Heide N. Schulz-Vogt

Sulfide induces phosphate release from polyphosphate in cultures of a marine Beggiatoa strain

Sulfur bacteria such as Beggiatoa or Thiomargarita have a particularly high capacity for storage because of their large size. In addition to sulfur and nitrate, these bacteria also store phosphorus in the form of polyphosphate. Thiomargarita namibiensis has been shown to release phosphate from internally stored polyphosphate in pulses creating steep peaks of phosphate in the sediment and thereby inducing the precipitation of phosphorus-rich minerals. Large sulfur bacteria populate sediments at the sites of recent phosphorite formation and are found as fossils in ancient phosphorite deposits. Therefore, it can be assumed that this physiology contributes to the removal of bioavailable phosphorus from the marine system and thus is important for the global phosphorus cycle. We investigated under defined laboratory conditions which parameters stimulate the decomposition of polyphosphate and the release of phosphate in a marine Beggiatoa strain. Initially, we tested phosphate release in response to anoxia and high concentrations of acetate, because acetate is described as the relevant stimulus for phosphate release in activated sludge. To our surprise, the Beggiatoa strain did not release phosphate in response to this treatment. Instead, we could clearly show that increasing sulfide concentrations and anoxia resulted in a decomposition of polyphosphate. This physiological reaction is a yet unknown mode of bacterial polyphosphate usage and provides a new explanation for high phosphate concentrations in sulfidic marine sediments.

A single-cell sequencing approach to the classification of large, vacuolated sulfur bacteria

The colorless, large sulfur bacteria are well known because of their intriguing appearance, size and abundance in sulfidic settings. Since their discovery in 1803 these bacteria have been classified according to their conspicuous morphology. However, in microbiology the use of morphological criteria alone to predict phylogenetic relatedness has frequently proven to be misleading. Recent sequencing of a number of 16S rRNA genes of large sulfur bacteria revealed frequent inconsistencies between the morphologically determined taxonomy of genera and the genetically derived classification. Nevertheless, newly described bacteria were classified based on their morphological properties, leading to polyphyletic taxa. We performed sequencing of 16S rRNA genes and internal transcribed spacer (ITS) regions, together with detailed morphological analysis of hand-picked individuals of novel non-filamentous as well as known filamentous large sulfur bacteria, including the hitherto only partially sequenced species Thiomargarita namibiensis, Thioploca araucae and Thioploca chileae. Based on 128 nearly full-length 16S rRNA-ITS sequences, we propose the retention of the family Beggiatoaceae for the genera closely related to Beggiatoa, as opposed to the recently suggested fusion of all colorless sulfur bacteria into one family, the Thiotrichaceae. Furthermore, we propose the addition of nine Candidatus species along with seven new Candidatus genera to the family Beggiatoaceae. The extended family Beggiatoaceae thus remains monophyletic and is phylogenetically clearly separated from other related families.

Anaerobic sulfide oxidation with nitrate by a freshwater Beggiatoa enrichment culture.

ABSTRACT 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.

The genus Pseudovibrio contains metabolically versatile bacteria adapted for symbiosis

The majority of strains belonging to the genus Pseudovibrio have been isolated from marine invertebrates such as tunicates, corals and particularly sponges, but the physiology of these bacteria is poorly understood. In this study, we analyse for the first time the genomes of two Pseudovibrio strains – FO-BEG1 and JE062. The strain FO-BEG1 is a required symbiont of a cultivated Beggiatoa strain, a sulfide-oxidizing, autotrophic bacterium, which was initially isolated from a coral. Strain JE062 was isolated from a sponge. The presented data show that both strains are generalistic bacteria capable of importing and oxidizing a wide range of organic and inorganic compounds to meet their carbon, nitrogen, phosphorous and energy requirements under both, oxic and anoxic conditions. Several physiological traits encoded in the analysed genomes were verified in laboratory experiments with both isolates. Besides the versatile metabolic abilities of both Pseudovibrio strains, our study reveals a number of open reading frames and gene clusters in the genomes that seem to be involved in symbiont–host interactions. Both Pseudovibrio strains have the genomic potential to attach to host cells, interact with the eukaryotic cell machinery, produce secondary metabolites and supply the host with cofactors.

