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.
Dreyfus
L A
LA
Department of Microbiology, University of Texas Medical Branch, Galveston 77550.
D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000528:radiation effects; D007875:Legionella / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D010957:Plasmids; D011938:Rec A Recombinases / Q000096:biosynthesis / Q000235:genetics; D015183:Restriction Mapping; D014466:Ultraviolet Rays
D004251:DNA Transposable Elements; D011938:Rec A Recombinases