Decolorization of azo dyes by Rhodobacter sphaeroides

Rhodobacter sphaeroides AS1.1737 decolorized more than 90% of several azo dyes (200 mg dyes l^–1) in 24 h. The optimal culture conditions were: anaerobic illumination (1990 lx), peptone as carbon source, temperature 35–40 °C and pH 7–8. Intracellular crude enzyme from this strain had azoreductase activity, optimized temperature as 45–50 °C, and decolorization kinetics which were consistent with a ping-pong mechanism.     

Bioremediation of cadmium by growing Rhodobacter sphaeroides: kinetic characteristic and mechanism studies

The removal kinetic characteristic and mechanism of cadmium by growing Rhodobacter sphaeroides were investigated. The removal data were fitted to the second-order equation, with a correlation coefficient, R2=0.9790-0.9916. Furthermore, it was found that the removal mechanism of cadmium was predominantly governed by bioprecipitation as cadmium sulfide with biosorption contributing to a minor extent. Also, the results revealed that the activities of cysteine desulfhydrase in strains grown in the presence of 10 and 20 mg/l of cadmium were higher than in the control, while the activities in the presence of 30 and 40 mg/l of cadmium were lower than in the control. Content analysis of subcellular fractionation showed that cadmium was mostly removed and transformed by precipitation on the cell wall.     

Expression,purification and characterisation of full-length histidine protein kinase RegB from Rhodobacter sphaeroides

The global redox switch between aerobic and anaerobic growth in Rhodobacter sphaeroides is controlled by the RegA/RegB two-component system, in which RegB is the integral membrane histidine protein kinase, and RegA is the cytosolic response regulator. Despite the global regulatory importance of this system and its many homologues, there have been no reported examples to date of heterologous expression of full-length RegB or any histidine protein kinases. Here, we report the amplified expression of full-length functional His-tagged RegB in Escherichia coli, its purification, and characterisation of its properties. Both the membrane-bound and purified solubilised RegB protein demonstrate autophosphorylation activity, and the purified protein autophosphorylates at the same rate under both aerobic and anaerobic conditions confirming that an additional regulator is required to control/inhibit autophosphorylation. The intact protein has similar activity to previously characterised soluble forms, but is dephosphorylated more rapidly than the soluble form (half-life ca 30 minutes) demonstrating that the transmembrane segment present in the full-length RegB may be an important regulator of RegB activity. Phosphotransfer from RegB to RegA (overexpressed and purified from E. coli) by RegB is very rapid, as has been reported for the soluble domain. Dephosphorylation of active RegA by full-length RegB has a rate similar to that observed previously for soluble RegB.     

Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides

Background

High intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the α-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen.

Results

One third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation.

Conclusion

Some R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a “core iron response” that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-794) contains supplementary material, which is available to authorized users.     

Acid denaturation of the B875 light-harvesting complex in membranes of Rhodobacter sphaeroides

The structural basis for the spectral red shift in the near-IR absorption band of the B875 light-harvesting complex was examined by treatment of membranes from Rhodobacter sphaeroides M21 with acid. This mutant strain lacks the overlapping spectral bands of the B800–850 light-harvesting antenna and gives rise to membrane fragments with both surfaces accessible to protons. At pH 2.2, about half the absorption at 876 nm was converted within 10 min to a free pigment band; the remaining absorption appeared at 880 nm and shifted to 845 nm over the next three hours. These spectral shifts could not be reversed by alkali. Approximately one-third of the characteristic near-IR CD signal of B875 was also lost initially and replaced by a broad trough centered near 854 nm. Thereafter, the CD spectrum was dominated by the strong conservative signal of the 845 nm absorbing component which was attributed to an oligomeric bacteriopheophytin a species, probably a dimer. A kinetic analysis of the acid-induced absorption changes suggested a multi-step model with rate constants of 0.37 min^-1 for the initial rapid change and 0.05 and 0.11 min^-1 for the respective subsequent steps. The non-conservative nature of the near-IR CD spectrum of the intact complex, together with the spectral changes observed after the initial loss of near-IR absorption and CD, suggest that pigment-pigment interactions are not solely responsible for the red shift in this complex.Abbreviations BChl bacteriochlorophyll a - BPheo bacteriopheophytin a     

