Fecal coliforms and enterococci are indicator organisms used worldwide to monitor water quality. These bacteria are used in microbial source tracking (MST) studies, which attempt to assess the contribution of various host species to fecal pollution in water. Ideally, all strains of a given indicator organism (IO) would experience equal persistence (maintenance of culturable populations) in water; however, some strains may have comparatively extended persistence outside the host, while others may persist very poorly in environmental waters. Assessment of the relative contribution of host species to fecal pollution would be confounded by differential persistence of strains. Here, freshwater and saltwater mesocosms, including sediments, were inoculated with dog feces, sewage, or contaminated soil and were incubated under conditions that included natural stressors such as microbial predators, radiation, and temperature fluctuations. Persistence of IOs was measured by decay rates (change in culturable counts over time). Decay rates were influenced by IO, inoculum, water type, sediment versus water column location, and Escherichia coli strain. Fecal coliform decay rates were significantly lower than those of enterococci in freshwater but were not significantly different in saltwater. IO persistence according to mesocosm treatment followed the trend: contaminated soil > wastewater > dog feces. E. coli ribotyping demonstrated that certain strains were more persistent than others in freshwater mesocosms, and the distribution of ribotypes sampled from mesocosm waters was dissimilar from the distribution in fecal material. These results have implications for the accuracy of MST methods, modeling of microbial populations in water, and efficacy of regulatory standards for protection of water quality.

UreG is a GTPase required for assembly of the nickel-containing active site of urease. Herein, a Strep-tagged Klebsiella aerogenes UreG (UreGStr) and selected site-directed variants of UreGStr were constructed for studying the in vivo effects on urease activation in recombinant Escherichia coli cells, characterizing properties of the purified proteins, and analysis of in vivo and in vitro protein-protein interactions. Whereas the Strep-tag had no effect on UreG’s ability to activate urease, enzyme activity was essentially abolished in the K20A, D49A, C72A, H74A, D80A, and S111A UreGStr variants, with diminished activity also noted with E25A, C28A, and S115A proteins. Lys20 and Asp49 are likely to function in binding/hydrolysis of GTP and binding of Mg, respectively. UreGStr binds one nickel or zinc ion per monomer (Kd = ~5 μM for each metal ion) at a binding site that includes Cys72, as shown by a 12-fold increased Kd for nickel ions using C72A UreGStr and by a thiolate-to-nickel charge-transfer band that is absent in the mutant protein. Based on UreG homology to HypB, a GTPase needed for hydrogenase assembly, along with the mutation results, His74 is likely to be an additional metal ligand. In vivo pull-down assays revealed Asp80 as critical for stabilizing UreGStr interaction with the UreABC-UreDF complex. In vitro pull-down assays demonstrated UreG binding to UreE, with the interaction enhanced by nickel or zinc ions. The metallochaperone UreE is suggested to transfer its bound nickel to UreG in the UreABC-UreDFG complex, with the metal ion subsequently transferring to UreD, and then into the nascent active site of urease in a GTP-dependent process.

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Viable methanogens have been detected in dry, aerobic environments such as dry reservoir sediment, dry rice paddies and aerobic desert soils, which suggests that methanogens have mechanisms for long-term survival in a desiccated state. In this study, we quantified the survival rates of the methanogenic archaeon Methanosarcina barkeri after desiccation under conditions equivalent to the driest environments on Earth and subsequent exposure to different stress factors. There was no significant loss of viability after desiccation for 28 days for cells grown with either hydrogen or the methylotrophic substrates, but recovery was affected by growth phase, with cells desiccated during the stationary phase of growth having a higher rate of recovery after desiccation. Synthesis of methanosarcinal extracellular polysaccharide (EPS) significantly increased the viability of desiccated cells under both anaerobic and aerobic conditions compared with that of non-EPS-synthesizing cells. Desiccated M. barkeri exposed to air at room temperature did not lose significant viability after 28 days, and exposure of M. barkeri to air after desiccation appeared to improve the recovery of viable cells compared with that of desiccated cells that were never exposed to air. Desiccated M. barkeri was more resistant to higher temperatures, and although resistance to oxidative conditions such as ozone and ionizing radiation was not as robust as in other desiccation-resistant microorganisms, the protection mechanisms are likely adequate to maintain cell viability during periodic exposure events. The results of this study demonstrate that after desiccation M. barkeri has the innate capability to survive extended periods of exposure to air and lethal temperatures.A s more microorganisms are discovered in extreme environments on Earth that were previously considered to be devoid of life, we have been redefining the physical and chemical parameters for life to exist. One such extreme is the long-term storage of viable cells in a desiccated state, which imposes physiological constraints that many species cannot tolerate. A common characteristic of known desiccation-tolerant microorganisms, which include spore-forming bacteria, heterocyst-forming cyanobacteria, heteropolysaccharide-forming Beijerinckia, and Deinococcus, is the formation of relatively thick outer cell layers (32). The synthesis of an outer cell layer often composed of an extracellular polysaccharide (EPS), in conjunction with other mechanisms such as robust DNA repair and compatible solute formation, enables the cells to retain the minimal intracellular water activity required for survival (32).The methanogenic Archaea, despite their preference for highly reduced, anoxic conditions in order to grow, are globally distributed in a wide range of anaerobic, aquatic environments, including Antarctic lakes, submarine hydrothermal vents, rice paddies, sewage digestors, and as symbionts in rumen, termites, protozoa, and human large intestines (38). Among the least anticipated environments wher...

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