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.     

A simple method to release polyphosphate from activated sludge for phosphorus reuse and recycling

In enhanced biological phosphorus removal processes, activated sludge microorganisms accumulate large quantities of polyphosphate (polyP). It was discovered that nearly all of the polyP could be released from activated sludge simply by heating it at 70 degrees C for about 1 h. The chain length of released polyP ranged from 100 to 200 phosphate (P(i)) residues. The addition of CaCl(2) precipitated approximately 75% of the total phosphorus without pH adjustment. The formed precipitate contained more P and less Ca than typical natural phosphorite deposits. Hence, in combination with enhanced biological phosphorus removal, the present method has potential for the development of a simple process for recovering phosphorus in a reusable form from wastewater.     

Regulation of bacterial phosphate taxis and polyphosphate accumulation in response to phosphate starvation stress

Phosphorus (P) is an essential constituent in all types of living organisms. Bacteria, which use inorganic phosphate (P_i), as the preferred P source, have evolved complex systems to survive during P_i starvation conditions. Recently, we found thatPseudomonas aeruginosa, a monoflagellated, obligately aerobic bacterium, is attracted to P_i. The evidence that the chemotactic response to P_i (P_i taxis) was observed only with cells grown in P_i-limiting medium suggests that P_i taxis plays an important role in scavenging P_i residues under conditions of P_i starvation. Many bacteria also exhibit rapid and extensive accumulation of polyphosphate (polyP), when P_i is added to cells previously subjected to P_i starvation stress. Since polyP can serve as a P source during P_i starvation conditions, it is likely that polyP accumulation is a protective mechanism for survival during P_i starvation. In the present review, we summarize our current knowledge on regulation of bacterial P_i taxis and polyP accumulation in response to P_i starvation stress.     

A comparison of phosphorus immobilization in sediments of freshwater and coastal marine systems

The extent to which sediments of aquatic systems immobilize or release phosphorus can affect dramatically the P content of overlying waters. Data from 48 different aquatic systems suggests that there may be a major difference between fresh- and salt-water systems in this immobilization. Under oxic conditions (water overlying sediments had dissolved oxygen > 0.5 mg/L) P is strongly immobilized in sediments of most fresh-water systems. In sediments of most salt-water systems P is released from sediments and behaves, essentially, as a conservative tracer of benthic decomposition. This difference in P cycling is large enough to have an influence on the often cited difference in phytoplankton nutrient limitation between fresh- and salt-water systems.     

Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment

A novel lipase was isolated from a metagenomic library of Baltic Sea sediment bacteria. Prokaryotic DNA was extracted and cloned into a copy control fosmid vector (pCC1FOS) generating a library of >7000 clones with inserts of 24-39 kb. Screening for clones expressing lipolytic activity based on the hydrolysis of tributyrin and p-nitrophenyl esters, identified 1% of the fosmids as positive. An insert of 29 kb was fragmented and subcloned. Subclones with lipolytic activity were sequenced and an open reading frame of 978 bp encoding a 35.4-kDa putative lipase/esterase h1Lip1 (DQ118648) with 54% amino acid similarity to a Pseudomonas putida esterase (BAD07370) was identified. Conserved regions, including the putative active site, GDSAG, a catalytic triad (Ser148, Glu242 and His272) and a HGG motif, were identified. The h1Lip1 lipase was over expressed, (pGEX-6P-3 vector), purified and shown to hydrolyse p-nitrophenyl esters of fatty acids with chain lengths up to C14. Hydrolysis of the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) confirmed that h1Lip1 was a lipase. The apparent optimal temperature for h1Lip1, by hydrolysis of p-nitrophenyl butyrate, was 35 degrees C. Thermal stability analysis showed that h1Lip1 was unstable at 25 degrees C and inactivated at 40 degrees C with t1/2 <5 min.     

