Differential regulation of periplasmic nitrate reductase gene (napKEFDABC) expression between aerobiosis and anaerobiosis with nitrate in a denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans

A denitrifying phototroph, Rhodobacter sphaeroides f. sp. denitrificans, has the ability to denitrify by respiring nitrate. The periplasmic respiratory nitrate reductase (Nap) catalyses the first step in denitrification and is encoded by the genes, napKEFDABC. By assaying the ss-galactosidase activity of napKEFD-lacZ fusions in wild type and nap mutant cells grown under various growth conditions, the environmental signal for inducing nap expression was examined. Under anoxic conditions with nitrate, nap genes expression in the wild-type strain was highest in the dark, and somewhat lowered by incident light, but that of the napA, napB, and napC mutant strains was low, showing that nap expression is dependent on nitrate respiration. Under oxic conditions, both the wild type and nap mutant cells showed high ss-galactosidase activities, comparable to the wild-type grown under anoxic conditions with nitrate. Myxothiazol, a specific inhibitor of the cytochrome bc (1) complex, did not affect the beta-galactosidase activity in the wild-type cells grown aerobically, suggesting that the redox state of the quinone pool was not a candidate for the activation signal for aerobic nap expression. These results suggested that the trans-acting regulatory signals for nap expression differ between anoxic and oxic conditions. Deletion analysis showed that the nucleotide sequence from -135 to -88 with respect to the translational start point is essential for nap expression either under anoxic or oxic conditions, suggesting that the same cis-acting element is involved in regulating nap expression under either anoxic with nitrate or oxic conditions.     

Involvement in denitrification of the napKEFDABC genes encoding the periplasmic nitrate reductase system in the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans

Seven genes, napKEFDABC, encoding the periplasmic nitrate reductase system were cloned from the denitrifying phototrophic bacterium Rhodobacter sphaeroides f. sp. denitrificans IL106. Two transmembrane proteins, NapK and NapE, an iron-sulfur protein NapF, a soluble protein NapD, a catalytic subunit of nitrate reductase precursor NapA, a soluble c-type diheme cytochrome precursor NapB, and a membrane-anchored c-type tetraheme cytochrome NapC were deduced as the gene products. Every mutant in which each nap gene was disrupted by omega-cassette insertion lost nitrate reductase activity as well as the ability of cells to grow with nitrate under anaerobic-dark conditions. A transconjugant of the napD-disrupted mutant with a plasmid bearing the napKEFDABC genes recovered both nitrate reductase activity and nitrate-dependent anaerobic-dark growth of cells. Denitrification activity, which was not observed in the napD mutant, was also restored by the conjugation. These results indicate that the periplasmic nitrate reductase encoded by the napKEFDABC genes is the enzyme responsible for denitrification in this phototroph, although the presence of a membrane-bound nitrate reductase has been reported in the same strain.     

Molybdenum requirement for translocation of dimethyl sulfoxide reductase to the periplasmic space in a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans.

Translocation of dimethyl sulfoxide (DMSO) reductase to the periplasmic space was studied in vivo with a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans, using immunoblotting analysis and radioactive labeling. A polypeptide with an apparent molecular mass about 2,000 Da higher than that of DMSO reductase accumulated during induction of the reductase with DMSO. An uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, inhibited the processing of the polypeptide after cells had been radioactively pulse-labeled with [35S]methionine. These results indicated that the higher-molecular-mass polypeptide was the precursor form of DMSO reductase. The precursor form accumulated in either the cytoplasm or the membrane, whereas the mature form accumulated in the periplasmic space. The membrane-bound precursor was sensitive to proteinase K treatment from both the cytoplasmic and periplasmic sides of the membrane, indicating that the polypeptide binds to the membrane, exposing it to both the outer and inner surfaces of the cytoplasmic membrane. Processing of the precursor was hampered by removal of molybdate from the medium and was restored by its readdition. It was also inhibited by the addition of tungstate in the medium.     

