Identification of a lexA Gene in, and Construction of a lexA Mutant of, Xanthomonas campestris pv. citri

The lexA gene of Xanthomonas campestris pathovar citri (X.c. pv. citri) was cloned and sequenced. The 639-bp open reading frame encodes a protein of 213 amino acids that shares substantial sequence homology with the products of previously characterized lexA genes, sharing 46% identity with the LexA protein of Escherichia coli. Amino acids required for autocatalytic cleavage of LexA are conserved in the X.c. pv. citri protein, whereas domains thought to mediate DNA binding differ markedly from those of LexA proteins from E. coli and other bacteria. The X.c. pv. citri LexA protein was overexpressed in E. coli, and SDS-polyacrylamide gel electrophoresis revealed a molecular size of 23 kDa for the purified protein. A lexA mutant of X.c. pv. citri was constructed by gene replacement, and the basal level of recA expression in this mutant was shown to be similar to that for wild-type cells exposed to a DNA-damaging agent. These results indicate that LexA functions as a repressor of recA expression in X.c. pv. citri. Received: 1 September 1999 / Accepted: 25 October 1999     

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacteriumAnabaena sp. strain PCC 7120

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacteriumAnabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of theAnabaena ADPGlc PPase gene and its expression inEscherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48347 Da which is in agreement with the molecular mass determined by SDS-PAGE for theAnabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and theE. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in theAnabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in anE. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the nativeAnabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be theAnabaena enzyme.     

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.     

LexA protein of cyanobacterium Anabaena sp. strain PCC7120 exhibits in vitro pH-dependent and RecA-independent autoproteolytic activity

The LexA protein of the nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 exhibits a RecA-independent and alkaline pH-dependent autoproteolytic cleavage. The autoproteolytic cleavage of Anabaena LexA occurs at pH 8.5 and above, stimulated by the addition of Ca²⁺ and in the temperature range of 30–57 °C. Mutational analysis of Anabaena LexA protein indicated that the cleavage occurred at the peptide bond between Ala-84 and Gly-85, and optimal cleavage required the presence of Ser-118 and Lys-159, as also observed for LexA protein of Escherichia coli. Cleavage of Anabaena LexA was affected upon deletion of three amino acids, ⁸⁶GLI. These three amino acids are unique to all cyanobacterial LexA proteins predicted to be cleavable. The absence of RecA-dependent cleavage at physiological pH, which has not been reported for other bacterial LexA proteins, is possibly due to the absence of RecA interacting sites on Anabaena LexA protein, corresponding to the residues identified in E. coli LexA, and low cellular levels of RecA in Anabaena. Exposure to SOS-response inducing stresses, such as UV-B and mitomycin C neither affected the expression of LexA in Anabaena nor induced cleavage of LexA in either Anabaena 7120 or E. coli overexpressing Anabaena LexA protein. Though the LexA may be acting as a repressor by binding to the LexA box in the vicinity of the promoter region of specific gene, their derepression may not be via proteolytic cleavage during SOS-inducing stresses, unless the stress induces increase in cytoplasmic pH. This could account for the regulation of several carbon metabolism genes rather than DNA-repair genes under the regulation of LexA in cyanobacteria especially during high light induced oxidative stress.     

The protein HU can displace the LexA repressor from its DNA-binding sites

The major bacterial histone-like protein HU is a small, basic, dimeric protein composed of two closely related subunits. HU is involved in several processes in the bacterial cell such as the initiation of replication, transposition, gene inversion and cell division. It has been suggested that HU could introduce structural changes to the DNA which would facilitate or inhibit the binding of regulatory proteins to their specific sites. In this study we investigated the effect of HU on the binding of LexA protein, the regulator of SOS functions, to three of its specific binding sites. We show that HU can displace LexA from its binding sites on the operators of the lexA, recA and sfiA genes. The lexA operator was the most sensitive while the higher affinity sfiA operator was the least sensitive. Since HU, like its homologue IHF, probably binds DNA in the minor groove we tested the effect of distamycin, a drug which binds to the minor groove, on LexA binding. Like HU, this drug disrupted LexA–operator complexes. These results suggest that distortion of the minor groove of the lexA operators excludes the binding of the repressor to the major groove.     

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     

DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Differential regulation of ssb genes in the nitrogen‐fixing cyanobacterium,Anabaena sp. strain PCC71201

