Spectral and Photochemical Properties of Rhodobacter sphaeroides R-26 Reaction Center Films in Vacuum.

Using absorption spectroscopy in the visible/near-IR and mid-IR regions, spectral and photochemical properties of isolated reaction centers (RCs) from Rhodobacter sphaeroides R-26 were studied in dried films on the inorganic support surface (quartz or CaF2 plates) under vacuum dehydration conditions (10-2 or 7·10-5 mm Hg). Three detergents, N,N-dimethyldodecylamine N-oxide (LDAO), Triton X-100 (TX100), and n-dodecyl-ß-D-maltoside (DM), were tested for their ability to stabilize the RC-detergent complexes in the vacuum-dried state. It was shown that in the presence of LDAO, RC complexes underwent destruction in vacuum. In contrast, DM provided an environment that minimized irreversible disruptive changes in the RCs in vacuum. The effects of vacuum dehydration on the RC-DM films included a small increase in the content of α-helices in the RC protein, a short-wavelength reversible shift in the optical transitions of pigments, and minor changes in the electronic structure of the P+ dimer. The films retained their photochemical activity upon excitation with high-intensity light (200 mW/cm2). TX100 also helped to maintain spectral and functional properties of the RCs in vacuum; however, in this case, the stabilizing effect was less pronounced than in the presence of DM, especially, at high detergent concentrations. The results are discussed within the framework of a model suggesting that the detergent-protein interactions and the properties of detergent micelles play a dominant role in maintaining the structure of the RCs upon vacuum dehydration of the RC complexes. The obtained data can be useful for developing hybrid photoconverting systems based on bacterial RCs.

Isotope discrimination by form IC RubisCO from Ralstonia eutropha and Rhodobacter sphaeroides, metabolically versatile members of 'Proteobacteria' from aquatic and soil habitats.

RubisCO, the CO2 fixing enzyme of the Calvin-Benson-Bassham (CBB) cycle, is responsible for the majority of carbon fixation on Earth. RubisCO fixes 12 CO2 faster than 13 CO2 resulting in 13 C-depleted biomass, enabling the use of δ13 C values to trace CBB activity in contemporary and ancient environments. Enzymatic fractionation is expressed as an ε value, and is routinely used in modelling, for example, the global carbon cycle and climate change, and for interpreting trophic interactions. Although values for spinach RubisCO (ε = ~29‰) have routinely been used in such efforts, there are five different forms of RubisCO utilized by diverse photolithoautotrophs and chemolithoautotrophs and ε values, now known for four forms (IA, B, D and II), vary substantially with ε = 11‰ to 27‰. Given the importance of ε values in δ13 C evaluation, we measured enzymatic fractionation of the fifth form, form IC RubisCO, which is found widely in aquatic and terrestrial environments. Values were determined for two model organisms, the 'Proteobacteria' Ralstonia eutropha (ε = 19.0‰) and Rhodobacter sphaeroides (ε = 22.4‰). It is apparent from these measurements that all RubisCO forms measured to date discriminate less than commonly assumed based on spinach, and that enzyme ε values must be considered when interpreting and modelling variability of δ13 C values in nature.

Bioremediation of lead contaminated soil with Rhodobacter sphaeroides.

Bioremediation with microorganisms is a promising technique for heavy metal contaminated soil. Rhodobacter sphaeroides was previously isolated from oil field injection water and used for bioremediation of lead (Pb) contaminated soil in the present study. Based on the investigation of the optimum culturing conditions and the tolerance to Pb, we employed the microorganism for the remediation of Pb contaminated soil simulated at different contamination levels. It was found that the optimum temperature, pH, and inoculum size for R. sphaeroides is 30-35 °C, 7, and 2 × 10(8) mL(-1), respectively. Rhodobacter sphaeroides did not remove the Pb from soil but did change its speciation. During the bioremediation process, more available fractions were transformed to less accessible and inert fractions; in particular, the exchangeable phase was dramatically decreased while the residual phase was substantially increased. A wheat seedling growing experiment showed that Pb phytoavailability was reduced in amended soils. Results inferred that the main mechanism by which R. sphaeroides treats Pb contaminated soil is the precipitation formation of inert compounds, including lead sulfate and lead sulfide. Although the Pb bioremediation efficiency on wheat was not very high (14.78% root and 24.01% in leaf), R. sphaeroides remains a promising alternative for Pb remediation in contaminated soil.

Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics.

Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu.

Phytoavailability and geospeciation of cadmium in contaminated soil remediated by Rhodobacter sphaeroides.

A microorganism was isolated from oil field injection water and identified as Rhodobacter sphaeroides. It was used for the remediation of simulated cadmium-contaminated soil. The phytoavailability of Cd was investigated through wheat seedling method to determine the efficiency of remediation. It was found that after remediation, the accumulation of Cd in wheat roots and leaves decreased by 67% and 53%, respectively. The Cd speciation in soil was determined with Tessier extraction procedure. It was found that the total Cd content in soil did not change during the experiments, but the geo-speciation of Cd changed remarkably. Among the five fractions, the concentration of exchangeable phases decreased by 27-46% and that of the phases bound to Fe-Mn oxides increased by 22-44%. The decrease of Cd accumulation in wheat showed significant positive correlation with the decrease of exchangeable phases. It could be concluded that the remediation of R. sphaeroides was carried out through the conversion of Cd to more stable forms. The decrease of sulfate concentration in supernatant indicated that the R. sphaeroides consumed sulfate.

Genome sequence of Rhodobacter sphaeroides Strain WS8N.

Rhodobacter sphaeroides is a metabolically diverse photosynthetic alphaproteobacterium found ubiquitously in soil and freshwater habitats. Here we present the annotated genome sequence of R. sphaeroides WS8N.

Proton motive force in Rhodobacter sphaeroides under anaerobic conditions in the dark.

In order to examine the mediatory role of proton motive force (∆p) or proton ATPase in H2 production by Rhodobacter sphaeroides, ∆p was determined under anaerobic conditions in the dark, and the ATPase activity has been studied in R. sphaeroides strain A-10, isolated from Arzni mineral springs in Armenia. Membrane potential (∆φ) was measured from the distribution of tetraphenylphosphonium cation; pH gradient (∆pH) was the difference between the external and cytoplasmic pH values, and the latter was measured by 9-aminoacridine (9-AA) fluorescence changes. At pH 7.5, ∆φ was of -94 mV and the reversed ∆pH was +30 mV, resulting in ∆p of -64 mV. The addition of N,N'-dicyclohexylcarbodiimide (DCCD), the F0F1-ATPase inhibitor, was not affect ∆φ. It was shown that ∆φ varies nearly linearly with ΔpH, ∆φ increased from -57.1 mV at pH 6.0 to -103.8 mV at pH 8.0; it was compensated at high external pH by a reversed ∆pH, resulting in a low ∆p under anaerobic-dark conditions. Intracellular ATP concentrations and energetic charge (EC) were measured to evaluate a metabolism activity of R. sphaeroides.

Isolation of a thermotolerant photosynthetic bacterium, Rhodobacter sphaeroides Strain, NAT, and its capacity for oil and chemical oxygen demand removal at high temperatures.

A thermotolerant photosynthetic bacterium NAT identified as Rhodobacter sphaeroides was isolated. When alginate-immobilized cells of strain NAT were used in high-temperature treatment of artificial sewage wastewater containing oil, the chemical oxygen demand (COD) decreased by 80% and 76% of the oil was removed after 96 h of treatment at 55 degrees C. Lipase activity was observed in the culture.

Molecular genetic analysis of a dimethylsulfoniopropionate lyase that liberates the climate-changing gas dimethylsulfide in several marine alpha-proteobacteria and Rhodobacter sphaeroides.

The alpha-proteobacterium Sulfitobacter EE-36 makes the gas dimethylsulfide (DMS) from dimethylsulfoniopropionate (DMSP), an abundant antistress molecule made by many marine phytoplankton. We screened a cosmid library of Sulfitobacter for clones that conferred to other bacteria the ability to make DMS. One gene, termed dddL, was sufficient for this phenotype when cloned in pET21a and introduced into Escherichia coli. Close DddL homologues exist in the marine alpha-proteobacteria Fulvimarina, Loktanella Oceanicola and Stappia, all of which made DMS when grown on DMSP. There was also a dddL homologue in Rhodobacter sphaeroides strain 2.4.1, but not in strain ATCC 17025; significantly, the former, but not the latter, emits DMS when grown with DMSP. Escherichia coli containing the cloned, overexpressed dddL genes of R. sphaeroides 2.4.1 and Sulfitobacter could convert DMSP to acrylate plus DMS. This is the first identification of such a 'DMSP lyase'. Thus, DMS can be made either by this DddL lyase or by a DMSP acyl CoA transferase, specified by dddD, a gene that we had identified in several other marine bacteria.

