Vibrio cholerae is both an environmental bacterium and a human intestinal pathogen. The attachment of bacteria to surfaces in biofilms is thought to be an important feature of the survival of this bacterium both in the environment and within the human host. Biofilm formation occurs when cell-surface and cell-cell contacts are formed to make a three-dimensional structure characterized by pillars of bacteria interspersed with water channels. In monosaccharide-rich conditions, the formation of the V. cholerae biofilm requires synthesis of the VPS exopolysaccharide. MbaA (locus VC0703), an integral membrane protein containing a periplasmic domain as well as cytoplasmic GGDEF and EAL domains, has been previously identified as a repressor of V. cholerae biofilm formation. In this work, we have studied the role of the protein NspS (locus VC0704) in V. cholerae biofilm development. This protein is homologous to PotD, a periplasmic spermidine-binding protein of Escherichia coli. We show that the deletion of nspS decreases biofilm development and transcription of exopolysaccharide synthesis genes. Furthermore, we demonstrate that the polyamine norspermidine activates V. cholerae biofilm formation in an MbaA-and NspS-dependent manner. Based on these results, we propose that the interaction of the norspermidine-NspS complex with the periplasmic portion of MbaA diminishes the ability of MbaA to inhibit V. cholerae biofilm formation. Norspermidine has been detected in bacteria, archaea, plants, and bivalves. We suggest that norspermidine serves as an intercellular signaling molecule that mediates the attachment of V. cholerae to the biotic surfaces presented by one or more of these organisms.

bSPOR domains are about 75 amino acids long and probably bind septal peptidoglycan during cell division. We mutagenized 33 amino acids with surface-exposed side chains in the SPOR domain from an Escherichia coli cell division protein named FtsN. The mutant SPOR domains were fused to Tat-targeted green fluorescent protein ( TT GFP) and tested for septal localization in live E. coli cells. Lesions at the following 5 residues reduced septal localization by a factor of 3 or more: Q251, S254, W283, R285, and I313. All of these residues map to a ␤-sheet in the published solution structure of FtsN SPOR . Three of the mutant proteins (Q251E, S254E, and R285A mutants) were purified and found to be defective in binding to peptidoglycan sacculi in a cosedimentation assay. These results match closely with results from a previous study of the SPOR domain from DamX, even though these two SPOR domains share <20% amino acid identity. Taken together, these findings support the proposal that SPOR domains localize by binding to septal peptidoglycan and imply that the binding site is associated with the ␤-sheet. We also show that FtsN SPOR contains a disulfide bond between ␤-sheet residues C252 and C312. The disulfide bond contributes to protein stability, cell division, and peptidoglycan binding.

Background: M. tuberculosis two-component systems converge to co-regulate gene expression during nitrate metabolism. Results: Analysis of nitrate/nitrite-responsive gene expression reveals novel interaction between NarL and DevR response regulators. Conclusion: Cooperative binding of NarL and DevR enables co-regulation of a subset of genes. Significance: The findings establish endogenous nitrite as a signal and highlight interplay between M. tuberculosis NarLS and DevRS/DosT signaling systems.

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Siderophores are microbially-produced ferric iron chelators. They are essential for microbial survival, but their presence and function for cave microorganisms have not been extensively studied. Siderophores are classified based on the common functional groups (catechols, hydroxamates, carboxylates, and mixed) that coordinate to ferric (Fe3+) iron. Cave environments are nutrient-limited and previous evidence suggests siderophore usage in carbonate caves. We hypothesize that siderophores are likely used as a mechanism in caves to obtain critical ferric iron. Cave bacteria were collected from long-term parent cultures (LT PC) or short-term parent cultures (ST PC) inoculated with ferromanganese deposits (FMD) and carbonate secondary minerals from Lechuguilla and Spider caves in Carlsbad Caverns National Park, NM. LT PC were incubated for 10−11 years to identify potential chemolithoheterotrophic cultures able to survive in nutrient-limited conditions. ST PC were incubated for 1−3 days to identify a broader diversity of cave isolates. A total of 170 LT and ST cultures, 18 pure and 152 mixed, were collected and used to classify siderophore production and type and to identify siderophore producers. Siderophore production was slow to develop (>10 days) in LT cultures with a greater number of weak siderophore producers in comparison to the ST cultures that produced siderophores in <10 days, with a majority of strong siderophore producers. Overall, 64% of the total cultures were siderophore producers, with the majority producing hydroxamate siderophores. Siderophore producers were classified into Proteobacteria (Alpha-, Beta-, or Gamma-), Actinobacteria, Bacteroidetes, and Firmicutes phyla using 16S rRNA gene sequencing. Our study supports our hypothesis that cave bacteria have the capability to produce siderophores in the subsurface to obtain critical ferric iron.