光合菌Rhodobacter sphaeroides 601hupT基因的克隆与功能分析

从紫色非硫光合细菌Rhodobacter sphaeroides 601的吸氢酶(hup)基因簇中,克隆了hupT基因,并对该基因进行了测序,分析了由其推测的氨基酸序列的同源性.hupT基因全长1 332 bp,编码一分子量约为48 23 kD的蛋白.将hupT基因引入大肠杆菌进行了体外表达.纯化基因产物HupT,并进行HupT的自身磷酸化分析.结果表明,HupT属于双组份调节系统中的组氧酸蛋白激酶.将hupT基因导入光合菌Rhodobacter capsulatus吸氢酶负调节基因突变株BSE8后.野生型吸氢酶的表型得以恢复,说明所克隆的R.sphaeroides 601中的hupT基因在吸氢酶的表达中起负调节作用.     

Interactions between the Rhodobacter sphaeroides ECF sigma factor, sigma(E), and its anti-sigma factor, ChrR

Rhodobacter sphaeroides sigma(E) is a member of the extra cytoplasmic function sigma factor (ECF) family, whose members have been shown to regulate gene expression in response to a variety of signals. The functions of ECF family members are commonly regulated by a specific, reversible interaction with a cognate anti-sigma factor. In R.sphaeroides, sigma(E) activity is inhibited by ChrR, a member of a newly discovered family of zinc containing anti-sigma factors. We used gel filtration chromatography to gain insight into the mechanism by which ChrR inhibits sigma(E) activity. We found that formation of the sigma(E):ChrR complex inhibits the ability of sigma(E) to form a stable complex with core RNA polymerase. Since the sigma(E):ChrR complex inhibits the ability of the sigma factor to bind RNA polymerase, we sought to identify amino acid substitutions in sigma(E) that altered the sensitivity of this sigma factor to inhibition by ChrR. This analysis identified single amino acid changes in conserved region 2.1 of sigma(E) that either increased or decreased the sensitivity of sigma(E) for inhibition by ChrR. Many of the amino acid residues that alter the sensitivity of sigma(E) to ChrR are located within regions known to be important for interacting with core RNA polymerase in other members of the sigma(70) superfamily. Our results suggest a model where solvent-exposed residues with region 2.1 of sigma(E) interact with ChrR to sterically occlude this sigma factor from binding core RNA polymerase and to inhibit target gene expression.     

Biological Synthesis of Semiconductor Zinc Sulfide Nanoparticles by Immobilized Rhodobacter sphaeroides

A novel, clean biological transformation reaction by immobilized Rhodobacter sphaeroides has been developed for the synthesis of zinc sulfide (ZnS) nanoparticles with an average diameter of 8 nm. The nanoparticles were examined by X-ray diffraction, transmission electron microscopy, energy dispersive analyses of X-rays, UV–vis optical absorption and photoluminescence spectra. The average diameter of ZnS nanoparticles varied according to the culture time.     

