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Cell-surface signalling (CSS) is a signal transfer system of Gram-negative bacteria used to detect extracellular signals and modulate gene transcription in response. These three-protein systems are formed by an outer membrane receptor, a cytoplasmic membrane-embedded anti-σ factor and a cytosolic extracytoplasmic function σ factor (σECF). In absence of an inducing signal, the anti-σ factor binds to and keeps the σECFfactor sequestered, thus preventing its interaction with the RNA polymerase and the transcription of signal response genes. Presence of the signal triggers a signalling cascade that extends from the outer membrane to the cytosol and results in σECFfactor activation. Recently, we and others have reported that CSS σECFfactor activation requires the regulated and sequential proteolysis of the cognate anti-σ factor, and the function of the Prc and RseP proteases. However, many features of this proteolytic cascade are still unclear. In this work, we have identified another protease that modulates CSS activity, namely the periplasmic carboxyl-terminal processing protease CtpA. We show that both CtpA and the previously identified protease Prc control CSS activation by modulating the levels of the anti-σ factor. CtpA functions upstream of Prc in the proteolytic cascade and seems to prevent the Prc-mediated proteolysis of the CSS anti-σ factor. Importantly, using zebrafish embryos and the A549 cell line as hosts, we show that mutants in thersePandctpAproteases of the human pathogenPseudomonas aeruginosaare considerably attenuated in virulence while theprcmutation increases virulence likely by enhancing the production of outer membrane vesicles. Because proteases are druggable proteins, the identification of regulatory proteases involved inP. aeruginosavirulence holds promise for the development of novel strategies to fight this clinically relevant pathogen.

The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive micro-organisms. Three T6SSs have recently been described in Pseudomonas putida KT2440 and it has been shown that one, K1-T6SS, is used to outcompete a wide range of phytopathogens, protecting plants from pathogen infections. Given the relevance of this system as a powerful and innovative mechanism of biological control, it is critical to understand the processes that govern its expression. Here, we experimentally defined two transcriptional units in the K1-T6SS cluster. One encodes the structural components of the system and is transcribed from two adjacent promoters. The other encodes two hypothetical proteins, the tip of the system and the associated adapters, and effectors and cognate immunity proteins, and it is also transcribed from two adjacent promoters. The four identified promoters contain the typical features of σ-dependent promoters. We have studied the expression of the system under different conditions and in a number of mutants lacking global regulators. P. putida K1-T6SS expression is induced in the stationary phase, but its transcription does not depend on the stationary σ factor RpoS. In fact, the expression of the system is indirectly repressed by RpoS. Furthermore, it is also repressed by RpoN and the transcriptional regulator FleQ, an enhancer-binding protein typically acting in conjunction with RpoN. Importantly, expression of the K1-T6SS gene cluster is positively regulated by the GacS–GacA two-component regulatory system (TCS) and repressed by the RetS sensor kinase, which inhibits this TCS. Our findings identified a complex regulatory network that governs T6SS expression in general and P. putida K1-T6SS in particular, with implications for controlling and manipulating a bacterial agent that is highly relevant in biological control., P.B.'s research was supported by the MCIN/AEI/10.13039/501100011033 Spanish agency through the Juan de la Cierva JCI-2010–06615 and Ramon y Cajal RYC2019-026551-I grants, and by EMBO through the short-term fellowship ASTF No 449–2014. This work was funded by the BBSRC with project BB/N002539/1 in A.F.'s laboratory, and by the MCIN/AEI/10.13039/501100011033 Spanish agency with projects BIO2017-83763-P and PID2020-115682GB-I00 in M.A.L.'s laboratory