In vitro assessment of gastrointestinal viability of two photosynthetic bacteria, Rhodopseudomonas palustris and Rhodobacter sphaeroides.

The objectives of this study were to assess the potential of two photosynthetic bacteria (PSB), Rhodopseudomonas palustris HZ0301 and Rhodobacter sphaeroides HZ0302, as probiotics in aquaculture. The viability of HZ0301 and HZ0302 in simulated gastric transit conditions (pH 2.0, pH 3.0 and pH 4.0 gastric juices) and in simulated small intestinal transit conditions (pH 8.0, with or without 0.3% bile salts) was tested. The effects of HZ0301 and HZ0302 on the viability and permeability of intestinal epithelial cell in primary culture of tilapias, Oreochromis nilotica, were also detected. All the treatments were determined with three replicates. The simulated gastric transit tolerance of HZ0301 and HZ0302 strains was pH-dependent and correspondingly showed lower viability at pH 2.0 after 180 min compared with pH 3.0 and pH 4.0. Both HZ0301 and HZ0302 were tolerant to simulated small intestine transit with or without bile salts in our research. Moreover, there was no significant difference (P>0.05) among three treatments including the control and the groups treated with HZ0301 or HZ0302 both in intestinal epithelial cell viability and membrane permeability, showing no cell damage. In summary, this study demonstrated that HZ0301 and HZ0302 had high capacity of upper gastrointestinal transit tolerance and were relatively safe for intestinal epithelial cells of tilapias.

Biosynthesis of intracellular 5-aminolevulinic acid by a newly identified halotolerant Rhodobacter sphaeroides.

Of 23 strains of halotolerant (up to 12% w/v NaCl) photosynthetic bacteria isolated from various sources, one isolate, SH5, accumulated intracellular 5-aminolevulinic acid (ALA) at 0.45 microg/g dry cell wt (DCW) growing aerobically in the dark. The strain was identified as Rhodobacter sphaeroides using 16S rDNA sequencing. Biosynthesis of ALA was enhanced to 14 microg/g DCW using modified glutamate/glucose (50 mM) medium with the addition of 10 mM levulinic acid after 24 h cultivation. Addition of 30 microM Fe(2+) to this medium increased the yield to 226 microg/g DCW.

Identification of two gene loci involved in poly-beta-hydroxybutyrate production in Rhodobacter sphaeroides FJ1.

BACKGROUND AND PURPOSE: Polyhydroxyalkanoates (PHAs), biopolyesters of hydroxyl fatty acids, are synthesized and deposited as cytoplasmic inclusions in many bacteria. We isolated a poly-beta-hydroxybutyrate (PHB)-producing bacterium designated Rhodobacter sphaeroides FJ1. To characterize PHB biosynthesis in this organism, we isolated the genes encoding proteins involved in PHB metabolism. METHODS: The genes responsible for the synthesis, accumulation, and degradation of PHB in R. sphaeroides FJ1 were cloned and characterized. RESULTS: Genes involved in the biosynthesis and metabolism of PHB were found to be located in 2 different loci in the genome of R. sphaeroides FJ1. One locus contained genes encoding PHB depolymerase (phbZ), PHB synthase (phbC), phasin (phbP) and the regulator protein (phbR). The other locus contained the beta-ketothiolase gene (phbA) and the acetoactyl-CoA reductase gene (phbB). The phbZ gene was orientated in an opposite direction to that of phbC, phbP and phbR genes that were located in the same cluster. R. sphaeroides FJ1 was able to grow in wastewater released from the human waste treatment plant of Fu-Jen University. Optimal growth and PHB production were achieved when R. sphaeroides FJ1 was grown in tryptic soy broth containing 50% wastewater. PHB production by R. sphaeroides FJ1 varied in media with different carbon to nitrogen ratios, but the level of PHB synthase was constant, suggesting that PHB production depends mainly on substrate supply. CONCLUSIONS: Six genes encoding proteins related to PHB metabolism are clustered in 2 separate loci, phbZCPR and phbAB, in a PHB-producing bacterium R. sphaeroides FJ1 isolated from wastewater. PHB synthase, the key enzyme for PHB synthesis, is constitutively expressed, and its expression level is not affected by different growth conditions.