Rhodopseudomonas sphaeroides forma sp. denitrificans,a denitrifying strain as a subspecies of Rhodopseudomonas sphaeroides

From polluted water of a lagoon pond a new type of denitrifying photosynthetic purple bacteria was isolated. With respect to morphology, fine structure, photopigments, requirement for growth factors, the range of utilization of organic substrates for phototrophic growth and DNA base ratio, the denitrifying strains show the closest resemblance to Rhodopseudomonas sphaeroides and were therefore described as a subspecies named R. sphaeroides forma sp. denitrificans. The new isolates grow well with nitrate anaerobically in the dark accompanying the evolution of nitrogen gas. They cannot assimilate nitrate as the nitrogen source for growth.     

Nitrite-reducing ability is related to growth inhibition by nitrite in Rhodobacter sphaeroides f. sp. denitrificans

Growth inhibition of Rhodobacter sphaeroides f. sp. denitrificans IL106 by nitrite under anaerobic-light conditions became less pronounced when the gene encoding nitrite reductase was deleted. Growth of another deletion mutant of the genes encoding nitric oxide reductase was severely suppressed by nitrite. Our results suggest that nitrite reductase increases the sensitivity to nitrite through the production of nitric oxide.     

Spectroscopic studies of the molybdenum-containing dimethyl sulfoxide reductase from Rhodobacter sphaeroides f. sp. denitrificans.

Absorption and EPR spectroscopic properties of purified dimethyl sulfoxide (Me2SO) reductase from Rhodobacter sphaeroides f. sp. denitrificans have been examined. The absence of prosthetic groups other than the molybdenum center in the enzyme has made it possible to study its absorption properties. The enzyme displays multiple absorbance peaks in both the oxidized and the dithionite-reduced forms. The oxidized enzyme has absorbance peaks at 280, 350, 470, 550, and 720 nm while the dithionite-reduced enzyme has peaks at 280, 374, and 645 nm with a shoulder at 430 nm. A comparison of the absorbance spectrum of oxidized Me2SO reductase with that of the molybdenum fragment of rat liver sulfite oxidase shows that the 350 and 470 peaks are common to both proteins. EPR studies of the Mo(V) form of Me2SO reductase show a rhombic signal with g1 = 1.988, g2 = 1.977, g3 = 1.961, and g(ave) = 1.975. The signal shows evidence of coupling to an exchangeable proton with A1 = 1.05, A2 = 1.13, A3 = 0.98, and Aave = 1.05 millitesla. These parameters are similar to those of other Mo enzymes, however, the epr signal of this enzyme differs from those of other Mo hydroxylases in showing only a slight sensitivity to pH and no detectable anion effect. EPR potentiometric titrations of Me2SO reductase gave midpoint potentials of +144 mV for the Mo(VI)/Mo(V) couple and +160 mV for the Mo(V)/Mo(IV) couple at room temperature and +141 mV for the Mo(VI)/Mo(V) couple and +200 mV for the Mo(V)/Mo(IV) couple at 173 K.     

A Novel Molybdenum Protein with a Low Molecular Mass in Rhodobacter sphaeroides f. sp. denitrificans

A novel molybdenum cofactor-containing protein with a low molecularmass of 20 kDa was found in a photodenitrifier, Rhodobactersphaeroides f. sp. denitrificans. The protein was located inthe cytoplasm and was produced constitutively. (Received March 25, 1993; Accepted May 14, 1993)     

SspA, an outer membrane protein, is highly induced under salt-stressed conditions and is essential for growth under salt-stressed aerobic conditions in Rhodobacter sphaeroides f. sp. denitrificans

