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.     

Methylotrophy in Freshwater Beggiatoa alba Strains

Two freshwater strains of the gammaproteobacterium Beggiatoa alba, B18LD and OH75-2a, are able to use methanol as a sole carbon and energy source under microoxic conditions. Genes encoding a methanol dehydrogenase large-subunit homolog and four enzymes of the tetrahydromethanopterin-dependent C₁ oxidation pathway were identified in B18LD. No evidence of methanotrophy was detected.     

High Nitrate Concentrations in Vacuolate, Autotrophic Marine Beggiatoa spp

Massive accumulations of very large Beggiatoa spp. are found at a Monterey Canyon cold seep and at Guaymas Basin hydrothermal vents. Both environments are characterized by high sediment concentrations of soluble sulfide and low levels of dissolved oxygen in surrounding waters. These filamentous, sulfur-oxidizing bacteria accumulate nitrate intracellularly at concentrations of 130 to 160 mM, 3,000- to 4,000-fold higher than ambient levels. Average filament widths range from 24 to 122 (mu)m, and individual cells of all widths possess a central vacuole. These findings plus recent parallel discoveries for Thioploca spp. (H. Fossing, V. A. Gallardo, B. B. Jorgensen, M. Huttel, L. P. Nielsen, H. Schulz, D. E. Canfield, S. Forster, R. N. Glud, J. K. Gundersen, J. Kuver, N. B. Ramsing, A. Teske, B. Thamdrup, and O. Ulloa, Nature (London) 374:713-715, 1995) suggest that nitrate accumulation may be a universal property of vacuolate, filamentous sulfur bacteria. Ribulose bisphosphate carboxylase-oxygenase and 2-oxoglutarate dehydrogenase activities in the Beggiatoa sp. from Monterey Canyon suggest in situ autotrophic growth of these bacteria. Nitrate reductase activity is much higher in the Monterey Beggiatoa sp. than in narrow, laboratory-grown strains of Beggiatoa spp., and the activity is found primarily in the membrane fraction, suggesting that the vacuolate Beggiatoa sp. can reduce nitrate coupled to electron flow through an electron transport system. Nitrate-concentrating and respiration potentials of these chemolithoautotrophs suggest that the Beggiatoa spp. described here are an important link between the sulfur, nitrogen, and carbon cycles at the Monterey Canyon seeps and the Guaymas Basin hydrothermal vents where they are found.     

Chemotaxis toward Nitrogenous Compounds by Swimming Strains of Marine Synechococcus spp.

Many of the open-ocean isolates of the marine unicellular cyanobacterium Synechococcus spp. are capable of swimming motility, whereas coastal isolates are nonmotile. Surprisingly, the motile strains do not display phototactic or photophobic responses to light, but they do demonstrate positive chemoresponses to several nitrogenous compounds. The chemotactic responses of Synechococcus strain WH8113 were investigated using blind-well chemotaxis chambers fitted with 3.0-μm-pore-size Nuclepore filters. One well of each chamber contained cells suspended in aged Sargasso Sea water, and the other well contained the potential chemoattractant in seawater. The number of cells that crossed the filter into the attractant-seawater mixture was measured by direct cell counts and compared with values obtained in chambers lacking gradients. Twenty-two compounds were tested, including sugars, amino acids, and simple nitrogenous substrates, at concentrations ranging from 10⁻⁵ to 10⁻¹⁰ M. Strain WH8113 responded positively only to ammonia, nitrate, β-alanine, glycine, and urea. Typically, there was a 1.5- to 2-fold increase in cell concentrations above control levels in chambers containing these compounds, which is comparable to results from similar experiments using enteric and photoheterotrophic bacteria. However, the threshold levels of 10⁻⁹ to 10⁻¹⁰ M found for Synechococcus spp. chemoresponses were lower by several orders of magnitude than those reported for other bacteria and fell within a range that could be ecologically significant in the oligotrophic oceans. The presence of chemotaxis in motile Synechococcus spp. supports the notion that regions of nutrient enrichment, such as the proposed microzones and patches, may play an important role in picoplankton nutrient dynamics.     

