Faecal indicator bacteria enumeration in beach sand: a comparison study of extraction methods in medium to coarse sands

Aims:  The absence of standardized methods for quantifying faecal indicator bacteria (FIB) in sand hinders comparison of results across studies. The purpose of the study was to compare methods for extraction of faecal bacteria from sands and recommend a standardized extraction technique.
Methods and Results:  Twenty-two methods of extracting enterococci and Escherichia coli from sand were evaluated, including multiple permutations of hand shaking, mechanical shaking, blending, sonication, number of rinses, settling time, eluant-to-sand ratio, eluant composition, prefiltration and type of decantation. Tests were performed on sands from California, Florida and Lake Michigan. Most extraction parameters did not significantly affect bacterial enumeration. anova revealed significant effects of eluant composition and blending; with both sodium metaphosphate buffer and blending producing reduced counts.
Conclusions:  The simplest extraction method that produced the highest FIB recoveries consisted of 2 min of hand shaking in phosphate-buffered saline or deionized water, a 30-s settling time, one-rinse step and a 10 : 1 eluant volume to sand weight ratio. This result was consistent across the sand compositions tested in this study but could vary for other sand types.
Significance and Impact of the Study:  Method standardization will improve the understanding of how sands affect surface water quality.     

Bacterial community of very wet and acidic subalpine forest and fire-induced grassland soils

The subalpine forest and grassland ecosystems at Tatajia in Yushan National Park, Taiwan, at an elevation of 2,700 m, mean annual precipitation of 4,100 mm, mean annual temperature of 9.5°C, and soil pH near 3.5, represent land types whose bacterial communities have not been previously characterized. To this end, small subunit (SSU) rRNA libraries were prepared from environmental DNA, and 319 clones were sequenced and characterized. Despite differences in vegetation, Acidobacteria, Proteobacteria and Firmicutes were the most abundant phyla in soil communities from the forest and grassland. Although not significantly different, on the basis of Chao1, Shannon and other indices and rarefaction analyses, the diversity of the bacterial community of grassland appeared higher than that of the forest. The composition of the most abundant operational taxonomic units (OTUs) also differed between the grassland and forest communities. Because the grassland was formed by fire 30 years ago from forest, these results indicated a different bacterial community could form within that time. Moreover, most of the OTUs abundant in Tatajia soils had been previously detected in other studies, but in lower numbers. Therefore, the bacterial communities in Tatajia differed in relative abundance but not in types of bacteria present. However, one acidobacterial OTU abundant in Tatajia had previously been found to be abundant in soils from around the world. Thus, this OTU may represent a particularly abundant and cosmopolitan bacterial phylotype.     

