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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. 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 nutrients such as 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 (CCNP), 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, which the majority preferred 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.

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Elevated levels of arsenic and uranium have been detected in water sources near abandoned uranium mines in the Southwest. Evidence suggests uranium exposure increases the likelihood of immune dysfunction and this study investigates the impact of arsenic and uranium on human immune cell lines. Concentration-dependent cytotoxicity occurred following exposure to arsenite, whereas cells remained viable after 48 -h treatment with up to 100 µM uranyl acetate despite uptake of uranium into cells. Arsenite stimulated an oxidative stress response as detected by Nrf-2 nuclear accumulation and induction of HMOX-1 and NQO1, which was not detected with up to 30 µM uranyl acetate. Cellular oxidative stress can promote DNA damage and arsenite, but not uranium, stimulated DNA damage as measured by pH2AX. Arsenic enhanced the cytotoxic response to etoposide suggesting an inhibition of DNA repair, unlike uranium. Similarly, uranium did not inhibit PARP-1 activity. Because uranium reportedly stimulates oxidative stress, DNA damage and cytotoxicity in adherent epithelial cells, the current study suggests distinct cell type differences in response to uranium that may relate to generation of oxidative stress and associated downstream consequences. Delineating the actions of uranium across different cell targets will be important for understanding the potential health effects of uranium exposures.

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Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M. tuberculosis NarL/NarS as a functional two-component system and identified His(241) and Asp(61) as conserved phosphorylation sites in NarS and NarL, respectively. Transcriptional profiling between M. tuberculosis H37Rv and a ΔnarL mutant strain during exponential growth in broth cultures with or without nitrate defined an ∼30-gene NarL regulon that exhibited significant overlap with DevR-regulated genes, thereby implicating a role for the DevR response regulator in the regulation of nitrate metabolism. Notably, expression analysis of a subset of genes common to NarL and DevR regulons in M. tuberculosis ΔdevR, ΔdevSΔdosT, and ΔnarL mutant strains revealed that in response to nitrite produced during aerobic nitrate metabolism, the DevRS/DosT regulatory system plays a primary role that is augmented by NarL. Specifically, NarL itself was unable to bind to the narK2, acg, and Rv3130c promoters in phosphorylated or unphosphorylated form; however, its interaction with DevR∼P resulted in cooperative binding, thereby enabling co-regulation of these genes. These findings support the role of physiologically derived nitrite as a metabolic signal in mycobacteria. We propose NarL-DevR binding, possibly as a heterodimer, as a novel mechanism for co-regulation of gene expression by the DevRS/DosT and NarL/NarS regulatory systems.

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SPOR 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 ß-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 ß-sheet. We also show that FtsN(SPOR) contains a disulfide bond between ß-sheet residues C252 and C312. The disulfide bond contributes to protein stability, cell division, and peptidoglycan binding.

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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.

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