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.     

Phylogeny and distribution of nitrate-storing Beggiatoa spp. in coastal marine sediments

Filamentous sulphide-oxidizing Beggiatoa spp. often occur in large numbers in the coastal seabed without forming visible mats on the sediment surface. We studied the diversity, population structure and the nitrate-storing capability of such bacteria in the Danish Limfjorden and the German Wadden Sea. Their distribution was compared to the vertical gradients of O2, NO3- and H2S as measured by microsensors. The main Beggiatoa spp. populations occurred in a 0.5-3 cm thick intermediate zone, below the depth of oxygen and nitrate penetration but above the zone of free sulphide. The Beggiatoa spp. filaments were found to store nitrate, presumably in liquid vacuoles up to a concentration of 370 mM NO3-, similar to the related large marine sulphur bacteria, Thioploca and Thiomargarita. The observations indicate that marine Beggiatoa spp. can live anaerobically and conserve energy by coupling sulphide oxidation with the reduction of nitrate to dinitrogen and/or ammonia. Calculations of the diffusive nitrate flux and the potential sulphide oxidation by Beggiatoa spp. show that the bacteria may play a critical role for the sulphur cycling and the nitrogen balance in these coastal environments. 16S rDNA sequence analysis shows a large diversity of these uncultured, nitrate-storing Beggiatoa spp. Smaller (9-17 micro m wide) and larger (33-40 micro m wide) Beggiatoa spp. represent novel phylogenetic clusters distinct from previously sequenced, large marine Beggiatoa spp. and Thioploca spp. Fluorescence in situ hybridization (FISH) of the natural Beggiatoa spp. populations showed that filament width is a conservative character of each phylogenetic species but a given filament width may represent multiple phylogenetic species in a mixed population.     

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.     

Chemotaxis toward Nitrogenous Compounds by Swimming Strains of Marine Synechococcus spp

Many of the open-ocean isolates of the marine unicellular cyanobacterium Synechococcus spp. are capable of swimming motility, whereas coastal isolates are nonmotile. Surprisingly, the motile strains do not display phototactic or photophobic responses to light, but they do demonstrate positive chemoresponses to several nitrogenous compounds. The chemotactic responses of Synechococcus strain WH8113 were investigated using blind-well chemotaxis chambers fitted with 3.0-mum-pore-size Nuclepore filters. One well of each chamber contained cells suspended in aged Sargasso Sea water, and the other well contained the potential chemoattractant in seawater. The number of cells that crossed the filter into the attractant-seawater mixture was measured by direct cell counts and compared with values obtained in chambers lacking gradients. Twenty-two compounds were tested, including sugars, amino acids, and simple nitrogenous substrates, at concentrations ranging from 10 to 10 M. Strain WH8113 responded positively only to ammonia, nitrate, beta-alanine, glycine, and urea. Typically, there was a 1.5- to 2-fold increase in cell concentrations above control levels in chambers containing these compounds, which is comparable to results from similar experiments using enteric and photoheterotrophic bacteria. However, the threshold levels of 10 to 10 M found for Synechococcus spp. chemoresponses were lower by several orders of magnitude than those reported for other bacteria and fell within a range that could be ecologically significant in the oligotrophic oceans. The presence of chemotaxis in motile Synechococcus spp. supports the notion that regions of nutrient enrichment, such as the proposed microzones and patches, may play an important role in picoplankton nutrient dynamics.     

Motility patterns of filamentous sulfur bacteria, Beggiatoa spp

The large sulfur bacteria, Beggiatoa spp., live on the oxidation of sulfide with oxygen or nitrate, but avoid high concentrations of both sulfide and oxygen. As gliding filaments, they rely on reversals in the gliding direction to find their preferred environment, the oxygen-sulfide interface. We observed the chemotactic patterns of single filaments in a transparent agar medium and scored their reversals and the glided distances between reversals. Filaments within the preferred microenvironment glided distances shorter than their own length between reversals that anchored them in their position as a microbial mat. Filaments in the oxic region above the mat or in the sulfidic, anoxic region below the mat glided distances longer than the filament length between reversals. This reversal behavior resulted in a diffusion-like spreading of the filaments. A numerical model of such gliding filaments was constructed based on our observations. The model was applied to virtual filaments in the oxygen- and sulfide-free zone of the sediment, which is a main habitat of Beggiatoa in the natural environment. The model predicts a long residence time of the virtual filament in the suboxic zone and explains why Beggiatoa accumulate high nitrate concentrations in internal vacuoles as an alternative electron acceptor to oxygen.     

