Cloned recA Gene Research Articles

Construction and Characterization of a recA Mutant of Xanthomonas campestris pv. citri

The recA gene of Xanthomonas campestris pathovar citri (X. c. pv. citri) was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame of 1,032 bp that encodes a 344-amino acid protein and which shows a high degree of homology to recA genes from other bacteria. The cloned recA gene of X. c. pv. citri restored to a recA deletion mutant of Escherichia coli the ability to resist killing induced by methylmethane sulfonate or ultraviolet radiation, indicating that the cloned gene was functional in E. coli. A recA mutant of X. c. pv. citri was constructed by gene replacement and was shown both not to possess detectable DNA recombination activity and to be markedly more sensitive to DNA-damaging agents than is wild-type X. c. pv. citri. Transformation of recA mutants of E. coli and X. c. pv. citri with a plasmid containing X. c. pv. citri recA conferred the ability to produce a 37-kDa protein that reacted with antiserum to E. coli RecA protein. Two additional open reading frames were also identified in the recA region of the X. c. pv. citri genome: One located immediately upstream from recA that encodes a 213-amino acid protein with a high degree of similarity to the LexA protein of E. coli, and another located immediately downstream of recA that encodes a 153-amino acid protein with sequence similarity to the RecX protein of Pseudomonas aeruginosa.

Sequence analysis of theGluconobacter oxydansRecA protein and construction of arecA-deficient mutant

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, and Pseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.

Sequence analysis of the <i>Gluconobacter oxydans</i> RecA protein and construction of a <i>rec</i>A-deficient mutant

The deduced amino acid sequence of Gluconobacter oxydans RecA protein shows 75.2, 69.4, and 66.2% homology with those from Aquaspirillum magnetotacticum, Escherichia coli, and Pseudomonas aeruginosa, respectively. The amino acid residues essential for function of the recombinase, protease, and ATPase in E. coli recA protein are conserved in G. oxydans. Of 24 amino acid residues believed to be the ATP binding domain of E. coli RecA, 17 are found to be identical in G. oxydans RecA. Interestingly, nucleotide sequence alignment between the SOS box of G. orphans recA gene and those from different microorganisms revealed that all the DNA sequences examined have dyad symmetry that can form a stem-loop structure. A G. oxydans recA-deficient mutant (LCC96) was created by allelic exchange using the cloned recA gene that had been insertionally inactivated by a kanamycin-resistance cassette. Such replacement of the wild-type recA with a kanamycin resistance gene in the chromosome was further verified by Southern hybridization. Phenotypically, the recA-deficient mutant is significantly more sensitive to UV irradiation than the wild-type strain, suggesting that the recA gene of G. oxydans ATCC9324 plays a role in repairing DNA damage caused by UV irradiation. Moreover, the mutant strain is much more plasmid transformable than its parent strain, illustrating that G. oxydans LCC96 could be used as a host to take up the recombinant plasmid for gene manipulation.

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 lambda-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.

Cloning of the Helicobacter pylori recA gene and functional characterization of its product.

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned a recA homolog from Helicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to the Campylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus sigma 70 promoter sequence was found upstream of recA. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream of recA. Compared to the wild-type strains, isogenic H. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinant H. pylori RecA protein produced in Escherichia coli strain GC6 (recA-) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein in H. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of the H. pylori mutant using the cloned recA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA in H. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced in E. coli, giving rise to a smaller but inactive protein.

Properties of R-plasmid pEB017, which confers both enhanced UV-radiation resistance and mutability to wild-type, recA and umuC strains of Escherichia coli K12

The R-plasmid, pEB017, restored recombination ability to recA56 and conferred enhanced resistance to UV-radiation and enhanced UV-radiation mutability to wild-type, recA56 and umuC36 strains of Escherichia coli K12. Comparatively, pEB017 enhanced UV-radiation mutability in a umuC strain, and also enhanced UV-radiation and nitrofuran mutability in a wild-type strain several-fold more than did another R-plasmid, pKM101. Plasmid pEB017 also mediated about a 3-fold enhancement of the SOS induction of ß-galactosidase synthesis in a recA strain, compared with the normal recA + gene of E. coli. A BamHI fragment of pEB017 DNA was cloned into plasmid vector pBR322 to yield pEB021. The BamHI fragment in pEB021 (3.5 kb) is about 170 bp longer between the BamHI and PstI sites on the left end of the recA-like fragment, compared with published data on a similarly cloned recA gene from E. coli. Plasmid pEB021 conferred enhanced resistance to UV-radiation and enhanced UV-radiation mutability in wild-type and recA strains, and restored recombination ability in a recA strain. The introduction of pEB021 into a umuC strain made the cells slightly more resistant to killing by UV-irradiation, and promoted a small amount of UV-mutability in an otherwise nonmutable strain. These results suggest that R-plasmid pEB017 has a recA-like gene that mediates the enhanced resistance to UV-radiation and enhanced UV-radiation mutability, but which seems different in several important aspects from the normal recA gene in E. coli.

Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene

A recombinant lambda 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.

Functional characteristics of the recA gene from Serratia marcescens strain Sb

The cloned recA gene from Serratia marcescens Sb was expressed and complemented defects in the UV repair, recombination, and SOS induction of an Escherkhia coli host deleted for recA. Moreover, the Serratia gene, recA (Sm), supported the same frequency of recombination per unit length of DNA as did the homologous Escherichia coli gene, recA(Ec).

Molecular cloning and characterization of the recA gene of Methylomonas clara and construction of recA deficient mutant.

The recA gene of the methylotrophic bacterium Methylomonas clara has been isolated from a genomic library by hybridization with the Escherichia coli recA gene. Its complete nucleotide sequence consists of 1029 bp encoding a polypeptide of 342 amino acids. Nucleotide sequence analysis of the M. clara recA gene revealed extensive homologies to recA genes from E. coli and Pseudomonas aeruginosa. Part of the physiological activity of the M. clara RecA protein has become evident in that E. coli recA mutant HB101 is complemented. The cloned recA gene has been modified in vitro by site-specific mutagenesis and by insertion of a kanamycin-resistance gene cassette into the recA coding sequence. M. clara recA mutants were obtained by replacement of the active recA gene by an in-vitro inactivated gene copy.

Cloning of the recA gene from a free-living leptospire and distribution of RecA-like protein among spirochetes

A recombinant plasmid carrying the recA gene of Leptospira biflexa serovar patoc was isolated from a cosmid library of genomic DNA by complementation of an Escherichia coli recA mutation. The cloned serovar patoc recA gene efficiently restored resistance to UV radiation and methyl methanesulfonate. Recombination proficiency was also restored, as measured by the formation of Lac+ recombinants from duplicated mutant lacZ genes. Additionally, the cloned recA gene increased the spontaneous and mitomycin C-induced production of lambda phage in lysogens of an E. coli recA mutant. The product of the cloned recA gene was identified in maxicells as a polypeptide with an Mr of 43,000. Antibodies prepared against the E. coli RecA protein cross-reacted with the serovar patoc RecA protein, indicating structural conservation. Southern hybridization data showed that the serovar patoc recA gene has diverged from the recA gene of L. interrogans, Leptonema illini, and E. coli. With the exception of the RecA protein of L. interrogans serovar hardjo, the RecA protein of the Leptospira serovars and L. illini were synthesized at elevated levels following treatment of cells with nalidixic acid. The level of detectable RecA correlated with previous studies demonstrating that free-living cells of L. biflexa serovars and L. illini were considerably more resistant to DNA-damaging agents than were those of parasitic L. interrogans serovars. RecA protein was not detected in cells of virulent Treponema pallidum or Borrelia burgdorferi.

Molecular analysis of the Bacteroides fragilis recA gene

The nucleotide sequence of a 2.5-kb DNA segment containing the Bacteroides fragilis recA gene was determined. The coding region of the recA gene specifies a protein of 318 amino acids. The RecA protein of B. fragilis shows significant homology with that of Escherichia coli, Thiobacillus ferooxidans, Pseudomonas aeruginosa and Proteus mirabilis. No SOS box characteristic of LexA-regulated promoters could be identified in the 5′-noncoding region of the B. fragilis recA gene. Promoter activity of the cloned recA gene in E. coli was located within a 113-bp fragment of the B. fragilis DNA by in vitro construction of operon fusions with a promoterless lacZ gene. The transcription start point for this gene in B. fragilis was determined by primer extension analysis.

Construction of an Azospirillum brasilense Sp7 recA mutant

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.

Expression and nucleotide sequence analysis of the Legionella pneumophila recA gene

The nucleotide sequence of the L pneumophila recA gene was determined. The coding region was 1044 nucleotides (348 codons), specifying a 37,934 Da protein. Preceding the recA gene was a tandem set of transcription regulatory sequences and putative LexA binding sites. When expressed in E. coli, the cloned recA gene yield two proteins with molecular weights of approximately 38,000 and 35,500 Da. The larger of these two proteins shared 70.4% and 74.6% identity with the E. coli and Pseudomonas aeruginosa RecA proteins, respectively. The 35,500 Da recA encoded protein was presumed to be the product of translation from Met26 which was preceded by an alternate ribosomal binding site.