Complete genome sequence of Desulfocapsa sulfexigens,a marine deltaproteobacterium specialized in disproportionating inorganic sulfur compounds

Desulfocapsa sulfexigens SB164P1 (DSM 10523) belongs to the deltaproteobacterial family Desulfobulbaceae and is one of two validly described members of its genus. This strain was selected for genome sequencing, because it is the first marine bacterium reported to thrive on the disproportionation of elemental sulfur, a process with a unresolved enzymatic pathway in which elemental sulfur serves both as electron donor and electron acceptor. Furthermore, in contrast to its phylogenetically closest relatives, which are dissimilatory sulfate-reducers, D. sulfexigens is unable to grow by sulfate reduction and appears metabolically specialized in growing by disproportionating elemental sulfur, sulfite or thiosulfate with CO₂ as the sole carbon source. The genome of D. sulfexigens contains the set of genes that is required for nitrogen fixation. In an acetylene assay it could be shown that the strain reduces acetylene to ethylene, which is indicative for N-fixation. The circular chromosome of D. sulfexigens SB164P1 comprises 3,986,761 bp and harbors 3,551 protein-coding genes of which 78% have a predicted function based on auto-annotation. The chromosome furthermore encodes 46 tRNA genes and 3 rRNA operons.     

Complete genome sequence of Marinobacter adhaerens type strain (HP15), a diatom-interacting marine microorganism

Marinobacter adhaerens HP15 is the type strain of a newly identified marine species, which is phylogenetically related to M. flavimaris, M. algicola, and M. aquaeolei. It is of special interest for research on marine aggregate formation because it showed specific attachment to diatom cells. In vitro it led to exopolymer formation and aggregation of these algal cells to form marine snow particles. M. adhaerens HP15 is a free-living, motile, rod-shaped, Gram-negative gammaproteobacterium, which was originally isolated from marine particles sampled in the German Wadden Sea. M. adhaerens HP15 grows heterotrophically on various media, is easy to access genetically, and serves as a model organism to investigate the cellular and molecular interactions with the diatom Thalassiosira weissflogii. Here we describe the complete and annotated genome sequence of M. adhaerens HP15 as well as some details on flagella-associated genes. M. adhaerens HP15 possesses three replicons; the chromosome comprises 4,422,725 bp and codes for 4,180 protein-coding genes, 51 tRNAs and three rRNA operons, while the two circular plasmids are ~187 kb and ~42 kb in size and contain 178 and 52 protein-coding genes, respectively.     

The relative importance of biological and chemical processes in the release of phosphorus from a highly organic sediment

Bacteria can play an important role in the process of anaerobic phosphorus release: they can act as a direct source of orthophosphates, or as a catalyst of iron hydroxyde reduction. We studied their influence on phosphorus release from highly organic sediments of a Canadian shield lake. Phosphorus and iron release were measured under aerobic and anaerobic conditions, with or without sterilization, and at different pH. We measured also the abundance and activity of bacteria in sediments. The increased P release after sterilization can be explained by cell lysis. Compared to sterilization, changing oxygen concentrations or acidification had little or no effect on P release. In these sediments, phosphorus and iron movements were independent. Most of the total dissolved iron seemed to be linked to humic acids, but not phosphorus.A contribution to the GRIL (Groupe de Recherche Interuniversitaire de Limnologie)     

Genomic properties of Marine Group A bacteria indicate a role in the marine sulfur cycle

