The role of soluble cytochrome c-551 in cyclic electron flow-driven active transport in Chromatium vinosum

Spheroplasts have been prepared from the photosynthetic purple sulfur bacterium Chromatium vinosum by lysozyme plus ethylenediaminetetraacetic acid treatment. These spheroplasts are able to take up alanine in the light, but light-dependent alanine uptake is lost upon subsequent washing of the spheroplasts. The observations that alanine uptake driven by a potassium plus valinomycin-induced membrane potential (outside positive) is not affected by washing and that light-dependent alanine uptake can be restored by addition of the supernatant from washing suggest that a soluble electron carrier is lost during washing. Light-dependent alanine uptake in washed spheroplasts could be restored by addition of C. vinosum cytochrome c-551. Other soluble electron carriers from C. vinosum (high-potential iron protein, cytochrome ‘f’, cytochrome c′ and the flavocytochrome c-552) did not restore alanine uptake nor did a variety of other soluble electron carrier proteins from other organisms. These results suggest that cytochrome c-551 functions as an electron carrier in the cyclic electron transfer chain of C. vinosum. Mitochondrial cytochrome c (equine heart) and cytochrome c-551 from Pseudomonas aeruginosa were highly effective in restoring light-dependent alanine uptake in washed spheroplasts, making it likely that C. vinosum cytochrome c-551 is related by evolution to the same cytochrome c family as these other two c cytochromes.     

Energy-dependent sodium efflux and sodium-dependent α-aminoisobutyrate transport in purple photosynthetic bacteria

Uptake of alanine and its nonmetabolizable analog α-aminoisobutyric acid (AIB) by the photosynthetic purple sulfur bacterium Chromatium vinosum is stimulated fivefold by Na⁺. Neither Li⁺ nor K⁺ have any stimulatory effect. AIB uptake can be supported by a Na⁺ gradient in the absence of other energy sources. AIB uptake is also accompanied by Na⁺ uptake. These results suggest that AIB is taken up by C. vinosum via a sodium symport. Cells of C. vinosum and the purple nonsulfur bacterium Rhodospirillum rubrum show energy-dependent Na⁺ efflux and Na⁺ uptake can be demonstrated with chromatophores prepared from these bacteria.     

Active transport of α-methylglucoside by the photosynthetic bacterium Chromatium vinosum

The photosynthetic purple sulfur bacterium Chromatium vinosum possesses an active transport system for glucose that also transports the nonmetabolizable glucose analog, αmethylglucoside. Transport is not accompanied by phosphorylation nor does it appear to require ATP. Rather α-methylglucoside uptake appears to be driven directly by the electrochemical proton gradient produced by light-driven cyclic electron flow.     

The pathway of cyclic electron transport in chromatophores of Chromatium vinosum. Evidence for a Q-cycle mechanism

Chromatophores of the purple sulfur bacterium Chromatium vinosum were shown to contain a cytochrome similar to cytochrome c₁ and two b cytochromes. Cytochrome b can be accumulated in the reduced form upon illumination at an ambient redox potential of +415 mV in the presence of the electron transport inhibitors antimycin A or HOQNO. The reductions of cytochrome b, of the high-potential cytochrome c₅₅₅ and of the primary electron donor P-870 are all inhibited by myxothiazol. Dark-adapted C. vinosum chromatophores show little cytochrome b reduction on the first flash. Considerable cytochrome b reduction (1 cytochrome b:8 P-870 present) is observed on the second flash. This observation and the 1:1 stoichiometry observed between cytochrome b reduction and P-870⁺ reduction after the second flash support a Q-cycle model for cyclic electron flow in C. vinosum.     

Cytochrome changes in control and concanavalin A-stimulated lymphocytes

α-Aminoisobutyrate (AIB) serves as a transportable, nonmetabolizable alanine analog in the purple sulfur bacterium Chromatium vinosum. AIB transport in C. vinosum appears to be catalyzed by an electrogenic Na⁺-alanine (AIB) symport without any direct participation of ATP-driven or H⁺-symport systems. In addition to Na⁺ being cotransported with AIB via the symport, a transmembrane Na⁺ gradient appears to increase the affinity of the symport of AIB. It appears that these two effects of Na⁺ involve different Na⁺-binding sites.     

