Beggiatoa leptomitoformis sp. nov., the first freshwater member of the genus capable of chemolithoautotrophic growth.

A strain of filamentous sulfur bacteria was isolated from freshwater spring contaminated with residential and agricultural wastewater in Moscow region, Russia. According to the results of phylogenetic analysis, strain D-402T belonged to the genus Beggiatoa within the family Beggiatoaceae of the class Gammaproteobacteria. Within the genus Beggiatoa, strain D-402T was most closely related to Beggiatoa alba strains. Strain D-402T had a DNA G+C content 42.1 mol%. The DNA-DNA hybridization value between strain D-402T and Beggiatoa alba strain B15LD was 33 %. Predominant fatty acids were C18 : 1 (46.1 and 53.3 %), C16 : 0 (15.5 and 16.2 %) and C16 : 1 (32.9 and 25.4 %) for strains D-402T and B15LD, respectively. In contrast to known representatives of Beggiatoa, strain D-402T was capable of chemolithoautotrophic growth with sulfide and thiosulfate as electron donors. Oxidation of sulfide and thiosulfate was accompanied by deposition of sulfur globules within the cells. Strain D-402T was capable of heterotrophic growth. The strain was capable of using different organic compounds, sulfur compounds and hydrogen as electron donors. Based on these observations, strain D-402T is considered as a representative of a species Beggiatoa leptomitoformis sp. nov. of the genus Beggiatoa. The type strain is D-402T (=DSM 14946T=UNIQEM U 779T).

Barite encrustation of benthic sulfur-oxidizing bacteria at a marine cold seep.

Crusts and chimneys composed of authigenic barite are found at methane seeps and hydrothermal vents that expel fluids rich in barium. Microbial processes have not previously been associated with barite precipitation in marine cold seep settings. Here, we report on the precipitation of barite on filaments of sulfide-oxidizing bacteria at a brine seep in the Gulf of Mexico. Barite-mineralized bacterial filaments in the interiors of authigenic barite crusts resemble filamentous sulfide-oxidizing bacteria of the genus Beggiatoa. Clone library and iTag amplicon sequencing of the 16S rRNA gene show that the barite crusts that host these filaments also preserve DNA of Candidatus Maribeggiatoa, as well as sulfate-reducing bacteria. Isotopic analyses show that the sulfur and oxygen isotope compositions of barite have lower δ(34)S and δ(18)O values than many other marine barite crusts, which is consistent with barite precipitation in an environment in which sulfide oxidation was occurring. Laboratory experiments employing isolates of sulfide-oxidizing bacteria from Gulf of Mexico seep sediments showed that under low sulfate conditions, such as those encountered in brine fluids, sulfate generated by sulfide-oxidizing bacteria fosters rapid barite precipitation localized on cell biomass, leading to the encrustation of bacteria in a manner reminiscent of our observations of barite-mineralized Beggiatoa in the Gulf of Mexico. The precipitation of barite directly on filaments of sulfide-oxidizing bacteria, and not on other benthic substrates, suggests that sulfide oxidation plays a role in barite formation at certain marine brine seeps where sulfide is oxidized to sulfate in contact with barium-rich fluids, either prior to, or during, the mixing of those fluids with sulfate-containing seawater in the vicinity of the sediment/water interface. As with many other geochemical interfaces that foster mineral precipitation, both biological and abiological processes likely contribute to the precipitation of barite at marine brine seeps such as the one studied here.

Two new Beggiatoa species inhabiting marine mangrove sediments in the Caribbean.

Beggiatoaceae, giant sulphur-oxidizing bacteria, are well known to occur in cold and temperate waters, as well as hydrothermal vents, where they form dense mats on the floor. However, they have never been described in tropical marine mangroves. Here, we describe two new species of benthic Beggiatoaceae colonizing a marine mangrove adjacent to mangrove roots. We combined phylogenetic and lipid analysis with electron microscopy in order to describe these organisms. Furthermore, oxygen and sulphide measurements in and ex situ were performed in a mesocosm to characterize their environment. Based on this, two new species, Candidatus Maribeggiatoa sp. and Candidatus Isobeggiatoa sp. inhabiting tropical marine mangroves in Guadeloupe were identified. The species identified as Candidatus Maribeggiatoa group suggests that this genus could harbour a third cluster with organisms ranging from 60 to 120 µm in diameter. This is also the first description of an Isobeggiatoa species outside of Arctic and temperate waters. The multiphasic approach also gives information about the environment and indications for the metabolism of these bacteria. Our study shows the widespread occurrence of members of Beggiatoaceae family and provides new insight in their potential role in shallow-water marine sulphide-rich environments such as mangroves.

Enrichment and identification of large filamentous sulfur bacteria related to Beggiatoa species from brackishwater ecosystems of Tamil Nadu along the southeast coast of India.