Marine Group A (MGA) is a deeply branching and uncultivated phylum of bacteria. Although their functional roles remain elusive, MGA subgroups are particularly abundant and diverse in oxygen minimum zones and permanent or seasonally stratified anoxic basins, suggesting metabolic adaptation to oxygen-deficiency. Here, we expand a previous survey of MGA diversity in O₂-deficient waters of the Northeast subarctic Pacific Ocean (NESAP) to include Saanich Inlet (SI), an anoxic fjord with seasonal O₂ gradients and periodic sulfide accumulation. Phylogenetic analysis of small subunit ribosomal RNA (16S rRNA) gene clone libraries recovered five previously described MGA subgroups and defined three novel subgroups (SHBH1141, SHBH391, and SHAN400) in SI. To discern the functional properties of MGA residing along gradients of O₂ in the NESAP and SI, we identified and sequenced to completion 14 fosmids harboring MGA-associated 16S RNA genes from a collection of 46 fosmid libraries sourced from NESAP and SI waters. Comparative analysis of these fosmids, in addition to four publicly available MGA-associated large-insert DNA fragments from Hawaii Ocean Time-series and Monterey Bay, revealed widespread genomic differentiation proximal to the ribosomal RNA operon that did not consistently reflect subgroup partitioning patterns observed in 16S rRNA gene clone libraries. Predicted protein-coding genes associated with adaptation to O₂-deficiency and sulfur-based energy metabolism were detected on multiple fosmids, including polysulfide reductase (psrABC), implicated in dissimilatory polysulfide reduction to hydrogen sulfide and dissimilatory sulfur oxidation. These results posit a potential role for specific MGA subgroups in the marine sulfur cycle.     

Controlling phosphate release from phosphate-enriched sediments by adding various iron compounds

In a laboratory experiment, different ironsalts (FeCl₂, FeCl₃, FeSO₄) andFe₂O₃ were added to a phosphateenriched silty loam sediment in order to studytheir effect on phosphate mobilisation.Phosphate concentrations in sediment pore waterwere not reduced by the addition ofFe₂O₃. Addition of both ironchlorides, however, resulted in a strongdecrease of phosphate levels in sediment porewater. A similar but less pronounced effect wascaused by the addition of iron as iron(II)sulphate. Sulphate appears to counteract theimmobilisation of phosphate brought about byiron(II). Phosphate release from the sedimentappeared to be determined by the iron/phosphateratio in the sediment pore water. The additionof Fe₂O₃ barely affected thephosphate release from the sediment whereas theaddition of iron salts was effective inpreventing phosphate release. Increased amountsof iron added to the sediment resulted in adecreased phosphate release.     

海洋沉积物微生物介导有机碳转化研究进展

海洋沉积物是地球上最大的有机碳库,其中生存的微生物总量大、分布范围广、类群多样、代谢方式复杂,并共同构成海洋沉积物微生物组。海洋沉积物微生物组介导的有机碳降解与矿化过程不但能为沉积物中的生命活动提供物质和能量,也能参与调控碳循环过程,并在长时间尺度上对地球气候系统产生重大影响。沉积物中的有机碳在复杂多样的微生物代谢活动下被逐步降解,其最终的矿化过程与不同的电子受体消耗相偶合,并形成对应的地球化学分区。研究海洋沉积物微生物及其介导的有机碳转化过程对我们深入认识沉积物中的元素循环过程,并进一步评估其对整个地球系统的影响具有重要科学意义。本文对海洋沉积物微生物组的体量、包含的微生物多样性、代谢活性以及在不同地球化学分区中主要的微生物类群和代谢机制进行综述,最后基于研究现状展望了海洋沉积物微生物组的未来研究方向。     

Rates of sulfate reduction and thiosulfate consumption in a marine microbial mat

Abstract The sulfur cycle in a microbial mat was studied by determining viable counts of sulfate-reducing bacteria, chemolithoautotrophic sulfur bacteria and anoxygenic phototrophic bacteria. All three functional groups of sulfur bacteria revealed a maximum population density in the uppermost 5 mm of the mat: 1.1 × 10⁸ cells of sulfate reducers cm⁻³ sediment, 2.0 × 10⁹ cells of chemolithoautotrophs cm⁻³ sediment, and 4.0 × 10⁷ cells of anoxygenic phototrophs cm⁻³ sediment. Bacterial dynamics were studied by sulfate reduction rate measurements, both under anoxic conditions (dark incubation) and oxic conditions (incubation in the light), and determination of the vertical distribution of the potential rate of thiosulfate consumption under oxic conditions. Sulfate reduction rates in the top 5 mm of the sediment were 566 nmol cm⁻³ d⁻¹ in the absence of oxygen, and 123 nmol cm⁻³ d⁻¹ in the presence of oxygen. In the latter case, the maximum rate was found in the 5–10-mm depth horizon (361 nmol cm⁻³ d⁻¹). Biological consumption of amended thiosulfate was rapid and decreased with depth, while in the presence of molybdate, thiosulfate consumption decreased to 10–30% of the original rate.     

