LeuO antagonizes H‐NS and StpA‐dependent repression in Salmonella enterica ompS1

Abstract

SummaryThe ompS1 gene encodes a quiescent porin in Salmonella enterica. We analysed the effects of H-NS and StpA, a paralogue of H-NS, on ompS1 expression. In an hns single mutant expression was derepressed but did not reach the maximum level. Expression in an stpA single mutant showed the same low repressed level as the wild type. In contrast, in an hns stpA background, OmpS1 became abundant in the outer membrane. The expression of ompS1 was positively regulated by LeuO, a LysR-type quiescent regulator that has been involved in pathogenesis. Upon induction of the cloned leuO gene into the wild type, ompS1 was completely derepressed and the OmpS1 porin was detected in the outer membrane. LeuO activated the P1 promoter in an OmpR-dependent manner and P2 in the absence of OmpR. LeuO bound upstream of the regulatory region of ompS1 overlapping with one nucleation site of H-NS and StpA. Our results are thus consistent with a model where H-NS binds at a nucleation site and LeuO displaces H-NS and StpA.

New insights into transcriptional regulation by H-NS

“…Thus, a single protein may counter silencing by more than one mechanism. The LysR family regulator LeuO counters H-NS silencing of the putative virulence gene yjjQ in avian pathogenic E. coli [52] and H-NS/StpA silencing of the ompS1 porin gene in Salmonella [53]. The importance of H-NS in regulating horizontal gene transfer is not limited to pathogenesis.…”

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

“…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).…”

“…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).…”

“…The advantage of the software used was that it allowed the evaluation of the effect on DNA curvature of every double point mutation that encompassed the region from 2226 to 276 (fragment Fb-wt) (Fig. 1A, B).The mutations that affected the DNA curvature in silico were introduced into the wild-type ompS1 59 upstream regulatory region near the H-NS binding site as defined previously by EMSA and DNase footprinting analysis (De la Cruz et al, 2007). First, a double mutation was introduced to diminish the extent of DNA curvature (pRO310-mt).…”

“…DNA fragments generated by PCR (100 ng) were incubated with increasing concentrations of H-NS, StpA or LeuO for 20 min at 4 uC in 20 ml. Binding buffers for each protein were described previously (De la Cruz et al, 2007). The DNA fragments were separated by electrophoresis in 6 % native polyacrylamide gels in 0.56 Tris/borate/EDTA buffer at 4 uC.…”