Carl O. Wirsen

Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments.

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein. DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the other sample, which probably correspond to the dominant bacterial populations in these communities. Three of the four 16S rDNA fragments were sequenced. By comparison with 16S rRNA sequences of the Ribosomal Database Project, two of the DGGE-separated fragments were assigned to the genusThiomicrospira. To identify these ‘phylotypes’ in more detail, a phylogenetic framework was created by determining the nearly complete 16S rRNA gene sequence (approx. 1500 nucleotides) from three describedThiomicrospira species, viz.,Tms. crunogena, Tms. pelophila, Tms. denitrificans, and from a new isolate,Thiomicrospira sp. strain MA2-6. AllThiomicrospira species exceptTms. denitrificans formed a monophyletic group within the gamma subdivision of the Proteobacteria.Tms. denitrificans was assigned as a member of the epsilon subdivision and was distantly affiliated withThiovulum, another sulfur-oxidizing bacterium. Sequences of two dominant 16S rDNA fragments obtained by DGGE analysis fell into the gamma subdivisionThiomicrospira. The sequence of one fragment was in all comparable positions identical to the 16S rRNA sequence ofTms. crunogena. Identifying a dominant molecular isolate asTms. crunogena indicates that this species is a dominant community member of hydrothermal vent sites. Another ‘phylotype’ represented a newThiomicrospira species, phylogenetically in an intermediate position betweenTms. crunogena andTms. pelophila. The third ‘phylotype’ was identified as aDesulfovibrio, indicating that sulfate-reducing bacteria, as sources of sulfide, may complement sulfur- and sulfide-oxidizing bacteria ecologically in these sulfide-producing hydrothermal vents.

Isolation and Characterization of Novel Psychrophilic, Neutrophilic, Fe-Oxidizing, Chemolithoautotrophic α- and γ-Proteobacteria from the Deep Sea

ABSTRACT We report the isolation and physiological characterization of novel, psychrophilic, iron-oxidizing bacteria (FeOB) from low-temperature weathering habitats in the vicinity of the Juan de Fuca deep-sea hydrothermal area. The FeOB were cultured from the surfaces of weathered rock and metalliferous sediments. They are capable of growth on a variety of natural and synthetic solid rock and mineral substrates, such as pyrite (FeS2), basalt glass (∼10 wt% FeO), and siderite (FeCO3), as their sole energy source, as well as numerous aqueous Fe substrates. Growth temperature characteristics correspond to the in situ environmental conditions of sample origin; the FeOB grow optimally at 3 to 10°C and at generation times ranging from 57 to 74 h. They are obligate chemolithoautotrophs and grow optimally under microaerobic conditions in the presence of an oxygen gradient or anaerobically in the presence of nitrate. None of the strains are capable of using any organic or alternate inorganic substrates tested. The bacteria are phylogenetically diverse and have no close Fe-oxidizing or autotrophic relatives represented in pure culture. One group of isolates are γ-Proteobacteria most closely related to the heterotrophic bacterium Marinobacter aquaeolei (87 to 94% sequence similarity). A second group of isolates are α-Proteobacteria most closely related to the deep-sea heterotrophic bacterium Hyphomonas jannaschiana (81 to 89% sequence similarity). This study provides further evidence for the evolutionarily widespread capacity for Fe oxidation among bacteria and suggests that FeOB may play an unrecognized geomicrobiological role in rock weathering in the deep sea.

Characterization of an Autotrophic Sulfide-Oxidizing Marine Arcobacter sp. That Produces Filamentous Sulfur

ABSTRACT A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4′,6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is “Candidatus Arcobacter sulfidicus.” For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO2 fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO2 fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by “Candidatus Arcobacter sulfidicus” or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.

Deep-Sea Primary Production at the Galapagos Hydrothermal Vents

Dense animal populations surrounding recently discovered hydrothermal vents at the Galapagos Rift sea-floor spreading center, 2550 meters deep, are probably sustained by microbial primary production. Energy in the form of geothermically reduced sulfur compounds emitted from the vents is liberated during oxidation and used for the reduction of carbon dioxide to organic matter by chemosynthetic bacteria.

Stable isotope studies of the carbon, nitrogen and sulfur cycles in the Black Sea and the Cariaco Trench

Abstract Samples for stable isotope studies of possible chemosynthesis in anoxic basins were collected in 1986 in the Cariaco Trench and May 1988 in the Black Sea. POM (particulate organic matter) collected at oxic/anoxic interfaces in the water column showed no distinctive carbon or nitrogen isotopic compositions that could be associated with chemosynthetic bacteria. Carbon and nitrogen isotopic compositions at POM concentration maxima near the top of the sulfide zone were −23%o and 4.5%o, respectively, in both the Black Sea and the Cariaco Trench. Measurements of dissolved inorganic carbon (DIC) in the Black Sea indicated that carbon respired during decomposition at depth had an isotopic composition of −23%o and was isotopically similar to phytoplankton, with no distinctive component that could be attributed to chemosynthetic carbon. These results indicate that either the biomass of chemosynthetic bacteria in the oxic/anoxic interface zones is low relative to sinking phytoplankton or that chemoautotrophic bacteria have isotopic compositions similar to those of phytoplankton. In the uppermost 50 m of sulfidic waters in the Black Sea, sulfide isotopic compositions changed significantly in a region of sulfide consumption, increasing up to 5%o vs deep-water background values of −40.5%o. These increases in sulfide isotopic compositions may be due to sulfide oxidation mediated by MnO 2 or oxygen, but are not consistent with sulfide oxidation by photosynthetic bacteria. Growth experiments with sulfate-reducing bacteria suggested that part of the increase in sulfide isotopic compositions could be due to rapid rates of sulfate reduction in the oxic/anoxic interface regions.