The chemotaxis response regulator CheY can catalyze its own acetylation

One of the processes by which CheY, the excitatory response regulator of chemotaxis in Escherichia coli, can be activated to generate clockwise flagellar rotation is by acetyl-CoA synthetase (Acs)-mediated acetylation. Deletion of Acs results in defective chemotaxis, indicating the involvement of Acs-mediated acetylation in chemotaxis. To investigate whether Acs is the sole acetylating agent of CheY, we purified the latter from a delta acs mutant. Mass spectrometry analysis revealed that this protein is partially acetylated in spite of the absence of Acs, suggesting that CheY can be post-translationally acetylated in vivo by additional means. Using [14C]AcCoA in the absence of Acs, we demonstrated that one of these means is autoacetylation, with AcCoA serving as an acetyl donor and with a rate similar to that of Acs-mediated acetylation. Biochemical characterization of autoacetylated CheY and mass spectrometry analysis of its tryptic digests revealed that its acetylated lysine residues are those found in CheY acetylated by Acs, but the acetylation-level distribution among the acetylation sites was different. Like CheY acetylated by Acs, autoacetylated CheY could be deacetylated by Acs. Also similarly to the case of Acs-mediated acetylation, the phosphodonors of CheY, CheA and acetyl phosphate, each inhibited the autoacetylation of CheY, whereas the phosphatase of CheY, CheZ, enhanced it. A reduced AcCoA level interfered with chemotaxis to repellents, suggesting that CheY autoacetylation may be involved in chemotaxis of E. coli. Interestingly, this interference was restricted to repellent addition and was not observed with attractant removal, thus endorsing our earlier suggestion that the signaling pathway triggered by repellent addition is not identical to that triggered by attractant removal.     

Effects of light/dark cycle, mixing pattern and partial pressure of H2 on biohydrogen production by Rhodobacter sphaeroides ZX-5

The effects of light/dark cycle, mixing pattern and partial pressure of H₂ on the growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated. The results from light/dark cycle culture showed that little or no hydrogen production was observed during the dark periods, and the hydrogen production immediately recovered once illumination was resumed. Also, it was found that the optimum condition of shaking velocity was 120 rpm for hydrogen photo-fermentation. Meanwhile, shaking during H₂ production phase (i.e., cell growth stationary phase) of photo-fermentation played a crucial role on effectively enhancing the phototrophic hydrogen production, rather than that during cell exponential growth phase. The other factor evaluated was hydrogen partial pressure in the culture system. The substrate conversion efficiency increased from 86.07% to 95.56% along with the decrease of the total pressure in the photobioreactor from 1.082 × 10⁵ to 0.944 × 10⁵ Pa, which indicated that reduction of H₂ partial pressure by lowering the operating pressure substantially improved H₂ production in an anaerobic, photo-fermentation process.     

Site-directed mutagenesis of substrate binding sites of azoreductase from Rhodobacter sphaeroides

Comparison of three-dimensional structures of flavin-dependent azoreductases revealed two conserved loops around the flavin mononucleotide (FMN) cofactor. Tyr74, His75 and Lys109 in the two loops of azoreductase AZR from Rhodobacter sphaeroides were replaced with Trp, Asn and Ala/His by site-directed mutagenesis, respectively. The optimal pH values of K109H and H75N were pH 6, and those of K109A and Y74W were pH 9. The optimal temperature (30°C) was not affected by mutation. Positively charged residues at position 109 is critical for the binding of methyl red. K109 might only be involved in the binding of the 2′-phosphate group of NADPH and have no effect on the binding of NADH. Y74W and H75N mutations decreased the binding of methyl red/nitrofurazone and had no affect on the binding of NADPH.     

Isolation and characterization of a virulent phage for Rhodobacter sphaeroides

A new virulent bacteriophage, termed øRsV, was isolated from a local sewage plant on the facultative phototrophic bacterium Rhodobacter sphaeroides DSM 159 as the host organism. Electron microscopic studies revealed that in general morphology phage øRsV resembles the T-even Escherichia coli phages. The host range of phage øRsV was restricted to strains of R. sphaeroides. E. coli strains B and K 12 were not infected. The phage genome was characterized on the basis of thermal denaturation profiles and restriction analyses indicating that it consists of about 160 kb of double-stranded DNA lacking cohesive ends. The G+C content was determined to be 46.8 mol%.     

