Transcription of the oprF Gene of Pseudomonas aeruginosa Is Dependent Mainly on the SigX Sigma Factor and Is Sucrose Induced

Abstract

The OprF porin is the major outer membrane protein of Pseudomonas aeruginosa. OprF is involved in several crucial functions, including cell structure, outer membrane permeability, environmental sensing, and virulence. The oprF gene is preceded by the sigX gene, which encodes the poorly studied extracytoplasmic function (ECF) sigma factor SigX. Three oprF promoters were previously identified. Two intertwined promoters dependent on 70 and SigX are located in the sigX-oprF intergenic region, whereas a promoter dependent on the ECF AlgU lies within the sigX gene. An additional promoter was found in the cmpX-sigX intergenic region. In this study, we dissected the contribution of each promoter region and of each sigma factor to oprF transcription using transcriptional fusions. In Luria-Bertani (LB) medium, the oprF-proximal region (sigX-oprF intergenic region) accounted for about 80% of the oprF transcription, whereas the AlgU-dependent promoter had marginal activity. Using the sigX mutant PAOSX, we observed that the SigX-dependent promoter was largely predominant over the 70 -dependent promoter. oprF transcription was increased in response to low NaCl or high sucrose concentrations, and this induced transcription was strongly impaired in the absence of SigX. The lack of OprF itself increased oprF transcription. Since these conditions led to cell wall alterations, oprF transcription could be activated by signals triggered by perturbation of the cell envelope.

Identification of the Alternative Sigma Factor SigX Regulon and Its Implications for Pseudomonas aeruginosa Pathogenicity

“…In a previous study, Bouffartigues and colleagues demonstrated that in the P. aeruginosa strain PAO1, expression of the outer membrane porin OprF is dependent on SigX when the bacteria is cultivated in LB medium. The oprF gene is located 108 bp downstream of the sigX gene and was suggested before to be transcribed both monocistronically and bicistronically (10). Although we observed a slight downregulation of oprF in our PA14 sigX mutant (log 2 fold change of Ϫ0.726; P ϭ 0.075), oprF is not listed in our transcriptomic data (see Table S2 in the supplemental material) due to the stringent filter criteria.…”

“…In P. aeruginosa deletion of sigX led to impaired growth under low-salt concentrations and reduced oprF expression (9). Later, Bouffartigues and colleagues confirmed these data and presented a link between lowered sodium chloride concentrations and the transcription of oprF due to the activation of the sigX promoter (10). As the ECF factor SigX was shown to be essential for survival under lowosmolarity-medium conditions and seems to be involved in responses to osmotic and cell wall stresses (9,10), it was suggested that the SigX regulon might be larger than anticipated.…”

“…1B). A growth defect of a ⌬sigX mutant in the PAO1 strain has been described previously by Brinkman et al (9), and an involvement of SigX in the regulation and adaptation to osmotic stress and, in particular, to low osmolarity has been suggested (9,10). In line with these previous observations, the PA14 ⌬sigX mutant exhibited delayed growth with an extended lag phase in liquid LB cultures, which became most apparent under low-sodium-chloride-medium (8 mM NaCl) conditions (Fig.…”

“…So far, cell envelope stress, iron limitation, and oxidative stress have been demonstrated to play a pivotal role during host infection and were described to activate ECF factors (7,8). In addition to the best-studied P. aeruginosa ECF factors AlgU and PvdS, SigX has been investigated in recent studies in the context of transcriptional regulation of the outer membrane protein OprF (9,10). P. aeruginosa SigX shares 49% sequence similarity to w of Bacillus subtilis, which is induced by osmotic stress, phage infection, or interruption of cell wall biosynthesis following antibiotic treatment (2,11).…”

Otopathogenic Pseudomonas aeruginosa Enters and Survives Inside Macrophages

“…Since bacterial outer membrane proteins (OMPs) play a crucial role in interaction of pathogens with immune cells (Confer and Ayalew, 2013), we determined the role of P. aeruginosa OprF in bacterial survival inside macrophages. OprF is the major OMP of P. aeruginosa and has been demonstrated to play an important role in interaction of this pathogen with host cells (Nestorovich et al, 2006; Bouffartigues et al, 2012; Reusch, 2012; Mishra et al, 2015). We observed that survival of otopathogenic P. aeruginosa inside macrophages requires bacterial oprF expression.…”