Isolation and characterization of chitinase from a marine bacterium Vibrio sp.

A chitinase was separated from the culture broth of Vibrio sp. 11211 isolated from sediment from the South China Sea. The chitinase was purified 18.3-fold with 33% recovery by ammonium sulphate precipitation and chromatography. The subunit molecular weight of the enzyme was estimated by SDS-PAGE to be about 30kDa. The enzyme showed optimum pH at 6.5 and optimum temperature at 50^°C, and was stable in the pH range of 4 to 9 and at the temperature below 40^°C.     

The effect of deposition of organic matter on phosphorus dynamics in experimental marine sediment systems

The effect of deposition of organic matter on phosphorus dynamics in sandy marine sediments was evaluated using an experimental system (boxcosms) and three different strategies: (1) no supply (2) one single addition (3) weekly additions of a suspension of algal cells (Phaeocystis spec.). Macrofauna (3 species, 6 individuals of each) were added to half of the boxes. Both in the case of the single and weekly additions a clear effect of increased organic matter loading on phosphorus dynamics was found. Following the organic matter addition, porewater phosphate concentrations in the upper sediment layer increased, phosphate release rates from the sediment increased by a factor 3–5 and in the boxes to which a single addition was applied NaOH-extractable phosphorus increased substantially. The increase in phosphate release rates from the sediment was attributed to mineralization of the added material and to direct release from the algal cells. No clear effect of the presence of macrofauna on sediment-water exchange of phosphate could be discovered. The macrofauna were very effective at reworking the sediment, however, as illustrated by the organic carbon profiles. It is hypothesized that the sediment-water exchange rates of phosphate were regulated by the layer of algal material which was present on the sediment surface in the fed boxes. In the boxes to which the single addition was applied porewater phosphate concentrations were lower and NaOH-extractable phosphorus was higher in the presence of macrofauna, suggesting that macrofauna can stimulate phosphate binding in the sediment.Publication no. 40 of the project Applied Scientific Research Netherlands Institute for Sea Research (BEWON)     

Purification and characterization of antibacterial substances produced by a marine bacterium Pseudoalteromonas haloplanktis strain

Aims: To purify and characterize compounds with antimicrobial activity from Pseudoalteromonas haloplanktis inhibition (INH) strain. Methods and Results: The P. haloplanktis isolated from a scallop hatchery was used to analyse antibacterial activities. Crude extracts were obtained with ethyl acetate of the cultured broth, after separation of bacterial cells, and assays against six strains of marine bacteria and nine clinically important pathogenic bacteria. The active compounds were purified from ethyl acetate extracts, by a combination of SiO₂ column and thin layer chromatography. Two active fractions were isolated, and chemical structures of two products from the major one were unambiguously identified as isovaleric acid (3-methylbutanoic acid) and 2-methylbutyric acid (2-methylbutanoic acid), by comparing their mass spectra and ¹H- and ¹³C-nuclear magnetic resonance spectra to those of authentic compounds. Conclusions: In the antibacterial activity of P. haloplanktis INH strain, extra cell compounds are involucred, mainly isovaleric and 2-methylbutyric acids. Significance and Impact of the Study: Production of antimicrobial compounds by marine micro-organisms has been widely reported; however, the efforts not always are conducted to purification and applications of these active compounds. This study is a significant contribution to the knowledge of compounds unique from marine bacteria as potential sources of new drugs in the pharmacological industry.     

