Burkholderia insulsa sp. nov., a facultatively chemolithotrophic bacterium isolated from an arsenic-rich shallow marine hydrothermal system.

Enrichment cultures inoculated with hydrothermally influenced nearshore sediment from Papua New Guinea led to the isolation of an arsenic-tolerant, acidophilic, facultatively aerobic bacterial strain designated PNG-April(T). Cells of this strain were Gram-stain-negative, rod-shaped, motile and did not form spores. Strain PNG-April(T) grew at temperatures between 4 °C and 40 °C (optimum 30-37 °C), at pH 3.5 to 8.3 (optimum pH 5-6) and in the presence of up to 2.7% NaCl (optimum 0-1.0%). Both arsenate and arsenite were tolerated up to concentrations of at least 0.5 mM. Metabolism in strain PNG-April(T) was strictly respiratory. Heterotrophic growth occurred with O2 or nitrate as electron acceptors, and aerobic lithoautotrophic growth was observed with thiosulfate or nitrite as electron donors. The novel isolate was capable of N2-fixation. The respiratory quinones were Q-8 and Q-7. Phylogenetically, strain PNG-April(T) belongs to the genus Burkholderia and shares the highest 16S rRNA gene sequence similarity with the type strains of Burkholderia fungorum (99.8%), Burkholderia phytofirmans (98.8%), Burkholderia caledonica (98.4%) and Burkholderia sediminicola (98.4%). Differences from these related species in several physiological characteristics (lipid composition, carbohydrate utilization, enzyme profiles) and DNA-DNA hybridization suggested the isolate represents a novel species of the genus Burkholderia, for which we propose the name Burkholderia insulsa sp. nov. The type strain is PNG-April(T) ( = DSM 28142(T) = LMG 28183(T)).

Molecular tools for the detection of nitrogen cycling Archaea.

Archaea are widespread in extreme and temperate environments, and cultured representatives cover a broad spectrum of metabolic capacities, which sets them up for potentially major roles in the biogeochemistry of their ecosystems. The detection, characterization, and quantification of archaeal functions in mixed communities require Archaea-specific primers or probes for the corresponding metabolic genes. Five pairs of degenerate primers were designed to target archaeal genes encoding key enzymes of nitrogen cycling: nitrite reductases NirA and NirB, nitrous oxide reductase (NosZ), nitrogenase reductase (NifH), and nitrate reductases NapA/NarG. Sensitivity towards their archaeal target gene, phylogenetic specificity, and gene specificity were evaluated in silico and in vitro. Owing to their moderate sensitivity/coverage, the novel nirB-targeted primers are suitable for pure culture studies only. The nirA-targeted primers showed sufficient sensitivity and phylogenetic specificity, but poor gene specificity. The primers designed for amplification of archaeal nosZ performed well in all 3 criteria; their discrimination against bacterial homologs appears to be weakened when Archaea are strongly outnumbered by bacteria in a mixed community. The novel nifH-targeted primers showed high sensitivity and gene specificity, but failed to discriminate against bacterial homologs. Despite limitations, 4 of the new primer pairs are suitable tools in several molecular methods applied in archaeal ecology.

Ribosomal tag pyrosequencing of DNA and RNA from benthic coral reef microbiota: community spatial structure, rare members and nitrogen-cycling guilds.

Ribosomal tag libraries based on DNA and RNA in coral reef sediment from Hawaii show the microbial community to be dominated by the bacterial phyla Proteobacteria, Firmicutes and Actinobacteria, the archaeal order Nitrosopumilales and the uncultivated divisions Marine Group III (Euryarchaeota) and Marine Benthic Group C (Crenarchaeota). Operational taxonomic units (OTUs) number in the high thousands, and richness varies with site, presence or absence of porewater sulfide (sediment depth), and nucleotide pool. Rank abundance curves of DNA-based libraries, but not RNA-based libraries, possess a tail of low abundance taxa, supporting the existence of an inactive 'rare' biosphere. While bacterial libraries from two oxic samples differ markedly, those from two anoxic (sulfidic) samples are similar. The four dominant bacterial OTUs are members of genera that include pathogens, but are found in marine environments, and include facultative anaerobes, i.e. dissimilatory nitrate reducers and denitrifiers. This may explain their abundance in both oxic and anoxic samples. A numerous archaeon is closely related to the lithoautotrophic ammonia oxidizer Nitrosopumilus maritimus. Known bacterial ammonia oxidizers are essentially absent, but bacterial nitrite oxidizers are abundant. Although other studies of this reef found evidence for anaerobic ammonia oxidizers, primer bias rendered that clade invisible to this study.

An oligarchic microbial assemblage in the anoxic bottom waters of a volcanic subglacial lake.

