Identification and Characterization of a SecondlexAGene ofXanthomonas axonopodisPathovar citri

Abstract

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 g/ml and 1 order of magnitude more resistant to another DNA-damaging agent, methylmethane sulfonate at 30 g/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 g/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.

Role of Stress Response Sigma Factor SigG in Mycobacterium tuberculosis

“…MMC causes cell death during DNA damage, but there is also rescue due to DNA repair and the SOS response (error-prone response system). However, in the absence of LexA repression, the SOS system is constitutively expressed, and therefore lexA mutants have been demonstrated to display relative MMC resistance compared to the wild type (24). Therefore, we tested the MMC resistance of the M. tuberculosis ⌬sigG strain.…”

Identification and characterization of two uvrA genes of Xanthomonas axonopodis pathovar citri

“…citri wild-type strain XW47 and lexA mutants were described previously (Yang et al 2005). The uvrA1, uvrA2, and uvrA1uvrA2 mutants XUVR-A1, XUVR-A2 (XUVR-A2K), and XUVR-A1A2, were constructed in this study.…”

“…Immune complexes on Western blots were detected with horseradish peroxidase--conjugated goat anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and an enhanced chemiluminescence substrate (EZ-Link kit, Pierce, Rockford, IL, USA). The intensity of bands representing immunoreactive proteins was quantiWed by densitometry as described previously (Yang et al 2005). …”

“…LexA1 and LexA2 proteins were puriWed from lysates of E. coli (lexA -strain DM1187) cells harboring pLexA1E or pLexA2E (Yang et al 2005). Electrophoresis mobility shift assay (EMSA) was performed using a DIG gel shift kit (Roche Applied Science, Indianapolis, IN, USA).…”

A Constitutively Expressed, Truncated umuDC Operon Regulates the recA -Dependent DNA Damage Induction of a Gene in Acinetobacter baylyi Strain ADP1

“…The number of lexA genes present in bacteria is variable, ranging from zero in Rickettsia prowazekii (1), Borrelia burgdorferi, Chlamydia pneumoniae, and Helicobacter pylori (9) to two in, for example, Geobacter sulfurreducens (25) and Xanthomonas axonopodis pv. citri (47). The specific sequences of SOS boxes also vary between and within bacterial classes (7,8,9,15,18,25,46).…”