Rhodovulum Steppense Research Articles

Nitrogen Fixation Activity and Genome Analysis of a Moderately Haloalkaliphilic Anoxygenic Phototrophic Bacterium Rhodovulum tesquicola

The genome of the moderately haloalkaliphilic diazotrophic anoxygenic phototrophic bacterium Rhodovulum tesquicola A-36sT isolated from an alkaline lake was analyzed and compared to the genomes of the closest species Rhodovulum steppense A-20sT and Rhodovulum strictum DSM 11289T. The genomic features of three organisms are quite similar, reflecting their ecological and physiological role of facultative photoheterotrophs. Nevertheless, the nitrogenase activity of the pure cultures of the studied bacteria differed significantly: the highest rate (4066 nmoles C2H2/mg of dry weight per hour) was demonstrated by Rhodovulum strictum while the rates in Rhodovulum tesquicola and Rhodovulum steppense were an order of magnitude lower (278 and 523 nmoles C2H2/mg of dry weight per hour, respectively). This difference can be attributed to the presence of an additional nitrogenase operon found exclusively in R. strictum and to the structural variation in nitrogenase operon in R. tesquicola.

Purification and Investigation of Physicochemical and Regulatory Properties of Homogeneous L-Lactate: Cytochrom c Oxidoreductase Obtained from the Nonsulfur Purple Bacterium Rhodovulum steppense

L-Lactate: cytochrome c oxidoreductase activity was detected in cells of strain A-20s of the nonsulfur haloalkalophilic purple bacterium Rhodovulum steppense. An electrophoretically homogeneous preparation of the enzyme was obtained by purification. The enzyme had a specific activity of 4.75 U/mg protein, a 81.9-fold purification degree, and a 2.2% yield. The kinetic and physicochemical characteristics were determined. The value of the Michaelis constant with lactate was 15 μM. The temperature optimum for the studied enzyme was 31°C; optimum of pH was 8.2. It was found that the enzyme was a homodimer with a molecular weight of ~140 kDa; the mass of individual subunit was 68 kDa.

Oligomeric forms of bacterial malate dehydrogenase: a study of the enzyme from the phototrophic non-sulfur bacterium Rhodovulum steppense A-20s.

Malate dehydrogenase (EC 1.1.1.37) was purified to homogeneity from the phototrophic purple non-sulfur bacterium Rhodovulum steppense A-20s. According to gel-chromatography and electrophoretic studies, malate dehydrogenase is present as a dimer, tetramer and octamer depending on cultivation conditions. In phototrophic aerobic conditions only the tetrameric form was present, in chemotrophic aerobic conditions all three forms were detected, while in the absence of oxygen the octameric form disappeared. The malate dehydrogenase oligomers are encoded by a single gene and composed of the same 35kDa polypeptide but differ in pH and temperature optimum, in affinities to malate, oxaloacetate, NADH and NAD+ and in regulation by cations and citrate. By modulating the cultivation conditions, it has been established that the dimer participates in the glyoxylate cycle; the tetramer operates in the tricarboxylic acid cycle, and the octamer may be involved in the adaptation to oxidative stress.

Isoformes of Malate Dehydrogenase from Rhodovulum Steppense A-20s Grown Chemotrophically under Aerobic Conditions

Three malate dehydrogenase isoforms (65-, 60-, and 71-fold purifications) with specific activities of 4.23, 3.88, and 4.56 U/mg protein were obtained in an electrophoretically homogenous state from Rhodovulum steppense bacteria strain A-20s chemotropically grown under aerobic conditions. The physicochemical and kinetic properties of malate dehydrogenase isoforms were determined. The molecular weight and the Michaelis constants were determined; the effect of hydrogen ions on the forward and reverse MDH reaction was studied. The results of the study demonstrated that the enzyme consists of subunits; the molecular weight of subunits was determined by SDS-PAGE.

Experimental Modeling of Bacterially-Induced Ca Carbonate Precipitation: New Insights on Possible Mechanisms

The contribution of microorganisms, particularly bacteria, in carbonate mineral formation, the main natural processes controlling CO2 level in the atmosphere, has played an important role since the Archean Eon. In this study we review our recent experimental work on CaCO3 precipitation induced by two anoxygenic phototrophic bacteria (APB), Rhodovulum steppense A-20sT and Rhodovulum sp. S-17-65, and by cyanobacteria Gloeocapsa sp. f-6gl. These bacteria are representatives of two important groups of photosynthetic organisms present at the Earth surface both in the past and at the present times. The mechanisms of organomineralization deriving from APB and cyanobacteria activities are drastically different and relate to the main physical and chemical processes controlling CaCO3 precipitation from aqueous solution, essentially local supersaturation with respect to carbonates induced by photosynthesis outside the living cells and Ca adsorption onto cell surface and the associated extracellular polymeric substances (EPS).The APB can physiologically control their surface potential to electrostatically attract nutrients at alkaline pH, while rejecting Ca ions to prevent Ca adsorption and subsequent CaCO3 precipitation in the vicinity of cell surface and thus, cell incrustation. In contrast to other previously-investigated calcifying bacteria, no cellular protection mechanism against Ca2+ adsorption and subsequent carbonate precipitation has been evidenced for cyanobacteria Gloeocapsa sp. f-6gl. This is most likely linked to the peculiar cellular organization of this species that involves several cells clustered in one single capsule. In this regard, colony-forming, EPS-rich, capsular cyanobacteria may be among the most efficient calcifying microorganisms all along the Earth history and can be of interest for various technological applications involving carbonation.

