Nitrogen fixation and nitrate utilization by marine and freshwater Beggiatoa

Four newly isolated marine strains of Beggiatoa and five freshwater strains were tested for nitrogen fixation in slush agar medium. All strains reduced acetylene when grown microaerobically in media containing a reduced sulfur source and lacking added combined nitrogen. The addition of 2 mmol N, as nitrate or ammonium salts, completely inhibited this reduction. Although not optimized for temperature or cell density, acetylene reduction rates ranged from 3.2 to 12 nmol·mg prot^-1 min^-1. Two freshwater strains did not grow well or reduce acetylene in medium lacking combined nitrogen if sulfide was replaced by thiosulfate. Two other strains grew well in liquid media lacking both combined nitrogen and reduced sulfur compounds but only under lowered concentrations of air. All freshwater strains grew well in medium containing nitrate as the combined nitrogen source. Since they did not reduce acetylene under these conditions, we infer that they can assimilate nitrate.     

Anaerobic Sulfide Oxidation with Nitrate by a Freshwater Beggiatoa Enrichment Culture

A lithotrophic freshwater Beggiatoa strain was enriched in O₂-H₂S gradient tubes to investigate its ability to oxidize sulfide with NO₃⁻ as an alternative electron acceptor. The gradient tubes contained different NO₃⁻ concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O₂, H₂S, pH, and NO₃⁻ were determined with microsensors. The more NO₃⁻ that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO₃⁻ to oxidize sulfide at depths below the depth that O₂ penetrated. In the presence of NO₃⁻ Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO₃⁻ was added. These thick mats spatially separated O₂ and sulfide but not NO₃⁻ and sulfide, and therefore NO₃⁻ must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O₂ flux and a twofold-higher sulfide flux into the NO₃⁻-exposed mats compared to the fluxes for controls without NO₃⁻. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S⁰ with NO₃⁻ as the electron acceptor.     

Oxygen Responses and Mat Formation by Beggiatoa spp

The behavioral response of single Beggiatoa sp. filaments moving on a gas-permeable membrane was studied by the combined use of microscopy and oxygen microelectrodes during controlled oscillations of oxygen tension. The bacteria reacted to increasing oxygen by reversing the direction of movement. The same step-up phobic response to oxygen was observed when a filament tip or loop glided into a stable microgradient of increasing oxygen. The response was sensitive to a change in oxygen tension of <5% of air saturation min⁻¹. The response time was 20 to 50 s. Frequently, only part of the filament responded, which led to the formation of sharp bends, loops, and coils. This partial response facilitated the positioning of the long filaments within the narrow O₂-H₂S interface. The structure of whole Beggiatoa mats on sediment surfaces varied from loose to dense in relation to shallow or steep oxygen gradients in the 0.3- to 2-mm-thick, unstirred boundary layer. In an illuminated sediment Beggiatoa spp. lived together with photosynthetic organisms and migrated vertically in accordance with light/dark variations. The combined effect of phobic responses to light and oxygen can explain this migration.     

Improved Enrichment and Isolation Procedures for Obtaining Pure Cultures of Beggiatoa

Scoring agar surfaces with alginate swabs before placing washed filaments of Beggiatoa on the agar has greatly increased the rate at which single filaments move from contaminated areas. Numerous morphological types of pure cultures have been grown in organic media supplemented with either catalase or reducing agents. Aerated sewage was used as the enrichment source.     

Enumeration, Isolation, and Characterization of Beggiatoa from Freshwater Sediments

An accurate most-probable-number enumeration method was developed for counting the number of Beggiatoa trichomes from various freshwater sediments. The medium consisted of extracted hay, diluted soil extract, 0.05% acetate, and 15 to 35 U of catalase per ml. The same enrichment medium, but without the acetate, was the best enrichment medium from which to obtain pure cultures because it supported good growth of the beggiatoas without allowing them to be overgrown by other bacteria. A total of 32 strains of Beggiatoa were isolated from seven different freshwater habitats and partially characterized. The strains were separated into five groups based on several preliminary characteristics. Four of the groups contained cells with trichomes of approximately the same diameter (1.5 to 2.7 μm) and may be Beggiatoa leptomitiformis or an unnamed species. The fifth group appeared to be Beggiatoa alba. With the exception of three strains, all of the strains deposited sulfur in the presence of hydrogen sulfide, and all strains grew heterotrophically and deposited poly-β-hydroxybutyrate and volutin when grown on acetate supplemented with low concentrations of other organic nutrients. Thin sections of sulfur-bearing trichomes indicated that the sulfur granules were external to the cytoplasmic membrane and that they were surrounded by an additional membrane.     

