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.     

Relationship between the functional regions of the RecA protein and ATP hydrolysis in UV-irradiated Escherichia coli cells.

The time course of the intracellular ATP concentration in several UV-irradiated RecA protease constitutive (Cptc) mutants of E. coli has been studied. All Cptc mutants harboring a mutation in region 3 of the RecA protein (including amino acid residues 298-301) increased ATP after UV damage but without any subsequent decrease. Nevertheless, these mutants induced the SOS response after UV irradiation. Likewise, truncated RecA proteins lacking region 3 are also unable to carry out massive ATP hydrolysis in UV-irradiated cells. On the other hand, mutants in region 1 (including amino acids 25-39) or 2 (amino acids 157-184) of the RecA protein showed an increase in ATP concentration during the first 20 min following UV irradiation, which dropped afterwards to the basal level. All these data indicate that region 3 of the RecA protein must be involved in the ATP hydrolysis process. Furthermore, a relationship between the quantity of the UV-mediated ATP produced and the strength of the different RecA Cptc mutants has also been found. Accordingly, both lexA71::Tn5 and null lexA mutants of E. coli only show a cellular ATP increase after UV irradiation when containing a multicopy plasmid carrying either a wild-type lexA or a lexA (Ind-) gene.     

recA operator mutations and their usefulness

Mutations at three positions in the operator for recA have been detected, cloned and sequenced. Derepressed amounts of recA protein vary over a forty-fold range and correlate well with reduced affinities for lexA repressor of the mutant operators. One mutant confirms the region of major groove interaction between repressor and operator. Another has been used to demonstrate that RecF pathway genes other than recA are under lexA control.     

Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities.

Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDCEc). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants.     

Physiology of the SOS response: kinetics of lexA and recA transcriptional activity following induction

The products of the lexA and recA genes play central roles in the regulation of the Escherichia coli SOS response. We have measured the rate of mRNA synthesis from each gene at intervals following various inducing treatments in order to obtain a more precise timing of the induction process. Further, we provide quantitative evidence for kinetics of decay from fully induced levels of mRNA synthesis to basal levels as the cells shut down the SOS response which are in agreement with previously published data on the expression of specific SOS functions. The induction kinetics of lexA and recA gene expression are parallel except for nalidixic acid (NAL) treatment, with the actual levels of lexA mRNA synthesis being about 10-fold lower than that of recA. Reestablishment of repression from RecA commenced over 30 min earlier than from lexA. These results are fully consistent with the model that the functions result from the increased gene expression.     

Effect of tsl (thermosensitive suppressor of lex) mutation on postreplication repair in Escherichia coli K-12.

Cells of Escherichia coli K-12 carrying lexA or recA mutations are more sensitive to UV radiation than corresponding wild-type cells and are defective in postreplication repair. Supressor mutations (tsl) have been described previously which increase the UV resistance of lexA uvr+, lexA uvrA, and recAI uvr+ strains, but not the resistance of recA1 uvrA strains. We have studied the effect of the tsl-1 mutation on postreplication repair and find that the enhanced survival conferred by this mutation is correlated with an increased capacity for postreplication repair.     

Allele replacement in Escherichia coli by use of a selectable marker for resistance to spectinomycin: replacement of the lexA gene.

We replaced the Escherichia coli lexA gene by a segment of DNA coding for resistance to spectinomycin and streptomycin. The use of this segment expands the range of selectable markers usable for allele replacement. The availability of this null lexA mutation will facilitate genetic analysis of lexA and the SOS regulon.     

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.     

Analysis of cell division in single clones of the Escherichia coli K-12 lexA mutant.

The growth of clones of lexA mutant and lexA+ cells was analyzed. During normal growth lexA mutant clones frequently divided early, producing smaller newborn cells than the lexA+ clones. Some newborn cells in the lexA clones did not elongate or divide at all, a response that was never observed in the lexA+ clones. When starved for thymidine, most of the lexA mutant clones elongated and subsequently divided. The majority of lexA+ clones also elongated but did not divide. The above results suggest that one of the functions of the lexA+ gene is coordination of DNA repair with cell division.     

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.     

Phenotypes of lexA mutations in Salmonella enterica: evidence for a lethal lexA null phenotype due to the Fels-2 prophage

The LexA protein of Escherichia coli represses the damage-inducible SOS regulon, which includes genes for repair of DNA. Surprisingly, lexA null mutations in Salmonella enterica are lethal even with a sulA mutation, which corrects lexA lethality in E. coli. Nine suppressors of lethality isolated in a sulA mutant of S. enterica had lost the Fels-2 prophage, and seven of these (which grew better) had also lost the Gifsy-1 and Gifsy-2 prophages. All three phage genomes included a homologue of the tum gene of coliphage 186, which encodes a LexA-repressed cI antirepressor. The tum homologue of Fels-2 was responsible for lexA lethality and had a LexA-repressed promoter. This basis of lexA lethality was unexpected because the four prophages of S. enterica LT2 are not strongly UV inducible and do not sensitize strains to UV killing. In S. enterica, lexA(Ind(-)) mutants have the same phenotypes as their E. coli counterparts. Although lexA null mutants express their error-prone DinB polymerase constitutively, they are not mutators in either S. enterica or E. coli.