Alice Boulanger, Guillaume Déjean, Roland Barriot, Kounthéa Phok, Claude Gutierrez, Patricia Bordes, Laure Lavatine, Anke Becker, Emmanuelle Lauber, Marie-Pierre Castanié-Cornet, Matthieu Arlat, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Unité mixte de recherche interactions plantes-microorganismes, Institut National de la Recherche Agronomique (INRA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), University of Freiburg [Freiburg], Centre National de la Recherche Scientifique (CNRS), Universite de Toulouse (UPS), Bundesministerium fur Bildung und Forschung, Germany 0313805A, and German Research Foundation SPP 1316
Bacteria often encounter diverse and rapidly changing environments. To overcome harmful situations, they must be capable of sensing external changes and transmitting this information across biological membranes into the cell, which results in the appropriate redirection of gene expression to prevent or repair cellular damages caused by stress. Extracytoplasmic function (ECF) σ factors provide one common means of bacterial signal transduction to regulate gene expression in response to various extracellular changes (65). ECF σ factors represent the largest and most diverse subfamily of σ70 proteins. They generally recognize a −35 box with a clear bias toward a GAAC in their target promoters, while the −10 region tends to be highly variable between ECF subfamily members (65). One of the best-studied ECF σ factors is the key regulator of the extracytoplasmic stress response factor σE from Escherichia coli, encoded by the rpoE gene (56). ECF proteins were recently divided into 43 major phylogenetically distinct groups named ECF01 to ECF43 (65). RpoE-like ECF σ factors are part of one predominant subgroup found in most bacterial phyla and comprise ECF01 to -04 proteins. RpoE-like ECF σ factors are autoregulated and are required for a wide range of functions. For instance, the E. coli σE factor is essential for growth and promotes the expression of factors that help to preserve and/or restore cell envelope integrity (2). Salmonella enterica serovar Typhimurium σE is required for protection against reactive oxygen species and antimicrobial peptides and for stationary-phase survival (20, 67). Bacillus subtilis σW seems to constitute an antibiosis regulon acting against cell envelope stress (34). S. Typhimurium σE, Pseudomonas aeruginosa AlgU, and Vibrio cholerae σE are required for virulence (5). ECFs can thus be considered models to understand how bacteria sense and respond to their environment both during their interaction with their host and in their free-living state. RpoE-like ECF σ factors are tightly regulated in order to coordinate their activation with the appropriate environmental cues. In most cases, the σE factor is cotranscribed with a cognate transmembrane anti-σ factor possessing an extracytoplasmic domain and an intracellular σ-binding domain. In the absence of stimulus, the membrane-bound anti-σ binds tightly to the σ factor, thereby keeping it inactive (33). Upon receiving a proper signal, the anti-σ factor is inactivated by regulated intramembrane proteolysis (RIP), resulting in the release and subsequent activation of the σE factor. This mechanism has been well studied for the anti-σ factors RseA, MucA, and RsiW, regulating the activity of E. coli σE, P. aeruginosa AlgU, or B. subtilis σW, respectively (1, 32, 75). In E. coli, the accumulation of C-terminal domains of unfolded porins is the activating signal of the RpoE response by triggering the activation of the inner-membrane-anchored protease, DegS (site 1 protease), and the subsequent cleavage of RseA within its periplasmic domain by DegS. The resulting truncated anti-σ factor is then a suitable substrate for a second inner-membrane protease, RseP/YaeL (site 2 protease), which cleaves RseA near the cytoplasmic face of the inner membrane, releasing an RseAcyto-σE complex into the cytoplasm, where the remaining RseA fragment is degraded by cytoplasmic proteases, resulting in the active σE (1). Another important mediator of the extracytoplasmic stress response is the periplasmic protease DegP, also known as HtrA and DO in E. coli or MucD in P. aeruginosa (22, 55). DegP binds to and degrades misfolded proteins and acts as a chaperone to direct the proper folding of some envelope proteins (66). As such, this family of proteases regulates the σE stress response system by removing misfolded proteins in the periplasm that could activate the degradation pathway of the anti-σE factor, even in the absence of stress (27). The Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris is an epiphytic bacterium that can become a vascular pathogen, causing black rot disease of crucifers (52). The bacterium produces a large amount of extracellular polysaccharide (EPS) that plays an important role during bacterial infection, and X. campestris pv. campestris has been used as a model organism for investigating the mechanism of bacterial pathogenesis. X. campestris pv. campestris flourishes in and adapts to a wide range of habitats: during epiphytic life, X. campestris pv. campestris is exposed to harsh stresses, such as oligotrophic conditions, desiccation, or large changes in temperature. Upon entry into plant tissues, X. campestris pv. campestris cells must face defense reactions of the host, including oxidative conditions. Finally, the natural life cycle of X. campestris pv. campestris includes long periods of survival on seeds or plant scraps or in the soil, where again it must survive a variety of stressful conditions before it can infect a new host plant. Its ability to manage variable and often lethal external conditions can be partly attributed to its large repertoire of alternative σ factors. Of the 4,179 open reading frames (ORFs) comprising the large 5.1-Mb X. campestris pv. campestris strain ATCC 33913 genome, 15 ORFs encode characterized or putative σ factors, 10 of which belong to the ECF subfamily (23). Little is known about which σ factors are required for the survival of X. campestris pv. campestris under stress and the contribution of these factors to virulence. The classification of ECF σ factors strongly suggested that the XCC1267 gene encodes the σE factor of X. campestris pv. campestris (65). Moreover, previous work by Cheng et al. (17) described the biochemical characterization of the σE factor of X. campestris pv. campestris strain 11 and suggested that it could have a role in the heat shock response. Therefore, we aimed at deciphering the roles and regulation mechanisms of the extracytoplasmic stress response regulator σE in X. campestris pv. campestris. In the present work we characterized the rpoE operon genes, rpoE, rseA, and mucD. Using primer extension and lacZ transcriptional reporter fusions, we show that rpoE transcription is autoregulated and that RseA and MucD are negative regulators of σE activity. We identified 45 putative members of the σE regulon by a transcriptome analysis, including the heat stress σ factor σH and a number of periplasmic or membrane proteins. We provided evidence that σE is an important regulator of stress responses in X. campestris pv. campestris, since it has a role in heat adaptation, resistance to cadmium, and stationary-phase survival. Furthermore, our results strongly suggest that σE is regulated by a RIP mechanism involving RseP (XCC1366) and DegS (XCC3898) putative proteases, as in many other bacteria. However, our data suggest that the RIP proteases RseP and DegS are not only dedicated to RseA cleavage and that the proteolytic cascade of RseA could involve other proteases.