Lipid Biomarker Patterns of Phosphogenic Sediments from Upwelling Regions

Sediments of upwelling regions off Namibia, Peru, and Chile contain dense populations of large nitrate-storing sulfide-oxidizing bacteria, Thiomargarita, Beggiatoa, and Thioploca. Increased contents of monounsaturated C16 and C18 fatty acids have been found at all stations studied, especially when a high density of sulfide oxidizers in the sediments was observed. The distribution of lipid biomarkers attributed to sulfate reducers (10MeC16:0 fatty acid, ai-C15:0 fatty acid, and mono-O-alkyl glycerol ethers) compared to the distribution of sulfide oxidizers indicate a close association between these bacteria. As a consequence, the distributions of sulfate reducers in sediments of Namibia, Peru, and Chile are closely related to differences in the motility of the various sulfide oxidizers at the three study sites. Depth profiles of mono-O-alkyl glycerol ethers have been found to correlate best with the occurrence of large sulfide-oxidizing bacteria. This suggests a particularly close link between mono-O-alkyl glycerol ether-synthesizing sulfate reducers and sulfide oxidizers. The interaction between sulfide-oxidizing bacteria and sulfate-reducing bacteria reveals intense sulfur cycling and degradation of organic matter in different sediment depths.

Multiple self-splicing introns in the 16S rRNA genes of giant sulfur bacteria

The gene encoding the small subunit rRNA serves as a prominent tool for the phylogenetic analysis and classification of Bacteria and Archaea owing to its high degree of conservation and its fundamental function in living organisms. Here we show that the 16S rRNA genes of not-yet-cultivated large sulfur bacteria, among them the largest known bacterium Thiomargarita namibiensis, regularly contain numerous self-splicing introns of variable length. The 16S rRNA genes can thus be enlarged to up to 3.5 kb. Remarkably, introns have never been identified in bacterial 16S rRNA genes before, although they are the most frequently sequenced genes today. This may be caused in part by a bias during the PCR amplification step that discriminates against longer homologs, as we show experimentally. Such length heterogeneity of 16S rRNA genes has so far never been considered when constructing 16S rRNA-based clone libraries, even though an elongation of rRNA genes due to intervening sequences has been reported previously. The detection of elongated 16S rRNA genes has profound implications for common methods in molecular ecology and may cause systematic biases in several techniques. In this study, catalyzed reporter deposition–fluorescence in situ hybridization on both ribosomes and rRNA precursor molecules as well as in vitro splicing experiments were performed and confirmed self-splicing of the introns. Accordingly, the introns do not inhibit the formation of functional ribosomes.

Exo-metabolome of Pseudovibrio sp. FO-BEG1 analyzed by ultra-high resolution mass spectrometry and the effect of phosphate limitation.

Oceanic dissolved organic matter (DOM) is an assemblage of reduced carbon compounds, which results from biotic and abiotic processes. The biotic processes consist in either release or uptake of specific molecules by marine organisms. Heterotrophic bacteria have been mostly considered to influence the DOM composition by preferential uptake of certain compounds. However, they also secrete a variety of molecules depending on physiological state, environmental and growth conditions, but so far the full set of compounds secreted by these bacteria has never been investigated. In this study, we analyzed the exo-metabolome, metabolites secreted into the environment, of the heterotrophic marine bacterium Pseudovibrio sp. FO-BEG1 via ultra-high resolution mass spectrometry, comparing phosphate limited with phosphate surplus growth conditions. Bacteria belonging to the Pseudovibrio genus have been isolated worldwide, mainly from marine invertebrates and were described as metabolically versatile Alphaproteobacteria. We show that the exo-metabolome is unexpectedly large and diverse, consisting of hundreds of compounds that differ by their molecular formulae. It is characterized by a dynamic recycling of molecules, and it is drastically affected by the physiological state of the strain. Moreover, we show that phosphate limitation greatly influences both the amount and the composition of the secreted molecules. By assigning the detected masses to general chemical categories, we observed that under phosphate surplus conditions the secreted molecules were mainly peptides and highly unsaturated compounds. In contrast, under phosphate limitation the composition of the exo-metabolome changed during bacterial growth, showing an increase in highly unsaturated, phenolic, and polyphenolic compounds. Finally, we annotated the detected masses using multiple metabolite databases. These analyses suggested the presence of several masses analogue to masses of known bioactive compounds. However, the annotation was successful only for a minor part of the detected molecules, underlining the current gap in knowledge concerning the biosynthetic ability of marine heterotrophic bacteria.