Anabaena sp. PCC7120 possesses three genes coding for single‐stranded DNA‐binding (SSB) protein, of which ssb1 was a single gene, and ssb2 and ssb3 are the first genes of their corresponding operons. Regulation of the truncated ssb genes, ssb1 (alr0088) and ssb2 (alr7559), was unaffected by N‐status of growth. They were negatively regulated by the SOS‐response regulatory protein LexA, as indicated by the (i) binding of Anabaena LexA to the LexA box of regulatory regions of ssb1 and ssb2, and (ii) decreased expression of the downstream gfp reporter gene in Escherichia coli upon co‐expression of LexA. However, the full‐length ssb gene, ssb3 (all4779), was regulated by the availability of Fe²⁺ and combined nitrogen, as indicated by (i) increase in the levels of SSB3 protein on Fe²⁺‐depletion and decrease under Fe²⁺‐excess conditions, and (ii) 1.5‐ to 1.6‐fold decrease in activity under nitrogen‐fixing conditions compared to nitrogen‐supplemented conditions. The requirement of Fe²⁺ as a co‐factor for repression by FurA and the increase in levels of FurA under nitrogen‐deficient conditions in Anabaena (Lopez‐Gomollon et al. 2007) indicated a possible regulation of ssb3 by FurA. This was substantiated by (i) the binding of FurA to the regulatory region of ssb3, (ii) repression of the expression of the downstream gfp reporter gene in E. coli upon co‐expression of FurA, and (iii) negative regulation of ssb3 promoter activity by the upstream AT‐rich region in Anabaena. This is the first report on possible role of FurA, an important protein for iron homeostasis, in DNA repair of cyanobacteria.     

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.     

Genetic analysis of the LexA repressor: Isolation and characterization of LexA(Def) mutant proteins

Summary We report the isolation of LexA mutant proteins with impaired repressor function. These mutant proteins were obtained by transforming a LexA-deficient recA-lacZ indicator strain with a randomly mutagenized plasmid harbouring the lexA gene and subsequent selection on MacConkey-lactose indicator plates. A total of 24 different lexA(Def) missense mutations were identified. All except three mutant proteins are produced in near-normal amounts suggesting that they are fairly resistant to intracellular proteases. All lexA(Def) missense mutations are situated within the first 67 amino acids of the amino-terminal DNA binding domain. The properties of an intragenic deletion mutant suggest that the part of the amino-terminal domain important for DNA recognition or domain folding should extent at least to amino acids 69 or 70. A recent 2D-NMR study (Lamerichs et al. 1989) has identified three a helices in the DNA binding domain of LexA. The relative orientation of two of them (helices 2 and 3) is reminiscent of, but not identical to, the canonical helix-turn-helix motif suggesting nevertheless that helix 3 might be involved in DNA recognition. The distribution of the lexA(Def) missense mutations along the first 67 amino-terminal amino acids indeed shows some clustering within helix 3, since 8 out of the 24 different missense mutations are found in this helix. However one mutation in front of helix 1 and five mutations between amino acids 61 and 67 suggest that elements other than helices 2 and 3 may be important for DNA binding.     

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.     

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     

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.     

A pAO1-encoded molybdopterin cofactor gene (moaA) ofArthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein

A gene homologous tomoaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor ofEscherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 ofArthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encodedmoaA gene fromA. nicotinovorans was expressed inE. coli as an active protein that functionally complementedmoaA mutants. Its reduced amino acid sequence shows 43% identity to theE. coli MoaA, 44% to the NarAB gene product fromBacillus subtilis, and 42% to the gene product of two contiguous ORFs fromMethanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in thefixZ gene product fromRhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement themoaA mutation inE. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy ofmoaA may not be unique in theA. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.     

Effects of mutations altering SOS regulation on a nalidixic acid-inducible system for the production of heterologous proteins inEscherichia coli

Summary The major leftward early promoter of phage p _L, has frequently been used to drive expression of heterologous genes inEscherichia coli.p _L is typically maintained fully repressed by the lambda cl protein. When induction of heterologous protein synthesis is desired, one of several potential mechanisms of destroying cl function is employed and the expression of the foreign gene commences. One method of derepressingp _L involves exposing cells to nalidixic acid, which results in the activation of RecA protein and the subsequent RecA-mediated proteolytic cleavage of cl. Activated RecA also mediates the cleavage of theE. coli LexA protein, resulting in induction of the SOS regulon (at least 15E. coli genes, includingrec A). We have examined the effect of two chromosomal mutations on the productivity of nalidixic acid inductions. One of the tested mutations (recA o) increased the intracellular concentration of RecA prior to induction; the other (lexAind^–) resulted in a mutated lexA protein insensitive to RecA-mediated cleavage. These mutations were introduced into a strain carrying acl⁺ defective lysogen. Synthesis of two heterologous proteins, human 1-antitrypsin and a fusion protein partially derived from thePlasmodium falciparum circumsporozooite surface antigen, was examined in the wild-type and mutant strains. The maximum -1 antitrypsin concentration achieved was improved by 50% when therecA o strain was used rather than the wild type; however; only smaller changes (20% or less) in the maximum concentration of the malaria fusion protein wer observed. Use of thelexAind^– strain resulted in a decrease in the maximum concentration attained for both heterologous products.     

Weigle reactivation and mutagenesis of bacteriophage lambda in lexA(Def) mutants of E. coli K12

Summary The SOS response in UV-irradiated bacteria enhances the survival and mutagenesis of infecting damaged bacteriophage . In a lexA(Def) strain, SOS bacterial genes are fully derepressed by an inactivating mutation in the LexA repressor gene. We tested several lexA(Def) derivative strains for their capacity to constitutively promote high survival and mutagenesis of irradiated . We showed that UV irradiation of the lexA(Def) host bacteria is still necessary for optimal efficiency of both these SOS functions, which are dependent on the umuC gene product and an activated form of RecA protein.     

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