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PubMed2007

Growth yields in bacterial denitrification and nitrate ammonification.

Denitrification and nitrate ammonification are considered the highest-energy-yielding respiration systems in anoxic environments after oxygen has been consumed. The corresponding free energy changes are 7 and 35% lower than that of aerobic respiration, respectively. Growth yield determinations with pure cultures of Paracoccus denitrificans and Pseudomonas stutzeri revealed that far less energy is converted via ATP into cell mass than expected from the above calculations. Denitrification with formate or hydrogen as electron donor yielded about 2.4 to 3.0 g dry matter per mol formate or hydrogen and 15 to 18 g dry matter per mol acetate. Similar yields with acetate were obtained with Pseudomonas stutzeri. Wolinella succinogenes and Sulfurospirillum deleyianum, which reduce nitrate to ammonia, both exhibited similar yield values with formate or H2 plus nitrate. The results indicate that ATP synthesis in denitrification is far lower than expected from the free energy changes and even lower than in nitrate ammonification. The results are discussed against the background of our present understanding of electron flow in denitrification and with respect to the importance of denitrification and nitrate ammonification in the environment.

Acetates↗
PubMed1994

Secretion of human epidermal growth factor (EGF) in autotrophic culture by a recombinant hydrogen-utilizing bacterium, Pseudomonas pseudoflava, carrying broad-host-range EGF secretion vector pKSEGF2.

We constructed the broad-host-range human epidermal growth factor (EGF) secretion plasmid pKSEGF2 by inserting the Escherichia coli tac promoter, the signal sequence of Pseudomonas stutzeri amylase, and the synthesized EGF gene into the broad-host-range vector pKT230. E. coli JM109 carrying pKSEGF2 secreted EGF into the periplasm and the culture medium under the control of the tac promoter. Pseudomonas aeruginosa PAO1161 carrying pKSEGF2 and Pseudomonas putida AC10 carrying pKSEGF2 secreted EGF into the culture medium constitutively. Four hydrogen-utilizing bacteria, Pseudomonas pseudoflava, Alcaligenes eutrophus, Alcaligenes paradoxus, and Paracoccus denitrificans, were transconjugated with pKSEGF2 from eight hydrogen-utilizing bacteria tested. In these transconjugated hydrogen-utilizing bacteria, P. pseudoflava carrying pKSEGF2 grew autotrophically and secreted EGF, confirmed by Western blot (immunoblot) analysis, into the culture medium constitutively.

Alcaligenes↗
PubMed1997

Identification and molecular genetic analysis of multiple loci contributing to high-level tellurite resistance in Rhodobacter sphaeroides 2.4.1.

The ability of the facultative photoheterotroph Rhodobacter sphaeroides to tolerate and reduce high levels of tellurite in addition to at least 10 other rare earth metal oxides and oxyanions has considerable potential for detoxification and bioremediation of contaminated environments. We report the identification and characterization of two loci involved in high-level tellurite resistance. The first locus contains four genes, two of which, trgAB, confer increased tellurite resistance when introduced into the related bacterium Paracoccus denitrificans. The trgAB-derived products display no significant homology to known proteins, but both are likely to be membrane-associated proteins. Immediately downstream of trgB, the cysK (cysteine synthase) and orf323 genes were identified. Disruption of the cysK gene resulted in decreased tellurite resistance in R. sphaeroides, confirming earlier observations on the importance of cysteine metabolism for high-level tellurite resistance. The second locus identified is represented by the telA gene, which is separated from trgAB by 115 kb. The telA gene product is 65% similar to the product of the klaB (telA) gene from the tellurite-resistance-encoding kilA operon from plasmid RK2. The genes immediately linked to the R. sphaeroides telA gene have no similarity to other components of the kilA operon. R. sphaeroides telA could not functionally substitute for the plasmid RK2 telA gene, indicating substantial functional divergence between the two gene products. However, inactivation of R. sphaeroides telA resulted in a significant decrease in tellurite resistance compared to the wild-type strain. Both cysK and telA null mutations readily gave rise to suppressors, suggesting that the phenomenon of high-level tellurite resistance in R. sphaeroides is complex and other, as yet uncharacterized, loci may be involved.

Amino Acid Sequence↗
PubMed2004

Use of Sinorhizobium meliloti as an indicator for specific detection of long-chain N-acyl homoserine lactones.