Filamentous bacteria inhabiting the sheaths of marine Thioploca spp. on the Chilean continental shelf

A new component of the benthic Thioploca mat microbial ecosystem on the Chilean continental shelf was detected by epifluorescence microscopy: filamentous, bacterial endobionts of 4–5-μm filament diameter and length sometimes exceeding 1 mm. These filaments were identified as growing within Thioploca sheaths located between the sediment surface and c . 5 cm depth. Their location coincided with maximal biomass and biovolume of Thioploca filaments in surficial sediments, and with maximal abundance and activity of sulfate-reducing bacterial populations near the sediment/water interface. FISH and environmental characteristics support the working hypothesis that these endobiont populations are members of the filamentous, sulfate-reducing bacterial genus Desulfonema . Found at several sampling stations over a decade-long interval (1994–2006), these populations appear to be a stable component of the Chilean Thioploca mat ecosystem.     

Effects of added organic residues and calcium carbonate on polyphosphate hydrolysis in four Quebec soils

Polyphosphate hydrolysis was studied in three surface samples and one subsurface sample of Quebec soil treated with alfalfa residues (44.8 t ha⁻¹) and farmyard manure (FYM; 44.8 t ha⁻¹); and in two acid soil samples treated with CaCO₃ (12.5 t ha⁻¹). The polyphosphates used were Na₄P₂O₇. 10H₂O (NaPP) and PolyN (a triammonium pyrophosphate-orthophosphate mixture). Each polyphosphate was added at a rate of 1.00g P kg⁻¹ soil. Addition of alfalfa residues promoted polyphosphate hyrolysis of both NaPP and PolyN through reduced polyphosphate sorption and increasing pyrophosphatase activity. Application of FYM increased polyphosphate hydrolysis in Uplands topsoil and retarded hydrolysis in the other soils. Hydrolysis was probably reduced because of orthophosphate (OP) introduced with the FYM. Added CaCO₃ accelerated polyphosphate hydrolysis in an acid topsoil sample through reduced sorption, but slowed hydrolysis in the subsoil sample, due to a reduction in enzyme activity.     

Sulfide pulsing as the controlling factor of spinae production in Chlorobium limicola strain UdG 6038

Chlorobium limicola UdG 6038, a green sulfur bacterium, was isolated from anoxic sediments. Cells were gram-negative, non-motile, ovoid shaped, and contained chlorobactene and bacteriochlorophyll c as the main photosynthetic pigments. The DNA G+C content was 56.4 mol%. Ultrastructural studies revealed the presence of abundant spinae (45–110 spinae per cell) attached to the cell wall. India-ink-stained cells observed under the optical microscope were surrounded by a large capsule (5–11 μm total diameter). The presence of this capsule was coincident with the presence of a large number of spinae (> 30 spinae per cell). The mucilaginous capsule was attached to the spinae without penetrating it. In batch culture, the synthesis of spinae in strain UdG 6038 was not affected by changes in temperature, pH, salt concentration, or illumination at physiological ranges and hence, the cells remained spined. The control of spinae production was experimentally confirmed using a semicontinuous batch culture refed by sulfide pulsing. The culture remained at a low spination level (> 30 spinae per cell) only when the duration of sulfide starvation between pulses was less than 5 h. After longer sulfide starvation periods, the cells remained spined (more than 38 ± 6.3 spinae per cell). This observation supports the idea that the duration of sulfide limitation in the culture plays a key role in controlling the spination process in strain C. limicola UdG 6038. Chlorobium spinae may play an eco-physiological role in buoyancy capacity and adhesion of sulfur globules to the cells in natural environments where sulfide concentrations are expected to be highly variable. Revision received: 13 November 1995 / Accepted: 19 January 1996     

A comparative study of specificity of fucoidanases from marine microorganisms and invertebrates

Specificities of actions of fucoidanases from the marine microorganism Pseudoalteromonas citrea KMM 3296 and the marine mollusk Littorina kurila were studied. The enzymes possess similar specificities and catalyze the cleavage of accessible α-(1→3)-fucoside bonds in fucoidans with highly sulfated α-(1→4; 1→3)-L-fucooligosaccharides. A high degree of sulfation of the fucose residues in fucoidans makes α-(1→3)-L-fucoside bonds inaccessible for the action of the studied enzymes. The maximum degree of cleavage of fucoidan was achieved by the fucoidanase from the marine bacterium Pseudoalteromonas citrea KMM 3296.