Structural and functional characterization of the lexA gene of Xanthomonas campestris pathovar citri
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O R I GI N A L P A P E R
Y.-C. Yang á M.-K. Yang á T.-T. Kuo á J. Tu
Structural and functional characterization of the lexA gene of Xanthomonas campestris pathovar citri
Received: 16 August 2000 / Accepted: 10 November 2000 / Published online: 22 February 2001 Ó Springer-Verlag 2001
Abstract The role of the LexA protein and, speci®cally, its eect on recA expression were analyzed in Xanthomonas campestris pathovar citri (X.c. pv. citri). Overexpression of LexA from X.c. pv. citri, in the plant pathogen, as well as in Escherichia coli, results in increased sensitivity to the DNA-damaging agents mitomycin C and ultraviolet radiation, indicating that the recombinant X.c. pv. citri LexA protein is functional in a dierent bacterial species. Immunoblot analysis revealed that the overexpressed LexA protein functioned as a repressor of recA expression in X.c. pv. citri, and that the mitomycin C-induced increase in the abundance of RecA was accompanied by speci®c proteolysis of LexA that required RecA. Although the LexA protein from X.c. pv. citri also blocked the expression of recA in E. coli, the E. coli RecA protein was not able to support the autocatalytic cleavage of LexA from the plant pathogen. The transcription start site of the X.c. pv. citri lexA gene was identi®ed, and the region upstream of this gene was shown to confer responsiveness to mitomycin C on a luciferase reporter gene construct. Electrophoretic mobility-shift assays demonstrated that X.c. pv.
Communicated by R. Devoret Y.-C. Yang Institute of Life Science, National Defense Medical Center, Taipei, Taiwan, Republic of China M.-K. Yang Department of Biology, Fu Jen University, Taipei, Taiwan, Republic of China T.-T. Kuo Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China J. Tu (&) Institute of Botany, Academia Sinica, Nankang 115, Taipei, Taiwan, Republic of China E-mail: [email protected] Tel.: +886-2-27821258 ext. 425 Fax: +886-2-27827954
citri LexA interacts with the promoter region of X.c. pv. citri lexA, as well as with those of the recA genes of X.c. pv. citri and E. coli. These results indicate that LexA functions as a repressor of gene expression in X.c. pv. citri just as it does in E. coli. Key words Xanthomonas campestris pathovar citri á LexA repressor á LexA cleavage á DNA-binding protein
Introduction We have previously isolated and characterized a gene located immediately upstream of recA in Xanthomonas campestris pathovar citri (X.c. pv. citri) that is homologous to lexA of Escherichia coli (Yang et al. 2000). Characterization of an X.c. pv. citri lexA mutant (XLE22) has suggested that the LexA protein is a negative regulator of the expression of recA. The role of LexA in repressing the expression of various genes, including both recA and lexA, has been studied extensively in E. coli with regard to the SOS response to DNA damage (Little et al. 1981; Little and Mount 1982; Walker 1984; Humayun 1998). Damage to DNA induces the activation of RecA, which then promotes the autocatalytic cle
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