Isolation and characterization of heterotrophic bacteria able to grow aerobically with quaternary ammonium alcohols as sole source of carbon and nitrogen.

The quaternary ammonium alcohols (QAAs) 2,3-dihydroxypropyl-trimethyl-ammonium (TM), dimethyl-diethanol-ammonium (DM) and methyl-triethanol-ammonium (MM) are hydrolysis products of their parent esterquat surfactants, which are widely used as softeners in fabric care. We isolated several bacteria growing with QAAs as the sole source of carbon and nitrogen. The strains were compared with a previously isolated TM-degrading bacterium, which was identified as a representative of the species Pseudomonas putida (Syst. Appl. Microbiol. 24 (2001) 252). Two bacteria were isolated with DM, referred to as strains DM 1 and DM 2, respectively. Based on 16S-rDNA analysis, they provided 97% (DM 1) and 98% (DM 2) identities to the closest related strain Zoogloea ramigera Itzigsohn 1868AL. Both strains were long, slim, motile rods but only DM 1 showed the floc forming activity, which is typical for representatives of the genus Zoogloea. Using MM we isolated a Gram-negative, non-motile rod referred to as strain MM 1. The 16S-rDNA sequence of the isolated bacterium revealed 94% identities (best match) to Rhodobacter sphaeroides only. The strains MM 1 and DM 1 exclusively grew with the QAA which was used for their isolation. DM 2 was also utilizing TM as sole source of carbon and nitrogen. However, all of the isolated bacteria were growing with the natural and structurally related compound choline.

Phenotypic characterisation and genetic complementation of dimethylsulfoxide respiratory mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus.

Two chlorate resistant mutants of Rhodobacter sphaeroides were isolated which were deficient in dimethylsulfoxide reductase activity. Immunoblotting experiments showed that the phenotype of these mutants and that of Rhodobacter capsulatus strain DK9, a mutant unable to reduce dimethylsulfoxide, was correlated with low or undetectable levels of the dimethylsulfoxide reductase apoprotein. All three mutants were complemented by a cosmid from a library of Rhodobacter sphaeroides genomic DNA. Further genetic complementation analysis revealed that functions required for restoration of dimethylsulfoxide reductase activity in the Rhodobacter sphaeroides mutants were encoded on an 9 kb EcoR1 DNA fragment derived from this cosmid. Expression of this 9 kb DNA fragment in Escherichia coli showed that it encoded the dimethylsulfoxide reductase structural gene of Rhodobacter sphaeroides.

Reductive pentose phosphate-independent CO2 fixation in Rhodobacter sphaeroides and evidence that ribulose bisphosphate carboxylase/oxygenase activity serves to maintain the redox balance of the cell.

Whole-cell CO2 fixation and ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity were determined in Rhodobacter sphaeroides wild-type and mutant strains. There is no obvious difference in the levels of whole-cell CO2 fixation for the wild type, a form I RubisCO deletion mutant, and a form II RubisCO deletion mutant. No ribulose 1,5-bisphosphate-dependent CO2 fixation was detected in a form I-form II RubisCO double-deletion mutant (strain 16) or strain 16PHC, a derivative from strain 16 which was selected for the ability to grow photoheterotrophically with CO2 as an electron acceptor. However, significant levels of whole-cell CO2 fixation were detected in both strains 16 and 16PHC. Strain 16PHC exhibited CO2 fixation rates significantly higher than those of strain 16; the rates found for strain 16PHC were 30% of the level found in photoheterotrophically grown wild-type strain HR containing both form I and form II RubisCO and 10% of the level of the wild-type strain grown photolithoautotrophically. Strain 16PHC could not grow photolithoautotrophically in a CO2-H2 atmosphere; however, CO2 fixation catalyzed by photoheterotrophically grown strain 16PHC was repressed by addition of the alternate electron acceptor dimethyl sulfoxide. Dimethyl sulfoxide addition also influenced RubisCO activity under photolithoautotrophic conditions; 40 to 70% of the RubisCO activity was reduced without significantly influencing growth. Strain 16PHC and strain 16 contain nearly equivalent but low levels of pyruvate carboxylase, indicating that CO2 fixation enzymes other than pyruvate carboxylase contribute to the ability of strain 16PHC to grow with CO2 as an electron acceptor.

