The widely conserved phage-shock-protein A (and operon expression is mediated through a promoter upstream of that depends on sigma factor RpoN (54) and the enhancer binding protein PspF. to the identification of RpoN-independent promoters both upstream and downstream of was also determined. The discovery of these RpoN-independent promoters reveals yet another level of transcriptional complexity for the operon that may function to allow low-level constitutive expression of genes and/or additional regulation under some conditions. INTRODUCTION The phage-shock-protein system (Psp) may help the bacterial cell to survive during dissipation of the proton motive force (PMF) and is conserved in many Gram-negative bacteria (for a recent review see Darwin, 2005). It was originally identified in K-12 (Brissette regulon (and regulon of (and operon expression has been observed following overexpression of secretins, some 175013-84-0 supplier cytoplasmic 175013-84-0 supplier membrane proteins, and upon disruption of the F0F1-ATPase (Maxson & Darwin, 2004). The Psp system of is also essential for virulence in a mouse model of infection (Darwin & Miller, 1999; Darwin & Miller, 2001). This is probably because Psp is essential during production of the Ysc type III secretion system (Darwin & Miller, 2001). Apparently, the Psp system must respond to a stress caused by mislocalization of the YscC secretin component of the type III secretion system (Darwin & Miller, 2001). Consequently, null mutations of some genes cause severe growth defects when is overexpressed (Darwin & Miller, 2001; Green & Darwin, 2004; Maxson 175013-84-0 supplier & Darwin, 2006). Regulation of gene expression has been well studied, and much of the current understanding is probably applicable to the homologous regulon. The and control regions each contain an RpoN (54)-dependent promoter, as well as binding sites for integration host factor (IHF) and PspF, a member of the enhancer binding protein family (Jovanovic regulon is completely dependent on PspF. Regulation is also mediated by several of the other Psp proteins. The peripheral cytoplasmic membrane protein PspA acts as a negative regulator, by directly interacting with PspF and inhibiting its activity (Dworkin expression, together with examination of the DNA sequence of its control region, indicated that it appeared to have a PspF/RpoN-dependent promoter (Darwin & Miller, 2001; Green & Darwin, 2004). Here, we confirm the presence of this promoter and determine its exact location. In addition, earlier genetic experiments raised the possibility of PspF- and, therefore, RpoN-independent expression of at least some of the genes within the operon (Darwin & Miller, 2001). In this study, we strengthened this hypothesis by constructing an null mutation and using interposon analysis of (genes. METHODS Bacterial strains, plasmids and routine growth conditions Bacterial strains and plasmids used in this study are listed in Table 1. For routine plasmid manipulations the host strain was DH5. Plasmids with an R6K were maintained in CC118 from either S17-1 or SM10 strains were grown at 37C, and strains were grown at 26C or 37C as noted. All strains were grown in Luria-Bertani (LB) broth and on LB agar (Miller, 1972). Antibiotics were used as before (Maxson & Darwin, 2004). Table 1 Bacterial strains and plasmids Strain constructions The red recombinase gene replacement system (Datsenko & Wanner, 2000), adapted for use in (Maxson & Darwin, 2004) was used to construct an in frame deletion mutation. Briefly, a mutation was constructed using Red recombinase-mediated allelic exchange. The kanamycin resistance gene was then removed by FLP recombinase mediated excision and the in frame deletion mutation was confirmed by colony PCR and Southern hybridization analysis (data not shown). The null mutant was grown in the presence of 5 mM alanyl-glutamine to alleviate a minor growth defect in LB media. The growth and regulatory phenotypes of deletion mutants 175013-84-0 supplier could be fully complemented by a plasmid encoding RpoN (data not shown). Single copy operon fusions were constructed, integrated into the locus and confirmed by colony PCR analysis as described previously (Maxson & Darwin, 2005). When necessary, strains were cured of the virulence plasmid as described (Darwin & Miller, 2001). Interposon analysis of (insert fragment of pAJD457 (Table 1) has a unique as a blunt ended as a and insertions, clones were chosen with the -Sp in the same orientation. Following insertion of the -Sp cassette, the and fragments were cloned into pAJD905 (Table 1). Each operon fusion was integrated into the locus of strains and confirmed by colony PCR analysis as Rabbit Polyclonal to Akt (phospho-Thr308) described previously (Maxson & Darwin, 2005). -Galactosidase assays The effect of YscC production on the.