Abstract
Extracytoplasmic function (ECF) sigma factors control expression of large numbers of genes in bacteria. Most ECF sigma factors are inhibited by antisigma proteins, with inhibition being relieved by environmental signals that lead to inactivation of the antisigma protein and consequent sigma factor activity. In cell surface signaling (CSS) systems in Gram negative bacteria antisigma activity is controlled by an outer membrane protein receptor and its ligand. In Pseudomonas aeruginosa one such system controls expression of genes for secretion and uptake of a siderophore, pyoverdine. In this system the activities of two sigma factors σFpvI and σPvdS are inhibited by antisigma protein FpvR20 that binds to the sigma factors, preventing their interaction with core RNA polymerase. Transport of ferripyoverdine by its outer membrane receptor FpvA causes proteolytic degradation of FpvR20, inducing expression of σFpvI- and σPvdS-dependent target genes. Here we show that degradation of FpvR20 and induction of target gene expression was initiated within 1 min of addition of pyoverdine. FpvR20 was only partially degraded in a mutant lacking the intracellular ClpP protease, resulting in an FpvR20 subfragment (FpvR12) that inhibited σFpvI and σPvdS. The translation inhibitor chloramphenicol did not prevent induction of an σFpvI-dependent gene, showing that degradation of FpvR20 released pre-existing σFpvI in an active form. However, chloramphenicol inhibited induction of σPvdS-dependent genes showing that active σPvdS is not released when FpvR20 is degraded and instead, σPvdS must be synthesized in the absence of FpvR20 to be active. These findings show that sigma factor activation occurs rapidly following addition of the inducing signal in a CSS pathway and requires ClpP protease. Induction of gene expression that can arise from release of active sigma from an antisigma protein but can also require new sigma factor synthesis.
Highlights
Extracytoplasmic function sigma factors are the largest and most diverse family of sigma factors in bacteria, directing expression of genes in response to a wide range of environmental stimuli (Staron et al, 2009; Ho and Ellermeier, 2012; Mascher, 2013)
One of the best-characterized cell surface signaling (CSS) systems controls expression of genes for synthesis of a siderophore pyoverdine and subsequent uptake of ferripyoverdine in the opportunistic pathogen Pseudomonas aeruginosa (Figure 1). In this system sigma factors σFpvI and σPvdS are inhibited by antisigma protein FpvR20 that is formed by cleavage of a 37 kDa precursor protein (Draper et al, 2011)
DNA fragments required for strain construction were amplified from genomic DNA of P. aeruginosa PAO1 by PCR with FirePol DNA Polymerase (Solis Biodyne) or Taq DNA Polymerase Reddymix (ThermoPrime) using appropriate primers (Supplementary Table S1) that were designed on the basis of the P. aeruginosa PAO1 genome sequence1 (Winsor et al, 2011)
Summary
Extracytoplasmic function sigma factors are the largest and most diverse family of sigma factors in bacteria, directing expression of genes in response to a wide range of environmental stimuli (Staron et al, 2009; Ho and Ellermeier, 2012; Mascher, 2013). In CSS systems antisigma protein activity (and that of the cognate ECF sigma factor) is controlled by an outer membrane protein receptor in response to an extracellular chemical signal, commonly a ferrisiderophore (Visca et al, 2002; Braun et al, 2006; Llamas et al, 2014). One of the best-characterized CSS systems controls expression of genes for synthesis of a siderophore pyoverdine and subsequent uptake of ferripyoverdine in the opportunistic pathogen Pseudomonas aeruginosa (Figure 1). In this system sigma factors σFpvI and σPvdS are inhibited by antisigma protein FpvR20 that is formed by cleavage of a 37 kDa precursor protein (Draper et al, 2011). The rate of induction of target gene expression in response to the appropriate environmental signal has not been determined for this or any other CSS pathway
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