B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis

Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, β, for H_A [Kirmaier et al. (1991) Science 251: 922–927] with two mutations, G(M203)D and Y(M210)W, near B_A, we have created a double and a triple mutant with long lifetimes of the excited state P^* of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P⁺Q_A ⁻ formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P⁺H_B ⁻ formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P^* lifetime of 15 ps [Heller et al. (1995) Science 269: 940–945]. We conclude that the lifetime of P^* is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q_B, was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q_A does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9–26]. It is shown that B-branch electron transfer leading to P⁺Q_B ⁻ formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P⁺Q_B ⁻ formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P⁺Q_B ⁻ state under continuous illumination were not successful. Charge recombination of P⁺Q_B ⁻ formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P⁺Q_B ⁻ trapped in wildtype RCs (10⁵ s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780–5786]. This difference is discussed in light of the different binding sites for Q_B and Q_B ⁻ that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812–816]. We present the first low-temperature absorption difference spectrum due to P⁺Q_B ⁻. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization and localization of phosphatidylglycerophosphate and phosphatidylserine synthases in Rhodobacter sphaeroides

Catalytic properties and membrane associations of the phosphatidylglycerophosphate (PGP) and phosphatidylserine (PS) synthases of Rhodobacter sphaeroides were examined to further characterize sites of phospholipid biosynthesis. In preparations of cytoplasmic membrane (CM) enriched in these activities, apparent K _m values of PGP synthase were 90 M for sn-glycerol-3-phosphate and 60 M for CDP-diacylglycerol; the apparent K _m of PS synthase for l-serine was near 165 M. Both enzymes required Triton X-100 with optimal PS synthase activity at a detergent/CDP-diacylglycerol (mol/mol) ratio of 7.5:1.0, while for optimal PGP synthase, a range of 10–50:1.0 was observed. Unlike the enzyme in Escherichia coli and several other Gram-negative bacteria, the PS synthase activity had a specific requirement for magnesium and was tightly associated with membranes rather than ribosomes in crude cell extracts. Sedimentation studies suggested that the PGP synthase ws distributed uniformly over the CM in both chemoheterotrophically and photoheterotrophically grown cells, while the PS synthase was confined mainly to a vesicular CM fraction. Solubilized PGP synthase activity migrated as a single band with a pI value near 5.5 in a chromatofocusing column and 5.8 on isoelectric focusing; in the latter procedure, the pI was shifted to 5.3 in the presence of CDP-diacylglycerol. The PGP synthase activity gave rise to a single polypeptide band in lithium dodecyl sulfatepolyacrylamide gel electrophoresis at 4°C.Abbreviations CM cytoplasmic membrane - ICM intracytoplasmic photosynthetic membrane - OM outer membrane - PGP phosphatidylglycerophosphate - PS phosphatidylserine - TLC thin-layer chromatography Supported in part by a Fellowship Awards from the Charles and Johanna Busch Memorial Fund Award to the Rutgers Bureau of Biological Research     

Characterization of a novel enoyl-acyl carrier protein reductase of diazaborine-resistant Rhodobacter sphaeroides mutant

Rhodobacter sphaeroides contains two enoyl-acyl carrier protein (ACP) reductases, FabI(1) and FabI(2). However, FabI(1) displays most of the cellular enzyme activity. The spontaneous diazaborine-resistant mutation was mapped as substitution of glutamine for proline 155 (P155Q) of FabI(1). The mutation of FabI(1)[P155Q] increased the specificity constants (k(cat)/K(m)) for crotonyl-ACP and NADH by more than 2-fold, while the site-directed mutation G95S (FabI(1)[G95S]), corresponding to the well-known G93 mutation of Escherichia coli FabI, rather decreased the values. Inhibition kinetics of the enzymes revealed that triclosan binds to the enzyme in the presence of NAD(+), while the diazaborine appears to interact with NADH and NAD(+) in the enzyme active site. The apparent inhibition constant K(i)(') of triclosan for FabI(1)[P155Q] and FabI(1)[G95S] at saturating NAD(+) were approximately 80- and 3-fold higher than that for the wild-type enzyme, respectively, implying that the inhibition was remarkably impaired by the P155Q mutation. The similar levels of K(i)(') of diazaborine for the mutant enzymes were also observed with respect to NAD(+). Thus, the novel mutation P155Q appears to disturb the binding of inhibitors to the enzyme without affecting the catalytic efficiency.     