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.     

Salmonella spp., Vibrio spp., Clostridium perfringens, and Plesiomonas shigelloides in Marine and Freshwater Invertebrates from Coastal California Ecosystems

The coastal ecosystems of California are highly utilized by humans and animals, but the ecology of fecal bacteria at the land–sea interface is not well understood. This study evaluated the distribution of potentially pathogenic bacteria in invertebrates from linked marine, estuarine, and freshwater ecosystems in central California. A variety of filter-feeding clams, mussels, worms, and crab tissues were selectively cultured for Salmonella spp., Campylobacter spp., Escherichia coli-O157, Clostridium perfringens, Plesiomonas shigelloides, and Vibrio spp. A longitudinal study assessed environmental risk factors for detecting these bacterial species in sentinel mussel batches. Putative risk factors included mussel collection near higher risk areas for livestock or human sewage exposure, adjacent human population density, season, recent precipitation, water temperature, water type, bivalve type, and freshwater outflow exposure. Bacteria detected in invertebrates included Salmonella spp., C. perfringens, P. shigelloides, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio alginolyticus. Overall, 80% of mussel batches were culture positive for at least one of the bacterial species, although the pathogens Campylobacter, E. coli-O157, and Salmonella were not detected. Many of the same bacterial species were also cultured from upstream estuarine and riverine invertebrates. Exposure to human sewage sources, recent precipitation, and water temperature were significant risk factors for bacterial detection in sentinel mussel batches. These findings are consistent with the hypothesis that filter-feeding invertebrates along the coast concentrate fecal bacteria flowing from land to sea and show that the relationships between anthropogenic effects on coastal ecosystems and the environmental niches of fecal bacteria are complex and dynamic.     

In Situ Reproductive Rate of Freshwater Caulobacter spp.

Electron microscope grids were submerged in Lake Washington, Seattle, Wash., in June 1996 as bait to which Caulobacter sp. swarmers would attach and on which they would then reproduce in situ. Enumeration of bands in the stalks of attached cells implied that the caulobacters were completing approximately three reproductive cycles per day. A succession of morphological types of caulobacters occurred, as well as an episode of bacteriovore grazing that slowed the accumulation of caulobacters and prevented the aging of the population.     

Regulation of Metabolic and Electron Transport Pathways in the Freshwater Bacterium Beggiatoa leptomitiformis D-402

The biomass yield of freshwater filamentous sulfur bacteria of the genus Beggiatoa, when grown lithoheterotrophically or mixotrophically, has been shown to increase 2 to 2.5 times under microaerobic conditions (0.12 mg/l oxygen) as compared to aerobic conditions (9 mg/l oxygen). The activity of the glyoxylate cycle key enzymes have been found to increase two to three times under microaerobic conditions (at an O₂ concentration of 2 mg/l), and the activities of the sulfur metabolism enzymes increased three to five times (at an O₂ concentration of 0.1–0.5 mg/l). It has also been found that, under microaerobic conditions, thiosulfate was almost completely oxidized to sulfate by the bacteria, without accumulation of intermediate metabolites. At the same time, a 2- to 15-fold decrease in the activities of the tricarboxylic acid cycle enzymes involved in the reduction of NAD and FAD was observed. Reorganization of the respiratory chain after changes in aeration and type of nutrition was also observed. It has been found that, in cells grown heterotrophically, the terminal part of the respiratory chain contained an aa ₃-type oxidase, whereas, during mixotrophic, lithoheterotrophic, and autotrophic growth, aa ₃-type oxidase synthesis was inhibited, and the synthesis of a cbb ₃-type oxidase, which is induced under microaerobic conditions, was activated. The gene of the catalytic subunit CcoN of the cbb ₃-type oxidase was sequenced and proved to be highly homologous to the corresponding genes of other proteobacteria.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 452–459.Original Russian Text Copyright © 2005 by Muntyan, Grabovich, Patritskaya, Dubinina.     

