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     

Construction of a recA mutant of Burkholderia (formerly Pseudomonas), cepacia

A recA mutant was constructed of a soil isolate of Burkholderia cepacia, strain ATCC 17616. Prior to mutagenesis, the recA gene was cloned from this strain by its ability to complement the methyl methanesulfonate sensitivity of an Escherichia coli recA mutant. Sequence analysis of the strain showed high sequence similarity (94% nucleic acid and 99% amino acid identity) with the recA gene previously cloned from a clinical isolate of B. cepacia, strain JN25. The subcloned recA gene from B. cepacia ATCC 17616 restored UV resistance and recombination proficiency to recA mutants of E. coli and Pseudomonas aeruginosa, as well as restoring the ability of D3 prophages to be induced to lytic growth from a RecA⁻ strain of P. aeruginosa. The recA mutant of B. cepacia ATCC 17616 was constructed by λ-mediated Tn5 mutagenesis of the cloned recA gene in E. coli, followed by replacement of the Tn5-interrupted gene for the wild-type allele in the chromosome of B. cepacia by marker exchange. The RecA⁻ phenotype of the mutant was demonstrated by the loss of UV resistance as compared to the parental strain. Southern hybridization analysis of chromosomal DNA from the mutant indicated the presence of Tn5 in the recA gene, and the location of the Tn5 insertion in the recA allele was identified by nucleotide sequence analysis. A test using the recA mutant to see if acquired resistance to d-serine toxicity in B. cepacia might be a result of RecA-mediated activities proved negative; nevertheless, RecA activity potentially contributes to the overall genomic plasticity of B. cepacia and a recA mutant will be useful in bioengineering of this species. Received: 24 January / Received revision: 11 July 1997 / Accepted: 25 August 1997     

Cloning, sequencing and expression of a recA gene from Amycolatopsis mediterranei

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter.     

The sequence and function of the recA gene and its protein in Pseudomonas aeruginosa PAO

Summary The recA gene of Pseudomonas aeruginosa has been isolated and its nucleotide sequence has been determined. The coding region of the recA gene has 1038 bp specifying 346 amino acids. The recA protein of P. aeruginosa showed a striking homology with that of Escherichia coli except for the carboxy-terminal region both at the nucleotide and amino acid level. The recA ⁺-carrying plasmids restored the UV sensitivity and recombination ability of several rec mutants of P. aeruginosa. The precise location of the recA gene on the chromosome was deduced from the analysis of R plasmids.     

Construction of an Azospirillum brasilense Sp7 recA mutant

Summary Cosmid clones encoding the recA gene of Azospirillum brasilense were isolated by intergeneric complementation of an Escherichia coli recA mutant. Site-directed Tn5 mutagenesis and subcloning of one complementing cosmid clone allowed us to localize the A. brasilense recA gene on a 1.2 kb DNA fragment. One Tn5 insertion that inactivates the cloned recA gene was crossed into the chromosome of A. brasilense by marker exchange. The resulting A. brasilense recA mutant showed increased sensitivity to the DNA methylating agent methyl methanesulfonate and to ultraviolet light and had at least one hundredfold reduced recombinational activity compared to the parent strain.     

Cloning and characterization of the recA gene of Paracoccus denitrificans and construction of a recA-deficient mutant

The recA gene of Paracoccus denitrificans has been isolated from a genomic library by hybridization with the Rhodobacter sphaeroides recA gene. Its complete nucleotide sequence consists of 1071 bp encoding a polypeptide of 356 amino acids. Nucleotide sequence analysis of the P. denitrificans recA gene revealed the closest identities with the R. sphaeroides and the Rhodobacter capsulatus recA genes. Nevertheless, and surprisingly, recA genes of these two phototrophic bacteria are not DNA damage-inducible when introduced into P. denitrificans cells, whereas recA genes of both P. denitrificans and Rhizobium etli are. These results suggest that the promoters of P. denitrificans and R. etli recA genes have a similar regulatory sequence. A recA-defective mutant of P. denitrificans has also been constructed by replacement of the active recA gene by an in vitro inactivated gene copy.     

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.     

Construction and characterization of a plasmid coding for a fragment of the Escherichia coli recA protein.

Summary The E. coli recA gene was cloned from the phage precA into the vector pBR313. A plasmid, pJL3, was also isolated by cloning a portion of the recA gene into the vector pBR322. pJL3 coded for a fragment of the recA protein 34 Kd (kilodaltons) in size (compared to 40 Kd for the intact protein). This fragment was antigenically related to the recA protein and its synthesis was subject to the same controls as that of the recA protein. The fragment did not express any detectable recA function. When wild-type cells with pJL3 were treated with nalidixic acid, the 34 Kd fragment and the -lactamase, made from a gene located downstream from the recA segment, were expressed at very high levels. Moreover, in these cells the rate of synthesis of intact recA protein from the chromosome was inhibited about 2-fold, relative to other chromosomal proteins, when compared to wild-type cells with the pBR322 vector. High level expression of the recA protein fragment and/or the -lactamase appeared to be lethal. The size of the 34 Kd fragment, taken together with the location of chain-termination codons in pJL3, localizes the regulatory region of the recA gene within 100 base pairs.     