Beggiatoa species are filamentous sulfide-oxidizing bacteria belonging to the family Beggiatoaceae that contains several largest bacteria known today. These large sulfur bacteria occur in diverse ecosystems and play an important role in the global sulfur, nitrogen and phosphorus cycle. In this study, sediment samples from brackishwater shrimp culture ponds and other brackishwater ecosystems from Tamil Nadu, southeast coast of India, were enriched for Beggiatoa species. Extracted hay medium supplemented with catalase was used and were incubated for two weeks at 28°C. Out of seven set-ups, four yielded positive growth of filamentous sulfide-oxidizing bacteria. The filaments were several millimeters long, ranged in width between 2 and 15 µm and exhibited typical gliding motility. The 16S rRNA gene of four single filaments representing the four positive enrichments was subjected to PCR-DGGE followed by sequencing. All four filaments were affiliated to the Beggiatoaceae, but showed less than 89% identity with the Beggiatoa type strain Beggiatoa alba and less than 93% identity with any other sequence of the family. One of the four filaments revealed a nearly full-length 16S rDNA sequence (1411bp) and it formed a monophyletic cluster with two of the partial DGGE-16S rRNA gene sequences (99-100% identity) within the Beggiatoa species cluster. These organisms could possibly represent a novel genus within the family Beggiatoaceae. The fourth partial sequence affiliated with less than 93% sequence identity to the genera Parabeggiatoa, Thioploca and Thiopilula, and was likewise strongly delineated from any sequence published in the family.

Unusual polyphosphate inclusions observed in a marine Beggiatoa strain.

Sulfide-oxidizing bacteria of the genus Beggiatoa are known to accumulate phosphate intracellularly as polyphosphate but little is known about the structure and properties of these inclusions. Application of different staining techniques revealed the presence of unusually large polyphosphate inclusions in the marine Beggiatoa strain 35Flor. The inclusions showed a co-occurrence of polyphosphate, calcium and magnesium when analyzed by scanning electron microscopy and energy dispersive X-ray analysis. Similar to polyphosphate-enriched acidocalcisomes of prokaryotes and eukaryotes, the polyphosphate inclusions in Beggiatoa strain 35Flor are enclosed by a lipid layer and store cations. However, they are not notably acidic. 16S rRNA gene sequence-based phylogenetic reconstruction showed an affiliation of Beggiatoa strain 35Flor to a monophyletic branch, comprising other narrow vacuolated and non-vacuolated Beggiatoa species. The polyphosphate inclusions represent a new type of membrane surrounded storage compartment within the genus Beggiatoa, distinct from the mostly nitrate-storing vacuoles known from other marine sulfide-oxidizing bacteria of the family Beggiatoaceae.

Thermophilic anaerobic oxidation of methane by marine microbial consortia.

The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor. AOM is performed by microbial consortia of archaea (ANME) associated with partners related to sulfate-reducing bacteria. In vitro enrichments of AOM were so far only successful at temperatures ≤25 °C; however, energy gain for growth by AOM with sulfate is in principle also possible at higher temperatures. Sequences of 16S rRNA genes and core lipids characteristic for ANME as well as hints of in situ AOM activity were indeed reported for geothermally heated marine environments, yet no direct evidence for thermophilic growth of marine ANME consortia was obtained to date. To study possible thermophilic AOM, we investigated hydrothermally influenced sediment from the Guaymas Basin. In vitro incubations showed activity of sulfate-dependent methane oxidation between 5 and 70 °C with an apparent optimum between 45 and 60 °C. AOM was absent at temperatures ≥75 °C. Long-term enrichment of AOM was fastest at 50 °C, yielding a 13-fold increase of methane-dependent sulfate reduction within 250 days, equivalent to an apparent doubling time of 68 days. The enrichments were dominated by novel ANME-1 consortia, mostly associated with bacterial partners of the deltaproteobacterial HotSeep-1 cluster, a deeply branching phylogenetic group previously found in a butane-amended 60 °C-enrichment culture of Guaymas sediments. The closest relatives (Desulfurella spp.; Hippea maritima) are moderately thermophilic sulfur reducers. Results indicate that AOM and ANME archaea could be of biogeochemical relevance not only in cold to moderate but also in hot marine habitats.

Vacuolated Beggiatoa-like filaments from different hypersaline environments form a novel genus.

In this study, members of a specific group of thin (6-14 µm filament diameter), vacuolated Beggiatoa-like filaments from six different hypersaline microbial mats were morphologically and phylogenetically characterized. Therefore, enrichment cultures were established, filaments were stained with fluorochromes to show intracellular structures and 16S rRNA genes were sequenced. Morphological characteristics of Beggiatoa-like filaments, in particular the presence of intracellular vacuoles, and the distribution of nucleic acids were visualized. In the intracellular vacuole nitrate reached concentrations of up to 650 mM. Fifteen of the retrieved 16S rRNA gene sequences formed a monophyletic cluster and were phylogenetically closely related (≥ 94.4% sequence identity). Sequences of known filamentous sulfide-oxidizing genera Beggiatoa and Thioploca that comprise non-vacuolated and vacuolated filaments from diverse habitats clearly delineated from this cluster. The novel monophyletic cluster was furthermore divided into two sub-clusters: one contained sequences originating from Guerrero Negro (Mexico) microbial mats and the other comprised sequences from five distinct Spanish hypersaline microbial mats from Ibiza, Formentera and Lake Chiprana. Our data suggest that Beggiatoa-like filaments from hypersaline environments displaying a thin filament diameter contain nitrate-storing vacuoles and are phylogenetically separate from known Beggiatoa. Therefore, we propose a novel genus for these organisms, which we suggest to name 'Candidatus Allobeggiatoa'.

