Richard Devereux

Genus- and group-specific hybridization probes for determinative and environmental studies of sulfate-reducing bacteria

Summary A set of six oligonucleotides, complementary to conserved tracts of 16S rRNA from phylogenetically-defined groups of sulfate-reducing bacteria, was characterized for use as hybridization probes in determinative and environmental microbiology. Four probes were genus specific and identified Desulfobacterium spp., Desulfobacter spp., Desulfobulbus spp., or Desulfovibrio spp. The other two probes encompassed more diverse assemblages. One probe was specific for the phylogenetic lineage composed of Desulfococcus mutlivorans, Desulfosarcina variabilis, and Desulfobotulus sapovorans. The remaining probe was specific for Desulfobacterium spp., Desulfobacter spp., D. multivorans, D. variabilis, and D. sapovorans. Temperature of dissociation was determined for each probe and the designed specificities of each were evaluated by hybridizations against closely related nontargeted species. In addition, each probe was screened by using a “phylogrid” membrane which consisted of nucleic acids from sixtyfour non-targeted organisms representing a diverse collection of eukarya, archaea, and bacteria. The application of these probes to studies in environmental microbiology was evaluated by hybridizations to 16S rRNAs of sulfate-reducing bacteria present in marine sediments.

Amplification of ribosomal RNA sequences

Emergence of the major outlines of bacterial phylogenetic relationships marks a defining advancement in the recent history of microbiology. This was made possible through the work of Woese and his colleagues who pioneered the comparison of rRNA sequences [30]. The ability to measure bacterial phylogenetic relationships, and those among microscopic eukaryotes, has had a direct impact on systematics that extends well into microbial ecology. While the driving force has been basic phylogenetic research, application of molecular techniques based on rRNA sequence comparisons to determinative and environmental microbiology is a blossoming area [9,26].

Characterization of a new thermophilic sulfate-reducing bacterium

A thermophilic sulfate-reducing vibrio isolated from thermal vent water in Yellowstone Lake, Wyoming, USA is described. The gram-negative, curved rod-shaped cells averaged 0.3 μm wide and 1.5 μm long. They were motile by means of a single polar flagellum. Growth was observed between 40° and 70 °C with optimal growth at 65 °C. Cultures remained viable for one year at 27 °C although spore-formation was not observed. Sulfate, thiosulfate and sulfite were used as electron acceptors. Sulfur, fumarate and nitrate were not reduced. In the presence of sulfate, growth was observed only with lactate, pyruvate, hydrogen plus acetate, or formate plus acetate. Pyruvate was the only compound observed to support fermentative growth. Pyruvate and lactate were oxidized to acetate. Desulfofuscidin and c-type cytochromes were present. The G+C content was 29.5 mol%. The divergence in the 16S ribosomal RNA sequences between the new isolate and Thermodesulfobacterium commune suggests that these two thermophilic sulfate-reducing bacteria represent different genera. These two bacteria depict a lineage that branches deeply within the Bacteria domain and which is clearly distinct from previously defined phylogenetic lines of sulfate-reducing bacteria. Strain YP87 is described as the type strain of the new genus and species Thermodesulfovibrio yellowstonii. Yellowstone Lake (Wyoming, USA) is located within one of the most tectonically active regions in the world (Klump et al. 1988; Remsen et al. 1990). Hydrothermal springs, hot gas fumaroles and elevated substrata temperatures have been observed within the lake itself (e.g., Remsen et al. 1990). Hydrothermal vent waters were reported to be anoxic, high in dissolved nutrients relative to the lake water and to have temperatures in excess of 80 °C (Klump et al. 1988; Remsen et al. 1990). Sulfate concentrations averaged 380 μM in vent waters and 80 μM in bulk lake water (Klump et al. 1988; Remsen et al. 1990). On the basis of on these physical and chemical characteristics, and the observation (e.g., Zeikus et al. 1983) that microbial sulfate reduction is prevalent in the thermal aquatic environments of Yellowstone National Park, we hypothesized that hydrothermal vent waters in Yellowstone Lake could support the growth of thermophilic sulfate reducers.Here we describe the general characteristics of a new thermophilic sulfate reducing bacterium, Thermodesulfovibrio yellowstonii, which was isolated from hydrothermal vent water in Sedge Bay of Yellowstone Lake, Wyoming, USA. In addition, we report on the phylogenetic relationship of this new isolate with other thermophilic and mesophilic sulfate-reducing bacteria.