Physiological Adaptation of a Nitrate-Storing Beggiatoa sp. to Diel Cycling in a Phototrophic Hypersaline Mat

The aim of this study was to investigate the supposed vertical diel migration and the accompanying physiology of Beggiatoa bacteria from hypersaline microbial mats. We combined microsensor, stable-isotope, and molecular techniques to clarify the phylogeny and physiology of the most dominant species inhabiting mats of the natural hypersaline Lake Chiprana, Spain. The most dominant morphotype had a filament diameter of 6 to 8 μm and a length varying from 1 to >10 mm. Phylogenetic analysis by 16S rRNA gene comparison revealed that this type appeared to be most closely related (91% sequence identity) to the narrow (4-μm diameter) nonvacuolated marine strain MS-81-6. Stable-isotope analysis showed that the Lake Chiprana species could store nitrate intracellularly to 40 mM. The presence of large intracellular vacuoles was confirmed by fluorescein isothiocyanate staining and subsequent confocal microscopy. In illuminated mats, their highest abundance was found at a depth of 8 mm, where oxygen and sulfide co-occurred. However, in the dark, the highest Beggiatoa densities occurred at 7 mm, and the whole population was present in the anoxic zone of the mat. Our findings suggest that hypersaline Beggiatoa bacteria oxidize sulfide with oxygen under light conditions and with internally stored nitrate under dark conditions. It was concluded that nitrate storage by Beggiatoa is an optimal strategy to both occupy the suboxic zones in sulfidic sediments and survive the dark periods in phototrophic mats.     

Growth Pattern and Yield of a Chemoautotrophic Beggiatoa sp. in Oxygen-Sulfide Microgradients

Recently developed techniques involving opposed, gel-stabilized gradients of O₂ and H₂S permit cultivation of a marine Beggiatoa strain as a chemolithoautotroph which uses gliding motility to precisely track the interface between H₂S and O₂. In the current study with microelectrodes, vertical profiles of H₂, O₂, and pH were measured in replicate cultures grown for various intervals. After an initial period of exponential biomass increase (doubling time, 11 h), linear growth prevailed throughout much of the time course. This H₂S-limited growth was followed by a transition to stationary phase when the declining H₂S flux was sufficient only to supply maintenance energy. During late-exponential and linear growth phases, the Beggiatoa sp. consumed a constant 0.6 mol of H₂S for each 1.0 mol of O₂, the ratio anticipated for balanced lithoautotrophic growth at the expense of complete oxidation of H₂S to SO₄²⁻. Over the entire range of conditions studied, this consumption ratio varied by approximately twofold. By measuring the extent to which the presence of the bacterial plate diminished the overlap of O₂ and H₂S, we demonstrated that oxidation of H₂S by Beggiatoa sp. is approximately 3 orders of magnitude faster than spontaneous chemical oxidation. By integrating sulfide profiles and comparing sulfide consumed with biomass produced, a growth yield of 8.4 g (dry weight) mol⁻¹ of H₂S was computed. This is higher than that found for sulfide-grown thiobacilli, indicating very efficient growth of Beggiatoa sp. as a chemoautotroph. The methods used here offer a unique opportunity to determine the yield of H₂S-oxidizing chemolithoautotrophs while avoiding several problems inherent in the use of homogeneous liquid culture. Finally, by monitoring time-dependent formation of H₂S profiles under anoxic conditions, we demonstrate a method for calculating the molecular diffusion coefficient of soluble substrates in gel-stabilized media.     

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 δ³⁴S and δ¹⁸O 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.     

Colorless Sulfur Bacteria, Beggiatoa spp. and Thiovulum spp., in O2 and H2S Microgradients

The interactions between colorless sulfur bacteria and the chemical microgradients at the oxygen-sulfide interface were studied in Beggiatoa mats from marine sediments and in Thiovulum veils developing above the sediments. The gradients of O₂, H₂S, and pH were measured by microelectrodes at depth increments of 50 μm. An unstirred boundary layer in the water surrounding the mats and veils prevented microturbulent or convective mixing of O₂ and H₂S. The two substrates reached the bacteria only by molecular diffusion through the boundary layer. The bacteria lived as microaerophiles or anaerobes even under stirred, oxic water. Oxygen and sulfide zones overlapped by 50 μm in the bacterial layers. Both compounds had concentrations in the range of 0 to 10 μmol liter⁻¹ and residence times of 0.1 to 0.6 s in the overlapping zone. The sulfide oxidation was purely biological. Diffusion calculations showed that formation of mats on solid substrates or of veils in the water represented optimal strategies for the bacteria to achieve a stable microenvironment, a high substrate supply, and an efficient competition with chemical sulfide oxidation. The continuous gliding movement of Beggiatoa cells in mats or the flickering motion of Thiovulum cells in veils were important for the availability of both O₂ and H₂S for the individual bacteria.     

