Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon

Abstract

The Salmonella typhimurium PhoP-PhoQ two-component regulatory system controls the expression of several genes, some of which are necessary for virulence. During a screening for PhoP-regulated genes, we identified the phoPQ operon as a PhoP-activated locus. ␤-Galactosidase activity originating from phoPQ-lac transcriptional fusions required the presence of both the transcriptional regulator PhoP and its cognate sensor-kinase PhoQ. At low concentrations, PhoQ stimulated expression of phoPQ-lac transcriptional fusions. However, larger amounts of PhoQ protein without a concomitant increase in PhoP failed to activate phoPQ-lac fusions. Two different transcripts are produced from the phoPQ operon during exponential growth. These transcripts define two promoters: phoPp 1 , which requires both PhoP and PhoQ for activity and which is environmentally regulated, and phoPp 2 , which remains active in the absence of PhoP and PhoQ but which is slightly stimulated by these proteins. The pattern of transcriptional autoregulation was also observed at the protein level with anti-PhoP antibodies. In sum, autoregulation of the phoPQ operon provides several levels of control for the PhoP-PhoQ regulon. First, environmental signals would stimulate PhoQ to phosphorylate the PhoP protein that is produced at basal levels from the PhoP-PhoQ-independent promoter. Then, phospho-PhoP would activate transcription of phoPp 1 , resulting in larger amounts of PhoP and PhoQ and increased expression of PhoP-activated genes. A return to basal levels could be mediated by a posttranscriptional mechanism by which translation of the mRNA produced from phoPp 1 is inhibited.Salmonellae are facultative intracellular pathogens responsible for several disease syndromes in a wide variety of animal species. In humans, they have been implicated in four pathological conditions: typhoid fever, gastroenteritis (food poisoning), bacteremia, and the asymptomatic carrier state (14). Certain Salmonella serotypes have a very narrow host range, while others are poorly host adapted and cause distinct diseases in different hosts. For example, typhoid fever is primarily caused by the human-adapted Salmonella typhi, while Salmonella typhimurium, the leading serotype associated with gastroenteritis in humans, causes a typhoid-like disease in susceptible mice (16). The ability to genetically manipulate S. typhimurium and the availability of excellent models of infection have allowed the identification of many of the virulence determinants that enable Salmonella spp. to adapt and prosper within different host environments.In S. typhimurium, virulence is controlled at the transcriptional level by several proteins, including the RpoS sigma factor (7), the cyclic AMP-binding protein CRP (4), and the two-component regulatory systems OmpR-EnvZ (6) and PhoPPhoQ (8,11,13,28). The PhoP-PhoQ system was originally identified as a virulence determinant by the intramacrophage survival defect of S. typhimurium phoP mutants (8, 9). A virulence role for phoP was independently demonstrated b...

Temperature and Mg2+ Sensing by a Novel PhoP-PhoQ Two-component System for Regulation of Virulence in Edwardsiella tarda

“…Our EMSA results showed that PhoP binds to the promoter regions of both esrB and phoP, suggesting that it may be involved in regulating the expression levels of both itself and EsrB. Transcriptional autoregulation was also observed in the phoPQ operon of S. typhimurium (35). The data herein demonstrated that the PhoP-PhoQ system senses temperature and Mg 2ϩ concentration.…”

“…Similar procedures were followed to generate the phoP LacZ reporter gene system using the primer pair pRWphoP (supplemental Table 1). For the ␤-galactosidase assays, E. tarda cells were grown in N-minimal medium (15) overnight at 20,23,25,30,35, or 37°C with 1 or 10 mM Mg 2ϩ . The overnight culture (5%) was inoculated into fresh medium and grown at the specified temperatures until the cell density reached 0.5, as measured by optical density at 600 nm (A 600 ).…”

Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides

“…However, the Mg# + regulation of catechol 2,3-dioxygenase expressed from the oprH ::xylE transcriptional fusion in strain H855 (Table 2), together with our observation that transcription of oprH is wholly dependent on the presence of PhoP (Macfarlane et al, 1999), provided indirect evidence for a very low level of phoP-phoQ transcription in this strain from an as yet unidentified second promoter. The presence of two promoters -one constitutive and one inducibleallowing a basal level of transcription is a common feature of two-component regulatory systems and has been reported for phoP-phoQ in E. coli (Kato et al, 1999), and for both phoP-phoQ and pmrA-pmrB in S. typhimurium (Gunn & Miller, 1996 ;Soncini et al, 1995). For the latter system, the second promoter lies within the 3h region of pmrC, the first gene of the pmrCAB operon (Gunn & Miller, 1996).…”

“…The sensor protein PhoQ responds to extracellular concentrations of Mg# + ions and, under conditions of Mg# + starvation, first autophosphorylates at a conserved histidine residue, then activates the regulator protein PhoP by a phosphotransfer reaction (Garcia Vescovi et al, 1996). The phoP-phoQ locus is subject to auto-regulation, such that low extracellular Mg# + concentrations upregulate transcription of this operon from an inducible promoter in a PhoP-dependent manner (Soncini et al, 1995). Other regulators under the control of PhoP-PhoQ include PmrA-PmrB (Gunn & Miller, 1996 ;, a two-component regulatory system responsible for structural changes in the outer-membrane lipopolysaccharide that lead to polymyxin B resistance (Gunn et al, 1998 ;Helander et al, 1994), and the transcriptional activator HilA, which is a key regulator of Salmonella pathogenicity island 1 and thus controls expression of several virulence factors (Bajaj et al, 1996).…”

Role for the Salmonella Flavohemoglobin in Protection from Nitric Oxide

“…A native hmp expression construct was created by PCR using SKO1 (213 bases upstream of the hmp start codon) and SH4: (5Ј-GTAAAAGAAAGGCATA-AAAAGCAGC-3Ј), an antisense oligo starting 48 bp downstream of a predicted 12-bp inverted repeat, which is centered 37 bp downstream of the hmp stop codon. The product was cloned into the XhoI/HindIII site of pUHE21-2lacI q (25), making pShmp. The hmp nucleotide sequence was deduced from sequencing three separate clones of pShmp (26), all of which were identical and deposited into GenBank.…”