Isolation of transposon Tn5 insertion mutants of Rhodobacter capsulatus unable to reduce trimethylamine-N-oxide and dimethylsulphoxide

1) Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) strain 37b4 was subjected to transposon Tn5 mutagenesis. 2) Kanamycin-resistant transconjugants were screened for their inability to reduce trimethylamine-N-oxide (TMAO) as judged by the lack of alkali production during anaerobic growth on plates containing glucose as carbon source and cresol red as pH indicator. 3) Of 6 mutants examined, all were found to have considerably decreased levels of methylviologen-dependent TMAO reductase activity and dimethylsulphoxide (DMSO) reductase activity. 4) Periplasmic fractions of one of these mutants (DK9) and of the parent strain were subjected to sodium dodecylsulphate polyacrylamide gel electrophoresis. The gels were stained for TMAO-reductase and DMSO-reductase. With the wild-type strain, only a single polypeptide band, M_r=46,000, stained for TMAO and DMSO reductase activity. In mutant DK9 this band was not detectable. 5) In contrast to the parent strain, harvested washed cells of mutant DK9 were unable to generate a cytoplasmic membrane potential in the presence of TMAO or DMSO under dark anaerobic conditions. 6) In contrast to the parent strain, DK9 was unable to grow in dark anaerobic culture with fructose as the carbon source and TMAO as oxidant.Abbreviations TMAO trimethylamine-N-oxide - DMSO dimethylsulphoxide - PMS phenazine methosulphate - cytoplasmic membrane potential     

Reduction of N-oxides and sulfoxide by the same terminal reductase inProteus mirabilis

Proteus mirabilis can grow anaerobically on the fermentable substrate, glucose. When the glucose medium was supplemented with an electron acceptor, growth doubled. However, the organism failed to grow anaerobically on the oxidizable substrate glycerol unless the medium was supplemented with an external electron acceptor. Dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nicotinamide N-oxide (NAMO), and nitrate (NO₃) can serve this function. Cell-free extracts ofP. mirabilis can reduce these compounds in the presence of various electron donors. In order to determine whether the same or different terminal reductase(s) are involved in the reduction of these compounds, we isolated mutants unable to grow on glycerol/DMSO medium. When these mutants were tested on glycerol medium containing TMAO, NAMO, and NO₃ as electron acceptors, it was found that there were two groups. Group I mutants were unable to grow with DMSO, TMAO, and NAMO, while their growth was unaffected with NO₃. Group II mutants were unable to grow on any electron acceptor including NO₃. Enzyme assays using reduced benzyl viologen with both groups of mutants were in agreement with growth studies. On the basis of these results, we conclude that the same terminal reductase is involved in the reduction of DMSO, TMAO, and NAMO (group I) and that the additional loss of NO₃ reductase in group II mutants is probably owing to a defect in the synthesis or insertion of molybdenum cofactor.     

Anaerobic respiratory growth of Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi with trimethylamine N-oxide, nitrate and fumarate: ecological implications

Two symbiotic species, Photobacterium leiognathi and Vibrio fischeri, and one non-symbiotic species, Vibrio harveyi, of the Vibrionaceae were tested for their ability to grow by anaerobic respiration on various electron acceptors, including trimethylamine N-oxide (TMAO) and dimethylsulphoxide (DMSO), compounds common in the marine environment. Each species was able to grow anaerobically with TMAO, nitrate or fumarate, but not with DMSO, as an electron acceptor. Cell growth under microaerophilic growth conditions resulted in elevated levels of TMAO reductase, nitrate reductase and fumarate reductase activity in each strain, whereas growth in the presence of the respective substrate for each enzyme further elevated enzyme activity. TMAO reductase specific activity was the highest of all the reductases. Interestingly, the bacteria-colonized light organs from the two squids, Euprymna scolopes and Euprymna morsei, and the light organ of the ponyfish, Leiognathus equus, also had high levels of TMAO reductase enzyme activity, in contrast to non-symbiotic tissues. The ability of these bacterial symbionts to support cell growth by respiration with TMAO may conceivably eliminate the competition for oxygen needed for both bioluminescence and metabolism.     