Sulfur Respiration in a Marine Chemolithoautotrophic Beggiatoa Strain

The chemolithoautotrophic strain Beggiatoa sp. 35Flor shows an unusual migration behavior when cultivated in a gradient medium under high sulfide fluxes. As common for Beggiatoa spp., the filaments form a mat at the oxygen–sulfide interface. However, upon prolonged incubation, a subpopulation migrates actively downward into the anoxic and sulfidic section of the medium, where the filaments become gradually depleted in their sulfur and polyhydroxyalkanoates (PHA) inclusions. This depletion is correlated with the production of hydrogen sulfide. The sulfur- and PHA-depleted filaments return to the oxygen–sulfide interface, where they switch back to depositing sulfur and PHA by aerobic sulfide oxidation. Based on these observations we conclude that internally stored elemental sulfur is respired at the expense of stored PHA under anoxic conditions. Until now, nitrate has always been assumed to be the alternative electron acceptor in chemolithoautotrophic Beggiatoa spp. under anoxic conditions. As the medium and the filaments were free of oxidized nitrogen compounds we can exclude this metabolism. Furthermore, sulfur respiration with PHA under anoxic conditions has so far only been described for heterotrophic Beggiatoa spp., but our medium did not contain accessible organic carbon. Hence the PHA inclusions must originate from atmospheric CO2 fixed by the filaments while at the oxygen–sulfide interface. We propose that the directed migration of filaments into the anoxic section of an oxygen–sulfide gradient system is used as a last resort to preserve cell integrity, which would otherwise be compromised by excessive sulfur deposition occurring in the presence of oxygen and high sulfide fluxes. The regulating mechanism of this migration is still unknown.

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.

Phosphate Limitation Induces Drastic Physiological Changes, Virulence-Related Gene Expression, and Secondary Metabolite Production in Pseudovibrio sp. Strain FO-BEG1

ABSTRACT Phosphorus is a vital nutrient for living organisms and is obtained by bacteria primarily via phosphate uptake. However, phosphate is often scarcely accessible in nature, and there is evidence that in many areas of the ocean, its concentration limits bacterial growth. Surprisingly, the phosphate starvation response has been extensively investigated in different model organisms (e.g., Escherichia coli), but there is a dearth of studies on heterotrophic marine bacteria. In this work, we describe the response of Pseudovibrio sp. strain FO-BEG1, a metabolically versatile alphaproteobacterium and potential symbiont of marine sponges, to phosphate limitation. We compared the physiology, protein expression, and secondary metabolite production under phosphate-limited conditions to those under phosphate surplus conditions. We observed that phosphate limitation had a pleiotropic effect on the physiology of the strain, triggering cell elongation, the accumulation of polyhydroxyalkanoate, the degradation of polyphosphate, and the exchange of membrane lipids in favor of phosphorus-free lipids such as sulfoquinovosyl diacylglycerols. Many proteins involved in the uptake and degradation of phospho-organic compounds were upregulated, together with subunits of the ABC transport system for phosphate. Under conditions of phosphate limitation, FO-BEG1 secreted compounds into the medium that conferred an intense yellow coloration to the cultures. Among these compounds, we identified the potent antibiotic tropodithietic acid. Finally, toxin-like proteins and other proteins likely involved in the interaction with the eukaryotic host were also upregulated. Altogether, our data suggest that phosphate limitation leads to a pronounced reorganization of FO-BEG1 physiology, involving phosphorus, carbon, and sulfur metabolism; cell morphology; secondary metabolite production; and the expression of virulence-related genes.