Characterization of Energy-Conserving Hydrogenase B in Methanococcus maripaludis

The Methanococcus maripaludis energy-conserving hydrogenase B (Ehb) generates low potential electrons required for autotrophic CO₂ assimilation. To analyze the importance of individual subunits in Ehb structure and function, markerless in-frame deletions were constructed in a number of M. maripaludis ehb genes. These genes encode the large and small hydrogenase subunits (ehbN and ehbM, respectively), a polyferredoxin and ferredoxin (ehbK and ehbL, respectively), and an ion translocator (ehbF). In addition, a gene replacement mutation was constructed for a gene encoding a putative membrane-spanning subunit (ehbO). When grown in minimal medium plus acetate (McA), all ehb mutants had severe growth deficiencies except the ΔehbO::pac strain. The membrane-spanning ion translocator (ΔehbF) and the large hydrogenase subunit (ΔehbN) deletion strains displayed the severest growth defects. Deletion of the ehbN gene was of particular interest because this gene was not contiguous to the ehb operon. In-gel activity assays and Western blots confirmed that EhbN was part of the membrane-bound Ehb hydrogenase complex. The ΔehbN strain was also sensitive to growth inhibition by aryl acids, indicating that Ehb was coupled to the indolepyruvate oxidoreductase (Ior), further supporting the hypothesis that Ehb provides low potential reductants for the anabolic oxidoreductases in M. maripaludis.Hydrogenotrophic methanococci specialize in utilizing H₂ as an electron donor, and these organisms possess six different Ni-Fe hydrogenases. These enzymes include two F_420--reducing hydrogenases, two non-F₄₂₀-reducing hydrogenases, and two membrane-bound hydrogenases (Eha and Ehb [5]). The F₄₂₀-reducing hydrogenases reduce coenzyme F₄₂₀, which subsequently reduces methenyltetrahydromethanopterin and methylenetetrahydromethanopterin, intermediates in the pathway of methanogenesis. In Methanococcus voltae, the F₄₂₀-reducing hydrogenase is also reported to reduce the 2-mercaptoethanesulfonate:7-mercaptoheptanoylthreonine phosphate heterodisulfide formed in the final step of methanogenesis (2). In contrast, Methanothermobacter marburgensis utilizes the non-F₄₂₀-reducing hydrogenase to reduce the heterodisulfide (22, 25).The two membrane-bound hydrogenases couple the chemiosmotic energy of ion gradients to H₂ oxidation and ferredoxin reduction. In the aceticlastic methanogen Methanosarcina barkeri, the homologous enzyme is called energy conserving hydrogenase or Ech and performs a variety of physiological functions, including the generation of a proton motive force during CO oxidation and concomitant proton reduction in aceticlastic methanogenesis and the generation of low potential electron donors for CO₂ reduction to formylmethanofuran in the first step of methanogenesis and the reductive carboxylation of acetyl coenzyme A (acetyl-CoA) to pyruvate in carbon assimilation (11, 12). In the hydrogenotrophic methanogens, it is predicted that the two energy-conserving hydrogenases (Eha and Ehb) have distinct roles (26). The Ehb appears to reduce low potential electron carriers utilized in autotrophic CO₂ fixation (16). Anabolic enzymes likely to be coupled to Ehb in this manner include (i) the carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) and the pyruvate oxidoreductase (Por), which catalyze the first two steps of carbon assimilation; (ii) the α-ketoglutarate oxidoreductase (Kor), which catalyzes the final step in the incomplete reductive tricarboxylic acid cycle; and (iii) the indolepyruvate oxidoreductase (Ior) and the 2-oxoisovalerate oxidoreductase (Vor), which are involved in amino acid biosynthesis from aryl and branched-chain acids, respectively. Support for these conclusions comes in large part from the phenotype of an M. maripaludis ehb gene replacement mutant S40, which was only capable of limited growth in the absence of acetate and amino acids (16). Furthermore, expression of CODH/ACS, Por, and Vor were significantly upregulated in the mutant, providing further evidence for a role of Ehb in these processes (16). In contrast, there is no direct evidence for the role of Eha. By analogy with the Methanosarcina Ech, it could be involved in generating reducing equivalents for the reduction of CO₂ to formylmethanofuran. Alternatively, hydrogenotrophic methanogens may have an alternative method of CO₂ reduction (27), and Eha could have another function entirely.In spite of some functional similarities between the Ech of the aceticlastic methanogens and Eha or Ehb of hydrogenotrophs, the structures of their operons are very different (Fig. ​(Fig.1).1). Based upon sequence comparisons, all of these membrane-bound hydrogenases possess conserved large and small hydrogenase subunits, a 2[4Fe-4S] ferredoxin, and an integral membrane ion translocator (3, 8, 26). Otherwise, the structures are very different. The purified Ech from Methanosarcina barkeri contains six polypeptides encoded by the six genes of the ech operon (8, 11). The Eha and Ehb hydrogenases have never been purified. The eha and ehb operons from the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus comprise 20 and 17 genes, respectively (23, 26). Most of these genes are predicted to encode transmembrane proteins, although there are also several polyferredoxins and hydrophilic proteins (26). Many of these genes are not homologous to the M. barkeri ech genes. The Methanococcus maripaludis genome contains homologs to the M. thermautotrophicus eha and ehb genes, although only nine of the ehb genes are contiguous on the genome (Fig. ​(Fig.1).1). In the present study, the Ehb from the hydrogenotrophic methanogen Methanococcus maripaludis was analyzed. M. maripaludis is a model organism that can be easily genetically modified. Furthermore, its genome has been sequenced, and many of its biochemical pathways have been characterized.Open in a separate windowFIG. 1.Genetic map of Methanosarcina barkeri ech (A), Methanothermobacter marburgensis ehb (MTH1235-1251) (B), and Methanococcus maripaludis ehb (MMP1631-1629) (C) operons. Genes encoding integral membrane proteins found only in Ehb are indicated in blue, integral membrane proteins conserved in both Ech and Ehb are blue with diagonal stripes, hydrogenase small subunits are yellow, hydrogenase large subunits are red, 4Fe-4S motif-containing proteins are brown, and other hydrophilic proteins present in Ehb but absent from Ech are gray. Notably, M. maripaludis contains homologs to all of the M. marburgensis ehb genes, but many are unlinked to the major gene cluster and not shown. Based upon references 5, 8, 11, and 26.     

