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A new regulatory DNA motif of the gamma subclass Proteobacteria: identification of the LexA protein binding site of the plant pathogen Xylella fastidiosa

Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain¹
Fundo de Defesa da Citricultura (Fundecitrus), 14807-040, VI. Melhado- C. P. 391, Araraquara, Sao Paulo, Brazil²
Centre de Recerca en Sanitat Animal (CReSA), Universitat Autònoma de Barcelona-Institut de Recerca i Tecnologia Agroalimentària (UAB-IRTA), Bellaterra, 08193 Barcelona, Spain³

Author for correspondence: Jordi Barbé. Tel: +34 93 581 1837. Fax: +34 93 581 2387. e-mail: jordi.barbe{at}uab.es

Escherichia coli LexA protein is the repressor of a gene network whose members are directly involved in the repair of damaged DNA and in the survival of bacterial cells until DNA lesions have been eliminated. The lexA gene is widely present in bacteria, although the sequences of only three LexA-binding sites are known: Gram-positive, alpha Proteobacteria and some members of gamma Proteobacteria represented by E. coli. Taking advantage of the fact that the genome sequence of the plant-pathogenic bacterium Xylella fastidiosa has been determined, its lexA gene has been cloned and overexpressed in E. coli to purify its product. After demonstration that X. fastidiosa lexA and recA genes are co-transcribed, gel mobility shift assays and directed mutagenesis experiments using the promoter of the lexA–recA transcriptional unit demonstrated that the X. fastidiosa LexA protein specifically binds the imperfect palindrome TTAGN₆TACTA. This is the first LexA binding sequence identified in the gamma Proteobacteria differing from the E. coli-like LexA box. Although a computational search has revealed the presence of TTAGN₆TACTA-like motifs upstream of X. fastidiosa genes other than lexA, X. fastidiosa LexA only binds the promoter of one of them, XF2313, encoding a putative DNA-modification methylase. Moreover, X. fastidiosa LexA protein does not bind any of the other genes whose homologues are regulated by the LexA repressor in E. coli (uvrA, uvrB, ssb, ruvAB, ftsK, dinG, recN and ybfE). RT-PCR quantitative analysis has also demonstrated that lexA–recA and XF2313 genes, as well as the X. fastidiosa genes which are homologues to those of E. coli belonging to the LexA regulon, with the exception of ssb, are DNA damage-inducible in X. fastidiosa.

Keywords: DNA damage, gene expression, SOS system

Abbreviations: DIG, digoxigenin

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