Cloning of the Serratia marcescens recA gene and construction of a Serratia marcescens recA mutant

A recombinant plasmid, pSM2513, containing an 8.5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2.7 kb Bg/II-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.     

Construction of an Agrobacterium tumefaciens C58 recA mutant.

Clones encoding the recA gene of Agrobacterium tumefaciens C58 were isolated from a cosmid bank by complementation of an Escherichia coli recA mutation. Subcloning and mutagenesis with the lacZ fusion transposon Tn3HoHo1 located the Agrobacterium recA gene to a 1.3-kilobase segment of DNA. beta-Galactosidase expression from the fusions established the direction in which the gene was transcribed. The gene restored homologous recombination as well as DNA repair functions in E. coli recA mutants. Similar complementation of DNA repair functions was observed in the UV-induced Rec- Agrobacterium mutant, LBA4301. The Agrobacterium recA gene was disrupted by insertion of a cassette encoding resistance to erythromycin, and the mutated gene was marker exchanged into the chromosome of strain NT-1. The resulting strain, called UIA143, was sensitive to UV irradiation and methanesulfonic acid methyl ester and unable to carry out homologous recombination functions. The mutation was stable and had no effect on other genetic properties of the Agrobacterium strain, including transformability and proficiency as a conjugal donor or recipient. Furthermore, strain UIA143 became tumorigenic upon introduction of a Ti plasmid, indicating that tumor induction is independent of recA functions. Sequence homology was detected between the recA genes of strain C58 and E. coli as well as with DNA isolated from agrobacteria representing the three major biochemically differentiated biovars of this genus. In some cases, biovar-specific restriction fragment length polymorphisms were apparent at the recA locus.     

Isolation and characterization of the recA gene of Rhizobium meliloti.

Interspecific complementation of an Escherichia coli recA mutant with plasmids containing a gene bank of Rhizobium meliloti DNA was used to identify a clone which contains the recA gene of R. meliloti. The R. meliloti recA protein can function in recombination and in response to DNA damage when expressed in an E. coli recA host, and hybridization studies have shown that DNA sequence homology exists between the recA gene of E. coli and that of R. meliloti. The isolated R. meliloti recA DNA was used to construct a recA R. meliloti, and this bacterium was not deficient in its ability to carry out symbiotic nitrogen fixation.     

Construction and characterization of a recA mutant of Thiobacillus ferrooxidans by marker exchange mutagenesis

To construct Thiobacillus ferrooxidans mutants by marker exchange mutagenesis, a genetic transfer system is required. The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from Escherichia coli to two private T. ferrooxidans strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed. To knock out the T. ferrooxidans recA gene, a mobilizable suicide plasmid carrying the ATCC 33020 recA gene disrupted by a kanamycin resistance gene was transferred from E. coli to T. ferrooxidans ATCC 33020 by conjugation under the best conditions determined. The two kanamycin-resistant clones, which have retained the kanamycin-resistant phenotype after growth for several generations in nonselective medium, were shown to have the kanamycin resistance gene inserted within the recA gene, indicating that the recA::Omega-Km mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination. These mutants exhibited a slightly reduced growth rate and an increased sensitivity to UV and gamma irradiation compared to the wild-type strain. However, the T. ferrooxidans recA mutants are less sensitive to these physical DNA-damaging agents than the recA mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair in T. ferrooxidans.     

Cloning and characteristics of recA gene in Pseudomonas aeruginosa

A recA-like gene from Pseudomonas aeruginosa was cloned and identified by means of interspecific complementation of gene recA repair defect in Escherichia coli. The gene was mapped in the PvuII-HindIII Ps. aeruginosa chromosome fragment of 1.5 kbp in length. Having been recloned in pUC18 or 19 plasmids in either of possible orientations, this fragment was shown to complement three different defects of E. coli recA mutants: in repair, recombination and SOS functions.     

Isolation and characterization of the Vibrio cholerae recA gene

A 3.6-kilobase PstI fragment was isolated from a Vibrio cholerae chromosomal DNA library and shown to encode RecA-like activity in complementation studies with Escherichia coli recA mutants. Although DNA hybridization experiments failed to detect any homology between the E. coli and V. cholerae recA genes, hyperimmune antiserum produced against purified E. coli RecA protein recognized epitopes shared by the V. cholerae protein. The V. cholerae chromosomal fragments, when cloned and transferred to E. coli, provided the missing recA functions, including resistance to the alkylating agent methyl methanesulfonate, resistance to UV irradiation, and promotion of homologous recombination in Hfr mating experiments.     

