Abbreviated Summary: Virulence gene regulation in Vibrio cholerae is highly complex, comprising several transcriptional activators and decision check points. The ToxRS complex is a key regulator that is subjected to regulated intramembrane proteolysis (RIP). The participating interaction partners were characterized, addressing DegS, DegP, ToxS and factors influencing ToxR-RIP, such as bile acids and the cysteine redox state of ToxR. In Vibrio cholerae, virulence gene expression is regulated by a transmembrane-localized transcription factor complex designated as ToxRS. ToxR harbours two cysteines in the periplasmic domain that can form inter- and intramolecular disulfide bonds. In this study, we investigated the σE−dependent inner membrane proteolysis of ToxR, which occurs via the periplasmic-localized proteases DegS and DegP. Both proteases respond to the redox state of the two cysteine thiol groups of ToxR. Interestingly, in the presence of sodium deoxycholate, ToxR proteolysis is blocked independently of ToxS, whereas ToxR activation by bile salts requires ToxS function. From these data, we identified at least two levels of control for ToxR activation by sodium-deoxycholate. First, bile inhibits ToxR degradation under starvation and alkaline pH or under conditions in which DegPS responds to the reduced disulfide bonds of ToxR. The second level links bile to ToxRS complex formation and further activation of its transcription factor activity. Overall, our data suggest a comprehensive bile sensory function for the ToxRS complex during host colonization.

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Virulence factor production in Vibrio cholerae is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation.

Prokaryotes are unicellular organisms that require sensory networks for their survival in rapidly changing habitats. In the course of evolution, transmembrane signaling systems have evolved to transmit signals from the extracellular environment across the cytoplasmic membrane into the cell. One-component signaling systems represent the oldest and simplest solution for such signal transmission, whereas two-component systems are evolutionarily younger (Ulrich et al., 2005). Although one-component systems are widely distributed among bacteria, only 3% are directly integrated into cytoplasmic membranes (Ulrich et al., 2005). A literature search revealed a non-exhaustive list of signaling molecules that includes ToxRS,

The facultative human pathogen Vibrio cholerae transits between the gastrointestinal tract of its host and aquatic reservoirs. V. cholerae adapts to different situations by the timely coordinated expression of genes during its life cycle. We recently identified a subclass of genes that are induced at late stages of infection. Initial characterization demonstrated that some of these genes facilitate the transition of V. cholerae from host to environmental conditions. Among these genes are uptake systems lacking detailed characterization or correct annotation. In this study, we comprehensively investigated the function of the VCA0682-to-VCA0687 gene cluster, which was previously identified as in vivo induced. The results presented here demonstrate that the operon encompassing open reading frames VCA0685 to VCA0687 encodes an ABC transport system for hexose-6-phosphates with K m values ranging from 0.275 to 1.273 M for glucose-6P and fructose-6P, respectively. Expression of the operon is induced by the presence of hexose-6P controlled by the transcriptional activator VCA0682, representing a UhpA homolog. Finally, we provide evidence that the operon is essential for the utilization of hexose-6P as a C and P source. Thereby, a physiological role can be assigned to hexose-6P uptake, which correlates with increased fitness of V. cholerae after a transition from the host into phosphate-limiting environments.T he life cycle of the facultative human pathogen Vibrio cholerae is marked by repetitive transitions between aquatic environments and the host gastrointestinal tract. Besides many other variable conditions, V. cholerae has to adjust to different qualities and quantities of nutrient sources. This variability is emphasized by the fact that utilization of nutrients under laboratory conditions, such as in full broth or a chemically defined minimal medium, represents no growth limitation for clinical V. cholerae isolates (1).In the open sea, bacteria such as V. cholerae face C, N, and P limitation and are restricted to limited nutrients on a picomolar or nanomolar scale, whereby substrates become available and accessible only in specific habitats (2). Therefore, marine bacteria are found close to organic particles ranging from micrometer-to millimeter-sized aggregates. These organic particles derive from different sources, such as lysed or dead phytoplankton, zooplankton, or fecal pellets. They deliver organic and inorganic substrates in concentrations that are up to 4 orders of magnitude greater than those found in particle-free open seawater (for a recent review, see reference 2). Therefore, many plants and animals in the ocean serve as microbial niches for V. cholerae (3-6). For example, copepods or other crustaceans contain or are covered with chitin, a ␤,1-4-linked polymer of 2-acetamido-2-deoxy-␤-D-glucopyranoside (GlcNAc) n and its deacetylated form, chitosan. These substrates are utilized by Vibrio sp. as C and N sources (7-9). In addition, biofilm formation on chitinous surfaces plays a crucial role in V. choler...