Filamentous sulfur bacteria, Beggiatoa spp., in arctic marine sediments (Svalbard, 79 degrees N).

Fjord sediments on the west coast of the arctic archipelago Svalbard were surveyed to understand whether large filamentous sulfur bacteria of the genus Beggiatoa thrive at seawater temperatures permanently near freezing. Two sediments had abundant populations of Beggiatoa, while at six sites, only sporadic occurrences were observed. We conclude that Beggiatoa, although previously unnoticed, are widespread in these arctic fjord sediments. Beggiatoa ranged in diameter from 2 to 52 microm and, by those tested, stored nitrate in vacuoles at up to 260 mM. The 16S rRNA gene sequence of a 20-microm-wide filament is closely associated with other large, marine, nitrate-storing Beggiatoa. The Beggiatoa mostly occurred in the upper 2-5 cm of oxidized surface sediment between oxygen and the deeper sulfidic zone. In spite of a very low or an undetectable sulfide concentration, sulfate reduction provided abundant H(2)S in this zone. The total living biomass of Beggiatoa filaments at one study site varied over 3 years between 1.13 and 3.36 g m(-2). Because of their large size, Beggiatoa accounted for up to 15% of the total prokaryotic biomass, even though the filament counts at this site were rather low, comprising <1/10,000 of the bacterial numbers on a cell basis.

Comparative analysis of Beggiatoa from hypersaline and marine environments.

The main criterion to classify a microorganism as belonging to the genus Beggiatoa is its morphology. All multicellular, colorless, gliding bacterial filaments containing sulfur globules described so far belong to this genus. At the ultrastructural level, they show also a very complex cell envelope structure. Here we describe uncultured vacuolated and non-vacuolated bacteria from two different environments showing all characteristics necessary to assign a bacterium to the genus Beggiatoa. We also intended to investigate whether narrow and vacuolate Beggiatoa do differ morphologically as much as they do phylogenetically. Both large, vacuolated trichomes and narrow filaments devoid of vacuoles were observed. We confirmed the identity of the narrow filaments by 16S rRNA phylogenetic analysis. The diameters of the trichomes ranged from 2.4 to 34 microm, and their lengths ranged from 10 microm to over 30 mm. Narrow trichomes moved by gliding at 3.0 microm/s; large filaments moved at 1.5 microm/s. Periplasmic sulfur inclusions were observed in both types of filaments, whereas phosphorus-rich bodies were found only in narrow trichomes. On the other hand, nitrate vacuoles were observed only in large trichomes. Ultra-thin section transmission electron microscopy showed differences between the cell ultrastructure of narrow (non-vacuolated) and large (vacuolated) Beggiatoa. We observed that cell envelopes from narrow Beggiatoa consist of five layers, whereas cell envelopes from large trichomes contain four layers.

Diversity and abundance of aerobic and anaerobic methane oxidizers at the Haakon Mosby Mud Volcano, Barents Sea.

Submarine mud volcanoes are formed by expulsions of mud, fluids, and gases from deeply buried subsurface sources. They are highly reduced benthic habitats and often associated with intensive methane seepage. In this study, the microbial diversity and community structure in methane-rich sediments of the Haakon Mosby Mud Volcano (HMMV) were investigated by comparative sequence analysis of 16S rRNA genes and fluorescence in situ hybridization. In the active volcano center, which has a diameter of about 500 m, the main methane-consuming process was bacterial aerobic oxidation. In this zone, aerobic methanotrophs belonging to three bacterial clades closely affiliated with Methylobacter and Methylophaga species accounted for 56%+/-8% of total cells. In sediments below Beggiatoa mats encircling the center of the HMMV, methanotrophic archaea of the ANME-3 clade dominated the zone of anaerobic methane oxidation. ANME-3 archaea form cell aggregates mostly associated with sulfate-reducing bacteria of the Desulfobulbus (DBB) branch. These ANME-3/DBB aggregates were highly abundant and accounted for up to 94%+/-2% of total microbial biomass at 2 to 3 cm below the surface. ANME-3/DBB aggregates could be further enriched by flow cytometry to identify their phylogenetic relationships. At the outer rim of the mud volcano, the seafloor was colonized by tubeworms (Siboglinidae, formerly known as Pogonophora). Here, both aerobic and anaerobic methane oxidizers were found, however, in lower abundances. The level of microbial diversity at this site was higher than that at the central and Beggiatoa species-covered part of the HMMV. Analysis of methyl-coenzyme M-reductase alpha subunit (mcrA) genes showed a strong dominance of a novel lineage, mcrA group f, which could be assigned to ANME-3 archaea. Our results further support the hypothesis of Niemann et al. (54), that high methane availability and different fluid flow regimens at the HMMV provide distinct niches for aerobic and anaerobic methanotrophs.