Interaction between Sox proteins of two physiologically distinct bacteria and a new protein involved in thiosulfate oxidation

Organisms using the thiosulfate-oxidizing Sox enzyme system fall into two groups: group 1 forms sulfur globules as intermediates (Allochromatium vinosum), group 2 does not (Paracoccus pantotrophus). While several components of their Sox systems are quite similar, i.e. the proteins SoxXA, SoxYZ and SoxB, they differ by Sox(CD)₂ which is absent in sulfur globule-forming organisms. Still, the respective enzymes are partly exchangeable in vitro: P. pantotrophus Sox enzymes work productively with A. vinosum SoxYZ whereas A. vinosum SoxB does not cooperate with the P. pantotrophus enzymes. Furthermore, A. vinosum SoxL, a rhodanese-like protein encoded immediately downstream of soxXAK, appears to play an important role in recycling SoxYZ as it increases thiosulfate depletion velocity in vitro without increasing the electron yield.     

Isolation and characterization of two soluble heme c-containing proteins from Chromatium vinosum

The photosynthetic purple sulfur bacterium Chromatium vinosum has been shown to possess two previously undetected heme c-containing, soluble proteins. One is an acidic, c-type cytochrome with a molecular weight of 12 300 and an oxidation-reduction midpoint potential (at pH 8.0) of ?82 mV. The other protein is a basic protein with a molecular weight of 11 900 and an oxidation-reduction midpoint potential (at pH 8.0) of ?110 mV. The basic protein, in both oxidized and reduced forms, has optical spectra similar to those of myoglobin and the oxidized C. vinosum protein exhibits a high-spin heme EPR spectrum similar to that of metmyoglobin. Furthermore, the basic C. vinosum protein binds CO and O₂. The spectra of the CO and O₂ complexes show significant similarities with the respective myoglobin complexes. Possible functions for an O₂-binding protein in C. vinosum are discussed.     

Insight into the molecular mechanism of the sulfur oxidation process by reverse sulfite reductase (rSiR) from sulfur oxidizer <Emphasis Type="Italic">Allochromatium vinosum</Emphasis>

Sulfur metabolism is one of the oldest known biochemical processes. Chemotrophic or phototrophic proteobacteria, through the dissimilatory pathway, use sulfate, sulfide, sulfite, thiosulfate or elementary sulfur by either reductive or oxidative mechanisms. During anoxygenic photosynthesis, anaerobic sulfur oxidizer Allochromatium vinosum forms sulfur globules that are further oxidized by dsr operon. One of the key redox enzymes in reductive or oxidative sulfur metabolic pathways is the DsrAB protein complex. However, there are practically no reports to elucidate the molecular mechanism of the sulfur oxidation process by the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum. In the present context, we tried to analyze the structural details of the DsrAB protein complex from sulfur oxidizer Allochromatium vinosum by molecular dynamics simulations. The molecular dynamics simulation results revealed the various types of molecular interactions between DsrA and DsrB proteins during the formation of DsrAB protein complex. We, for the first time, predicted the mode of binding interactions between the co-factor and DsrAB protein complex from Allochromatium vinosum. We also compared the binding interfaces of DsrAB from sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris. This study is the first to provide a comparative aspect of binding modes of sulfur oxidizer Allochromatium vinosum and sulfate reducer Desulfovibrio vulgaris.     

ATP-dependent K+ uptake by a photosynthetic purple sulfur bacterium

Chromatium vinosum contains an ATP-dependent K⁺ uptake system which is light independent and sensitive to DCCD. Kinetic measurements show K_m = 0.27 mM for K⁺ and K_m = 1.3 mM for Tl⁺, an alternate substrate. The internal K⁺ content of the cell increases with increasing medium osmolality and is unaffected by changes in external K⁺ concentration.     