Functional properties of Borrelia burgdorferi recA

Functions of the Borrelia burgdorferi RecA protein were investigated in Escherichia coli recA null mutants. Complementation with B. burgdorferi recA increased survival of E. coli recA mutants by 3 orders of magnitude at a UV dose of 2,000 microJ/cm(2). The viability at this UV dose was about 10% that provided by the homologous recA gene. Expression of B. burgdorferi recA resulted in survival of E. coli at levels of mitomycin C that were lethal to noncomplemented hosts. B. burgdorferi RecA was as effective as E. coli RecA in mediating homologous recombination in E. coli. Furthermore, E. coli lambda phage lysogens complemented with B. burgdorferi recA produced phage even in the absence of UV irradiation. The level of phage induction was 55-fold higher than the level in cells complemented with the homologous recA gene, suggesting that B. burgdorferi RecA may possess an enhanced coprotease activity. This study indicates that B. burgdorferi RecA mediates the same functions in E. coli as the homologous E. coli protein mediates. However, the rapid loss of viability and the absence of induction in recA expression after UV irradiation in B. burgdorferi suggest that recA is not involved in the repair of UV-induced damage in B. burgdorferi. The primary role of RecA in B. burgdorferi is likely to be a role in some aspect of recombination.     

Overcoming a defect in generalized recombination in the marine fish pathogen Vibrio anguillarum 775: construction of a recA mutant by marker exchange.

A defect in generalized recombination has prevented the use of marker exchange for the construction of specific chromosomal mutations in the marine fish pathogen Vibrio anguillarum 775. Through the use of large segments of homologous DNA, we were successful in overcoming this defect and used marker exchange to construct a recA mutant of V. anguillarum H775-3. A recombinant cosmid carrying the recA gene of V. anguillarum 775 in the center of a 25-kb cloned DNA insert was isolated by complementation of methyl methanesulfonate (MMS) sensitivity in Escherichia coli HB101. The recA gene was inactivated by inserting a kanamycin resistance gene into recA, and the mutant gene was subsequently introduced into V. anguillarum H775-3 by conjugal mobilization. Isolation of recombinants between cosmid-borne recA::kan sequences and chromosomal DNA was facilitated by the introduction of an incompatible plasmid, and Southern hybridization was used to verify the presence of recA::kan in the chromosomal DNA of the recA mutant. V. anguillarum carrying recA::kan was considerably more sensitive to UV radiation and to MMS than was its parent, and near wild-type levels of resistance to MMS and UV light were restored by introduction of cloned recA genes from both E. coli and V. anguillarum. These results indicate that recA is required for DNA repair in V. anguillarum and demonstrate the utility of this modified marker exchange technique for the construction of mutations in this economically important fish pathogen.     

The recA gene of Chlamydia trachomatis: Cloning, sequence, and characterization in Escherichia coli

Abstract The recA gene of Chlamydia trachomatis was isolated by complementation of an Escherichia coli recA mutant. The cloned gene restored resistance to methyl methanesulfonate in E. coli recA mutants. The DNA sequence of the chlamydial gene was determined and the deduced protein sequence compared with other RecA proteins. In E. coli recA deletion mutants, the cloned gene conferred moderate recombinational activity as assayed by Hfr matings. The chlamydial recA gene was efficient in repairing alkylated DNA but less so in repairing of UV damage when compared with the E. coli homologue. As detected by an SOS gene fusion, a small but measurable amount of LexA co-cleavage was indicated.     

Cloning and organization of seven arginine biosynthesis genes from Neisseria gonorrhoeae.

A genomic library for Neisseria gonorrhoeae, constructed in the lambda cloning vector EMBL4, was screened for clones carrying arginine biosynthesis genes by complementation of Escherichia coli mutants. Clones complementing defects in argA, argB, argE, argG, argIF, carA, and carB were isolated. An E. coli defective in the acetylornithine deacetylase gene (argE) was complemented by the ornithine acetyltransferase gene (argJ) from N. gonorrhoeae. This heterologous complementation is reported for the first time. The carAB operon from E. coli hybridized with the gonococcal clones that carried carA or carB genes under conditions of high stringency, detecting 80% or greater similarity and showing that the nucleotide sequence of the carbamoylphosphate synthetase genes is very similar in these two organisms. Under these conditions for hybridization, the gonococcal clones carrying argB or argF genes did not hybridize with plasmids containing the corresponding E. coli gene. Cocomplementation experiments established gene linkage between carA and carB. Clones complementing a gene defect in argE were also able to complement an argA mutation. This suggests that the enzyme ornithine acetyltransferase from N. gonorrhoeae (encoded by argJ) may be able to complement both argA and argE mutations in E. coli. The arginine biosynthesis genes in N. gonorrhoeae appear to be scattered as in members of the family Pseudomonadaceae.     