Horizontal and vertical migration patterns of Phorrnidium corallyticum and Beggiatoa spp. associated with black-band disease of corals

An in situ field study of the motility patterns exhibited by Phormidium corallyticum and Beggiatoa spp. in black-band disease of corals was conducted over a 5-day period. Measurements were made at a spatial resolution of 50 m to document the horizontal migration of black-band across living coral tissue, while vertical migrations within the band were documented by observation and macrophotography of the black-band surface. It was determined that horizontal migration occurred both day and night, with the fastest movements by the front of the band during the day and the back of the band at night. Beggiatoa would rise to the band surface at night, and would often remain above the cyanobacterial population during extended periods of illumination the following day. The migration patterns are discussed in terms of motility cues and microbial physiology.     

Characterization of Large, Autotrophic Beggiatoa spp. Abundant at Hydrothermal Vents of the Guaymas Basin

Filamentous bacteria, identified as members of the genus Beggiatoa by gliding motility and internal globules of elemental sulfur, occur in massive aggregations at the deep-sea hydrothermal vents of the Guaymas Basin, Gulf of California. Cell aggregates covering the surface of sulfide-emanating sediments and rock chimneys were collected by DS R/V Alvin and subjected to shipboard and laboratory experiments. Each sample collected contained one to three discrete width classes of this organism usually accompanied by a small number of "flexibacteria" (width, 1.5 to 4 mum). The average widths of the Beggiatoa classes were 24 to 32, 40 to 42, and 116 to 122 mum. As indicated by electron microscopy and cell volume/protein ratios, the dominant bacteria are hollow cells, i.e., a thin layer of cytoplasm surrounding a large central liquid vacuole. Activities of Calvin-cycle enzymes indicated that at least two of the classes collected possess autotrophic potential. Judging from temperature dependence of enzyme activities and whole-cell CO(2) incorporation, the widest cells were mesophiles. The narrowest Beggiatoa sp. was either moderately thermophilic or mesophilic with unusually thermotolerant enzymes. This was consistent with its occurrence on the flanks of hot smoker chimneys with highly variable exit temperatures. In situ CO(2) fixation rates, sulfide stimulation of incorporation, and autoradiographic studies suggest that these Beggiatoa spp. contribute significantly as lithoautrophic primary producers to the Guaymas Basin vent ecosystems.     

Mixotrophic Protists In Marine and Freshwater Ecosystems

ABSTRACT Some protists from both marine and freshwater environments function at more than one trophic level by combining photosynthesis and panicle ingestion. Photosynthetic algae from several taxa (most commonly chrysomonads and dinoflagellates) have been reported to ingest living prey or nonliving particles, presumably obtaining part of their carbon and/or nutrients from phagocytosis. Conversely, some ciliates and sarcodines sequester chloroplasts after ingestion of algal prey. Plastid retention or "chloroplast symbiosis" by protists was first demonstrated < 20 years ago in a benthic foraminiferan. Although chloroplasts do not divide within these mixotrophic protists, they continue to function photosynthetically and may contribute to nutrition. Sarcodines and ciliates that harbor endosymbiotic algae could be considered mixotrophic but are not covered in detail here. the role of mixotrophy in the growth of protists and the impact of their grazing on prey populations have received increasing attention. Mixotrophic protists vary in their photosynthetic and ingestion capabilities, and thus, in the relative contribution of photosynthesis and phagotrophy to their nutrition. Abundant in both marine and freshwaters, they are potentially important predators of algae and bacteria in some systems. Mixotrophy may make a stronger link between the microbial and classic planktonic food webs by increasing trophic efficiency.