Population-density-dependent gene expression in gram-negative bacteria involves the production of signal molecules characterized as N-acyl homoserine lactones (AHLs). The synthesis of AHLs by numerous microorganisms has been identified by using biosensor strains based on the Agrobacterium tumefaciens and Chromobacterium violaceum quorum-sensing systems. The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti is rapidly becoming a model organism for the study of quorum sensing. This organism harbors at least three different quorum-sensing systems (Sin, Mel, and Tra), which play a role in its symbiotic relationship with its host plant, alfalfa. The Sin system is distinguished among them for the production of long-chain AHLs, including C(18)-HL, the longest AHL reported so far. In this work, we show that construction of a sinI::lacZ transcriptional fusion results in a strain that detects long-chain AHLs with exquisite sensitivity. Overexpression of the SinR regulator protein from a vector promoter increases its sensitivity without loss of specificity. We also show that the resulting indicator strain can recognize long-chain AHLs produced by unrelated bacteria such as Paracoccus denitrificans and Rhodobacter capsulatus. This S. meliloti indicator strain should serve as a tool for the specific detection of long-chain AHLs in new systems.

4-Butyrolactone↗
PubMed2005

Inhibition of NO3- and NO2- reduction by microbial Fe(III) reduction: evidence of a reaction between NO2- and cell surface-bound Fe2+.

A recent study (D. C. Cooper, F. W. Picardal, A. Schimmelmann, and A. J. Coby, Appl. Environ. Microbiol. 69:3517-3525, 2003) has shown that NO(3)(-) and NO(2)(-) (NO(x)(-)) reduction by Shewanella putrefaciens 200 is inhibited in the presence of goethite. The hypothetical mechanism offered to explain this finding involved the formation of a Fe(III) (hydr)oxide coating on the cell via the surface-catalyzed, abiotic reaction between Fe(2+) and NO(2)(-). This coating could then inhibit reduction of NO(x)(-) by physically blocking transport into the cell. Although the data in the previous study were consistent with such an explanation, the hypothesis was largely speculative. In the current work, this hypothesis was tested and its environmental significance explored through a number of experiments. The inhibition of approximately 3 mM NO(3)(-) reduction was observed during reduction of a variety of Fe(III) (hydr)oxides, including goethite, hematite, and an iron-bearing, natural sediment. Inhibition of oxygen and fumarate reduction was observed following treatment of cells with Fe(2+) and NO(2)(-), demonstrating that utilization of other soluble electron acceptors could also be inhibited. Previous adsorption of Fe(2+) onto Paracoccus denitrificans inhibited NO(x)(-) reduction, showing that Fe(II) can reduce rates of soluble electron acceptor utilization by non-iron-reducing bacteria. NO(2)(-) was chemically reduced to N(2)O by goethite or cell-sorbed Fe(2+), but not at appreciable rates by aqueous Fe(2+). Transmission and scanning electron microscopy showed an electron-dense, Fe-enriched coating on cells treated with Fe(2+) and NO(2)(-). The formation and effects of such coatings underscore the complexity of the biogeochemical reactions that occur in the subsurface.

Ferric Compounds↗
PubMed2006

Autoregulator protein PhaR for biosynthesis of polyhydroxybutyrate [P(3HB)] possibly has two separate domains that bind to the target DNA and P(3HB): Functional mapping of amino acid residues responsible for DNA binding.

PhaR from Paracoccus denitrificans functions as a repressor or autoregulator of the expression of genes encoding phasin protein (PhaP) and PhaR itself, both of which are components of polyhydroxyalkanoate (PHA) granules (A. Maehara, S. Taguchi, T. Nishiyama, T. Yamane, and Y. Doi, J. Bacteriol. 184:3992-4002, 2002). PhaR is a unique regulatory protein in that it also has the ability to bind tightly to an effector molecule, PHA polyester. In this study, by using a quartz crystal microbalance, we obtained direct evidence that PhaR binds to the target DNA and poly[(R)-3-hydroxybutyrate] [P(3HB)], one of the PHAs, at the same time. To identify the PhaR amino acid residues responsible for DNA binding, deletion and PCR-mediated random point mutation experiments were carried out with the gene encoding the PhaR protein. PhaR point mutants with decreased DNA-binding abilities were efficiently screened by an in vivo monitoring assay system coupled with gene expression of green fluorescent protein in Escherichia coli. DNA-binding abilities of the wild-type and mutants of recombinant PhaR expressed in E. coli were evaluated using a gel shift assay and a surface plasmon resonance analysis. These experiments revealed that basic amino acids and a tyrosine in the N-terminal region, which is highly conserved among PhaR homologs, are responsible for DNA binding. However, most of the mutants with decreased DNA-binding abilities were unaffected in their ability to bind P(3HB), strongly suggesting that PhaR has two separate domains capable of binding to the target DNA and P(3HB).