The extracytoplasmic function sigma factor σVreI of the human pathogen Pseudomonas aeruginosa promotes transcription of potential virulence determinants, including secretion systems and secreted proteins. Its activity is modulated by the VreR anti-σ factor that inhibits the binding of σVreI to the RNA polymerase in the absence of a (still unknown) inducing signal. The vreI-vreR genes are expressed under inorganic phosphate (Pi) starvation, a physiological condition often encountered in the host that increases P. aeruginosa pathogenicity. However, whether or not σVreI is active in vivo during infection and contributes to the Pi starvation-induced virulence of this pathogen has not been analyzed yet. Using zebrafish embryos and a human alveolar basal epithelial cell line as P. aeruginosa hosts, we demonstrate in this work that σVreI is active during infection and that lack of σVreI considerably reduces the Pi starvation-induced virulence of this pathogen. Surprisingly, lack of the σVreI inhibitor, the VreR anti-σ factor, also diminishes the virulence of P. aeruginosa. By transcriptomic analyses we show that VreR modulates gene expression not only in a σVreI-dependent but also in a σVreI-independent manner. This includes potential virulence determinants and transcriptional regulators that could be responsible for the reduced virulence of the ΔvreR mutant., We thank W. Koudstaal for critical review of the manuscript, A. van der Sar and T. Verboom (AUMC, Amsterdam) for facilitating the work on zebrafish embryos, F. Madrazo (Instituto de Investigación Sanitaria Valdecilla) for assistance with the confocal fluorescence imaging, and R. Tobes (Era7 Bioinformatics) for the RNA-seq bioinformatics analysis. This work was funded by FEDER and the Spanish Ministry of Economy with grants SAF2015-68873-P and BIO2017-83763-P. JOA was supported by the Spanish Ministry of Economy through a FPI fellowship (BES-2013-066301).

Pathogens have developed several strategies to obtain iron during infection, including the use of iron-containing molecules from the host. Haem accounts for the vast majority of the iron pool in vertebrates and thus represents an important source of iron for pathogens. Using a proteomic approach, we have identified in this work a previously uncharacterized system, which we name Hxu, that together with the known Has and Phu systems, is used by the human pathogen Pseudomonas aeruginosa to respond to haem. We show that the Has and Hxu systems are functional signal transduction pathways of the cell-surface signalling class and report the mechanism triggering the activation of these signalling systems. Both signalling cascades involve an outer membrane receptor (HasR and HxuA respectively) that upon sensing haem in the extracellular medium produces the activation of an σ factor in the cytosol. HxuA has a major role in signalling and a minor role in haem acquisition in conditions in which the HasR and PhuR receptors or other sources of iron are present. Remarkably, P. aeruginosa compensates the lack of the HasR receptor by increasing the production of HxuA, which underscores the importance of haem signalling for this pathogen., This work was funded by the European Regional Development Fund and the Spanish Ministry of Economy with grants SAF2015‐68873‐P and BIO2017‐83763‐P. JOA is supported by the Spanish Ministry of Economy through a FPI fellowship (BES‐2013‐066301) and MAL through a Ramon&Cajal grant (RYC2011‐08874).

Gene regulation in bacteria is primarily controlled at the level of transcription initiation by modifying the affinity of the RNA polymerase (RNAP) for the promoter. This control often occurs through the substitution of the RNAP sigma (σ) subunit. Next to the primary σ factor, most bacteria contain a variable number of alternative σ factors of which the extracytoplasmic function group (σECF) is predominant. Pseudomonas aeruginosa contains nineteen σECF, including the virulence regulator σVreI. σVreI is encoded by the vreAIR operon, which also encodes a receptor-like protein (VreA) and an anti-σ factor (VreR). These three proteins form a signal transduction pathway known as PUMA3, which controls expression of P. aeruginosa virulence functions. Expression of the vreAIR operon occurs under inorganic phosphate (Pi) limitation and requires the PhoB transcription factor. Intriguingly, the genes of the σVreI regulon are also expressed in low Pi despite the fact that the σVreI repressor, the anti-σ factor VreR, is also produced in this condition. Here we show that although σVreI is partially active under Pi starvation, maximal transcription of the σVreI regulon genes requires the removal of VreR. This strongly suggests that an extra signal, probably host-derived, is required in vivo for full σVreI activation. Furthermore, we demonstrate that the activity of σVreI is modulated not only by VreR but also by the transcription factor PhoB. Presence of this regulator is an absolute requirement for σVreI to complex the DNA and initiate transcription of the PUMA3 regulon. The potential DNA binding sites of these two proteins, which include a pho box and −10 and −35 elements, are proposed., This work has been supported by the EU Seventh Framework Programme through a Marie Curie CIG grant (3038130), and the Spanish Ministry of Economy with grants inside the Ramón&Cajal (RYC2011-08874 to ML) and the Plan Nacional for I+D+i (SAF2012-31919 and SAF2015-68873-P) programs., USD 2212,20 APC fee funded by the EC FP7 Post-Grant Open Access Pilot