Sequence analysis of the Gluconobacter oxydans RecA protein and construction of a recA-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.
Liu
Y T
YT
Institute of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Chen
C G
CG
Chao
D C
DC
Lee
F
F
Liao
C L
CL
Sytwu
H K
HK
Chou
C F
CF
Ji
D D
DD
D016947:Acetobacteraceae / Q000737:chemistry* / Q000235:genetics* / Q000528:radiation effects; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA; D004260:DNA Repair; D018274:Electroporation; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D011938:Rec A Recombinases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013014:SOS Response, Genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
D004247:DNA; D011938:Rec A Recombinases