Interspecies regulation of the recA gene of gram-negative bacteria lacking an E. coli-like SOS operator

The recA genes of Agrobacterium tumefaciens, Rhizobium meliloti, Rhizobium phaseoli and Rhodobacter sphaeroides, species belonging to the alpha-group bacteria of the Proteobacteria class, have been fused in vitro to the lacZ gene of Escherichia coli. By using a mini-Tn5 transposon derivative, each of these recA-lacZ fusions was introduced into the chromosome of each of the four species, and into that of E. coli. The recA genes of three of the alpha bacteria are induced by DNA damage when inserted in A. tumefaciens, R. phaseoli or R. meliloti chromosomes. The expression of the recA gene of R. sphaeroides is DNA damage-mediated only when present in its own chromosome; none of the genes is induced in E. coli. Likewise, the recA gene of E. coli is not induced in any of the four alpha species. These data indicate that A. tumefaciens, R. meliloti and R. phaseoli possess a LexA-like repressor, which is able to block the expression of their recA genes, as well as that of R. sphaeroides, but not the recA gene of E. coli. The LexA repressor of R. sphaeroides does not repress the recA gene of A. tumefaciens, R. meliloti, R. phaseoli or E. coli.     

Cloning and characterization of the Azotobacter vinelandii recA gene and construction of a recA deletion mutant

Summary The recA gene of Azotobacter vinelandii was isolated from a genomic library by heterologous complementation of an Escherichia coli recA mutation for resistance to UV radiation. The A. vinelandii recA gene was localized on adjacent PstI fragments of 1.3 and 1.7 kb. The cloned A. vinelandii recA gene was functionally analogous to the E. coli recA gene. It was also able to complement the E. coli recA mutation for homologous recombination. A recA deletion mutant of A. vinelandii was constructed. This mutant was sensitive to DNA-damaging agents like UV rays, methyl methane sulfonate (MMS) and nalidixic acid and was deficient in homologous recombination.     

Gene sequence of recA + and construction of recA mutants of Vibrio cholerae

The recA ⁺ gene of Vibrio cholerae O1 has been cloned, its nucleotide sequence determined and the product characterized. A deletion mutation was constructed in the recA gene and mutants showed the typical sensitivity to UV and to DNA-damaging agents, as well as an inability to mediate homologous DNA recombination. The chromosomal recA deletion mutants in V. cholerae do not show altered virulence in the infant mouse cholera model and are thus ideal strains for use in complementation studies.     

Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene

Summary A recombinant phage carrying the recA gene of Rhizobium phaseoli was isolated from a R. phaseoli genomic library by complementation of the Fec^– phenotype of the recombinant phage in Escherichia coli. When expressed in E. coli, the cloned recA gene was shown to restore resistance to both UV irradiation and the DNA alkylating agent methyl methanesulphonate (MMS). The R. phaseoli recA gene also promoted homologous recombination in E. coli. The cloned recA gene was only weakly inducible in E. coli recA strains by DNA damaging agents. The DNA sequence of the R. phaseoli recA gene was determined and compared with published recA sequences. No LexA-binding site was detected in the R. phaseoli recA upstream region.     

Ribosomal abnormality in recA mutants of Escherichia coli.

Summary The tif-1 mutation has been shown to affect protein synthesis in vitro by increasing translational ambiguity (Ephrati-Elizur, Luther-Davies and Hayes, 1976). It is demonstrated here that some recA mutations confer similar abnormality. By comparing suitable combinations of ribosomes and soluble proteins from recA ⁺ and recA cells the defect is shown to be associated with ribosomes. The recA mutation, which suppresses most phenotypic characteristics of the tif-1 mutation (Castellazzi, George and Buttin, 1972(b)) does not suppress the ribosomal abnormality. Sience the closely linked tif-1 and recA mutations lead to the expression of a common property they may be in the same gene.     

Evaluation of recA sequencing for the classification of Aeromonas strains at the genotype level

Aims: To evaluate the usefulness of partial recA sequences for the identification of Aeromonas strains at the genotype level. Methods and Results: A partial recA sequence was obtained from 21 type or reference strains and 33 Aeromonas isolates, collected in the South of Switzerland from human, animal and aquatic environments. The 272 bp long recA fragments showed a mean interspecies divergence of 7·8% and allowed the classification of strains at genotype level. However, some discrepancies could be observed with other gene sequence based analyses in the classification of some strains. Conclusions: The 272 bp long recA fragment is a good molecular marker to infer taxonomy of members of the genus Aeromonas, even if the primers we chose for the amplification did not allow its direct sequencing. Significance and Impact of the Study: In the genus Aeromonas, nucleotide sequences of some protein‐encoding genes have already been evaluated as molecular markers to be used in taxonomical and epidemiological researches. This study suggests the usefulness of a recA fragment as a further sequence to investigate for these purposes.     

Evidence that a system similar to the recA system of Escherichia coli exists in Vibrio cholerae

Summary Two lines of evidence suggest that a gene analogous to the recA gene of Escherichia coli exists in Vibrio cholerae and that its product serves a proteolytic function in the SOS response. Firstly, Southern blot hybridization using the recA gene of E. coli as a probe revealed a genomic sequence in V. cholerae which hybridized with the probe. Secondly, the SOS-like response in V. cholerae (as measured by beta phage induction) triggered by DNA damaging agents like Furazolidone could be blocked by Antipain, a protease inhibitor known to inhibit RecA protease action in E. coli. Maximal blocking effect of Antipain on beta phage induction occurred at 1 mM. At this concentration neither the viability of the host bacterium nor the lytic growth of a clear plaque mutant of the phage was affected by Antipain.     

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.     

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