Osmoregulation in Rhodobacter sphaeroides.

Betaine (N,N,N-trimethylglycine) functioned most effectively as an osmoprotectant in osmotically stressed Rhodobacter sphaeroides cells during aerobic growth in the dark and during anaerobic growth in the light. The presence of the amino acids L-glutamate, L-alanine, or L-proline in the growth medium did not result in a significant increase in the growth rate at increased osmotic strengths. The addition of choline to the medium stimulated growth at increased osmolarities but only under aerobic conditions. Under these conditions choline was converted via an oxygen-dependent pathway to betaine, which was not further metabolized. The initial rates of choline uptake by cells grown in media with low and high osmolarities were measured over a wide range of concentrations (1.9 microM to 2.0 mM). Only one kinetically distinguishable choline transport system could be detected. Kt values of 2.4 and 3.0 microM and maximal rates of choline uptake (Vmax) of 5.4 and 4.2 nmol of choline/min.mg of protein were found in cells grown in the minimal medium without or with 0.3 M NaCl, respectively. Choline transport was not inhibited by a 25-fold excess of L-proline or betaine. Only one kinetically distinguishable betaine transport system was found in cells grown in the low-osmolarity minimal medium as well as in a high-osmolarity medium containing 0.3 M NaCl. In cells grown and assayed in the absence of NaCl, betaine transport occurred with a Kt of 15.1 microM and a Vmax of 3.2 nmol/min . mg of protein, whereas in cells that were grown and assayed in the presence of 0.3 M NaCl, the corresponding values were 18.2 microM and 9.2 nmol of betaine/min . mg of protein. This system was also able to transport L-proline, but with a lower affinity than that for betaine. The addition of choline of betaine to the growth medium did not result in the induction of additional transport systems.     

Phosphotransfer in Rhodobacter sphaeroides chemotaxis

The two-component sensing system controlling bacterial chemotaxis is one of the best studied in biology. Rhodobacter sphaeroides has a complex chemosensory pathway comprising two histidine protein kinases (CheAs) and eight downstream response regulators (six CheYs and two CheBs) rather than the single copies of each as in Escherichia coli. We used in vitro analysis of phosphotransfer to start to determine why R.sphaeroides has these multiple homologues. CheA(1) and CheA(2) contain all the key motifs identified in the histidine protein kinase family, except for conservative substitutions (F-L and F-I) within the F box of CheA(2), and both are capable of ATP-dependent autophosphorylation. While the K(m) values for ATP of CheA(1) and CheA(2) were similar to that of E.coli, the k(cat) value was three times lower, but similar to that measured for the related Sinorhizobium meliloti CheA. However, the two CheAs differed both in their ability to phosphorylate the various response regulators and the rates of phosphotransfer. CheA(2) phosphorylated all of the CheYs and both CheBs, whilst CheA(1) did not phosphorylate either CheB and phosphorylated only the response regulators encoded within its own genetic locus (CheY(1), CheY(2), and CheY(5)) and CheY(3). The dephosphorylation rates of the R.sphaeroides CheBs were much slower than the E.coli CheB. The dephosphorylation rate of CheY(6), encoded by the third chemosensory locus, was ten times faster than that of the E.coli CheY. However, the dephosphorylation rates of the remaining R.sphaeroides CheYs were comparable to that of E.coli CheY.     

Gramicidin in chromatophores of Rhodobacter sphaeroides

Chromatophores of Rhodobacter sphaeroides were excited with light flashes to generate a transmembrane electrical potential difference. The electric relaxation was measured by electrochromic absorption changes as a function of added gramicidin. At low gramicidin/bacteriochlorophyll (BChl) molar ratios the decay of the electrochromic absorption changes showed a biphasic behaviour, with a fast phase relaxing at some s, and a slow phase relaxing at more than 100 ms. This was attributable to a mixture of vesicles containing gramicidin dimers with others containing none. The concentration dependence of this effect was linear. This implied full dimerization of gramicidin. The data were interpreted to yield an average bacteriochlorophyll content per chromatophore of 770(±150) and the conductance of a single gramicidin dimer in the chromatophore membrane of 15(±4) pS (in about 115 mM KCl).Abbreviations BChl Bacteriochlorphyll - tricine N-Tris[hydroxymethyllmethylglycine Offprint requests to: W. Junge     