Physiological ecology of Clostridium glycolicum RD-1, an aerotolerant acetogen isolated from sea grass roots

ABSTRACT An anaerobic, H2-utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass Halodule wrightii. Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments. Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H2-CO2, formate, lactate, or pyruvate. Growth on sugars or ethylene glycol yielded acetate and ethanol as end products. RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% [vol/vol]) of O2 in the headspace of static, horizontally incubated culture tubes; the concentration of O2 decreased during growth in such cultures. Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O2. In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H2 became significant end products when RD-1 was grown on glucose in the presence of O2. Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H2 were produced. When the concentration of O2 in the headspace exceeded 1% (vol/vol), supplemental H2 was not utilized. The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that ofClostridium glycolicum DSM 1288T, an organism characterized as a fermentative anaerobe. Comparative experiments with C. glycolicum DSM 1288T demonstrated that it had negligible H2- and formate-utilizing capacities. However, carbon monoxide dehydrogenase was detected in both RD-1 and C.glycolicum DSM 1288T. A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that ofC. glycolicum DSM 1288Tconfirmed that RD-1 was a strain of C.glycolicum. These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O2, (iii) oxic conditions favor the production of ethanol, lactate, and H2by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O2 might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O2.

Rapid separation of microbial lipids using solid phase extraction columns

Abstract A method was developed to rapidly separate lipid classes commonly found in microorganisms. The method is based on the use of aminopropyl solid phase extraction columns to separate polyhydroxyalkanoates (PHA), phospholipids, sterols, triglycerides, diglycerides, monoglycerides, and steryl esters. Recoveries of all lipid classes, with the exception of PHA and sterols, ranged from 91% to greater than 99%. PHA were recovered at 69% of the standard, and sterols from 82–84% of the standard. When applied to the analysis of lipids extracted from the cyanobacterium Spirulina platensis , the method afforded excellent recovery and separation of phospholipids and diglycerides including saturated, monounsaturated and polyunsaturated fatty acids. The S. platensis lipids also contained hydrocarbons and phytol recovered in the steryl ester and diglyceride fractions, respectively. This method provided a high yield, specific and rapid separation of microbial lipids with little contamination from other lipid groups, and will be useful for the characterization of microbial communities in environmental samples.

Nonnutrient anthropogenic chemicals in seagrass ecosystems: fate and effects.

Impacts of human-related chemicals, either alone or in combination with other stressors, are important to understand to prevent and reverse continuing worldwide seagrass declines. This review summarizes reported concentrations of anthropogenic chemicals in grass bed-associated surface waters, sediments, and plant tissues and phytotoxic concentrations. Fate information in seagrass-rooted sediments and overlying water is most available for trace metals. Toxicity results in aqueous exposures are available for at least 13 species and a variety of trace metals, pesticides, and petrochemicals. In contrast, results for chemical mixtures and chemicals in sediment matrices are uncommon. Contaminant bioaccumulation information is available for at least 23 species. The effects of plant age, tissue type, and time of collection have been commonly reported but not biological significance of the chemical residues. Experimental conditions have varied considerably in seagrass contaminant research and interspecific differences in chemical residues and chemical tolerances are common, which limits generalizations and extrapolations among species and chemicals. The few reported risk assessments have been usually local and limited to a few single chemicals and species representative of the south Australian and Mediterranean floras. Media-specific information describing exposure concentrations, toxic effect levels, and critical body burdens of common near-shore contaminants is needed for most species to support integrated risk assessments at multiple geographical scales and to evaluate the ability of numerical effects-based criteria to protect these marine angiosperms at risk.