Coexistence of organisms competing for the same substrate: An example among the purple sulfur bacteria

The purpose of this study was to find a possible explanation for the coexistence of large and small purple sulfur bacteria in natural habitats. Experiments were carried out withChromatium vinosum SMG 185 andChromatium weissei SMG 171, grown in both batch and continuous cultures. The data may be summarized as follows: (a) In continuous light, with sulfide as growth rate-limiting substrate, the specific growth rate ofChr. vinosum exceeds that ofChr. weissei regardless of the sulfide concentration employed. Consequently,Chr. weissei is unable to compete successfully and is washed out in continuous cultures. (b) With intermittant light-dark illumination, the organisms showed balanced coexistence when grown in continuous cultures. The “steady-state” abundance ofChr. vinosum was found to be positively related to the length of the light period, and that ofChr. weissei to the length of the dark period. (c) Sulfide added during darkness is rapidly oxidized on subsequent illumination, resulting in the intracellular storage of reserve substances, which are later utilized for growth. The rate of sulfide oxidation/mg cell N/hr was found to be over twice as high inChr. weissei as inChr. vinosum. The observed coexistence may be explained as follows. In the light, with both strains growing, most of the sulfide will be oxidized byChr. vinosum [see (a)]. In the dark, sulfide accumulates. On illumination, the greater part of the accumulated sulfide will be oxidized byChr. weissei [see (c)]. A changed light-dark regimen should then have the effect as observed [see (b)]. These observations suggest that intermittant illumination may, at least in part explain the observed coexistence of both types of purple sulfur bacteria in nature.     

The genus Allochromatium (Chromatiales Chromatiaceae) revisited: a study on its intragenic structure based on multilocus sequence analysis (MLSA) and DNA-DNA hybridization (DDH)

In this study, the taxonomic status of anoxygenic photosynthetic bacteria belonging to the genus Allochromatium is revisited. The inter- and intraspecies relationship of seven Allochromatium strains, including a set of well described type strains, were examined by DNA-DNA hybridization (DDH) and multilocus sequence analysis (MLSA) using segments of seven protein-coding genes. The re-sequencing of the 16S rRNA, the internal transcriber spacer (ITS), multi-gene analysis and DDH comparison indicated that both type strains Allochromatium vinosum DSM 180^T and Allochromatium minutissimum DSM 1376^T are closely related to each other forming an independent cluster together with the strains A. vinosum DSM 183 and DSM 1686. The internal comparison of members of this A. vinosum phylogroup showed values of DDH relatedness above 80% and concatenated sequence similarities (4744 bp) above 98%. In contrast, the MLSA scheme has identified A. vinosum strain BH-2 as a separate lineage. Strain BH-2 was first classified as a member of the species A. vinosum based on DDH comparison. However, this strain showed the lowest similarity values of the 16S rRNA gene and concatenated sequences, as well as amino acid identity (AAI) when compared to other Allochromatium strains, suggesting that strain BH-2 may represent a new species.     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

15N resonance assignments of oxidized and reducedChromatium vinosum high-potential iron protein

The¹⁵N resonances in reduced and oxidizedChromatium vinosum high-potential iron protein have been assigned by use of¹H-¹H COSY spectra and¹H-¹⁵N HMQC, HMQC-COSY, and HMQC-NOESY spectra. Unambiguous assignment of 70 of 85 backbone¹⁵N resonances in the reduced protein and 62 of 85 resonances in the oxidized protein are made, as are 12 of 21 side-chain¹⁵N resonances.     

Cyanide-linked dimer-monomer equilibrium of Chromatium vinosum ferric cytochrome c′

Cyanide binding to Chromatium vinosum ferricytochrome c′ has been studied to further investigate possible allosteric interactions between the subunits of this dimeric protein. Cyanide binding to C. vinosum cytochrome c′ appears to be cooperative. However, the cyanide binding reaction is unusual in that the overall affinity of cyanide increases as the concentration of cytochrome c′ decreases and that cyanide binding causes the ligated dimer to dissociate to monomers as shown by gel-filtration chromatography. Therefore, the cyanide binding properties of C. vinosum ferricytochrome c′ are complicated by a cyanide-linked dimer to monomer dissociation equilibrium of the complexed protein. The dimer to monomer dissociation constant is 20-fold smaller than that for CO linked dissociation constant of ferrocytochrome c′. Furthermore, the pH dependence of both the intrinsic equilibrium binding constant and the dimer to monomer equilibrium dissociation constant was investigated over the pH range of 7.0 to 9.2 to examine the effect of any ionizable groups. The equilibrium constants did not exhibit a significant pH dependence over this pH range.     