Effects of Precise Deletions in Rhodobacter sphaeroides Reaction Center Genes on Steady-state Levels of Reaction Center Proteins: A Revised Model for Reaction Center Assembly

Possible interactions between photosynthetic reaction center (RC) proteins that protect these membrane proteins from proteolytic digestion in RC complex assembly were evaluated by use of translationally in-frame (nonpolar) RC gene-specific deletions. The RC H, RC M and RC L proteins were produced from plasmids, either alone or in concert with one or both of the others, in a strain of Rhodobacter sphaeroides that contained chromosomal deletions of all three RC genes. The steady-state amounts of these proteins in cell membrane and soluble fractions were assessed in western blots. The data are used to propose a model of RC assembly in which the RC M protein accumulates in the cell membrane regardless of the presence of the RC H and RC L proteins, and the RC M protein is a nucleus for addition of RC L followed by RC H in assembly of the RC holocomplex.     

Redox-responsive in vitro modulation of the signalling state of the isolated PrrB sensor kinase of Rhodobacter sphaeroides NCIB 8253

Prr is a global regulatory system that controls a large and diverse range of genes in Rhodobacter sphaeroides in response to changing conditions of environmental redox potential. PrrB is the membrane-bound sensor kinase and previously we showed that the purified, detergent-solubilised intact membrane protein is functional in autophosphorylation, phosphotransfer and phosphatase activities. Here we confirm that it also senses and responds directly to its environmental signal, redox potential; strong autophosphorylation of PrrB occurred in response to dithiothreitol (DTT)-induced reducing conditions (and levels increased in response to a wide 0.1-100 mM DTT range), whilst under oxidising conditions, PrrB exhibited low, just detectable levels of autophosphorylation. The clear response of PrrB to changes in reducing conditions confirmed its suitability for in vitro studies to identify modulators of its phosphorylation signalling state, and was used here to investigate whether PrrB might sense more than one redox-related signal, such as signals of cell energy status. NADH, ATP and AMP were found to exert no detectable effect on maintenance of the PrrB-P signalling state. By contrast, adenosine diphosphate produced a very strong increase in PrrB-P dephosphorylation rate, presumably through the back-conversion of PrrB-P to PrrB.     

Rhodethrin: a novel indole terpenoid ether produced by Rhodobacter sphaeroides has cytotoxic and phytohormonal activities

A novel metabolite was isolated from the culture supernatants of Rhodobacter sphaeroides OU5 when grown on l-tryptophan as sole source of nitrogen under photoheterotrophic conditions. It was identified by IR, NMR (¹H, ¹³C) and MS as an indole terpenoid ether [3-hydroxy-6-(1H-indol-3-yloxy)-4-methylhexanoic acid] and is named as rhodethrin. Rhodethrin at 0.5 μM gave positive test in auxin bioassay and initiated early rooting in tissue-cultured plants than IAA at 5 μM. Rhodethrin has cytotoxic activity against Sup-T₁ lymphoma and Colo-125 cancer cell lines at 10 nM.     

Response regulation in bacterial chemotaxis

The signal transduction system that mediates bacterial chemotaxis allows cells to moduate their swimming behavior in response to fluctuations in chemical stimuli. Receptors at the cell surface receive information from the surroundings. Signals are then passed from the receptors to cytoplasmic chemotaxis components: CheA, CheW, CheZ, CheR, and CheB. These proteins function to regulate the level of phosphorylation of a response regulator designated CheY that interacts with the flagellar motor switch complex to control swimming behavior. The structure of CheY has been determined. Magnesium ion is essential for activity. The active site contains highly conserved Asp residues that are required for divalent metal ion binding and CheY phosphorylation. Another residue-at the active site, Lys109, is important in the phosphorylation-induced conformational change that facilitates communication with the switch complex and another chemotaxis component, CheZ. CheZ facilitates the dephosphorylation of phospho-CheY. Defects in CheY and CheZ can be suppressed by mutations in the flagellar switch complex. CheZ is thought to modulate the switch bias by varying the level of phospho-CheY. © 1993 Wiley-Liss, Inc.