Population structure, persistence, and seasonality of autochthonous Escherichia coli in temperate, coastal forest soil from a Great Lakes watershed

The common occurrence of Escherichia coli in temperate soils has previously been reported, however, there are few studies to date to characterize its source, distribution, persistent capability and genetic diversity. In this study, undisturbed, forest soils within six randomly selected 0.5 m2 exclosure plots (covered by netting of 2.3 mm2 mesh size) were monitored from March to October 2003 for E. coli in order to describe its numerical and population characteristics. Culturable E. coli occurred in 88% of the samples collected, with overall mean counts of 16 MPN g(-1), ranging from < 1 to 1657 (n = 66). Escherichia coli counts did not correlate with substrate moisture content, air, or soil temperatures, suggesting that seasonality were not a strong factor in population density control. Mean E. coli counts in soil samples (n = 60) were significantly higher inside than immediately outside the exclosures; E. coli distribution within the exclosures was patchy. Repetitive extragenic palindromic polymerase chain reaction (Rep-PCR) demonstrated genetic heterogeneity of E. coli within and among exclosure sites, and the soil strains were genetically distinct from animal (E. coli) strains tested (i.e. gulls, terns, deer and most geese). These results suggest that E. coli can occur and persist for extended periods in undisturbed temperate forest soils independent of recent allochthonous input and season, and that the soil E. coli populations formed a cohesive phylogenetic group in comparison to the set of fecal strains with which they were compared. Thus, in assessing E. coli sources within a stream, it is important to differentiate background soil loadings from inputs derived from animal and human fecal contamination.     

Diet-induced obesity in rats leads to a decrease in sperm motility

Background  

Obesity is rapidly becoming a worldwide epidemic that affects children and adults. Some studies have shown a relationship between obesity and infertility, but until now it remains controversial. Thus, the aim of the present study was to investigate the effect of high-fat diet-induced obesity on male reproductive parameters.     

Complete genome sequence of the genetically tractable hydrogenotrophic methanogen Methanococcus maripaludis

The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. Unusual features included the absence of a Cdc6 homolog, implying a variation in replication initiation, and the presence of a bacterial-like RNase HI as well as an RNase HII typical of the Archaea. The presence of alanine dehydrogenase and alanine racemase, which are uniquely present among the Archaea, explained the ability of the organism to use L- and D-alanine as nitrogen sources. Features that contrasted with the related organism Methanocaldococcus jannaschii included the absence of inteins, even though close homologs of most intein-containing proteins were encoded. Although two-thirds of the ORFs had their highest Blastp hits in Methanocaldococcus jannaschii, lateral gene transfer or gene loss has apparently resulted in genes, which are often clustered, with top Blastp hits in more distantly related groups.     

Two biosynthetic pathways for aromatic amino acids in the archaeon Methanococcus maripaludis

Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon. Aromatic amino acids (AroAAs) are biosynthesized in this autotroph either by the de novo pathway, with chorismate as an intermediate, or by the incorporation of exogenous aryl acids via indolepyruvate oxidoreductase (IOR). In order to evaluate the roles of these pathways, the gene that encodes the third step in the de novo pathway, 3-dehydroquinate dehydratase (DHQ), was deleted. This mutant required all three AroAAs for growth, and no DHQ activity was detectible in cell extracts, compared to 6.0 +/- 0.2 mU mg(-1) in the wild-type extract. The growth requirement for the AroAAs could be fulfilled by the corresponding aryl acids phenylacetate, indoleacetate, and p-hydroxyphenylacetate. The specific incorporation of phenylacetate into phenylalanine by the IOR pathway was demonstrated in vivo by labeling with [1-(13)C]phenylacetate. M. maripaludis has two IOR homologs. A deletion mutant for one of these homologs contained 76, 74, and 42% lower activity for phenylpyruvate, p-hydoxyphenylpyruvate, and indolepyruvate oxidation, respectively, than the wild type. Growth of this mutant in minimal medium was inhibited by the aryl acids, but the AroAAs partially restored growth. Genetic complementation of the IOR mutant also restored much of the wild-type phenotype. Thus, aryl acids appear to regulate the expression or activity of the de novo pathway. The aryl acids did not significantly inhibit the activity of the biosynthetic enzymes chorismate mutase, prephenate dehydratase, and prephenate dehydrogenase in cell extracts, so the inhibition of growth was probably not due to an effect on these enzymes.     

p53 and TGF-beta in development: prelude to tumor suppression?

Recent work in Xenopus embryos reveals an unexpected developmental role for the tumor suppressor gene p53. This finding may have implications for the evolution of p53, its interaction with Smads in TGF-beta dependent mesoderm specification, and the cooperation among p53 family members.     

Continuous culture of Methanococcus maripaludis under defined nutrient conditions

To study global regulation in the methanogenic archaeon Methanococcus maripaludis, we devised a system for steady-state growth in chemostats. New Brunswick Bioflo 110 bioreactors were equipped with controlled delivery of hydrogen, nitrogen, carbon dioxide, hydrogen sulfide, and anaerobic medium. We determined conditions and media compositions for growth with three different limiting nutrients, hydrogen, phosphate, and leucine. To investigate leucine limitation we constructed and characterized a mutant in the leuA gene for 2-isopropylmalate synthase, demonstrating for the first time the function of this gene in the Archaea. Steady state specific growth rates in these studies ranged from 0.042 to 0.24 h(-1). Plots of culture density vs. growth rate for each condition showed the behavior predicted by growth modeling. The results show that growth behavior is normal and reproducible and validate the use of the chemostat system for metabolic and global regulation studies in M. maripaludis.