Cloning of the recA gene of Bordetella pertussis and characterization of its product.

A recA gene of Bordetella pertussis was identified in a plasmid library by complementation of a recA mutation in E coli and subcloned as a 2.1-kb Sph I DNA fragment. Southern hybridization experiments showed no similarity to the E coli recA gene, but very strong similarity to other Bordetella species. E coli recA mutant cells containing the B pertussis recA gene at high gene dosage were resistant to DNA-damaging agents such as methyl methane sulfonate or 4-nitroquinoline-N-oxide, displayed induction of SOS functions, and were able to promote DNA recombination, but not induction of phage lambda. The latter phenotype distinguishes the B pertussis recA gene product from the corresponding proteins from most other Gram-negative organisms. Amino acid sequence comparisons revealed a high degree of structural conservation between prokaryotic RecA proteins.     

Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant

A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.     

Degenerate oligonucleotide primers for enzymatic amplification of recA sequences from gram-positive bacteria and mycoplasmas.

RecA protein in gram-negative bacteria, especially in Escherichia coli, has been extensively studied, but little is known about this key enzyme in other procaryotes. Described here are degenerate oligonucleotide primers that have been used to amplify by the polymerase chain reaction (PCR) recA sequences from several gram-positive bacteria and mycoplasmas. The DNA sequences of recA PCR products from Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, and Mycoplasma pulmonis were determined and compared. These data indicate that the M. pulmonis recA gene has diverged significantly from recA genes of other eubacteria. It should be possible to use cloned recA PCR products to construct recA mutants, thereby providing the means of elucidating homologous genetic recombination and DNA repair activities in these organisms.     

Molecular cloning and characterization of the recA gene from the cyanobacterium Synechococcus sp. strain PCC 7002.

The recA gene of Synechococcus sp. strain PCC 7002 was detected and cloned from a lambda gtwes genomic library by heterologous hybridization by using a gene-internal fragment of the Escherichia coli recA gene as the probe. The gene encodes a 38-kilodalton polypeptide which is antigenically related to the RecA protein of E. coli. The nucleotide sequence of a portion of the gene was determined. The translation of this region was 55% homologous to the E. coli protein; allowances for conservative amino acid replacements yield a homology value of about 74%. The cyanobacterial recA gene product was proficient in restoring homologous recombination and partial resistance to UV irradiation to recA mutants of E. coli. Heterologous hybridization experiments, in which the Synechococcus sp. strain PCC 7002 recA gene was used as the probe, indicate that a homologous gene is probably present in all cyanobacterial strains.     

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.     

Escherichia coli K-12 mutants in which viability is dependent on recA function.

A gene required for growth and viability in recA mutants of Escherichia coli K-12 was identified. This gene, rdgB (for Rec-dependent growth), mapped near 64 min on the E. coli genetic map. In a strain carrying a temperature-sensitive recA allele, recA200, and an rdgB mutation, DNA synthesis but not protein synthesis ceased after 80 min of incubation at 42 degrees C, and there was extensive DNA degradation. The rdgB mutation alone had no apparent effect on DNA synthesis or growth; however, mutant strains did show enhanced intrachromosomal recombination and induction of the SOS regulon. The rdgB gene was cloned and its-gene product identified through the construction and analysis of deletion and insertion mutations of rdgB-containing plasmids. The ability of a plasmid to complement an rdgB recA mutant was correlated with its ability to produce a 25-kilodalton polypeptide as detected by the maxicell technique.     

Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant.

A recombinant plasmid carrying the recA gene of Aeromonas caviae was isolated from an A. caviae genomic library by complementation of an Escherichia coli recA mutant. The plasmid restored resistance to both UV irradiation and to the DNA-damaging agent methyl methanesulfonate in the E. coli recA mutant strain. The cloned gene also restored recombination proficiency as measured by the formation of lac+ recombinants from duplicated mutant lacZ genes and by the ability to propagate a strain of phage lambda (red gam) that requires host recombination functions for growth. The approximate location of the recA gene on the cloned DNA fragment was determined by constructing deletions and by the insertion of Tn5, both of which abolished the ability of the recombinant plasmid to complement the E. coli recA strains. A. caviae recA::Tn5 was introduced into A. caviae by P1 transduction. The resulting A. caviae recA mutant strain was considerably more sensitive to UV light than was its parent. Southern hybridization analysis indicated that the A. caviae recA gene has diverged from the recA genes from a variety of gram-negative bacteria, including A. hydrophila and A. sobria. Maxicell labeling experiments revealed that the RecA protein of A. caviae had an Mr of about 39,400.