Polysulfides as Intermediates in the Oxidation of Sulfide to Sulfate by Beggiatoa spp.

Zero-valent sulfur is a key intermediate in the microbial oxidation of sulfide to sulfate. Many sulfide-oxidizing bacteria produce and store large amounts of sulfur intra- or extracellularly. It is still not understood how the stored sulfur is metabolized, as the most stable form of S⁰ under standard biological conditions, orthorhombic α-sulfur, is most likely inaccessible to bacterial enzymes. Here we analyzed the speciation of sulfur in single cells of living sulfide-oxidizing bacteria via Raman spectroscopy. Our results showed that under various ecological and physiological conditions, all three investigated Beggiatoa strains stored sulfur as a combination of cyclooctasulfur (S₈) and inorganic polysulfides (S_n²⁻). Linear sulfur chains were detected during both the oxidation and reduction of stored sulfur, suggesting that S_n²⁻ species represent a universal pool of bioavailable sulfur. Formation of polysulfides due to the cleavage of sulfur rings could occur biologically by thiol-containing enzymes or chemically by the strong nucleophile HS⁻ as Beggiatoa migrates vertically between oxic and sulfidic zones in the environment. Most Beggiatoa spp. thus far studied can oxidize sulfur further to sulfate. Our results suggest that the ratio of produced sulfur and sulfate varies depending on the sulfide flux. Almost all of the sulfide was oxidized directly to sulfate under low-sulfide-flux conditions, whereas only 50% was oxidized to sulfate under high-sulfide-flux conditions leading to S⁰ deposition. With Raman spectroscopy we could show that sulfate accumulated in Beggiatoa filaments, reaching intracellular concentrations of 0.72 to 1.73 M.     

Influence of oxygen on sulfate reduction and growth of sulfate-reducing bacteria

The ambivalent relations of sulfate-reducing bacteria to molecular O₂ have been studied with ten freshwater and marine strains. Generally, O₂ was reduced prior to sulfur compounds and suppressed the reduction of sulfate, sulfite or thiosulfate to sulfide. Three strains slowly formed sulfide at O₂ concentrations of below 15 M (6% air saturation). In homogeneously aerated cultures, two out of seven strains tested, Desulfovibrio desulfuricans and Desulfobacterium autotrophicum, revealed weak growth with O₂ as electron acceptor (up to one doubling of protein). However, O₂ was concomitantly toxic. Depending on its concentration cell viability and motility decreased with time. In artificial oxygen-sulfide gradients with sulfide-containing agar medium and also in sulfide-free agar medium under an oxygen-containing gas phase, sulfate reducers grew in bands close to the oxic/anoxic interface. The specific O₂ tolerance and respiration capacity of different strains led to characteristically stratified gradients. The maximum O₂ concentration at the surface of a bacterial band (determined by means of microelectrodes) was 9 M. The specific rates of O₂ uptake per cell were in the same order of magnitude as the sulfate reduction rates in pure cultures. The bacteria stabilized the gradients, which were rapidly oxidized in the absence of cells or after killing the cells by formaldehyde. The motile strain Desulfovibrio desulfuricans CSN slowly migrated in the gradients in response to changing O₂ concentrations in the gas phase.     