The CheYs of Rhodobacter sphaeroides

The Escherichia coli two-component chemosensory pathway has been extensively studied, and its response regulator, CheY, has become a paradigm for response regulators. However, unlike E. coli, most chemotactic nonenteric bacteria have multiple CheY homologues. The roles and cellular localization of the CheYs in Rhodobacter sphaeroides were determined. Only two CheYs were required for chemotaxis, CheY(6) and either CheY(3) or CheY(4). These CheYs were partially localized to either of the two chemotaxis signaling clusters, with the remaining protein delocalized. Interestingly, mutation of the CheY(6) phosphorylatable aspartate to asparagine produced a stopped motor, caused by phosphorylation on alternative site Ser-83 by CheA. Extensive mutagenesis of E. coli CheY has identified a number of activating mutations, which have been extrapolated to other response regulators (D13K, Y106W, and I95V). Analogous mutations in R. sphaeroides CheYs did not cause activation. These results suggest that although the R. sphaeroides and E. coli CheYs are similar in that they require phosphorylation for activation, they may differ in both the nature of the phosphorylation-induced conformational change and their subsequent interactions with the flagellar motor. Caution should therefore be used when projecting from E. coli CheY onto novel response regulators.     

Features of Rhodobacter sphaeroides CcmFH

In this study, the in vivo function and properties of two cytochrome c maturation proteins, CcmF and CcmH from Rhodobacter sphaeroides, were analyzed. Strains lacking CcmH or both CcmF and CcmH are unable to grow under anaerobic conditions where c-type cytochromes are required, demonstrating their critical role in the assembly of these electron carriers. Consistent with this observation, strains lacking both CcmF and CcmH are deficient in c-type cytochromes when assayed under permissive growth conditions. In contrast, under permissive growth conditions, strains lacking only CcmH contain several soluble and membrane-bound c-type cytochromes, albeit at reduced levels, suggesting that this bacterium has a CcmH-independent route for their maturation. In addition, the function of CcmH that is needed to support anaerobic growth can be replaced by adding cysteine or cystine to growth media. The ability of exogenous thiol compounds to replace CcmH provides the first physiological evidence for a role of this protein in thiol chemistry during c-type cytochrome maturation. The properties of R. sphaeroides cells containing translational fusions between CcmF and CcmH and either Escherichia coli alkaline phosphatase or beta-galactosidase suggest that they are each integral cytoplasmic membrane proteins with their presumed catalytic domains facing the periplasm. Analysis of CcmH shows that it is synthesized as a higher-molecular-weight precursor protein with an N-terminal signal sequence.     

Purification and properties of a polyol dehydrogenase from the phototrophic bacterium Rhodobacter sphaeroides

A polyol dehydrogenase was detected in cell extracts of the facultative phototrophic bacterium Rhodobacter sphaeroides strain Si 4 grown on D-glucitol (sorbitol) as the sole carbon source. The enzyme was purified 150-fold to apparent homogeneity by steps involving fractionated (NH4)2SO4 precipitation, chromatography on Q-Sepharose and phenyl-Sepharose, and FPLC on Superose 12. The relative molecular mass (Mr) of the native polyol dehydrogenase was 47,200 as calculated from its Stokes' radius (rs = 2.76 nm) and sedimentation coefficient (s20, w = 4.15 S). SDS/PAGE resulted in one single band representing a polypeptide with a Mr of 52,200, indicating that the native protein is a monomer. The isoelectric point of the polyol dehydrogenase was determined to be pH 4.3. The enzyme was specific for NAD+ and oxidized both D-glucitol and D-mannitol to D-fructose, as well as D-arabinitol to D-ribulose. The pH optimum of substrate oxidation was pH 9.0 in 0.1 M Tris/HCl and that of substrate reduction was pH 6.5 in 0.1 M potassium phosphate. The reactions exhibited normal Michaelis-Menten kinetics allowing the estimation of KM values for NAD+ (0.18 mM) in the presence of D-glucitol, and for D-glucitol (31.8 mM), D-mannitol (0.29 mM) and D-arabinitol (1.8 mM), respectively. The KM value for D-fructose was 16.3 mM and that for NADH 0.02 mM. The equilibrium constants determined for the conversion of D-mannitol, D-glucitol and D-arabinitol were 4.5 nM, 0.58 nM and 80 pM, respectively. Based on the catalytic preference of the polyol dehydrogenase for D-mannitol, an enzymatic assay for D-mannitol was elaborated.     