In 2006, we sampled the anoxic bottom waters of a volcanic lake beneath the Vatnajökull ice cap (Iceland). The sample contained 5 x 10(5) cells per ml, and whole-cell fluorescent in situ hybridization (FISH) and PCR with domain-specific probes showed these to be essentially all bacteria, with no detectable archaea. Pyrosequencing of the V6 hypervariable region of the 16S ribosomal RNA gene, Sanger sequencing of a clone library and FISH-based enumeration of four major phylotypes revealed that the assemblage was dominated by a few groups of putative chemotrophic bacteria whose closest cultivated relatives use sulfide, sulfur or hydrogen as electron donors, and oxygen, sulfate or CO(2) as electron acceptors. Hundreds of other phylotypes are present at lower abundance in our V6 tag libraries and a rarefaction analysis indicates that sampling did not reach saturation, but FISH data limit the remaining biome to <10-20% of all cells. The composition of this oligarchy can be understood in the context of the chemical disequilibrium created by the mixing of sulfidic lake water and oxygenated glacial meltwater.

Functional characterization of the microbial community in geothermally heated marine sediments.

The microbial population of geothermally heated sediments in a shallow bay of Vulcano Island (Italy) was characterized with respect to metabolic activities and the putatively catalyzing hyperthermophiles. Site-specific anoxic culturing media, most of which were amended with combinations of electron donors (glucose or carboxylic acids) and acceptors (sulfate), were used for selective enrichment of metabolically defined subpopulations. The mostly archaeal chemoautotrophs produced formate at rates of 3.25 and 0.46 fmol cell(-1) day(-1) with and without sulfate, respectively. The glucose fermenting heterotrophs produced acetate (18 fmol cell(-1) day(-1)) and lactate (2.6 fmol cell(-1) day(-1)) and were identified as predominantly Thermus sp. and coccoid archaea. These archaeal cells also metabolized lactate (5.6 fmol cell(-1) day(-1)), but neither formate nor acetate. The heterotrophic culture enriched on formate/ acetate/propionate/sulfate utilized mainly formate (27 fmol cell(-1) day(-1)) and lactate (89-195 fmol cell(-1) day(-1)), and consumed sulfate (38-68 fmol cell(-1) day(-1)). These formate or lactate consuming sulfate reducers were dominated by Archaeoglobales (7% in situ) and unidentified Archaea. The in situ benthic community comprised 15% Crenarchaeota, a significant group only in the autotrophic cultures, and 3% Thermus sp., the putatively predominant group involved in fermentative metabolism. The role of Thermoccales (4% in situ) remained undisclosed in our experiments. This first comprehensive data set established plausible links between several groups of hyperthermophiles in shallow marine hydrothermal systems, their metabolic function within the benthic microbial community, and biogeochemical turnover rates.

Order-specific 16S rRNA-targeted oligonucleotide probes for (hyper)thermophilic archaea and bacteria.

New oligonucleotide probes were designed and evaluated for application in fluorescence in situ hybridization (FISH) studies on (hyper)thermophilic microbial communities--Arglo32, Tcoc164, and Aqui1197 target the 16S rRNA of Archaeoglobales, Thermococcales, and Aquificales, respectively. Both sequence information and experimental evaluation showed high coverage and specificity of all three probes. The signal intensity of Aqui1197 was improved by addition of a newly designed, unlabeled "helper" oligonucleotide, hAqui1045. It was shown that in addition to its function as a probe for Aquificales, Aqui1197 is suitable as a supplementary probe to extend the coverage of the domain-specific bacterial probe EUB338. In sediments from two hydrothermal seeps on Vulcano Island, Italy, the microbial community structure was analyzed by FISH with both established and the new oligonucleotide probes, showing the applicability of Arglo32, Tcoc164, and Aqui1197/hAqui1045 to natural samples. At both sites, all major groups of (hyper)thermophiles, except for methanogens, were detected: Crenarchaeota (19%, 16%), Thermococcales (14%, 22%), Archaeoglobales (14%, 12%), Aquificales (5%, 8%), Thermotoga/Thermosipho spp. (12%, 9%), Thermus sp. (12%, none), and thermophilic Bacillus sp. (12%, 8%).

Activity and distribution of bacterial populations in Middle Atlantic Bight shelf sands.

Abstract Spatiotemporal variation and metabolic activity of the microbial community were studied in coarse-grained Middle Atlantic Bight shelf sediments in relation to pools of dissolved and particulate carbon. Algal cells were present 8->70 mum) fraction of the sediment held the major share (61-98%) of benthic bacteria. Bacterial and algal cell abundances, exoenzymatic activity, and [DOC] generally showed higher values in May/July 2001 than in August/December 2000. Carbohydrates and proteins were hydrolyzed at potential rates of 1-12 nmol cm(-3) h(-1) (beta-glucosidase) and 3-70 nmol cm(-3) h(-1) (aminopeptidase), respectively. Fluorescence in situ hybridization analyses of the benthic microbes assigned 45-56% of DAPI-stained cells to Eubacteria and less than 2% to Eukarya. The prokaryotic community was dominated by planctomycetes and members of the Cytophaga/Flavobacterium cluster. Near the sediment surface, iodonitrotetrazolium violet reducing cells, that are considered actively respiring, amounted to 15-29% of total bacteria. Despite a low organic content (particulate organic carbon <0.03%) and relatively low bacterial abundances (<10(9) cm(-3)), the Middle Atlantic Bight shelf sediments showed organic matter turnover rates that are comparable to those found in organic-rich finer-grained deposits. Our findings suggest a high biocatalytic filtration activity in these coarse permeable sediments.