Vibrio cholerae’s mysterious Seventh Pandemic island (VSP-II) encodes novel Zur-regulated zinc starvation genes involved in chemotaxis and cell congregation

Vibrio cholerae is the causative agent of cholera, a notorious diarrheal disease that is typically transmitted via contaminated drinking water. The current pandemic agent, the El Tor biotype, has undergone several genetic changes that include horizontal acquisition of two genomic islands (VSP-I and VSP-II). VSP presence strongly correlates with pandemicity; however, the contribution of these islands to V. cholerae’s life cycle, particularly the 26-kb VSP-II, remains poorly understood. VSP-II-encoded genes are not expressed under standard laboratory conditions, suggesting that their induction requires an unknown signal from the host or environment. One signal that bacteria encounter under both host and environmental conditions is metal limitation. While studying V. cholerae’s zinc-starvation response in vitro, we noticed that a mutant constitutively expressing zinc starvation genes (Δzur) congregates at the bottom of a culture tube when grown in a nutrient-poor medium. Using transposon mutagenesis, we found that flagellar motility, chemotaxis, and VSP-II encoded genes were required for congregation. The VSP-II genes encode an AraC-like transcriptional activator (VerA) and a methyl-accepting chemotaxis protein (AerB). Using RNA-seq and lacZ transcriptional reporters, we show that VerA is a novel Zur target and an activator of the nearby AerB chemoreceptor. AerB interfaces with the chemotaxis system to drive oxygen-dependent congregation and energy taxis. Importantly, this work suggests a functional link between VSP-II, zinc-starved environments, and energy taxis, yielding insights into the role of VSP-II in a metal-limited host or aquatic reservoir.

Vibrio cholerae’s mysterious Seventh Pandemic island (VSP-II) encodes novel Zur-regulated zinc starvation genes involved in chemotaxis and autoaggregation

Vibrio cholerae is the causative agent of cholera, a notorious diarrheal disease that is typically transmitted via contaminated drinking water. The current pandemic agent, the El Tor biotype, has undergone several genetic changes that include horizontal acquisition of two genomic islands (VSP-I and VSP-II). VSP-I and -2 presence strongly correlates with pandemicity; however, the contribution of these islands to V. cholerae’s life cycle, particularly the 26-kb VSP-II, remains poorly understood. VSP-II-encoded genes are not expressed under standard laboratory conditions, suggesting that their induction requires an unknown signal from the host or environment. One signal that bacteria encounter under both host and environmental conditions is metal limitation. While studying V. cholerae’s zinc-starvation response in vitro, we noticed that a mutant constitutively expressing zinc-starvation genes (Δzur) aggregates in nutrient-poor media. Using transposon mutagenesis, we found that flagellar motility, chemotaxis, and VSP-II encoded genes are required for aggregation. The VSP-II genes encode an AraC-like transcriptional activator (VerA) and a methyl-accepting chemotaxis protein (AerB). Using RNA-seq and lacZ transcriptional reporters, we show that VerA is a novel Zur target and activator of the nearby AerB chemoreceptor. AerB interfaces with the chemotaxis system to drive oxygen-dependent autoaggregation and energy taxis. Importantly, this work suggests a functional link between VSP-II, zinc-starved environments, and aerotaxis, yielding insights into the role of VSP-II in a metal-limited host or aquatic reservoir.Author SummaryThe Vibrio Seventh Pandemic island was horizontally acquired by El Tor pandemic strain, but its role in pathogenicity or environmental persistence is unknown. A major barrier to VSP-II study was the lack of stimuli favoring its expression. We show that zinc starvation induces expression of this island and describe a transcriptional network that activates a VSP-II encoded aerotaxis receptor. Importantly, aerotaxis may enable V. cholerae to locate more favorable microenvironments, possibly to colonize anoxic portions of the gut or environmental sediments.

