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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Recently, there has been growing concern about how to most effectively support the literacy development of beginning and struggling readers with regard to helping them learn to effortlessly identify the huge number of words that proficient readers ultimately learn to read with automaticity. Some, noting the critical importance of phonics instruction in learning to read in an alphabetic writing system, take the position that students should attend only to alphabetic information in word‐solving attempts. However, long‐standing theories of the development of word‐reading skills support the value of teaching students to use both alphabetic and contextual information in word solving in interactive and confirmatory ways. The authors summarize 25 years of research in which beginning and struggling readers were taught to use both code‐ and meaning/context‐based strategies for word solving and were provided with explicit, responsive instruction focused on the alphabetic code. The authors present brief summaries of theoretical explanations of the word‐learning process. Then, the authors summarize six experimental studies that, together, included students in kindergarten through fourth grade and involved the implementation of the Interactive Strategies Approach in the primary grades and an extension of the approach with middle elementary students with reading difficulties. The studies resulted in substantially improved reading outcomes among treatment versus business‐as‐usual groups. The authors contend that using both phonics‐ and context‐based information facilitates the ability to build sight vocabulary, which in turn enables readers to turn their attention to the most important goal of literacy learning: meaning construction.

In vivo expression of CO dehydrogenase/acetyl coenzyme A synthase in Methanosarcina spp. is coordinately regulated in response to substrate by at least two mechanisms: differential transcription initiation and early elongation termination near the 3 end of a 371-bp leader sequence. This is the first report of regulation of transcription elongation in the Archaea.Methanogenic species in the genus Methanosarcina are the most metabolically diverse among methanogens, with the ability to grow by CO 2 reduction with H 2 , methyl reduction with H 2 , aceticlastic fermentation of acetate, or methylotrophic catabolism of methanol, methylated amines, and dimethyl sulfide (34). Methanosarcina acetivorans is also reported to grow with carbon monoxide (23). These organisms preferentially use substrates with higher free energy, such as methanol and trimethylamine (TMA), before utilizing acetate, exhibiting diauxic growth in the presence of both types of substrates (4,10,16,21,26). Despite their preference for nonaceticlastic substrates and the relatively lower growth rates and yields of Methanosarcina spp. with acetate, approximately 70% of the biogenic methane produced globally is generated from the catabolism of acetate (19). As a result, regulation of genes involved in the aceticlastic pathway is likely to have a significant impact on anaerobic degradation processes and the global biogenesis of methane.Several enzymes associated with aceticlastic methanogenesis, including acetate kinase, phosphotransacetylase, carbonic anhydrase, and carbon monoxide dehydrogenase/acetyl coenzyme A (acetyl-CoA) synthase (CODH/ACS), are regulated in response to substrates (13,14,17). Expression of the CODH/ ACS operon (cdhABCDE), which catalyzes the catabolism of acetyl-CoA in the aceticlastic pathway, is highly regulated in response to growth on acetate compared with methanol and TMA, but the mechanism(s) of this regulation is not known (28, 31). Two CODH/ACS operons are detected in the annotated genome sequences of M. acetivorans (11), Methanosarcina barkeri (20), and Methanosarcina mazei (7). In contrast, only one cdhABCDE operon has been detected in Methanosarcina thermophila (12). In M. acetivorans, both of these operons appear to be regulated in response to substrate, although the extent of the regulation is greater in one (MA3680) than in the other (MA1016) (17). Northern analysis of CODH/ACS mRNA from Methanosarcina thermophila indicates that the regulation of this CODH/ACS ortholog in response to substrate occurs, at least in part, at the level of mRNA (28). The regulation of this ortholog was corroborated with translational fusions of the M. thermophila CODH/ACS promoter to lacZ (2) and by peptide fragment analysis of a CODH/ACS component, CdhA, in M. mazei (9). This CODH/ACS operon has an unusually long 371-bp 5Ј leader region (28). In contrast, the 5Ј leader sequence of CODH/ACS in the obligately aceticlastic Methanosaeta concilii (formerly "Methanothrix soehgenii") is less than 100 bases long and has no significant sequence simi...

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