We have previously shown that an outer membrane protein, SspA, is prominently induced by salt stress in a photosynthetic bacterium, Rhodobacter sphaeroides f. sp. denitrificans IL106 (R. sphaeroides). In this study, we investigated the physiological role of SspA under various stress conditions. Using recombinant SspA expressed in Escherichia coli as an antigen, the polyclonal antiserum of SspA was prepared. Western blot analysis demonstrated that SspA was highly induced by salt stress under both anaerobic and aerobic conditions. SspA was also induced, but to a lesser extent, by osmotic and acid stress. It is reduced under heat and cold compared to non-stressed conditions. While sspA-disrupted R. sphaeroides grew normally under anaerobic conditions in either the presence or absence of stress, it displayed significantly retarded growth under aerobic conditions in the dark, especially when osmotic or salt stress were imposed. In addition, the sspA disruptant, but not the wild type, formed cell aggregates when grown under both anaerobic and aerobic conditions, and this phenotype was significantly enhanced under salt-stressed aerobic conditions. Together, our findings suggest that SspA is critical under salt-stressed, aerobic growth conditions.     

Cytochrome c" from the obligate methylotroph Methylophilus methylotrophus, an unexpected homolog of sphaeroides heme protein from the phototroph Rhodobacter sphaeroides.

The complete primary structure of an unusual soluble cytochrome c isolated from the obligate methylotrophic bacterium Methylophilus methylotrophus has been determined to contain 124 amino acids and to have an average molecular mass of 14293.0 Da. The sequence has two unusual features: firstly, the location of the heme-binding cysteines is far downstream from the N-terminus, namely at positions 49 and 52; secondly, an extra pair of cysteine residues is present near the C-terminus. In both respects, cytochrome c" is similar to the oxygen-binding heme protein SHP from the purple phototrophic bacterium Rhodobacter sphaeroides. In contrast to SHP, cytochrome c" changes from low-spin to high-spin upon reduction, due to dissociation of a sixth heme ligand histidine which is identified as His-95 by analogy to the class I cytochromes c. The distance of His-95 from the heme (41 residues) and the presence of certain consensus residues suggests that cytochrome c" is the second example of a variant class I cytochrome c.     

Anaerobic purification and characterization of nitrous oxide reductase from Rhodobacter sphaeroides f. sp. denitrificans IL106

The nitrous oxide reductase from the photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans IL106, has been purified under anaerobic conditions. The specific activity of the enzyme was 78 micromol nitrous oxide reduced per min per mg protein, which was approximately 80% higher than that of the aerobic form. The enzyme purified anaerobically retained most of its activity after aerobic storage at 4 degrees C for 2 months without any additives. Visible absorption spectra of the Rhodobacter nitrous oxide reductase resembled those of the enzymes from other origins. The enzyme retained its activity after reduction with sodium dithionite, and the enzyme activity could be determined using dithionite-reduced benzyl viologen. Turnover-dependent inactivation of the enzyme was suppressed by complete removal of oxygen from the reaction mixture, and promoted by zinc ions.     

Induction by nitrate of cytoplasmic and periplasmic proteins in the photodenitrifier Rhodobacter sphaeroides forma sp. denitrificans under anaerobic or aerobic condition

The synthesis of nitrate, nitrite, and nitrous oxide reductases is highly enhanced by the addition of nitrate during growth of Rhodobacter sphaeroides forma sp. denitrificans. Contrary to what is observed in many denitrifiers, the synthesis of these enzymes is not repressed by oxygen at concentrations as high as 37% air saturation. When oxygen concentration is increased up to 100% air saturation, the synthesis of nitrite and nitrous oxide reductases is repressed while the nitrate reductase is still synthesized. Two proteins, one periplasmic (35kDa) and the other cytoplasmic (32kDa), are also induced by nitrate, but not by trimethylamine-N-oxide or oxygen. Although their function is not yet known, these two proteins appear to be specifically linked to the denitrification pathway. The amino acid sequences of tryptic peptides and of the N-terminal ends of these proteins indicate no significant similarity with the sequences in the Swiss Prot Data Bank. However, a very good alignment is obtained between the amino acid sequences of the periplasmic nitrate reductase of Alcaligenes eutrophus H16 and those of various tryptic peptides of the nitrate reductase of R. sphaeroides forma sp. denitrificans.Abbreviations 2D Two-dimensional - DTT Dithiotreitol - PAGE Polyacrylamide gel electrophoresis - TMAO Trimethylamine-N-oxide - DMSO Dimethylsulfoxide - TMPD N,N,N,N tetramethyl-p-phenylenediamine     