Amino Acid Motifs↗
PubMed2007

The AppA and PpsR proteins from Rhodobacter sphaeroides can establish a redox-dependent signal chain but fail to transmit blue-light signals in other bacteria.

The AppA protein of Rhodobacter sphaeroides has the unique ability to sense and transmit redox and light signals. In response to decreasing oxygen tension, AppA antagonizes the transcriptional regulator PpsR, which represses the expression of photosynthesis genes, including the puc operon. This mechanism, which is based on direct protein-protein interaction, is prevented by blue-light absorption of the BLUF domain located in the N-terminal part of AppA. In order to test whether AppA and PpsR are sufficient to transmit redox and light signals, we expressed these proteins in three different bacterial species and monitored oxygen- and blue-light-dependent puc expression either directly or by using a luciferase-based reporter construct. The AppA/PpsR system could mediate redox-dependent gene expression in the alphaproteobacteria Rhodobacter capsulatus and Paracoccus denitrificans but not in the gammaproteobacterium Escherichia coli. Analysis of a prrA mutant strain of R. sphaeroides strongly suggests that light-dependent gene expression requires a balanced interplay of the AppA/PpsR system with the PrrA response regulator. Therefore, the AppA/PpsR system was unable to establish light signaling in other bacteria. Based on our data, we present a model for the interdependence of AppA/PpsR signaling and the PrrA transcriptional activator.

Bacterial Proteins↗
PubMed1977

Reduction of iron and synthesis of protoheme by Spirillum itersonii and other organisms.

Membranes from Spirillum itersonii reduce ferric iron to ferrous iron with reduced nicotinamide adenine dinucleotide or succinate as a source of reductant. Iron reduction was measured spectrophotometrically at 562 nm using ferrozine, which chelates ferrous iron specifically. Reduced nicotinamide adenine dinucleotide or succinate was also effective as a source of iron. The effects of respiratory inhibitors suggested that reduction of iron occurs at one or more sites on the respiratory chain before cytochrome c. Reduction of iron and synthesis of protoheme with the physiological reductants were also observed with crude extracts of other bacteria, including Rhodopseudomonas spheroides, Rhodopseudomonas capsulata, Paracoccus denitrificans, and Escherichia coli. The effect of oxygen upon reduction of iron and formation of protoheme was examined with membranes from S. itersonii, using succinate as a source of reductant. Both systems were inhibited by oxygen, but this effect was completely reversed by addition of antimycin A. We conclude that reduced components of the respiratory chain serve as reductants for ferric iron, but with oxygen present they are oxidized preferentially by the successive members of the chain. This could be a mechanism for regulating synthesis of heme and cytochrome by oxygen.

Antimycin A↗
PubMed1979

Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane.

Escherichia coli grown anaerobically on nitrate exhibited the same transport barrier to reduction of chlorate, relative to nitrate, as that exhibited by Paracoccus denitrificans. This establishes that the nitrate binding site of nitrate reductase (EC 1.7.99.4) in E. coli must also lie on the cell side of the nitrate transporter which is associated with the plasma membrane. Because nitrate reductase is membrane bound, the nitrate binding site is thus located on the inner aspect of the membrane. Nitrate pulse studies on E. coli in the absence of valinomycin showed a small transient alkalinization (leads to H+/NO3- congruent to --0.07) which did not occur with oxygen pulses. By analogy with P. denitrificans, the alkaline transient is interpreted to arise from proton-linked nitrate uptake which is closely followed by nitrite efflux. The result is consistent with internal reduction of nitrate, whereas external reduction would be expected to give leads to H+/NO3-ratios approaching --2.

Anaerobiosis↗
PubMed1980

Inhibition, but not uncoupling, of respiratory energy coupling of three bacterial species by nitrite.

The effect of nitrite on respiratory energy coupling of three bacteria was studied in light of a recent report that nitrite acted as an uncoupling agent with Paracoccus denitrificans grown under denitrifying conditions. Our determinations of proton translocation stoichiometry of Pseudomonas putida (aerobically grown), Pseudomonas aeruginosa, and P. denitrificans (grown both aerobically and under denitrifying conditions) showed nitrite inhibition of proton-to-oxidant stoichiometry, but not uncoupling. Nitrite both reduced the H+/O ratio and decreased the rate of proton resorption. Increased proton resorption rates, characteristic of authentic uncoupling agents, were not observed. The lack of enhanced proton permeability due to nitrite was verified via passive proton permeability assays. The H+/O ratio of P. aeruginosa increased when growth conditions were changed from aerobic to denitrifying. This suggested the induction of an additional coupling site in the electron transport chain of denitrifying P. aeruginosa.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1982

Alterations in the phospholipid composition of Rhodopseudomonas sphaeroides and other bacteria induced by Tris.