Active transport of chloride by Anacystis nidulans

Chloride uptake by the cyanobacterium Anacystis nidulans at 38°C is energy dependent showing maximum rate (around 5.10^-7 mol Cl^-xml cell water^-1xmin^-1) and accumulation (up to 160 fold) in light and air. The respective values in air and darkness were 40–70% lower. In the dark under N₂ no uptake was found. Chloride transport had an optimum at pH 6.7 and a K _M of 2.10^-5 M which was pH-independent. It was inhibited by carbonyl cyanide m-chlorophenylhydrazone and N,N′-dicyclohexylcarbodiimide in the light and in the dark, and also to a lesser extent by valinomycin. 3-(3,4-dichlorophenyl)-1,1-dimethylurea in the light caused a moderate stimulation. To obtain information about the energy source of active chloride transport the action of the four inhibitors on membrane potential (determined through the distribution of triphenylmethylphosphonium) and ATP level (determined by the firefly method) was examined. It was found that a high negative membrane potential was unfavorable for chloride accumulation probably by stimulating passive efflux. On the other hand a good correlation between ATP level and chloride transport activity was obtained. Attempts to induce chloride uptake by sudden acidification of the external medium in presence of N,N′-dicyclohexyl-carbodiimide or during anaerobiosis were not successful. Two mechanisms of chloride uptake are discussed:

1. primary active transport by an ATP-dependent pump, and
2. “chemiosmotic” secondary active transport linked to a proton gradient, the present data favoring mechanism a.

     

Photophosphorylation Associated with Photosystem II: III. Characterization of Uncoupling, Energy Transfer Inhibition, and Proton Uptake Reactions Associated with Photosystem II Cyclic Photophosphorylation

A number of uncouplers and energy transfer inhibitors suppress photosystem II cyclic photophosphorylation catalyzed by either a proton/electron or electron donor. Valinomycin and 2,4-dinitrophenol also inhibit photosystem II cyclic photophosphorylation, but these compounds appear to act as electron transport inhibitors rather than as uncouplers. Only when valinomycin, KCl, and 2,4-dinitrophenol were added simultaneously to phosphorylation reaction mixtures was substantial uncoupling observed. Photosystem II noncyclic and cyclic electron transport reactions generate positive absorbance changes at 518 nm. Uncoupling and energy transfer inhibition diminished the magnitude of these absorbance changes. Photosystem II cyclic electron transport catalyzed by either p-phenylenediamine or N,N,N′,N′-tetramethyl-p-phenylenediamine stimulated proton uptake in KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts. Illumination with 640 nm light produced an extent of proton uptake approximately 3-fold greater than did 700 nm illumination, indicating that photosystem II-catalyzed electron transport was responsible for proton uptake. Electron transport inhibitors, uncouplers, and energy transfer inhibitors produced inhibitions of photosystem II-dependent proton uptake consistent with the effects of these compounds on ATP synthesis by the photosystem II cycle. These results are interpreted as indicating that endogenous proton-translocating components of the thylakoid membrane participate in coupling of ATP synthesis to photosystem II cyclic electron transport.     

AtPTR1 and AtPTR5 transport dipeptides in planta

Transporters for di- and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1) family. A new member of this gene family, AtPTR5, was isolated from Arabidopsis (Arabidopsis thaliana). Expression of AtPTR5 was analyzed and compared with tissue specificity of the closely related AtPTR1 to discern their roles in planta. Both transporters facilitate transport of dipeptides with high affinity and are localized at the plasma membrane. Mutants, double mutants, and overexpressing lines were exposed to several dipeptides, including toxic peptides, to analyze how the modified transporter expression affects pollen germination, growth of pollen tubes, root, and shoot. Analysis of atptr5 mutants and AtPTR5-overexpressing lines showed that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules, and seeds. In contrast, AtPTR1 plays a role in uptake of peptides by roots indicated by reduced nitrogen (N) levels and reduced growth of atptr1 mutants on medium with dipeptides as the sole N source. Furthermore, overexpression of AtPTR5 resulted in enhanced shoot growth and increased N content. The function in peptide uptake was further confirmed with toxic peptides, which inhibited growth. The results show that closely related members of the PTR/NRT1 family have different functions in planta. This study also provides evidence that the use of organic N is not restricted to amino acids, but that dipeptides should be considered as a N source and transport form in plants.     