Chromate reduction by Rhodobacter sphaeroides

Rhodobacter sphaeroides grew in the presence of up to 43 μM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of R. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration. The optimum of the reaction is at pH 7.0 and 30°C. The enzyme activity showed a hyperbolic dependence on the concentrations of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A K _m of 15±1.3 μM CrO₄ ²⁻ and a V _max of 420±50 μmol min⁻¹ mg protein⁻¹ was determined for the enzyme isolated from anaerobically grown cells and 29±6.4 μM CrO₄ ²⁻ and 100±9.6 μmol CrO₄ ²⁻ min⁻¹ mg protein⁻¹ for the one from aerobically grown ones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 198–203. Received 05 January 2000/ Accepted in revised form 27 May 2000     

浑球红假单胞菌氨同化途径的研究

本文测定了浑球红假单胞菌(Rhodobacter sphaeroides)菌株601谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、谷氨酸脱氢酶(GDH)和丙氨酸脱氢酶(ADH)的活性。低氨时,GS/GOGAT活力高,GDH活力低,高氨时,GS/GOGAT活力低,GDH活力高。在以分子氮或低浓度氨为氮源的培养条件下,加入GS抑制刑MSX(L—methionine—DL—sulphoximine),细菌生长受到抑制。但是,生长在以谷氨酸为氮源的细菌则不受影响。上述结果表明,浑球红假单胞菌菌株601氨同化是通过GS/GOGAT途径和GDH途径。     

含硒类球红细菌的研究

为了确定类球红细菌转硒培养的最佳条件 ,研究了无机硒的加入浓度、时间以及分批补料培养对菌体生长和转硒效率的影响。实验表明 ,无机硒的浓度低于 1× 10 -5mol/L时 ,对类球红细菌的生长基本没有影响 ,并能将6 3.9%的无机硒转化为有机硒。转硒的最佳时间是在接种后 12h左右 ,此时转硒效率最高。实验还表明 ,分批补料培养可以提高菌体浓度 ,可使转硒效率和绝对量增加。体内试验表明 ,用 5mL/kgbw和 10mL/kgbw剂量的含硒类球红细菌灌养小鼠 ,可以使其全血GSH Px酶活性提高 2 0 .9%和 2 5 .5 % ,使其血清丙二醛 (MDA)含量降低2 1.0 %和 2 3.2 %。     

Rhodobacter sphaeroides: complexity in chemotactic signalling

Most bacteria have much more complex chemosensory systems than those of the extensively studied Escherichia coli. Rhodobacter sphaeroides, for example, has multiple homologues of the E. coli chemosensory proteins. The roles of these homologues have been extensively investigated using a combination of deletion, subcellular localization and phosphorylation assays. These studies have shown that the homologues have specific roles in the sensory pathway, and they differ in their cellular localization and interactions with other components of the pathway. The presence of multiple chemosensory pathways might enable bacteria to tune their tactic responses to different environmental conditions.     

光合细菌Rhodobacter sphaeroides新型表达系统研究进展

利用当前商业化的表达系统生产重组蛋白时,在寄主培养过程中都不能快速实时地检测重组蛋白的表达水平。光合细菌Rb.sphaeroides是研究细菌光合作用和膜蛋白形成的重要模式生物,具有开发成为一种新型表达系统的潜能。介绍了光合细菌Rb.sphaeroides新型表达系统的优势和开发情况。     

Electron transport-dependent taxis in Rhodobacter sphaeroides.