Evidence for Autotrophic CO2 Fixation via the Reductive Tricarboxylic Acid Cycle by Members of the ε Subdivision of Proteobacteria

Based on 16S rRNA gene surveys, bacteria of the epsilon subdivision of proteobacteria have been identified to be important members of microbial communities in a variety of environments, and quite a few have been demonstrated to grow autotrophically. However, no information exists on what pathway of autotrophic carbon fixation these bacteria might use. In this study, Thiomicrospira denitrificans and Candidatus Arcobacter sulfidicus, two chemolithoautotrophic sulfur oxidizers of the epsilon subdivision of proteobacteria, were examined for activities of the key enzymes of the known autotrophic CO(2) fixation pathways. Both organisms contained activities of the key enzymes of the reductive tricarboxylic acid cycle, ATP citrate lyase, 2-oxoglutarate:ferredoxin oxidoreductase, and pyruvate:ferredoxin oxidoreductase. Furthermore, no activities of key enzymes of other CO(2) fixation pathways, such as the Calvin cycle, the reductive acetyl coenzyme A pathway, and the 3-hydroxypropionate cycle, could be detected. In addition to the key enzymes, the activities of the other enzymes involved in the reductive tricarboxylic acid cycle could be measured. Sections of the genes encoding the alpha- and beta-subunits of ATP citrate lyase could be amplified from both organisms. These findings represent the first direct evidence for the operation of the reductive tricarboxylic acid cycle for autotrophic CO(2) fixation in epsilon-proteobacteria. Since epsilon-proteobacteria closely related to these two organisms are important in many habitats, such as hydrothermal vents, oxic-sulfidic interfaces, or oilfields, these results suggest that autotrophic CO(2) fixation via the reductive tricarboxylic acid cycle might be more important than previously considered.

Microbial Degradation of Organic Matter in the Deep Sea

Food materials from the sunken and recovered research submarine Alvin were found to be in a strikingly well-preserved state after exposure for more than 10 months to deep-sea conditions. Subsequent experiments substantiated this observation and indicated that rates of microbial degradation were 10 to 100 times slower in the deep sea than in controls under comparable temperatures.

Chemosynthetic microbial activity at Mid‐Atlantic Ridge hydrothermal vent sites

Chemosynthetic production of microbial biomass, determined by 14CO2 fixation and enzymatic (RuBisCo) activity, at the Mid-Atlantic Ridge (MAR) 23° and 26°N vent sites was found in various niches: warm water emissions, loosely rock-attached flocculent material, dense morphologically diverse bacterial mats covering the surfaces of polymetal sulfide deposits, and filamentous microbes on the carapaces of shrimp (Rimicaris exoculata). The bacterial mats on polymetal sulfide surfaces contained unicellular and filamentous bacteria which appeared to use as their chemolithotrophic electron or energy source either dissolved reduced minerals from vent emissions, mainly sulfur compounds, or solid metal sulfide deposits, mainly pyrite. Moderately thermophilic Chemosynthetic activity was observed in carbon dioxide fixation experiments and in enrichments, but no thermophilic aerobic sulfur oxidizers could be isolated. Both obligate and facultative chemoautotrophs growing at mesophilic temperatures were isolated from all chemosynthetically active surface scrapings. The obligate autotrophs could oxidize sterilized MAR natural sulfide deposits as well as technical pyrite at near neutral pH, in addition to dissolved reduced sulfur compounds. While the grazing by shrimp on the surface mats of MAR metal sulfide deposits was observed and deemed important, the animals’ primary occurrence in dense swarms near vent emissions suggests that they were feeding at these sites, where conditions for Chemosynthetic growth of their filamentous microbial epiflora were optimal. The data show that the transformation of geothermal energy at the massive polymetal sulfide deposits of the MAR is based on the lithoautotrophic oxidation of soluble sulfides and pyrites into microbial biomass.

Thiomicrospira crunogena sp. nov., a Colorless, Sulfur-Oxidizing Bacterium from a Deep-sea Hydrothermal Vent?

A new species of the genus Thiomicrospira was isolated from the 21°N deep-sea (2,600-m) hydrothermal vent area of the East Pacific Rise. This organism is an obligate chemolithoautotrophic sulfur oxidizer and differs from the two other species of this genus by its deoxyribonucleic acid base composition and by its growth rate and optimal pH in thiosulfate medium. The new species is named Thiomicrospira crunogena and has been deposited in the American Type Culture Collection as strain ATCC 35932, as well as in the Delft Culture Collection as strain LMD 84.00.

Deep-Sea Microorganisms: In situ Response to Nutrient Enrichment

After inoculation of sterile organic materials on the deep-sea floor and in situ incubation for 1 year, relatively minute rates of microbial transformation were recorded. This extremely slow conversion rate, as well as the type and quantity of organic matter normally reaching the ocean floor, appear to characterize microbial life in the deep sea.