Small non-coding RNA CjNC110 influences motility, autoagglutination, AI-2 localization, hydrogen peroxide sensitivity and chicken colonization in Campylobacter jejuni

ABSTRACTSmall non-coding RNAs are involved in many important physiological functions in pathogenic microorganisms. Previous studies have identified the presence of non-coding RNAs in the major zoonotic pathogen Campylobacter jejuni, however, few have been functionally characterized to date. CjNC110 is a conserved ncRNA in C. jejuni, located downstream of the luxS gene which is responsible for the production of the quorum-sensing molecule autoinducer-2 (AI-2). In this study, we utilized strand specific high-throughput RNAseq to identify potential targets or interactive partners of CjNC110 in a sheep abortion clone of C. jejuni. This data was then utilized to focus further phenotypic evaluation of the role of CjNC110 in motility, autoagglutination, quorum sensing, hydrogen peroxide sensitivity and chicken colonization in C. jejuni. Inactivation of the CjNC110 ncRNA led to a statistically significant decrease in autoagglutination ability as well as increased motility and hydrogen peroxide sensitivity when compared to wild-type. Extracellular AI-2 detection was decreased in ΔCjNC110, however, intracellular AI-2 accumulation was significantly increased, suggesting a key role of CjNC110 in modulating the transport of AI-2. Notably, ΔCjNC110 also showed a decreased ability to colonize chickens. Complementation of CjNC110 restored all phenotypic changes back to wild-type levels. The collective results of the phenotypic and transcriptomic changes observed in our data provide valuable insights into the pathobiology of C. jejuni sheep abortion clone and strongly suggest that CjNC110 plays an important role in regulation of energy taxis, flagellar glycosylation, cellular communication via quorum sensing, oxidative stress tolerance and chicken colonization in this important zoonotic pathogen.

The Molecular Mechanism of Nitrate Chemotaxis via Direct Ligand Binding to the PilJ Domain of McpN

ABSTRACTChemotaxis and energy taxis permit directed bacterial movements in gradients of environmental cues. Nitrate is a final electron acceptor for anaerobic respiration and can also serve as a nitrogen source for aerobic growth. Previous studies indicated that bacterial nitrate taxis is mediated by energy taxis mechanisms, which are based on the cytosolic detection of consequences of nitrate metabolism. Here we show thatPseudomonas aeruginosaPAO1 mediates nitrate chemotaxis on the basis of specific nitrate sensing by the periplasmic PilJ domain of the PA2788/McpN chemoreceptor. The presence of nitrate reducedmcpNtranscript levels, and McpN-mediated taxis occurred only under nitrate starvation conditions. In contrast to the NarX and NarQ sensor kinases, McpN bound nitrate specifically and showed no affinity for other ligands such as nitrite. We report the three-dimensional structure of the McpN ligand binding domain (LBD) at 1.3-Å resolution in complex with nitrate. Although structurally similar to 4-helix bundle domains, the ligand binding mode differs since a single nitrate molecule is bound to a site on the dimer symmetry axis. As for 4-helix bundle domains, ligand binding stabilized the McpN-LBD dimer. McpN homologues showed a wide phylogenetic distribution, indicating that nitrate chemotaxis is a widespread phenotype. These homologues were particularly abundant in bacteria that couple sulfide/sulfur oxidation with nitrate reduction. This work expands the range of known chemotaxis effectors and forms the basis for the exploration of nitrate chemotaxis in other bacteria and for the study of its physiological role.IMPORTANCENitrate is of central importance in bacterial physiology. Previous studies indicated that movements toward nitrate are due to energy taxis, which is based on the cytosolic sensing of consequences of nitrate metabolism. Here we present the first report on nitrate chemotaxis. This process is initiated by specific nitrate binding to the periplasmic ligand binding domain (LBD) of McpN. Nitrate chemotaxis is highly regulated and occurred only under nitrate starvation conditions, which is helpful information to explore nitrate chemotaxis in other bacteria. We present the three-dimensional structure of the McpN-LBD in complex with nitrate, which is the first structure of a chemoreceptor PilJ-type domain. This structure reveals striking similarities to that of the abundant 4-helix bundle domain but employs a different sensing mechanism. Since McpN homologues show a wide phylogenetic distribution, nitrate chemotaxis is likely a widespread phenomenon with importance for the life cycle of ecologically diverse bacteria.