Physicochemical, catalytic, and regulatory properties of malate dehydrogenase from Rhodovulum steppense bacteria, strain A-20s

The physicochemical, regulatory, and kinetic properties of malate dehydrogenase (EC 1.1.1.37) from haloalkaliphilic purple nonsulfur Rhodovulum steppense bacteria, strain A-20s, were studied. The malate dehydrogenase (MDH) preparation with a specific activity of 0.775 ± 0.113 U/mg protein was obtained in an electrophoretically homogeneous state using multistep purification. Using homogenous preparations, the molecular weight and the Michaelis constant of the enzyme were determined; the effects of metal ions, the temperature effect, and the thermal stability of the MDH were studied. The dimer structure of the enzyme was demonstrated by DS-Na-electrophoresis.

Chemical and structural status of copper associated with oxygenic and anoxygenic phototrophs and heterotrophs: possible evolutionary consequences

Copper adsorption on the surface and intracellular uptake inside the cells of four representative taxons of soil and aquatic micro-organisms: aerobic rhizospheric heterotrophs (Pseudomonas aureofaciens), anoxygenic (Rhodovulum steppense) and oxygenic (cyanobacteria Gloeocapsa sp. and freshwater diatoms Navicula minima) phototrophs were studied in a wide range of pH, copper concentration, and time of exposure. Chemical status of adsorbed and assimilated Cu was investigated using in situ X-ray absorption spectroscopy. In case of adsorbed copper, XANES spectra demonstrated significant fractions of Cu(I) likely in the form of tri-coordinate complexes with O/N and/or S ligands. Upon short-term reversible adsorption at all four studied micro-organisms' cell surface, Cu(II) is coordinated by 4.0 ± 0.5 planar oxygens at an average distance of 1.97 ± 0.02 Å, which is tentatively assigned to the carboxylate groups. The atomic environment of copper incorporated into diatoms and cyanobacteria during long-term growth is similar to that of the adsorbed metal with slightly shorter distances to the first O/N neighbor (1.95 Å). In contrast to the common view of Cu status in phototrophic micro-organisms, XAFS failed to detect sulfur in the nearest atomic environment of Cu assimilated by freshwater plankton (cyanobacteria) and periphyton (diatoms). The appearance of S in Cu 1st coordination shell at 2.27-2.32 Å was revealed only after long-term interaction of Cu with anoxygenic phototrophs (and Cu uptake by soil heterotrophs), suggesting Cu scavenging in the form of sulfhydryl, histidine/carboxyl or a mixture of carboxylate and sulfhydryl complexes. These new structural constraints suggest that adsorbed Cu(II) is partially reduced to Cu(I) already at the cell surface, where as intracellular Cu uptake and storage occur in the form of both Cu(I)-S linked proteins and Cu(II) carboxylates. Obtained results allow to better understand how, in the course of biological evolution, micro-organisms elaborated various mechanisms of Cu uptake and storage, from passive adsorption and uptake to active, protein-controlled surface reduction, and intracellular storage.

Calcium carbonate precipitation by anoxygenic phototrophic bacteria

Carbonate biomineralization is considered as one of the main natural processes controlling CO 2 levels in the atmosphere both in the past and at present time. In contrast to extensive studies of cyanobacterial calcification, biomineralization of anoxygenic phototrophic bacteria (APB) remained largely underestimated, despite their potentially important role on CaCO 3 precipitation in the biomats, notably in the past. Haloalcaliphilic Rhodovulum steppense A-20s and halophilic neutrophilic Rhodovulum sp. S-17-65 were examined with respect to their ability to precipitate CaCO 3 under controlled laboratory conditions. To characterize the link between the rate of bacterial growth (biomass production) and the rate of CaCO 3 precipitation, batch kinetic experiments with live, dead and inactivated bacteria both in nutrient solution and in inert electrolyte were performed and produced precipitates were examined by SEM, TEM and XRD techniques. Active strains A-20s and S-17-65 precipitated calcite from initially supersaturated solutions ( Ω calcite = 10 to 40) via increasing Ω calcite to 80–120 before the precipitation. The amount of precipitated CaCO 3 (mole) was directly correlated with the amount of organic C in bacterial biomass produced with a slope of dependence ranging from 0.3 to 0.6 and from 0.1 to 0.3 for A-20s and S-17-65, respectively, depending on the initial solution composition. For both bacterial strains, only live actively photosynthetizing bacteria were capable of effectively decreasing Ca concentration and form CaCO 3 with apparent bulk precipitation rates ranging from 0.001 to 0.0150 mmol/h at 10–20 g wet/L of biomass, similar to rates reported for other bacteria. SEM and XRD analyses of precipitates reveal the dominance of calcite with some amount of vaterite and monohydrocalcite forming spheres up to 100 μm diameter. The TEM analysis of bacterial suspension at the end of precipitation experiments did not demonstrate the presence of CaCO 3 at the surface or in the vicinity of live cells. This suggests the existence of certain cell protection mechanism against carbonate precipitation at the cell surface. Given the lower efficiency of photoheterotrophic APB, compared to photoautotrophic cyanobacteria, to precipitate CaCO 3 in natural conditions, it is possible that the overall potential of phototrophic community to form massive carbonate deposits was strongly limited before the appearance of oxygenic phototrophs.

