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.     

Ammonium and methylammonium transport by the nitrogen-fixing bacterium Azotobacter vinelandii.

Azotobacter vinelandii, grown with NH4+ as nitrogen source, was shown to possess an active transport system which can take up NH4+ against a concentration gradient of 58-fold. The properties of the NH4+ uptake system were investigated with the NH4+ analog CH3NH3+. The use of this analog was justified on the basis of the conclusion that the uptake of NH4+ and CH3NH3 involves a common binding site, as shown by the competitive inhibition of CH3NH3+ uptake by NH4+ (Ki approximately 3 microM). A Lineweaver-Burk plot for CH3NH3+ uptake revealed a biphasic curve, suggesting the existence of two CH3NH3+ (NH4+) uptake systems with apparent Km's for CH3NH3+ equal to 61 microM and 661 microM. The uptake of CH3NH3+ was inhibited by arsenate, as well as by cyanide or carbonyl cyanide-m-chlorophenyl hydrazone, indicating that phosphate bond energy is required.     

Photoresponses in Rhodobacter sphaeroides: role of photosynthetic electron transport.

Rhodobacter sphaeroides responds to a decrease in light intensity by a transient stop followed by adaptation. There is no measurable response to increases in light intensity. We confirmed that photosynthetic electron transport is essential for a photoresponse, as (i) inhibitors of photosynthetic electron transport inhibit photoresponses, (ii) electron transport to oxidases in the presence of oxygen reduces the photoresponse, and (iii) the magnitude of the response is dependent on the photopigment content of the cells. The photoresponses of cells grown in high light, which have lower concentrations of light-harvesting photopigment and reaction centers, saturated at much higher light intensities than the photoresponses of cells grown in low light, which have high concentrations of light-harvesting pigments and reaction centers. We examined whether the primary sensory signal from the photosynthetic electron transport chain was a change in the electrochemical proton gradient or a change in the rate of electron transport itself (probably reflecting redox sensing). R. sphaeroides showed no response to the addition of the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, which decreased the electrochemical proton gradient, although a behavioral response was seen to a reduction in light intensity that caused an equivalent reduction in proton gradient. These results strongly suggest that (i) the photosynthetic apparatus is the primary photoreceptor, (ii) the primary signal is generated by a change in the rate of electron transport, (iii) the change in the electrochemical proton gradient is not the primary photosensory signal, and (iv) stimuli affecting electron transport rates integrate via the electron transport chain.     

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.     

Ammonium (methylammonium) transport by Klebsiella pneumoniae

Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 100 μ}\text{M}$, $\text{V = 40 μ}\text{mol/min}$ per g dry weight at 15°C) is competitively inhibited by ammonium (apparent $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7 μ}\text{M}$). The low $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase.     

Binding-protein-dependent alanine transport in Rhodobacter sphaeroides is regulated by the internal pH.

The properties of an L-alanine uptake system in Rhodobacter sphaeroides were studied and compared with those of H+/lactose symport in R. sphaeroides 4P1, a strain in which the lactose carrier of Escherichia coli has been cloned and functionally expressed (F. E. Nano, Ph.D. thesis, University of Illinois, Urbana, 1984). Previous studies indicated that both transport systems were active only when electron transfer took place in the respiratory or cyclic electron transfer chain, while uptake of L-alanine also required the presence of K+ (M. G. L. Elferink, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1986). The results presented in this paper offer an explanation for these findings. Transport of the nonmetabolizable L-alanine analog 2-alpha-aminoisobutyric acid (AIB) is mediated by a shock-sensitive transport system. The apparently unidirectional uptake of AIB results in accumulation levels which exceed 7 x 10(3). The finding of L-alanine-binding activity in the concentrated crude shock fluid indicates that L-alanine is taken up by a binding-protein-dependent transport system. Transport of the nonmetabolizable lactose analog methyl-beta-D-thiogalactopyranoside (TMG) by the lactose carrier under anaerobic conditions in the dark was observed in cells and membrane vesicles. This indicates that the H+/lactose symport system is active without electron transfer. Uptake of AIB, but not that of TMG, is inhibited by vanadate with a 50% inhibitory concentration of 50 microM, which suggests a role of a phosphorylated intermediate in AIB transport. Uptake of TMG and AIB is regulated by the internal pH. The initial rates of uptake increased with the internal pH, and and pKa values of 7.2 for TMG and 7.8 for AIB. At an internal pH of 7, no AIB uptake occurred, and the rate of TMG uptake was only 30% of the rate at an internal pH of 8. In a previous study, we found that K+ plays an essential role in regulating the internal pH (T. Abee, K. J. Hellingwerf, and W. N. Konings, J. Bacteriol. 170:5647-5653, 1988). The dependence of solute transport in R. sphaeroides on both K+ and activity of an electron transfer chain can be explained by an effect of the internal pH, which subsequently influences the activities of the lactose-and binding-protein-dependent L-alanine transport system.     

Characterization of a binding protein-dependent glutamate transport system of Rhodobacter sphaeroides.

The mechanism of L-glutamate uptake was studied in Rhodobacter sphaeroides. Uptake of L-glutamate is mediated by a high-affinity (Kt of 1.2 microM), shock-sensitive transport system that is inhibited by vanadate and dependent on the internal pH. From the shock fluid, an L-glutamate-binding protein was isolated and purified. The protein binds L-glutamate (apparent Kd of 1.3 microM) and L-glutamine (Ki of 15 microM) with high affinity. The expression level of this binding protein is maximal at limiting concentrations of glutamine in the growth medium. The glutamate-binding protein restores the uptake of L-glutamate in spheroplasts. L-Aspartate is a strong competitive inhibitor of L-glutamate uptake (Ki of 3 microM) but competes only poorly with L-glutamate for binding to the binding protein (Ki of > 200 microM). The uptake of L-aspartate in R. sphaeroides also involves a binding protein which is distinct from the L-glutamate-binding protein. These data suggest that in R. sphaeroides, the L-glutamate- and L-aspartate-binding proteins interact with the same membrane transporter.     

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.     

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.     

Ammonium (methylammonium) transport by heterocysts and vegetative cells of Anabaena variabilis

Transport of the ammonium analogue [(14)C]methylammonium was similar in non-growing, fully differentiated heterocysts as compared to vegetative, multiplying cells of the filamentous cyanobacterium Anabaena variabilis. NH(4)(+) inhibited uptake into the cells and released accumulated methylammonium from the cells. These observations suggest that the main function of ammonium transport in heterocysts may not be NH(4)(+) acquisition but cyclic retention of ammonia produced by nitrogenase.     

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.     

The photosynthetic apparatus of Rhodobacter sphaeroides.

Functional and ultrastructural studies have indicated that the components of the photosynthetic apparatus of Rhodobacter sphaeroides are highly organized. This organization favors rapid electron transfer that is unimpeded by reactant diffusion. The light-harvesting complexes only partially surround the photochemical reaction center, which ensures an efficient shuttling of quinones between the photochemical reaction center and the bc1 complex.     

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.     

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     

RsGDB, the Rhodobacter sphaeroides Genome Database.

This report provides a summary of the sequencing project of the small chromosome (CII) of Rhodobacter sphaeroides 2.4.1(T),and introduces the first version of the genome database of this bacterium. The database organizes and describes diverse sets of biological information. The main role of the R.sphaeroides genome database (RsGDB) is to provide public access to the collected genomic information for R.sphaeroides via the World-Wide Web at http://utmmg.med.uth.tmc.edu/sphaeroides. The database allows the user access to hundreds of low redundancy R.sphaeroides sequences for further database searching, a summary of our current search results, and other allied information pertaining to this bacterium.