Characterizing the Hexose-6-Phosphate Transport System of Vibrio cholerae, a Utilization System for Carbon and Phosphate Sources

Abstract

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...

Nucleoside uptake in Vibrio cholerae and its role in the transition fitness from host to environment

“…Binding protein dependent transport systems, such as maltose or hexose-6-phosphate uptake systems, are characterized as high affinity sugar transporters, showing K m values, e.g. for maltose of around 1 μM (Behrmann et al, 1998;Moisi et al, 2013). Notably, the herein characterized NS transporters show considerable high affinity substrate transport properties, although it belongs to the electrochemical gradient-coupled permease family, also termed as secondary active transporter (Pao et al, 1998).…”

“…V. cholerae likely faces a variety of carbon sources in the host. Besides amino acids and polysaccharides, hexose-6-phosphates or sialic acid have been previously described as suitable carbon sources (Sharma et al, 2011;Moisi et al, 2013). Thus, it is quite surprising to already observe a fourfold decrease in transition fitness by just blocking NS uptake.…”

“…E. coli strains DH5αλpir and SM10λpir (Kolter et al, 1978;Hanahan, 1983;Miller and Mekalanos, 1988) were used for genetic manipulations. Minimal media M9 was prepared according to standard recipe (Miller, 1972) with addition of Tris/HCl pH 8.0 (100 mM), which does not affect V. cholerae growth, but allows to maintain buffer capacity even under conditions lacking inorganic phosphate (von Kruger et al, 1999;Seper et al, 2011;Moisi et al, 2013). Antibiotics and other supplements were used in the following final concentrations: streptomycin (Sm, 100 μg ml −1 ), ampicillin (Ap, 50 μg ml −1 in combination with other antibiotics, otherwise 100 μg ml −1 ), chloramphenicol (Cm, 2 μg ml −1 ), isopropyl-β-thiogalactopyranoside (IPTG, 0.5 mM), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal, 30 μg ml −1 ), sucrose (10%), glucose (Glu, 0.2%), glycerol (Gly, 0.2%), casamino acids (CAA, 0.2%), adenosine and cytidine (4 mg ml −1 if used separately, or 2 mg ml −1 each if used in combination), AMP and CMP (as carbon source 2 mg ml −1 each, as phosphate source 12 mg ml −1 each), herring sperm DNA (2.5 mg ml −1 ).…”

“…Growth kinetics were essentially performed as previously described in transparent 24 well plates (Greiner) with 1 ml culture volume (Seper et al, 2011;Moisi et al, 2013). Briefly, the respective strains were grown in a pre-culture for 24 h in M9-Glu with aeration and shaking at 37°C.…”

Comparative genome analysis reveals important genetic differences among serotype O1 and serotype O2 strains of Y. ruckeri and provides insights into host adaptation and virulence

“…This region of 19,566 bp contains genes encoding for a Crp‐Fnr family transcriptional regulator, a hypothetical protein, an enzyme related to an enterobactin‐like siderophore and three different gene clusters: one formed by three genes involved in iron transport, a group of three genes related to hexose phosphate uptake; and a region containing nine genes involved in the uptake and metabolism of citrate. Since most of these genes are related to virulence (Gray, Freitag, & Boor, ; Moisi et al., ; Urbany & Neuhaus, ), it is possible that the absence of this region could explain, in some way, the attenuation of Y. ruckeri ATCC29473. This is important for future studies and may help to shed light on the virulence of the species.…”