Phylogenetic Characterization of the Energy-taxis Receptor Aer in Pseudomonas and Phenotypic Characterization in P. pseudoalcaligenes KF707

Chemotaxis allows bacteria to sense gradients in their environment and respond by directing their swimming. Aer is a receptor that, instead of responding to a specific chemoattractant, allows bacteria to sense cellular energy levels and move towards favourable environments. In Pseudomonas, the number of apparent Aer homologs differs between the only two species it had been characterized in, P. aeruginosa and P. putida. Here we combined bioinformatic approaches with deletional mutagenesis in P. pseudoalcaligenes KF707 to further characterize Aer. It was determined that the number of Aer homologs varies between 0-4 throughout the Pseudomonas genus, and they were phylogenetically classified into 5 subgroups. We also used sequence analysis to show that these homologous receptors differ in their HAMP signal transduction domains. Genetic analysis also indicated that some Aer homologs have likely been subject to horizontal transfer. P. pseudoalcaligenes KF707 was unique among species for having three Aer homologs as well as the receptors CttP and McpB. Phenotypic characterization in this species showed the most prevalent homolog of Aer was key, but not essential for energy-taxis. This study demonstrates that energy-taxis in Pseudomonas varies between species and provides a new naming convention and associated phylogenetic details for Aer chemoreceptors.

Energy Taxis toward Host-Derived Nitrate Supports a Salmonella Pathogenicity Island 1-Independent Mechanism of Invasion

ABSTRACT Salmonella enterica serovar Typhimurium can cross the epithelial barrier using either the invasion-associated type III secretion system (T3SS-1) or a T3SS-1-independent mechanism that remains poorly characterized. Here we show that flagellum-mediated motility supported a T3SS-1-independent pathway for entering ileal Peyer’s patches in the mouse model. Flagellum-dependent invasion of Peyer’s patches required energy taxis toward nitrate, which was mediated by the methyl-accepting chemotaxis protein (MCP) Tsr. Generation of nitrate in the intestinal lumen required inducible nitric oxide synthase (iNOS), which was synthesized constitutively in the mucosa of the terminal ileum but not in the jejunum, duodenum, or cecum. Tsr-mediated invasion of ileal Peyer’s patches was abrogated in mice deficient for Nos2 , the gene encoding iNOS. We conclude that Tsr-mediated energy taxis enables S . Typhimurium to migrate toward the intestinal epithelium by sensing host-derived nitrate, thereby contributing to invasion of Peyer’s patches. IMPORTANCE Nontyphoidal Salmonella serovars, such as S. enterica serovar Typhimurium, are a common cause of gastroenteritis in immunocompetent individuals but can also cause bacteremia in immunocompromised individuals. While the invasion-associated type III secretion system (T3SS-1) is important for entry, S . Typhimurium strains lacking a functional T3SS-1 can still cross the intestinal epithelium and cause a disseminated lethal infection in mice. Here we observed that flagellum-mediated motility and chemotaxis contributed to a T3SS-1-independent pathway for invasion and systemic dissemination to the spleen. This pathway required the methyl-accepting chemotaxis protein (MCP) Tsr and energy taxis toward host-derived nitrate, which we found to be generated by inducible nitric oxide synthase (iNOS) in the ileal mucosa prior to infection. Collectively, our data suggest that S . Typhimurium enhances invasion by actively migrating toward the intestinal epithelium along a gradient of host-derived nitrate emanating from the mucosal surface of the ileum.