Alterations in the phospholipid head group composition of most strains of Rhodopseudomonas sphaeroides, as well as Rhodopseudomonas capsulata and Paracoccus denitrificans, occurred when cells were grown in medium supplemented with Tris. Growth of R. sphaeroides M29-5 in Tris-supplemented medium resulted in the accumulation of N-acylphosphatidylserine (NAPS) to as much as 40% of the total whole-cell phospholipid, whereas NAPS represented approximately 28 an 33% of the total phospholipid when R. capsulata and P. denitrificans respectively, were grown in medium containing 20 mM Tris. The accumulation of NAPS occurred primarily at the expense of phosphatidylethanolamine in both whole cells and isolated membranes of R. sphaeroides and had no detectable effect on cell growth under either chemoheterotrophic or photoheterotrophic conditions. Yeast extract (0.1%) and Casamino Acids (1.0%) were found to be antagonistic to the Tris-induced (20 mM) alteration in the phospholipid composition of R. sphaeroides. The wild-type strains R. sphaeroides 2.4.1 and RS2 showed no alteration in their phospholipid composition when they were grown in medium supplemented with Tris. In all strains of Rhodospirillaceae tested, as well as in P. denitrificans, NAPS represented between 1.0 and 2.0% of the total phospholipid when cells were grown in the absence of Tris. [32P]orthophosphoric acid entered NAPS rapidly in strains of R. sphaeroides that do (strain M29-5) and do not (strain 2.4.1) accumulate this phospholipid in response to Tris. Our data indicate that the phospholipid head group composition of many Rhodospirillaceae strains, as well as P. denitrificans, is easily manipulated; thus, these bacteria may provide good model systems for studying the effects of these modifications on membrane structure and function in a relatively unperturbed physiological system.

Membrane Lipids↗
PubMed1983

Immunochemical relationship of the major outer membrane protein of Rhodopseudomonas sphaeroides 2.4.1 to proteins of other photosynthetic bacteria.

Immunoblots of sodium dodecyl sulfate-polyacrylamide gels derived from outer membrane preparations of various strains of Rhodopseudomonas sphaeroides revealed polypeptides which cross-reacted with antibody directed against the major outer membrane protein of R. sphaeroides 2.4.1. Immunochemical quantitation of the major outer membrane protein of strain 2.4.1 showed approximately 5.5 x 10(4) molecules per cell whether cells were grown chemoheterotrophically or photoheterotrophically. Rhodospirillum rubrum outer membranes contained a cross-reactive protein, whereas the outer membranes derived from Rhodopseudomonas capsulata and Paracoccus denitrificans showed no cross-reaction with the antibody prepared against the major outer membrane protein from R. sphaeroides 2.4.1.

Bacterial Outer Membrane Proteins↗
PubMed1985

Succinate dehydrogenase in Rhodopseudomonas sphaeroides: subunit composition and immunocross-reactivity with other related bacteria.

Antibodies were raised against the succinate dehydrogenase (SDH) present in the chromatophores of phototrophically grown Rhodopseudomonas sphaeroides. Crossed immunoelectrophoresis experiments indicated that the SDH present in the cytoplasmic membranes of heterotrophically grown R. sphaeroides is probably the same enzyme observed in the chromatophores. The enzyme was extracted by Triton X-100 in a form which consisted of only two subunits (molecular weight, 68,000 and 30,000) and was not associated with a cytochrome b. The antibodies directed against SDH from R. sphaeroides showed no immunocross-reactivity with SDH from phylogenetically related bacterial species, including Rhodopseudomonas capsulata, Paracoccus denitrificans, Rhodopseudomonas palustris, Rhodospirillum rubrum, and Rhodospirillum fulvum.

Bacterial Chromatophores↗
PubMed1985

Nitric oxide-dependent proton translocation in various denitrifiers.