Rhodobacter sphaeroides showed chemotaxis to the terminal electron acceptors oxygen and dimethyl sulfoxide, and the responses to these effectors were shown to be influenced by the relative activities of the different electron transport pathways. R. sphaeroides cells tethered by their flagella showed a step-down response to a decrease in the oxygen or dimethyl sulfoxide concentration when using them as terminal acceptors. Bacteria using photosynthetic electron transport, however, showed a step-down response to oxygen addition. Addition of the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone did not cause a transient behavioral response, although it decreased the electrochemical proton gradient (delta p) and increased the rate of electron transport. However, removal of the ionophore, which caused an increase in delta p and a decrease in the electron transport rate, resulted in a step-down response. Together, these data suggest that behavioral responses of R. sphaeroides to electron transport effectors are caused by changes in the rate of electron transport rather than changes in delta p.     

Intracytoplasmic membrane vesiculation in light-harvesting mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract In Chlamydomonas reinhardtii there are three glutamate dehydrogenase isozymes which can use both NADH and NADPH as cofactors and respond differently to different nitrogen sources and several stress conditions. From data of induction of isozymes in different metabolic situations, we propose a possible physiological role for each of them in algal carbon and nitrogen metabolism.     

Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides

Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by-passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha-4,11-diene after cultivation with [4-¹³C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance.     

Involvement of transport in Rhodobacter sphaeroides chemotaxis.

The chemotactic response to a range of chemicals was investigated in the photosynthetic bacterium Rhodobacter sphaeroides, an organism known to lack conventional methyl-accepting sensory transduction proteins. Strong attractants included monocarboxylic acids and monovalent cations. Results suggest that the chemotactic response required the uptake of the chemoeffector, but not its metabolism. If a chemoeffector could block the uptake of another attractant, it also inhibited chemotaxis to that attractant. Sodium benzoate was not an attractant but was a competitive inhibitor of the propionate uptake system. Binding in an active uptake system was therefore insufficient to cause a chemotactic response. At different concentrations, benzoate either blocked propionate chemotaxis or reduced the sensitivity of propionate chemotaxis, an effect consistent with its role as a competitive inhibitor of uptake. Bacteria only showed chemotaxis to ammonium when grown under ammonia-limited conditions, which derepressed the ammonium transport system. Both chemotaxis and uptake were sensitive to the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, suggesting an involvement of the proton motive force in chemotaxis, at least at the level of transport. There was no evidence for internal pH as a sensory signal. These results suggest a requirement for the uptake of attractants in chemotactic sensing in R. sphaeroides.     

DNA repair mutants of Rhodobacter sphaeroides.

The genome of the photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1 comprises two chromosomes and five endogenous plasmids and has a 65% G+C base composition. Because of these characteristics of genome architecture, as well as the physiological advantages that allow this organism to live in sunlight when in an anaerobic environment, the sensitivity of R. sphaeroides to UV radiation was compared with that of the more extensively studied bacterium Escherichia coli. R. sphaeroides was found to be more resistant, being killed at about 60% of the rate of E. coli. To begin to analyze the basis for this increased resistance, a derivative of R. sphaeroides, strain 2.4.1 delta S, which lacks the 42-kb plasmid, was mutagenized with a derivative of Tn5, and the transposon insertion mutants were screened for increased UV sensitivity (UVs). Eight UVs strains were isolated, and the insertion sites were determined by contour-clamped homogeneous electric field pulsed-field gel electrophoresis. These mapped to at least five different locations in chromosome I. Preliminary analysis suggested that these mutants were deficient in the repair of DNA damage. This was confirmed for three loci by DNA sequence analysis, which showed the insertions to be within genes homologous to uvrA, uvrB, and uvrC, the subunits of the nuclease responsible for excising UV damage.