Abstract
OprF is the major outer membrane porin in bacteria belonging to the Pseudomonas genus. In previous studies, we have shown that OprF is required for full virulence expression of the opportunistic pathogen Pseudomonas aeruginosa. Here, we describe molecular insights on the nature of this relationship and report that the absence of OprF leads to increased biofilm formation and production of the Pel exopolysaccharide. Accordingly, the level of c-di-GMP, a key second messenger in biofilm control, is elevated in an oprF mutant. By decreasing c-di-GMP levels in this mutant, both biofilm formation and pel gene expression phenotypes were restored to wild-type levels. We further investigated the impact on two small RNAs, which are associated with the biofilm lifestyle, and found that expression of rsmZ but not of rsmY was increased in the oprF mutant and this occurs in a c-di-GMP-dependent manner. Finally, the extracytoplasmic function (ECF) sigma factors AlgU and SigX displayed higher activity levels in the oprF mutant. Two genes of the SigX regulon involved in c-di-GMP metabolism, PA1181 and adcA (PA4843), were up-regulated in the oprF mutant, partly explaining the increased c-di-GMP level. We hypothesized that the absence of OprF leads to a cell envelope stress that activates SigX and results in a c-di-GMP elevated level due to higher expression of adcA and PA1181. The c-di-GMP level can in turn stimulate Pel synthesis via increased rsmZ sRNA levels and pel mRNA, thus affecting Pel-dependent phenotypes such as cell aggregation and biofilm formation. This work highlights the connection between OprF and c-di-GMP regulatory networks, likely via SigX (ECF), on the regulation of biofilm phenotypes.
Highlights
Pseudomonas aeruginosa is a Gram-negative bacterium found in almost every wet ecological niches including soils, water, and plants (Spiers et al, 2000)
We report here that the absence of OprF in the outer membrane (OM) of P. aeruginosa results in increased biofilm formation
The Lack of OprF Results in Increased EPS Production, Cell Aggregation and Biofilm Formation We investigated the role of OprF in biofilm formation and EPS production by comparing three strains, i.e., the P. aeruginosa H103 wild-type strain, the isogenic oprF mutant H636 (Woodruff and Hancock, 1988), and the oprF complemented control strain H636O (Woodruff and Hancock, 1988)
Summary
Pseudomonas aeruginosa is a Gram-negative bacterium found in almost every wet ecological niches including soils, water, and plants (Spiers et al, 2000). It is an opportunistic human pathogen, causing a variety of infections including chronic lung infections in cystic fibrosis patients (Ramsey and Wozniak, 2005). This requires a well-developed ability to adapt to changes in environmental conditions, in which outer membrane (OM) proteins may play an important function considering their role in communication with the extracellular medium. OprF expression is controlled by at least two extracytoplasmic function (ECFs) sigma factors, AlgU and SigX (Brinkman et al, 1999; Firoved et al, 2002; Bouffartigues et al, 2012), which are involved in cell envelope homeostasis (Wood et al, 2006; Boechat et al, 2013; Duchesne et al, 2013; Gicquel et al, 2013; Blanka et al, 2014)
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