The effect of oxygen on denitrification during steady-state growth of Paracoccus halodenitrificans

Dimethylsulphoxide (DMSO) and trimethylamine oxide (TMAO) sustained anaerobic growth of Proteus vulgaris with the non-fermentable substrate lactate. Cytoplasmic membrane vesicles energized by electron transfer from formate to DMSO displayed anaerobic uptake of serine, which was hindered by metabolic inhibitors known to destroy the proton motive force. This showed that DMSO reduction was coupled with a chemiosmotic mechanism of energy conversion; similar data for TMAO respiration have been presented previously. All biochemical tests applied indicated that the oxides were reduced by the same reductase system. The DMSO and TMAO reductase activities showed the same mobility on ion-exchange chromatography, and polyacrylamide disc gel electrophoresis (pH 8.9), gradient gel electrophoresis, and gel isoelectric focusing; mol. wt. and pI determined were 95,000 and 4.6, respectively. DMSO inhibited reduction of [¹⁴C]TMAO in vesicles. The reductase was inducible to a certain extent; both oxides being equally efficient as inducers. TMAO was reduced at a higher rate than DMSO, explaining faster growth of cells and increased uptake of serine in vesicles with TMAO as electron acceptor. Comparative studies with Escherichia coli also gave evidence for common TMAO and DMSO reductase systems.Abbreviations TMAO trimethylamine oxide - DMSO dimethylsulphoxide     

Characterization of genes encoding dimethyl sulfoxide reductase of Rhodobacter sphaeroides 2.4.1T: an essential metabolic gene function encoded on chromosome II.

Rhodobacter sphaeroides 2.4.1T is a purple nonsulfur facultative phototrophic bacterium which exhibits remarkable metabolic diversity as well as genomic complexity. Under anoxic conditions, in the absence of light and the presence of dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO), R. sphaeroides 2.4.1T utilizes DMSO or TMAO as the terminal electron acceptor for anaerobic respiration, which is mediated by the molybdoenzyme DMSO reductase. Sequencing of a 13-kb region of chromosome II revealed the presence of 10 putative open reading frames, of which 5 possess homology to genes encoding the TMAO reductase (the tor system) of Escherichia coli. The dorS and dorR genes encode a sensor-regulator pair of the two-component sensory transduction protein family, homologous to the torS and torR gene products. The dorC gene was shown to encode a 44-kDa DMSO-inducible c-type cytochrome. The dorB gene encodes a membrane protein of unknown function homologous to the torD gene product. The dorA gene encodes DMSO reductase, containing the molybdopterin active site. Mutations were constructed in each of these dor genes, and the resulting mutants were shown to be impaired for DMSO-dependent anaerobic growth in the dark. The mutant strains exhibited negligible levels of DMSO reductase activity compared to the wild-type strain under similar growth conditions. Further, no DorA protein was detected in DorS and DorR mutant strains with anti-DorA antisera, suggesting that the products of these genes are required for the positive regulation of dor expression in response to DMSO. This characterization of the dor gene cluster is the first evidence that genes of chromosome CII encode metabolic functions which are essential under particular growth conditions.     

Involvement of cyclic AMP (cAMP) and cAMP receptor protein in anaerobic respiration of Shewanella oneidensis

Shewanella oneidensis is a metal reducer that can use several terminal electron acceptors for anaerobic respiration, including fumarate, nitrate, dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nitrite, and insoluble iron and manganese oxides. Two S. oneidensis mutants, SR-558 and SR-559, with Tn5 insertions in crp, were isolated and analyzed. Both mutants were deficient in Fe(III) and Mn(IV) reduction. They were also deficient in anaerobic growth with, and reduction of, nitrate, fumarate, and DMSO. Although nitrite reductase activity was not affected by the crp mutation, the mutants failed to grow with nitrite as a terminal electron acceptor. This growth deficiency may be due to the observed loss of cytochromes c in the mutants. In contrast, TMAO reduction and growth were not affected by loss of cyclic AMP (cAMP) receptor protein (CRP). Fumarate and Fe(III) reductase activities were induced in rich medium by the addition of cAMP to aerobically growing wild-type S. oneidensis. These results indicate that CRP and cAMP play a role in the regulation of anaerobic respiration, in addition to their known roles in catabolite repression and carbon source utilization in other bacteria.     