Molecular Cloning and Expression in Escherichia coli of the recA Gene of Legionella pneumophila

Interspecific complementation of an Escherichia coli recA mutant with a Legionella pneumophila genomic library was used to identify a recombinant plasmid encoding the L. pneumophila recA gene. Recombinant E. coli strains harbouring the L. pneumophila recA gene were isolated by replica-plating bacterial colonies on medium containing methyl methanesulphonate (MMS). MMS-resistant clones were identified as encoding the L. pneumophila recA analogue by their ability to protect E. coli HB101 from UV exposure and promote homologous recombination. Subcloning of selected restriction fragments and Tn5 mutagenesis localized the recA gene to a 1.7 kb Bg/II-EcoRI fragment. Analysis of minicell preparations harbouring a 1.9 kb EcoRI fragment containing the recA coding segment revealed a single 37.5 kDa protein. Insertional inactivation of the cloned recA gene by Tn5 resulted in the disappearance of the 37.5 kDa protein, concomitant with the loss of RecA function. The L. pneumophila recA gene product did not promote induction of a lambda lysogen; instead, the presence of the heterologous recA gene caused a significant reduction in spontaneous and mitomycin-C-induced prophage induction in recA+ and recA E. coli backgrounds. Despite the lack of significant genetic homology between the L. pneumophila recA gene and the E. coli counterpart, the L. pneumophila RecA protein was nearly identical to that of E. coli in molecular mass, and the two proteins showed antigenic cross-reactivity. Western blot analysis of UV-treated L. pneumophila revealed a significant increase in RecA antigen in irradiated versus control cells, suggesting that the L. pneumophila recA gene is regulated in a manner similar to that of E. coli recA.

Method for gene replacement in Pseudomonas aeruginosa used in construction of recA mutants: recA-independent instability of alginate production

The availability of a technique for site-directed mutagenesis by gene replacement provides a powerful tool for genetic analysis in any bacterial species. We report here a general technique for gene replacement in Pseudomonas aeruginosa. Genes on fragments of cloned P. aeruginosa DNA, altered by transposon mutagenesis, can be transduced into a recipient strain and can replace homologous genes in the P. aeruginosa genome. In this study we applied this technique to the construction of recA mutants of P. aeruginosa. A cloned segment of P. aeruginosa FRD1 DNA was isolated which encoded a protein analogous to the recA gene product of Escherichia coli. The P. aeruginosa recA gene was able to complement several defects associated with recA mutation in E. coli. Transposon Tn1 and Tn501 insertions in the cloned recA gene of P. aeruginosa were used to generate chromosomal recA mutants by gene replacement. These recA strains of P. aeruginosa were more sensitive to UV irradiation and methyl methane sulfonate and showed reduced recombination proficiency compared with the wild type. Also examined was the effect of recA mutations on the expression of alginate, a virulence trait. Alginate is a capsulelike polysaccharide associated with certain pulmonary infections, and its expression is typically unstable. The genetic mechanism responsible for the instability of alginate biosynthesis was shown to be recA independent.

Lambda placMu: a transposable derivative of bacteriophage lambda for creating lacZ protein fusions in a single step.

We isolated a plaque-forming derivative of phage lambda, lambda placMu1 , that contains sequences from bacteriophage Mu enabling it to integrate into the Escherichia coli chromosome by means of the Mu transposition system. The Mu DNA carried by this phage includes both attachment sites as well as the cI, ner (cII), and A genes. Lambda placMu1 also contains the lacZ gene, deleted for its transcription and translation initiation signals, and the lacY gene of E. coli, positioned next to the terminal 117 base pairs from the S end of Mu. Because this terminal Mu sequence is an open reading frame fused in frame to lacZ, the phage can create lacZ protein fusions in a single step when it integrates into a target gene in the proper orientation and reading frame. To demonstrate the use of this phage, we isolated lacZ fusions to the malB locus. These showed the phenotypes and regulation expected for malB fusions and could be used to isolate specialized transducing phages carrying the entire gene fusion as well as an adjacent gene (malE). They were found to be genetically stable and rarely (less than 10(-7] gave rise to secondary Lac+ insertions. We also isolated insertions into high-copy-number plasmids. The physical structure of these phage-plasmid hybrids was that expected from a Mu-dependent insertion event, with the lambda placMu prophage flanked by the Mu attachment sites. Lac+ insertions into a cloned recA gene were found at numerous positions and produced hybrid proteins whose sizes were correlated with the position of the fusions in recA.

Cloned truncated recA genes in E. coli II. Effects of truncated gene products on in vivo recA+ protein activity.

The ability of plasmids carrying truncated recA genes to sensitize recA+ cells to UV-irradiation was dependent upon the size of the cloned recA gene fragment. Radiosensitization correlated with the inhibition of recombinational repair, and the in vivo reduction of recA protein recombinase activity, as measured by lambda bio 11 plating efficiency. W-reactivation was also abolished by the radiosensitizing plasmids, whilst DNA degradation control, naladixic acid induced filamentation and lambda induction were unaffected. UV-induced mutagenesis in excision proficient E. coli was unaffected, whilst excision deficient strains were hypermutable. It is suggested that these effects of plasmids bearing 22% or more of the recA gene are the result of the interaction of full-sized and truncated protein subunits to generate multimers unable to catalyze recombination.