift mutations (N-Acetoxy-N-2-acetylaminofluorene, N-AcO-AAF). In the recA441 lexA(Def) strain all the genes which are part of the lexA regulon, including recA itself, are expressed constitutively. The recA441 mutation allows RecA to acquire its activated form when the bacteria are grown at 42° C. We show that in such strains Ap sites or N-OH-AF induce a high level of mutations only when the bacteria are grown at 42° C. On the other hand, we show that N-AcO-AAF can promote mutations even at 30° C; the number of mutations being increased when the bacteria were grown at 42° C. Analysis of the mutants obtained at 30° C indicate that they belong to both type of mutations, UmuC-dependent or UmuC-independent. The much higher ability of N-AcO-AAF to induce RecA as compared to N-OH-AF strongly suggests that the former mutagen is able to induce at least partially the activated form of RexA441 even at 30°C in a strain which overproduces RecA, [lexA(Def)]. Furthermore, we show that the UmuC-independent type of mutagenesis induced by N-AcO-AAF depends on gene(s) that are part of the lexA regulon.     

Degradation of Escherichia coli DNA: evidence for limitation in vivo by protein X, the recA gene product.

Summary DNA is more extensively degraded after it is damaged in recA mutants of E. coli than in wild type cells. All data presented here are consistent with the recA gene product, protein X, being an inhibitor of nalidixic acid induced degradation of the bulk DNA (but not of newly replicated DNA). Production of protein X also is correlated with appearance of various S.O.S. repair functions. Evidence was obtained by comparing the rates of protein X synthesis and solubilization of uniformly-labeled DNA in intact cells, incubated in the presence of nalidixic acid. A set of mutants at the lexA locus produced protein X at different rates and degraded their DNA at rates which were inversely correlated to their rates of protein X production. A low concentration of rifampicin quite specifically inhibited protein X production by wild type E. coli, and allowed more rapid DNA degradation. After the DNA was damaged by the incubation of cells in the presence of nalidixic acid, cells preloaded with protein X degraded their DNA more slowly. We propose that protein X could protect DNA against degradation by binding to singlestranded regions, thereby inhibiting nuclease action.     

Induction of recA+-protein synthesis in Escherichia coli.

Summary Escherichia coli was infected with precA ⁺to determine the genetic and physiological factors controlling recA ⁺gene expression. When precA ⁺replication was prevented by superinfection immunity, recA ⁺protein synthesis was induced by UV radiation. The recA ⁺gene is negatively controlled by the lexA ⁺gene product because i) a dominant lexA mutation, lexA3, prevented induction of recA ⁺protein synthesis ii) a recessive lexA mutation, tsl-1, caused induction of recA ⁺protein synthesis. Conversely positive control of recA ⁺gene expression requires recA ⁺protein because i) a co-dominant tif-1 mutation (a recA mutation) caused induction of recA ⁺protein synthesis ii) a recessive mutation, recA1, prevented cis-induction of recA protein synthesis. recA ⁺protein and Protein X of UV irradiated bacteria co-migrated and were subject to the same physiological and genetic controls. It is concluded that Protein X is recA ⁺protein. lysogenic induction was prevented by TPCK, a protease inhibitor. However TPCK did not prevent induction of recA ⁺protein synthesis, indicating that induction of the two processes occurs in different ways. It is suggested that the lexA ⁺and recA ⁺proteins normally combine to repress the recA ⁺gene. Derepression might occur after DNA damaging treatments because the amount of this complex would be reduced by recA ⁺protein i) binding to single-stranded DNA and/or ii) being activated to function proteolytically towards regulatory molecules such as repressor.     

Identification of protein X of Escherichia coli as the recA+/tif+ gene product.

Summary Protein X, molecular weight 40,000, has been separated from the other proteins of E. coliby a two-dimensional gel electrophoretic technique which separates proteins according to isoelectric point (pI) in the firstdimension and according to molecular weight in the second. When protein X is induced in wild-type cells by mitomycin C treatmentit has a pI6.0. However, when protein X is induced in a tif-1 mutant, either by temperatureshift-up to 42° or by mitomycin C treatment at 30°, it has a pI6.2. The low level of protein X which is present inuninduced tif mutants at 30° also has a pI6.2. These results suggest thattif-1 is a mis-sense mutation in the gene coding for protein X. Since transduction andcomplementation studies indicate that tif-1 is a mutation of therecA ⁺ gene (Castellazzi, Morand, George and Buttin, 1977) it follows that protein X is the recA ⁺ gene product.A model has been formulated to account for the relationship between protein X synthesis and the recA ⁺ and lexA ⁺ genes. In this model, a repressor coded by lexA ⁺ binds to the operator of the recA ⁺ gene from whence it can normally only be removed by the combined action of an inducer and protein X, the recA ⁺ product. Thus, protein X controls its own synthesis. The tif-1 mutation leads to a temperature sensitive form of protein X which, at 42°, can spontaneously remove the repressor without the intervention of the inducer.