Interaction of the haloalkaliphilic purple bacteria Rhodovulum steppense with aluminosilicate minerals

The interaction was investigated between the haloalkaliphilic nonsulfur purple bacteria Rhodovulum steppense A-20sT and layered aluminosilicates: micas (biotite, phlogopite, and muscovite) and clay minerals (montmorillonite and kaolinite). The interaction between all components of this system (minerals, water, medium, and bacteria) resulted in the changes in the chemical composition of the minerals and solutions. These changes were especially significant in the presence of bacteria. By using some elements for growth and promoting their transfer into the exchange pool of the minerals, bacteria removed these elements from the medium. The content of exchange bases in the aluminosilicates incubated in the presence of bacteria was several times higher than in the minerals incubated in sterile medium. The observed saturation of the mineral phase with potassium and magnesium may be considered the initial phase of diagenesis of the aluminosilicates under study.

Zeta potential of anoxygenic phototrophic bacteria and Ca adsorption at the cell surface: Possible implications for cell protection from CaCO3 precipitation in alkaline solutions

Electrophoretic mobility measurements and surface adsorption of Ca on living, inactivated, and heat-killed haloalkaliphilic Rhodovulum steppense, A-20s, and halophilic Rhodovulum sp., S-17-65 anoxygenic phototrophic bacteria (APB) cell surfaces were performed to determine the degree to which these bacteria metabolically control their surface potential equilibria. Zeta potential of both species was measured as a function of pH and ionic strength, calcium and bicarbonate concentrations. For both live APB in 0.1M NaCl, the zeta potential is close to zero at pH from 2.5 to 3 and decreases to −30 to −40mV at pH of 5–8. In alkaline solutions, there is an unusual increase of zeta potential with a maximum value of −10 to −20mV at a pH of 9–10.5. This increase of zeta potential in alkaline solutions is reduced by the presence of NaHCO3 (up to 10mM) and only slightly affected by the addition of equivalent amount of Ca. At the same time, for inactivated (exposure to NaN3, a metabolic inhibitor) and heat-killed bacteria cells, the zeta potential was found to be stable (−30 to −60mV, depending upon the ionic strength) between pH 5 and 11 without any increase in alkaline solutions. Adsorption of Ca ions on A-20s cells surface was more significant than that on S-17-65 cells and started at more acidic pHs, consistent with zeta potential measurements in the presence of 0.001–0.01mol/L CaCl2. Overall, these results indicate that APB can metabolically control their surface potential to electrostatically attract nutrients at alkaline pH, while rejecting/avoiding Ca ions to prevent CaCO3 precipitation in the vicinity of cell surface and thus, cell incrustation.

Rhodovulum steppense sp. nov., an obligately haloalkaliphilic purple nonsulfur bacterium widespread in saline soda lakes of Central Asia

Seven strains of purple nonsulfur bacteria isolated from the shallow-water steppe soda lakes of the cryoarid zone of Central Asia formed a genetically homogeneous group within the genus Rhodovulum. The isolates were most closely related to Rhodovulum strictum, from which they differed at the species level (99.5 % 16S rRNA gene identity and 42-44 % DNA-DNA hybridization level). According to genotypic and phenotypic characteristics, the strains were assigned to a new species of the genus Rhodovulum, for which the name Rhodovulum steppense sp. nov. is proposed. Cells of all strains were ovoid to rod-shaped, 0.3-0.8 microm wide and 1-2.5 microm long, and motile by means of polar flagella. They contained internal photosynthetic membranes of the vesicular type and photosynthetic pigments (bacteriochlorophyll a and carotenoids of the spheroidene series). All strains were obligate haloalkaliphiles, growing within a wide range of salinity (0.3-10 %) and pH (7.5-10), with growth optima at 1-5 % NaCl and pH 8.5. Photo- and chemoheterotrophic growth occurred with a number of organic compounds and biotin, thiamine and niacin as growth factors. No anaerobic respiration on nitrite, nitrate or fumarate and no fermentation was demonstrated. Bacteria grew photo- and chemolithoautotrophically with sulfide, sulfur and thiosulfate, oxidizing them to sulfate. Sulfide was oxidized via deposition of extracellular elemental sulfur. No growth with H(2) as electron donor was demonstrated. The major fatty acid was 18 : 1 (81.0 %). The major quinone was Q-10. The DNA G+C content was 66.1 mol% (T(m)). The type strain, A-20s(T) (=VKM B-2489(T) =DSM 21153(T)), was isolated from soda lake Khilganta (Zabaikal'skii Krai, southern Siberia, Russia).