Analyses of binding sequences of the two LexA proteins of Xanthomonas axonopodis pathovar citri.

Xanthomonas axonopodis pv. citri (X. axonopodis pv. citri) possesses two lexA genes, designated lexA1 and lexA2. Electrophoretic mobility shift data show that LexA1 binds to both lexA1 and lexA2 promoters, but LexA2 does not bind to the lexA1 promoter, suggesting that LexA1 and LexA2 play different roles in regulating the expression of SOS genes. In this study, we have determined that LexA2 binds to a 14-bp dyad-spacer-dyad palindromic sequence, 5'-TGTACAAATGTACA-3', located at nucleotides -41 to -28 relative to the translation start site of lexA2 of X. axonopodis pv. citri. The two spacer nucleotides in this sequence can be changed from AA to TT without affecting LexA2 binding; all other base deletions or substitutions abolish LexA2 binding. The LexA1 binding sequence in the promoter region of lexA2 is TTAGTACTAAAGTTATAA and is located at -133 to -116, and that in the lexA1 gene is AGTAGTAATACTACT located at nucleotides -19 to -5 relative to the translation start site of lexA1. Any base change in the latter sequence abolishes LexA1 binding.

Identification and characterization of a second lexA gene of Xanthomonas axonopodis Pathovar citri.

We previously identified and characterized a lexA gene from Xanthomonas axonopodis pv. citri. For this study, we cloned and expressed a lexA homologue from X. axonopodis pv. citri. This gene was designated lexA2, and the previously identified lexA gene was renamed lexA1. The coding region of lexA2 is 606 bp long and shares 59% nucleotide sequence identity with lexA1. Analyses of the deduced amino acid sequence revealed that LexA2 has structures that are characteristic of LexA proteins, including a helix-turn-helix DNA binding domain and conserved amino acid residues required for the autocleavage of LexA. The lexA2 mutant, which was constructed by gene replacement, was 4 orders of magnitude more resistant to the DNA-damaging agent mitomycin C at 0.1 microg/ml and 1 order of magnitude more resistant to another DNA-damaging agent, methylmethane sulfonate at 30 microg/ml, than the wild type. A lexA1 lexA2 double mutant had the same degree of susceptibility to mitomycin C as the lexA1 or lexA2 single mutant but was 1 order of magnitude more resistant to methylmethane sulfonate at 30 microg/ml than the lexA1 or lexA2 single mutant. These results suggest that LexA1 and LexA2 play different roles in regulating the production of methyltransferases that are required for repairing DNA damage caused by methylmethane sulfonate. A mitomycin C treatment also caused LexA2 to undergo autocleavage, as seen with LexA1. The results of electrophoresis mobility shift assays revealed that LexA2 does not bind the lexA1 promoter. It binds to both the lexA2 and recA promoters. However, neither LexA2 nor LexA1 appears to regulate recA expression, as lexA1, lexA2, and lexA1 lexA2 mutants did not become constitutive for recA transcription and RecA production. These results suggest that recA expression in X. axonopodis pv. citri is regulated by mechanisms that have yet to be identified.