Use of Reduced Sulfur Compounds by Beggiatoa spp.: Enzymology and Physiology of Marine and Freshwater Strains in Homogeneous and Gradient Cultures

The marine Beggiatoa strains MS-81-6 and MS-81-1c are filamentous, gliding, colorless sulfur bacteria. They have traditionally been cultured in very limited quantities in sulfide gradient media, where they grow as chemolithoautotrophs, forming a thin horizontal plate well below the air-agar interface. There, the facultatively chemolithoautotrophic strain MS-81-6 quantitatively harvests the flux of sulfide diffusing from below and oxidizes it to sulfate by using oxygen as the electron acceptor. Only recently have these strains been cultivated in bulk in defined liquid media (K. D. Hagen and D. C. Nelson, Appl. Environ. Microbiol. 62:947-953, 1996). In the current study, the obligately chemolithoautotrophic strain MS-81-1c was shown to have, despite much greater storage of elemental sulfur, an apparent Y(infH)(inf(inf2))(infS) twice that of MS-81-6 when the two strains were grown in identical sulfide-limited gradient media. While the basis of this difference in energy conservation has not been established, differences in sulfur oxidation enzymes were noted. Strain MS-81-1c appeared to be able to oxidize sulfite by using either the adenosine phosphosulfate (APS) pathway or a sulfite:acceptor oxidoreductase. APS pathway enzymes (ATP sulfurylase and APS reductase) were present at relatively high and constant levels regardless of growth conditions, while the sulfite:acceptor oxidoreductase activity varied at least eightfold, with the highest activity produced in sulfide gradient medium. By contrast, strain MS-81-6 showed no detectable activity of the APS pathway enzymes and possessed a sulfite:acceptor oxidoreductase activity just sufficient to account for its observed rate of growth in sulfide gradient medium. Freshwater strain OH-75-2a showed activity and regulation of sulfite:acceptor oxidoreductase consistent with lithotrophic energy conservation, a feature not yet proven for any freshwater Beggiatoa strain.     

Cable Bacteria in Freshwater Sediments

In marine sediments cathodic oxygen reduction at the sediment surface can be coupled to anodic sulfide oxidation in deeper anoxic layers through electrical currents mediated by filamentous, multicellular bacteria of the Desulfobulbaceae family, the so-called cable bacteria. Until now, cable bacteria have only been reported from marine environments. In this study, we demonstrate that cable bacteria also occur in freshwater sediments. In a first step, homogenized sediment collected from the freshwater stream Giber Å, Denmark, was incubated in the laboratory. After 2 weeks, pH signatures and electric fields indicated electron transfer between vertically separated anodic and cathodic half-reactions. Fluorescence in situ hybridization revealed the presence of Desulfobulbaceae filaments. In addition, in situ measurements of oxygen, pH, and electric potential distributions in the waterlogged banks of Giber Å demonstrated the presence of distant electric redox coupling in naturally occurring freshwater sediment. At the same site, filamentous Desulfobulbaceae with cable bacterium morphology were found to be present. Their 16S rRNA gene sequence placed them as a distinct sister group to the known marine cable bacteria, with the genus Desulfobulbus as the closest cultured lineage. The results of the present study indicate that electric currents mediated by cable bacteria could be important for the biogeochemistry in many more environments than anticipated thus far and suggest a common evolutionary origin of the cable phenotype within Desulfobulbaceae with subsequent diversification into a freshwater and a marine lineage.     

Insights into the genome of large sulfur bacteria revealed by analysis of single filaments

Marine sediments are frequently covered by mats of the filamentous Beggiatoa and other large nitrate-storing bacteria that oxidize hydrogen sulfide using either oxygen or nitrate, which they store in intracellular vacuoles. Despite their conspicuous metabolic properties and their biogeochemical importance, little is known about their genetic repertoire because of the lack of pure cultures. Here, we present a unique approach to access the genome of single filaments of Beggiatoa by combining whole genome amplification, pyrosequencing, and optical genome mapping. Sequence assemblies were incomplete and yielded average contig sizes of approximately 1 kb. Pathways for sulfur oxidation, nitrate and oxygen respiration, and CO₂ fixation confirm the chemolithoautotrophic physiology of Beggiatoa. In addition, Beggiatoa potentially utilize inorganic sulfur compounds and dimethyl sulfoxide as electron acceptors. We propose a mechanism of vacuolar nitrate accumulation that is linked to proton translocation by vacuolar-type ATPases. Comparative genomics indicates substantial horizontal gene transfer of storage, metabolic, and gliding capabilities between Beggiatoa and cyanobacteria. These capabilities enable Beggiatoa to overcome non-overlapping availabilities of electron donors and acceptors while gliding between oxic and sulfidic zones. The first look into the genome of these filamentous sulfur-oxidizing bacteria substantially deepens the understanding of their evolution and their contribution to sulfur and nitrogen cycling in marine sediments.     

Preparation,Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more “temperate swarmers” that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. “Wettability”, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.     

