Molecular cloning of the recA analog from the marine fish pathogen Vibrio anguillarum 775

Abstract

The recA analog from Vibrio anguillarum 775 was isolated by complementation of recA mutations in Escherichia coli, and its protein product was identified. The recA analog promoted recombination between two partially deleted lactose operons, stimulated both spontaneous and mitomycin C-induced phage production in RecA-lambda lysogens, and restored near wild-type levels of resistance to UV radiation and methyl methanesulfonate.The Escherichia coli RecA protein is required for recombination (11,17,32,(35)(36)(37)41) and for cleavage of the LexA repressor that regulates the SOS repair network (24)(25)(26)(27)44). Although recA is not essential for survival of E. coli, RecAstrains are impaired with respect to recombination, DNA repair, resistance to UV radiation, and resistance to chemical DNA-damaging agents (9,31,46,48 (30,38). Transformants (7) of E. coli HB101 (recA13) (5), which were selected on L agar-ampicillin (100 jxg/ml), were screened for RecA+ recombinant plasmids on medium containing 1.5 mM methyl methanesulfonate (MMS) (3). Of 7,000 ampicillin-resistant (Ap') transformants, 8 were resistant to MMS and carried recombinant plasmids that also conferred MMS' phenotypes upon E. coli SS201 (recA56) (43) and XL1-Blue (recAl) (6).Restriction mapping of these plasmids suggested that the recA analog was located on a 2.4-kb HindIII fragment that was common to all plasmids. This fragment was isolated (28), ligated into the HindlIl site of pBR322, and subjected to further analysis (Fig. 1). ClaI-generated deletions that removed either half of the cloned fragment resulted in MMS sensitivity, indicating that recA spanned the central ClaI site of pJS60 (Fig. 1A). To determine whether recA spanned the BglII site, deletions were constructed by digesting pJS60 with BglII and filling in the 3' recesses in the absence of dCTP by using the Klenow fragment of DNA polymerase I (30). Plasmid pJSB2 suffered a 260-base-pair (bp) deletion (Fig. 1A), but Apr transformants of E. coli HB1O1(pJSB2) were MMSr, indicating that the BglII site does not lie within recA. TnS and TnJO00 insertions that interrupted the recA analog mapped within a 1.1-kb DNA region that began 230 bp to the right of the BglII site and ended 570 bp to the left of the HindIII site in pJSB2 (Fig. 1B).The ability of cloned V. anguillarum recA to complement the MMSS phenotype of recA E. coli mutants was measured ( Fig. 2A) Figure 2A shows that E. coli HB1O1(pJS60) and HB1O1(pJSB2) were resistant to high levels of MMS. Similar results were obtained in SS201 and XL1-Blue backgrounds (data not shown) and were unexpected since the results in Fig. 2A show that wild-type V. anguillarum 775 was sensitive to 1 to 2 mM MMS. Insertion mutations in V. anguillarum recA resulted in MMSS strains that displayed curves similar to those of HB1O1(pBR322) (Fig. 2A). The exception was E. coli HB1O1(pJSG20), carrying Tn1000 insertion 20 (Fig. 1B). HB1O1(pJSG20) displayed low-level resistance to MMS ( Fig. 2A), suggesting that the insertion present in pJSG20 may be very near to the carbox...

Cloning and expression in Escherichia coli of a recA homologue from Mycobacterium tuberculosis

“…tuberculosis recombinant plasmid were sensitive when exposed to short-wavelength UV radiation. Similar results were observed for the cloned recA homologues from the Gram-negative bacterium Vibrio anguillarum (Singer, 1989) and the obligate anaerobe Bacteroides fragilis (Goodman et al, 1987). The results obtained with Vibrio anguillarum indicate that complementation becomes less efficient when doses of UV radiation are higher.…”

“…Sancar et al (1980) found the E. coli recA gene product to be a single polypeptide with a calculated molecular mass of 37.8 kDa. Genes encoding RecA-like activity have been cloned from many Gram-negative bacteria, including Pseudomonas aeruginosa (Kokjohn & Miller, 1985), Shigella flexneri (Keener et al, 1984), Vibrw anguillarum (Singer, 1989), Proteus vulgaris (Keener et al, 1984), Agrobacterium tumefaciens (Farrand et al, 1989) and Haemophilus influenzae (Setlow et al, 1988). However, there has been only one report of isolation from a Gram-positive organism, namely Bacillus subtilis (Marrero & Yasbin, 1988), and to date there has been no report of the cloning of a recA gene from a member of the Actinomycetes.…”

A comprehensive review of Vibrio (Listonella) anguillarum: ecology, pathology and prevention

“…Genes in the ivi region are believed to play a role in virulence mechanisms, phospholipase signalling, genetic regulation, metabolism and outermembrane assembly, but such functions are left only to speculation at the present time and more research in this area is likely to emerge in future studies (Zou et al 2010). Furthermore, the gene recA is expressed to repair damaged DNA and the gene is required for recombination but not survival (Singer 1989;Singer et al 1996).…”

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

“…In contrast to the established way of cloning recA-like genes by interspecies complementation of E. coli recA mutations (e.g. Better and Helinski 1983;Goldberg and Mekalanos 1986;Koomey and Falkow 1987;Berson et al 1989;Singer 1989), identification and subcloning of the M. clara recA gene was performed by hybridization analysis.…”

“…No difference can be observed between the amino acid sequences of various functional domains that have been identified by either structural and functional analyses of E. coli recA gene mutations (Kawashima et al 1984;Wang and Tessman 1985;Ogawa and Ogawa 1986). As an example, the nucleotide sequence of the putative ATP-binding site (Gly 65 to Thr 72) of the E. coli RecA protein is completely conserved in its P. aeruyinosa and M. clara homologues, as well as in the recently published recA genes from Aquaspirillum tnaonetotacticum (Berson et al 1990) and Vibrio anouillarum (Singer 1989). The high similarity is further confirmed by the results of overexpression and protein-labelling studies showing the production of a 37-kDa polypeptide, as well as by complementation analysis of UV-irradiation-sensitive E. coli recA mutants.…”