Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 05 Aug 2019 01:44:04 Reconstruction of the evolutionary history of the LexA-binding sequence Gerard Mazo´n, 1 Ivan Erill, 2 Susana Campoy, 3 Pilar Corte´s, 1 Evelyne Forano 4 and Jordi Barbe´ 1,3 Correspondence Jordi Barbe´ jordi.barbe@uab.es 1 Departament de Gene` tica i Microbiologia, Universitat Auto` noma de Barcelona, 08193 Bellaterra, Spain 2 Biomedical Applications Group, Centro Nacional de Microelectro´ nica, 08193 Bellaterra, Spain 3 Centre de Recerca en Sanitat Animal (CReSA), 08193 Bellaterra, Spain 4 Unite´ de Microbiologie, INRA, Centre de Recherches de Clermont-Ferrand-Theix, 63122 Saint-Gene` s-Champanelle, France Received 10 May 2004 Revised 14 July 2004 Accepted 15 July 2004 In recent years, the recognition sequence of the SOS repressor LexA protein has been identified for several bacterial clades, such as the Gram-positive, green non-sulfur bacteria and Cyanobacteria phyla, or the ‘Alphaproteobacteria’, ‘Deltaproteobacteria’ and ‘Gammaproteobacteria’ classes. Nevertheless, the evolutionary relationship among these sequences and the proteins that recognize them has not been analysed. Fibrobacter succinogenes is an anaerobic Gram-negative bacterium that branched from a common bacterial ancestor immediately before the Proteobacteria phylum. Taking advantage of its intermediate position in the phylogenetic tree, and in an effort to reconstruct the evolutionary history of LexA-binding sequences, the F. succinogenes lexA gene has been isolated and its product purified to identify its DNA recognition motif through electrophoretic mobility assays and footprinting experiments. After comparing the available LexA DNA-binding sequences with the F. succinogenes one, reported here, directed mutagenesis of the F. succinogenes LexA-binding sequence and phylogenetic analyses of LexA proteins have revealed the existence of two independent evolutionary lanes for the LexA recognition motif that emerged from the Gram-positive box: one generating the Cyanobacteria and ‘Alphaproteobacteria’ LexA-binding sequences, and the other giving rise to the F. succinogenes and Myxococcus xanthus ones, in a transitional step towards the current ‘Gammaproteobacteria’ LexA box. The contrast between the results reported here and the phylogenetic data available in the literature suggests that, some time after its emergence as a distinct bacterial class, the ‘Alphaproteobacteria’ lost its vertically received lexA gene, but received later through lateral gene transfer a new lexA gene belonging to either a cyanobacterium or a bacterial species closely related to this phylum. This constitutes the first report based on experimental evidence of lateral gene transfer in the evolution of a gene governing such a complex regulatory network as the bacterial SOS system. INTRODUCTION Preservation of genetic material is one of the most fundamental functions of any living being, and it is perhaps in the Domain Bacteria where this aspect has been most thoroughly studied. As in the case of many other biological processes, Escherichia coli has been the principal subject of this research, and many E. coli genes involved in preserva- tion of genetic material have been identified through the years. Some of them encode proteins that are able to repair different types of DNA damage, whilst others aim at guaranteeing cell survival in the presence of such lesions. Many of these genes act in a coordinated manner, con- stituting specific DNA repair networks, and the broadest and the most thoroughly studied of these regulons is the LexA-mediated SOS response (Walker, 1984). In E. coli, the LexA protein controls the expression of some 40 genes (Ferna´ndez de Henestrosa et al., 2000; Courcelle et al., 2001), including both the lexA and recA genes, which are, respectively, the negative and positive regulators of the SOS response (Walker, 1984). The E. coli LexA protein specifically recognizes and binds to an imperfect 16 bp palindrome with consensus sequence CTGTN 8 ACAG, designated the E. coli SOS or LexA box (Walker, 1984). Both in vitro and in vivo experiments have shown that Abbreviations: EMSA, electrophoresis mobility-shift assay; GST, glutathione S-transferase; LGT, lateral gene transfer. 0002-7315 G 2004 SGM Printed in Great Britain 3783 Microbiology (2004), 150, 3783–3795 DOI 10.1099/mic.0.27315-0