Active transport of proline into washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans grown with nitrate

Washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans, prepared from cultures grown anaerobically in light with NO ₃ ^- as the terminal acceptor, readily incorporated [¹⁴C]-proline both in light and in the dark. The proline uptake was coupled to the reduction of either NO ₃ ^- , NO ₂ ^- , N₂O or O₂. Light stimulated the accumulation of proline in these cells. The addition of NO ₃ ^- to washed cells in light decreased the K _m for proline from 40 M to 5.7 M. Proline transport was inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide both in light and in the dark with nitrate indicating that electron transfer from both denitrification and photosynthesis are involved in this uptake. Inhibition by carbonyl cyanide-m-chlorophenyl hydrazone and 2.4-dinitrophenol indicate that proline transport is energy dependent. The H⁺/proline stoichiometry increased from 1 to 2.5 when the external pH was increased from 6.0 to 8.0. Under these conditions pro increased but p decreased markedly above pH 7.0.Abbreviations TPP⁺ Tetraphenylphosphonium bromide - EDTA ethylenediamine-tetra-acetic acid - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DNP 2,4-dinitrophenol - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - DBMIB dibromo-methyl-isopropyl-p-benzoquinone - DCCD N,N-dicyclohexylcarbodiimide     

Accumulation on the Cytoplasmic Membrane of the Precursor to Dimethyl Sulfoxide Reductase in Molybdenum Cofactor-Deficient Mutants of Rhodobacter sphaeroides f. sp. denitrificans

The role of the molybdenum cofactor (Mo cofactor) in the translocationof dimethyl sulfoxide (DMSO) reductase to the periplasmic spacewas studied in vivo by isolating chlorate-resistant mutantsof Rhodobacter sphaeroides f. sp. denitrificans. More than 50%of the chlorate-resistant mutants isolated were defective inthe biosynthesis of the Mo cofactor and all of these mutantsaccumulated the precursor form of the enzyme. About 45% of themutants contained the same level of Mo cofactor as the parentstrain and exhibited normal levels of DMSO reductase and nitratereductase activities when chlorate was absent from the medium,but the activities of these enzymes were depressed when chloratewas present. Much of the accumulated precursor form of the enzymein a Mo cofactor-deficient mutant was bound to the cytoplasmicmembrane and was sensitive to treatment with proteinase K fromthe periplasmic side of the membrane, an indication that theprecursor was exposed on the periplasmic surface of the membrane.The precursor accumulated on the membrane of the parent strainwhen molybdate was removed from the medium or upon additionof tungstate and this precursor was also sensitive to the treatmentwith proteinase K from the periplasmic side. These results suggestthat the Mo cofactor is necessary for proteolytic processingof the precursor to the mature enzyme on the periplasmic sideof the membrane, whereas binding of the precursor to the membraneand translocation across it can occur in the absence of thecofactor. Almost all of the Mo cofactor available for directreconstitution in vitro of nitrate reductase activity from thenit-l mutant of Neurospora crassa was present in the cytoplasmicfractions. (Received December 11, 1991; Accepted March 25, 1992)     

Periplasmic location of dissimilatory nitrate and nitrite reductases in a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans

The localization of dissimilatory nitrate and nitrite reductasesof a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroidesforma sp. denitrificans, was investigated. Nitrate and nitritereductases were located in the periplasmic space of the bacteriumgrown anaerobically in the presence of nitrate either in lightor in darkness. Chromatophores showed nitrate and nitrite reductaseactivities when dithionite-reduced benzyl viologen was an electrondonor; this suggests that the enzymes were trapped inside thevesicles. ¹Present address: Japanese Red Cross Central Blood Center, Hiroo4-1-31, Shibuyaku, Tokyo 150, Japan. ²Present address: Plant Growth Laboratory, University of California,Davis, California 95616, U.S.A. (Received November 7, 1979; )     

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.     

