The DNA static curvature has a role in the regulation of the ompS1 porin gene in Salmonella enterica serovar Typhi

Abstract

The DNA static curvature has been described to play a key role as a regulatory element in the transcription process of several bacterial genes. Here, the role of DNA curvature in the expression of the ompS1 porin gene in Salmonella enterica serovar Typhi is described. The web server MUTACURVE was used to predict mutations that diminished or restored the extent of DNA curvature in the 59 regulatory region of ompS1. Using these predictions, curvature was diminished by sitedirected mutagenesis of only two residues, and curvature was restored by further mutagenesis of the same two residues. Lowering the extent of DNA curvature resulted in an increase in ompS1 expression and in the diminution of the affinity of the silencer proteins H-NS and StpA for the ompS1 59 regulatory region. These mutations were in a region shown not to contain the H-NS nucleation site, consistent with the notion that the effect on expression was due to changes in DNA structural topology. INTRODUCTIONSalmonella enterica serovar Typhi (S. Typhi) is the aetiological agent of typhoid fever in humans (Pang et al., 1998). In our laboratory we have identified the S. Typhi ompS1 gene that encodes the OmpS1 quiescent porin belonging to the OmpC/OmpF superfamily (Fernández-Mora et al., 1995). OmpS1 has been reported to have a role in swarming motility, biofilm formation and virulence in mice (Toguchi et al., 2000;Mireles et al., 2001;Rodríguez-Morales et al., 2006). Expression of ompS1 is dependent on two overlapping promoters, P1 and P2. The P1 promoter is dependent on the OmpR response regulator. The P2 promoter does not require OmpR for activation, being active only in the absence of OmpR (Oropeza et al., 1999; Flores-Valdez et al., 2003;De la Cruz et al., 2007). Another key element in the transcriptional regulation of ompS1 is the global regulatory protein H-NS, a nucleoid protein of 137 amino acids (15 kDa) that negatively regulates its expression (FloresValdez et al., 2003;De la Cruz et al., 2007). In Salmonella, H-NS has been proposed to selectively silence horizontally acquired genes by targeting sequences with a GC content lower than the resident genome, regulating around 12 % of its genes (Lucchini et al., 2006; Navarre et al., 2006). StpA, an H-NS paralogue, was found to repress ompS1 in an hns background; and LeuO, a LysR-type regulator, positively regulates ompS1 expression by antagonizing H-NS and StpA (De la Cruz et al., 2007). LeuO has been implicated in several functions, such as stress resistance, virulence and biofilm formation (VanBogelen et al., 1996;Fang et al., 2000;Majumder et al., 2001;Tenor et al., 2004; Lawley et al., 2006;Moorthy & Watnick, 2005;Rodríguez-Morales et al., 2006). Recently, our group has described several genes regulated by LeuO in S. Typhi (Hernández-Lucas et al., 2008).In bacterial genomes, the recognition of their binding targets by regulatory proteins is commonly considered to be sequence-dependent, although DNA curvature plays a well-characterized role in many transcriptional regulation mechanisms in pr...

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