Mitogenomic Evidence for an Indo-West Pacific Origin of the Clupeoidei (Teleostei: Clupeiformes)

The clupeoid fishes are distributed worldwide, with marine, freshwater and euryhaline species living in either tropical or temperate environments. Regional endemism is important at the species and genus levels, and the highest species diversity is found in the tropical marine Indo-West Pacific region. The clupeoid distribution follows two general pattern of species richness, the longitudinal and latitudinal gradients. To test historical hypotheses explaining the formation of these two gradients, we have examined the early biogeography of the Clupeoidei in reconstructing the evolution of their habitat preferences along with their ancestral range distributions on a time-calibrated mitogenomic phylogeny. The phylogenetic results support the distinction of nine main lineages within the Clupeoidei, five of them new. We infer several independent transitions from a marine to freshwater environment and from a tropical to temperate environment that occurred after the initial diversification period of the Clupeoidei. These results combined with our ancestral range reconstruction hypothesis suggest that the probable region of origin and diversification of the Clupeoidei during the Cretaceous period was the tropical marine precursor to the present Indo-West Pacific region. Thus, our study favors the hypotheses of “Region of origin” and “Tropical conservatism” to explain the origins of the longitudinal and latitudinal gradients of clupeoid species richness, respectively. Additional geological and paleontological evidence further define the tropical marine paleo-region of origin as the eastern Tethys Sea region. The Cretaceous fossil record of the Clupeoidei is partially incongruent with the results here as it contains taxa found outside this region. We discuss three possible causes of conflict between our biogeographical hypothesis and the distributions of the Cretaceous clupeoid fossils: regional extinction, incomplete taxonomic sampling and incorrect timescale estimation.     

A Hidden Pitfall in the Preparation of Agar Media Undermines Microorganism Cultivability

Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the “great plate count anomaly,” that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture of Gemmatimonas aurantiaca T-27^T and three representative sources of environmental samples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium.     

Dominating Role of an Unusual Magnetotactic Bacterium in the Microaerobic Zone of a Freshwater Sediment

A combination of polymerase chain reaction-assisted rRNA sequence retrieval and fluorescent oligonucleotide probing was used to identify in situ a hitherto unculturable, big, magnetotactic, rod-shaped organism in freshwater sediment samples collected from Lake Chiemsee. Tentatively named “Magnetobacterium bavaricum,” this bacterium is evolutionarily distant from all other phylogenetically characterized magnetotactic bacteria and contains unusually high numbers of magnetosomes (up to 1,000 magnetosomes per cell). The spatial distribution in the sediment was studied, and up to 7 × 10⁵ active cells per cm³ were found in the microaerobic zone. Considering its average volume (25.8 ± 4.1 μm³) and relative abundance (0.64 ± 0.17%), “M. bavaricum” may account for approximately 30% of the microbial biovolume and may therefore be a dominant fraction of the microbial community in this layer. Its microhabitat and its high content of sulfur globules and magnetosomes suggest that this organism has an iron-dependent way of energy conservation which depends on balanced gradients of oxygen and sulfide.     

Isolation of Strains Belonging to the Cosmopolitan Polynucleobacter necessarius Cluster from Freshwater Habitats Located in Three Climatic Zones

More than 40 bacterial strains belonging to the cosmopolitan Polynucleobacter necessarius cluster (Betaproteobacteria) were isolated from a broad spectrum of freshwater habitats located in three climatic zones. Sequences affiliated with the freshwater P. necessarius cluster are among the most frequently detected in studies on bacterial diversity in freshwater ecosystems. Despite this frequent detection with culture-independent techniques and the cosmopolitan occurrence of members affiliated with this cluster, no isolates have been reported thus far. The isolated strains have been obtained from lakes, ponds, and rivers in central Europe, the People's Republic of China, and East Africa by use of the filtration-acclimatization method. The 16S rRNA gene sequences of the isolates are 98.8 to 100% identical to reference sequences obtained by various authors by use of culture-independent methods. The isolates, aerobic heterotrophs, grew on a wide range of standard complex media and formed visible colonies on agar plates. Thus, the previous lack of isolates cannot be explained by a lack of appropriate media. Most of the isolates possess, under a wide range of culture conditions, very small cells (<0.1 μm³), even when grown in medium containing high concentrations of organic substances. Thus, these strains are obligate ultramicrobacteria. The obtained strains have a C-shaped cell morphology which is very similar to that of recently isolated ultramicrobacterial Luna cluster strains (Actinobacteria) and the SAR11 cluster strains (Alphaproteobacteria).     

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

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.