Transgenic expression of RecA of the spirochetes Borrelia burgdorferi and Borrelia hermsii in Escherichia coli revealed differences in DNA repair and recombination phenotypes.
After unsuccessful attempts to recover a viable RecA-deficient mutant of the Lyme borreliosis agent Borrelia burgdorferi, we characterized the functional activities of RecA of B. burgdorferi, as well as RecA of the relapsing fever spirochete Borrelia hermsii and the free-living spirochete Leptospira biflexa, in a recA mutant of Escherichia coli. As a control, E. coli RecA was expressed from the same plasmid vector. DNA damage repair activity was assessed after exposure of the transgenic cells to UV light or the radiomimetic chemicals methyl methanesulfonate and mitomycin C. Recombination activity in the cells was assessed by using an assay for homologous recombination between repeats in the chromosome and by measuring the ability of the cells to foster lytic growth by red gam mutant bacteriophage lambda. Overall, we found that transgenic cells with recA genes of B. burgdorferi, B. hermsii, and L. biflexa had approximately equivalent activities in promoting homologous recombination in the lacZ duplication assay, but cells with B. burgdorferi recA and, most notably, B. hermsii recA were significantly less capable than cells with L. biflexa recA or E. coli recA in responding to DNA damage or in facilitating plaque formation in the phage assay. The comparatively poor function of Borrelia recA in the latter set of assays may be the consequence of impaired coordination in the loading of the transgenic RecA by RecBCD and/or RecFOR in E. coli.
Putteet-Driver
Adrienne D
AD
Departments of Microbiology & Molecular Genetics, University of California Irvine, Irvine, California 92697-4025, USA.
Zhong
Jianmin
J
Barbour
Alan G
AG
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000254:growth & development; D001898:Borrelia / Q000737:chemistry / Q000235:genetics* / Q000821:virology; D025065:Borrelia burgdorferi / Q000737:chemistry / Q000235:genetics / Q000821:virology; D004260:DNA Repair / Q000235:genetics*; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D015870:Gene Expression; D008969:Molecular Sequence Data; D011938:Rec A Recombinases / Q000096:biosynthesis / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D016415:Sequence Alignment; D013045:Species Specificity; D014162:Transfection
D004269:DNA, Bacterial; D011938:Rec A Recombinases