Cloning and sequencing of a gene encoding a novel salt stress-induced membrane protein from Rhodobacter sphaeroides f. sp. dentrificans

The gene encoding a membrane protein, SspA, induced under salt stress conditions was cloned and sequenced from a photosynthetic bacterium, Rhodobacter sphaeroides f. sp. denitrificans IL106. A single open reading frame consisting of 972 base pairs that encoded a polypeptide composed of a signal peptide of 24 amino acids and a mature protein of 300 amino acids (Mr 33,386) was found. A database search failed to detect any highly homologous sequences, indicating that SspA is a novel protein. The protein was present in the outer membrane as a transmembrane protein and was specifically induced by salt stress, but not by heat shock.     

Isolation of a Periplasmic Molecular Chaperone-Like Protein of Rhodobacter sphaeroides f. sp. denitrificans That Is Homologous to the Dipeptide Transport Protein DppA of Escherichia coli

A periplasmic protein has been found to prevent aggregation of the acid-unfolded dimethyl sulfoxide reductase (DMSOR), the periplasmic terminal reductase of dimethyl sulfoxide respiration in the phototroph Rhodobacter sphaeroides f. sp. denitrificans, in a manner similar to that of the Escherichia coli chaperonin GroEL (Matsuzaki et al., Plant Cell Physiol. 37:333–339, 1996). The protein was isolated from the periplasm of the phototroph. It had a molecular mass of 58 kDa and had no subunits. The sequence of 14 amino-terminal residues of the protein was completely identical to that of the periplasmic dipeptide transport protein (DppA) of E. coli. The 58-kDa protein prevented aggregation to a degree comparable to that of GroEL on the basis of monomer protein. The 58-kDa protein also decreased aggregation of guanidine hydrochloride-denatured rhodanese, a mitochondrial matrix protein, during its refolding upon dilution. The 58-kDa protein is a kind of molecular chaperone and could be involved in maintaining unfolded DMSOR, after secretion of the latter into the periplasm, in a competent form for its correct folding.     

Genetic and Biochemical Evidence for the Involvement of a Molybdenum-Dependent Enzyme in One of the Selenite Reduction Pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106

Selenite reduction in Rhodobacter sphaeroides f. sp. denitrificans was observed under photosynthetic conditions, following a 100-h lag period. This adaptation period was suppressed if the medium was inoculated with a culture previously grown in the presence of selenite, suggesting that selenite reduction involves an inducible enzymatic pathway. A transposon library was screened to isolate mutants affected in selenite reduction. Of the eight mutants isolated, two were affected in molybdenum cofactor synthesis. These moaA and mogA mutants showed an increased duration of the lag phase and a decreased rate of selenite reduction. When grown in the presence of tungstate, a well-known molybdenum-dependent enzyme (molybdoenzyme) inhibitor, the wild-type strain displayed the same phenotype. The addition of tungstate in the medium or the inactivation of the molybdocofactor synthesis induced a decrease of 40% in the rate of selenite reduction. These results suggest that several pathways are involved and that one of them involves a molybdoenzyme. Although addition of nitrate or dimethyl sulfoxide (DMSO) to the medium increased the selenite reduction activity of the culture, neither the periplasmic nitrate reductase NAP nor the DMSO reductase is the implicated molybdoenzyme, since the napA and dmsA mutants, with expression of nitrate reductase and DMSO reductase, respectively, eliminated, were not affected by selenite reduction. A role for the biotine sulfoxide reductase, another characterized molybdoenzyme, is unlikely, since its overexpression in a defective strain did not restore the selenite reduction activity.