S cycling: Characterization of natural communities of sulfate-reducing bacteria by 165 rRNA sequence comparisons

Past studies of microbial communities responsible for geochemical transformations have been limited by an inability to representatively cultivate, and then identify, the constituent members. Ribosomal RNA sequences, particularly 16S-like rRNAs, provide a measure of phylogenetic relationship that can now be used to examine the structure and diversity of microbial communities. Sulfate-reducing bacteria (SRB) play an important role in the sulfur cycle and the terminal mineralization of organic matter in estuarine and marine environments. Because the Gram-negative mesophilic SRB comprise a phylogenetically coherent assemblage, their communities are well suited to explorations through rRNA sequence-based methodologies. In this study we related molecular biological methods using rRNA probes to geochemical measurements at two different sites. At an unvegetated site in northwest Florida, rates of sulfate reduction were low and SRB rRNA comprised about 5% of the total rRNA extracted from the sediment. The other site, a salt marsh in New Hampshire, had higher rates of sulfate-reduction with SRB rRNA accounting for up to 30% of the total rRNA extracted from the sediment. SRB community structure differed dramatically between the two sites with Desulfobulbus rRNA much less abundant in the unvegetated site than in the salt marsh. The differences in these SRB communities reflect differences in the ecology of their habitats.

Effects of light reduction on growth of the submerged macrophyte Vallisneria americana and the community of root-associated heterotrophic bacteria

Abstract A shading experiment was conducted over a growing season to measure the effects of light reduction on Vallisneria americana in Perdido Bay on the Florida–Alabama border and to determine the response of heterotrophic bacteria in the rhizosphere. Plants subjected to 92% light reduction showed the most pronounced effects in chlorophyll a concentration, above- and below-ground biomass, and leaf dimensions. These results further suggested that the V. americana life cycle, as exhibited in temperate waters, was impaired. Heterotrophic bacteria were enumerated and identified (i) from the roots and sediments of fully illuminated plants and from unvegetated sediments at three intervals and (ii) from the roots of plants that have been subjected to 92% light reduction for 3 months. Up to two orders of magnitude greater numbers of bacteria were enumerated from root samples than sediment samples on a dry weight basis. Bacteria enumerated from the roots of plants subjected to light reduction (1.3±1.1×10 8 CFU g −1 ) were significantly higher than numbers of bacteria enumerated from the roots of fully illuminated plants (4.8±1.8×10 7 g −1 in the summer) or sediment samples (1.4±0.03×10 6 g −1 ). This suggests the roots of seagrasses stressed by light reduction provided more nutrients for bacterial growth. Higher percentages of Gram-negative bacteria were isolated from roots (up to 85% in the fall) than sediments (0–15%). Examination of isolates for traits characteristic of rhizosphere bacteria (siderophore production, formation of the phytohormone indole-3-acetic acid, and antifungal activity) did not show a clear distinction between root-associated and sediment isolates. Taxonomic identifications of root-associated bacteria based on MIDI analysis of fatty acid methyl esters were consistent with bacteria known to be associated with other plants or found at oxic–anoxic interfaces. In addition, the bacterial identifications showed most species were associated with only roots or only sediments. These results support studies suggesting seagrass rhizospheres harbor distinct bacterial communities.

Phylogeny of Sulfate-Reducing Bacteria and a Perspective for Analyzing Their Natural Communities

It has been 7 years since publication of the second edition of Postgate’s monograph on sulfate-reducing bacteria (Postgate, 1984a). That revision was prompted by the discovery of morphologically and nutritionally varied, sulfate-reducing genera within the bacterial domain (previously named the eubacterial kingdom; Woese et al., 1990; Widdel, 1988). [Note: The taxonomical concept of domain is rather new and refers to the highest ranking taxon of a group of organisms.] Until then, sulfate-reducing bacteria had been thought of as a relatively narrow and physiologically specialized collection united by their distinctive production of hydrogen sulfide from reduction of sulfate.

Reclassification of Desulfovibrio desulfuricans Norway 4 as Desulfomicrobium norvegicum comb. nov. and confirmation of Desulfomicrobium escambiense (corrig., formerly escambium) as a new species in the genus Desulfomicrobium

Desulfomicrobium escambiense, Desulfomicrobium baculatum, Desulfomicrobium apsheronum, and Desulfovibrio desulfuricans Norway 4 are closely related as determined by a 16S rRNA comparison (levels of relatedness, 0.976 to 0.997) and are distinct on the basis of levels of DNA-DNA similarity (11.1 to 27.4%), genomic restriction fragment length polymorphism patterns, and certain phenotypic characteristics. We proposed that Desulfovibrio desulfuricans Norway 4 be renamed Desulfomicrobium norvegicum comb. nov.