Respiration of NO resulted in transient proton translocation in anaerobically grown cells of four physiologically diverse denitrifiers. Paracoccus denitrificans, Rhodopseudomonas sphaeroides subsp. denitrificans, "Achromobacter cycloclastes," and Rhizobium japonicum gave, respectively, H+/NO ratios of 3.65, 4.96, 1.94, and 1.12. Antimycin A completely inhibited NO-dependent proton translocation in P. denitrificans and severely restricted translocation in the R. sphaeroides strain. Proton uptake during NO respiration with antimycin A-inhibited cells supplied with an artificial electron source provided evidence for the periplasmic consumption of protons. Values obtained were consistent with the expected ratios of 0.5 mol of H+/mol of NO for reduction of NO to N2O and 1.0 mol of H+/mol of NO for reduction of NO to N2. These data are consistent with the presence of a unique NO reductase found only in anaerobically grown denitrifying cells.

Alcaligenes↗
PubMed1999

Heterologous expression of correctly assembled methylamine dehydrogenase in Rhodobacter sphaeroides.

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits, mauB and mauA. The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds. To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of P. denitrificans, mauFBEDACJG, were placed under the regulatory control of the coxII promoter of Rhodobacter sphaeroides and introduced into R. sphaeroides by using a broad-host-range vector. The heterologous expression of MADH was constitutive with respect to carbon source, whereas the native mau promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources. The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from P. denitrificans. These results indicate that mauM and mauN are not required for MADH or TTQ biosynthesis and that mauFBEDACJG are sufficient for TTQ biosynthesis, since R. sphaeroides cannot synthesize TTQ. A similar construct introduced into Escherichia coli did not produce detectable MADH activity or accumulation of the mauB and mauA gene products but did lead to synthesizes of amicyanin, the mauC gene product. This finding suggests that active recombinant MADH is not expressed in E. coli because one of the accessory gene products is not functionally expressed. This study illustrates the potential utility of R. sphaeroides and the coxII promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in E. coli. These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other mau genes.

Bacterial Proteins↗
PubMed2000

Heterologous NNR-mediated nitric oxide signaling in Escherichia coli.

The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melR promoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO(+) donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a "positive control" phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.

Amino Acid Substitution↗
PubMed2001

Two conserved glutamates in the bacterial nitric oxide reductase are essential for activity but not assembly of the enzyme.

The bacterial nitric oxide reductase (NOR) is a divergent member of the family of respiratory heme-copper oxidases. It differs from other family members in that it contains an Fe(B)-heme-Fe dinuclear catalytic center rather than a Cu(B)-heme-Fe center and in that it does not pump protons. Several glutamate residues are conserved in NORs but are absent in other heme-copper oxidases. To facilitate mutagenesis-based studies of these residues in Paracoccus denitrificans NOR, we developed two expression systems that enable inactive or poorly active NOR to be expressed, characterized in vivo, and purified. These are (i) a homologous system utilizing the cycA promoter to drive aerobic expression of NOR in P. denitrificans and (ii) a heterologous system which provides the first example of the expression of an integral-membrane cytochrome bc complex in Escherichia coli. Alanine substitutions for three of the conserved glutamate residues (E125, E198, and E202) were introduced into NOR, and the proteins were expressed in P. denitrificans and E. coli. Characterization in intact cells and membranes has demonstrated that two of the glutamates are essential for normal levels of NOR activity: E125, which is predicted to be on the periplasmic surface close to helix IV, and E198, which is predicted to lie in the middle of transmembrane helix VI. The subsequent purification and spectroscopic characterization of these enzymes established that they are stable and have a wild-type cofactor composition. Possible roles for these glutamates in proton uptake and the chemistry of NO reduction at the active site are discussed.

Amino Acid Substitution↗
PubMed2002

Long-chain acyl-homoserine lactone quorum-sensing regulation of Rhodobacter capsulatus gene transfer agent production.

Many proteobacteria use acyl-homoserine lactones as quorum-sensing signals. Traditionally, biological detection systems have been used to identify bacteria that produce acyl-homoserine lactones, although the specificities of these detection systems can limit discovery. We used a sensitive approach that did not require a bioassay to detect production of long-acyl-chain homoserine lactone production by Rhodobacter capsulatus and Paracoccus denitrificans. These long-chain acyl-homoserine lactones are not readily detected by standard bioassays. The most abundant acyl-homoserine lactone was N-hexadecanoyl-homoserine lactone. The long-chain acyl-homoserine lactones were concentrated in cells but were also found in the culture fluid. An R. capsulatus gene responsible for long-chain acyl-homoserine lactone synthesis was identified. A mutation in this gene, which we named gtaI, resulted in decreased production of the R. capsulatus gene transfer agent, and gene transfer agent production was restored by exogenous addition of N-hexadecanoyl-homoserine lactone. Thus, long-chain acyl-homoserine lactones serve as quorum-sensing signals to enhance genetic exchange in R. capsulatus.

4-Butyrolactone↗