Uncoupler-Resistant Glucose Uptake by the Thermophilic Glycolytic Anaerobe Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum)

The transport of glucose across the bacterial cell membrane of Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum) Rt8.B1 was governed by a permease which did not catalyze concomitant substrate transport and phosphorylation and thus was not a phosphoenolpyruvate-dependent phosphotransferase. Glucose uptake was carrier mediated, could not be driven by an artificial membrane potential (Δψ) in the presence or absence of sodium, and was not sensitive to inhibitors which dissipate the proton motive force (Δp; tetrachlorosalicylanilide, N,N-dicyclohexylcarboiimide, and 2,4-dinitrophenol), and no uptake of the nonmetabolizable analog 2-deoxyglucose could be demonstrated. The glucokinase apparent K_m for glucose (0.21 mM) was similar to the K_t (affinity constant) for glucose uptake (0.15 mM), suggesting that glucokinase controls the rate of glucose uptake. Inhibitors of ATP synthesis (iodoacetate and sodium fluoride) also inhibited glucose uptake, and this effect was due to a reduction in the level of ATP available to glucokinase for glucose phosphorylation. These results indicated that T. thermosulfuricus Rt8.B1 lacks a concentrative uptake system for glucose and that uptake is via facilitated diffusion, followed by ATP-dependent phosphorylation by glucokinase. In T. thermosulfuricus Rt8.B1, glucose is metabolized by the Embden-Meyerhof-Parnas pathway, which yields 2 mol of ATP (G. M. Cook, unpublished data). Since only 1 mol of ATP is used to transport 1 mol of glucose, the energetics of this system are therefore similar to those found in bacteria which possess a phosphotransferase.     

Menaquinone is the sole quinone in the facultatively aerobic green photosynthetic bacterium Chloroflexus aurantiacus

The role of quinones was investigated in Chloroflexus aurantiacus, a thermophilic green bacterium capable of photosynthetic or respiratory growth. Thin-layer chromatography, ultraviolet difference spectroscopy and high-pressure liquid chromatography showed that menaquinone is the only quinone present in both photosynthetic and respiratory Chloroflexus cultures. Menaquinone-10 and menaquinone-8 are the predominant homologues in both cultures. For comparative purposes the quinone compositions in photoheterotrophic cultures of Chromatium vinosum and Chlorobium limicola were also analyzed. Chloroflexus is the only facultatively aerobic photosynthetic bacterium that does not possess ubiquinone. Menaquinone appears to be the only quinone involved in the photosynthetic and oxidative electron transport in this organism.     

Induction of nitrate transport in maize roots, and kinetics of influx, measured with nitrogen-13

Unlike phosphate or potassium transport, uptake of nitrate by roots is induced, in part, by contact with the substrate ion. Plasmalemma influx of ¹³N-labeled nitrate in maize roots was studied in relation to induction of the uptake system, and the influence of short-term N starvation. Maize (Zea mays) roots not previously exposed to nitrate had a constitutive transport system (state 1), but influx increased 250% during six hours of contact with 100 micromolar nitrate, by which time the transport mechanism appeared to be fully synthesized (state 2). A three-day period of N starvation prior to induction and measurement of nitrate influx resulted in a greater capacity to transport nitrate than in unstarved controls, but this was fully expressed only if roots were kept in contact with nitrate for the six hours needed for full induction (state 2E). A kinetic analysis indicated a 160% increase in maximum influx in N-starved, induced roots with a small decrease in K_m. The inducible component to nitrate influx was induced only by contact with nitrate. Full expression of the nitrate inducible transport system was dependent upon mRNA synthesis. An inhibitor of cytoplasmic protein synthesis (cycloheximide) eliminated the formation of the transport system while inhibition by chloramphenicol of mitochondrial- or plastid-coded protein synthesis had no effect. Poisoning of membrane-bound proteins effectively disabled both the constitutive and induced transport systems.