Isolation, characterization, and complementation of a paralyzed flagellar mutant of Rhodobacter sphaeroides WS8.

A paralyzed Rhodobacter sphaeroides mutant strain (PARA1) was isolated by a motility screening procedure following mutagenesis of wild-type R. sphaeroides WS8-N with the transposable element TnphoA (Tn5 IS50L::phoA). PARA1 synthesized a wild-type level of flagellin, as detected by Western immunoblotting with antiflagellar antiserum. Flagellar staining showed that flagellin was assembled into apparently normal external flagellar filaments. Electron micrographs of basal body structures from PARA1 showed that some ring structures that were present were similar to those in wild-type R. sphaeroides WS8-N. PARA1 cells were nonmotile under all growth conditions. No pseudorevertants to motility were seen when PARA1 was grown in the presence of kanamycin to select for the presence of the transposon. The presence of the single copy of TnphoA in the PARA1 chromosome was demonstrated by Southern blotting. Western blotting of cytoplasmic, periplasmic, and membrane fractions of PARA1 with anti-alkaline phosphatase antiserum showed that the transposon had been inserted in-frame into a gene encoding a membrane protein. A SalI restriction endonuclease fragment was cloned from the chromosome of PARA1; this fragment contained a portion of the transposon and R. sphaeroides DNA sequence 5' of the site of insertion. This flanking R. sphaeroides DNA sequence was used to probe an R. sphaeroides WS8 cosmid library. A cosmid designated c19 hybridized to the probe, and a SalI restriction endonuclease fragment derived from this cosmid restored wild-type motility to PARA1 when introduced into this mutant strain by conjugation. The significance of this finding in a bacterium with unidirectionally rotating flagella is discussed.

[Selective method of isolating mutants sensitive to ultraviolet radiation from a culture of Rhodopseudomonas sphaeroides]. / Selektivnyi metod vydeleniia mutantov, chuvstvitel'nykh k ul'trafioletovomu izlucheniiu, kul'tury Rhodopseudomonas sphaeroides.

The method of penicillin selection used after UV-irradiation (lambda = 254 nm) allows one to select UV-sensitive mutants (uvs-mutants) of the phototrophous bacterium Rhodopseudomonas sphaeroides induced by nitrosomethylurea with an effectiveness greater by an order of magnitude. Over 30% of the uvs-mutants obtained using this method had an elevated sensitivity not only to far-UV (F-UV, lambda = 254 nm) but also to near-UV (N-UV, lambda greater than 280 nm) UV-irradiation. No correlation was found in the degree of sensitivity to F-UV and N-UV-irradiation of the uvs-mutants. Mutants highly sensitive to the lethal action of N-UV were isolated.

[Isolation of auxotrophic mutants of Rhodopseudomonas sphaeroides]. / Vydelenie auksotrofnykh mutantov u Rhodopseudomonas sphaeroides.

A collection of stable auxotrophic mutants with a frequency of reversions to the wild type below 10(-8) was obtained from the wild strain of Rhodopseudomonas sphaeroides 2R under the action of nitrosoguanidine or nitrosomethylurea. The mutants required methionine, leucine, arginine, histidine, tryptophan and cytosine for their growth. Mutants carrying 2 or 3 auxotrophic labels in their genome were selected from the primary auxotrophs under the action of UV (lambda = 254 nm). The paper describes a semi-enriched medium in which the diameter of prototrophic colonies reaches 5 mm while that of auxotrophic colonies does not exceed 1-2 mm. The selection of microcolonies from this medium makes it possible to increase the yield of auxotrophs several times as compared with the technique of imprints.