Oxygen and nitrate-dependent regulation of <Emphasis Type="Italic">dmsABC</Emphasis> operon expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>: sites for Fnr and NarL protein interactions

Background  

Escherichia coli can respire anaerobically using dimethyl sulfoxide (DMSO) or trimethylamine-N-oxide (TMAO) as the terminal electron acceptor for anaerobic energy generation. Expression of the dmsABC genes that encode the membrane-associated DMSO/TMAO reductase is positively regulated during anaerobic conditions by the Fnr protein and negatively regulated by the NarL protein when nitrate is present.     

Redox-Dependent Gene Regulation in Rhodobacter sphaeroides 2.4.1T: Effects on Dimethyl Sulfoxide Reductase (dor) Gene Expression

The ability of Rhodobacter sphaeroides 2.4.1^T to respire anaerobically with the alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) is manifested by the molybdoenzyme DMSO reductase, which is encoded by genes of the dor locus. Previously, we have demonstrated that dor expression is regulated in response to lowered oxygen tensions and the presence of DMSO or TMAO in the growth medium. Several regulatory proteins have been identified as key players in this regulatory cascade: FnrL, DorS-DorR, and DorX-DorY. To further examine the role of redox potentiation in the regulation of dor expression, we measured DMSO reductase synthesis and β-galactosidase activity from dor::lacZ fusions in strains containing mutations in the redox-active proteins CcoP and RdxB, which have previously been implicated in the generation of a redox signal affecting photosynthesis gene expression. Unlike the wild-type strain, both mutants were able to synthesize DMSO reductase under strictly aerobic conditions, even in the absence of DMSO. When cells were grown photoheterotrophically, dorC::lacZ expression was stimulated by increasing light intensity in the CcoP mutant, whereas it is normally repressed in the wild-type strain under such conditions. Furthermore, the expression of genes encoding the DorS sensor kinase and DorR response regulator proteins was also affected by the ccoP mutation. By using CcoP-DorR and CcoP-DorY double mutants, it was shown that the DorR protein is strictly required for altered dor expression in CcoP mutants. These results further demonstrate a role for redox-generated responses in the expression of genes encoding DMSO reductase in R. sphaeroides and identify the DorS-DorR proteins as a redox-dependent regulatory system controlling dor expression.     

Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101.

Escherichia coli grew anaerobically on a minimal medium with glycerol as the carbon and energy source and dimethyl sulfoxide (DMSO) as the terminal electron acceptor. DMSO reductase activity, measured with an artificial electron donor (reduced benzyl viologen), was preferentially associated with the membrane fraction (77 +/- 10% total cellular activity). A Km for DMSO reduction of 170 +/- 60 microM was determined for the membrane-bound activity. Methyl viologen, reduced flavin mononucleotide, and reduced flavin adenine dinucleotide also served as electron donors for DMSO reduction. Methionine sulfoxide, a DMSO analog, could substitute for DMSO in both the growth medium and in the benzyl viologen assay. DMSO reductase activity was present in cells grown anaerobically on DMSO but was repressed by the presence of nitrate or by aerobic growth. Anaerobic growth on DMSO coinduced nitrate, fumarate, and and trimethylamine-N-oxide reductase activities. The requirement of a molybdenum cofactor for DMSO reduction was suggested by the inhibition of growth and a 60% reduction in DMSO reductase activity in the presence of 10 mM sodium tungstate. Furthermore, chlorate-resistant mutants chlA, chlB, chlE, and chlG were unable to grow anaerobically on DMSO. DMSO reduction appears to be under the control of the fnr gene.     

Anaerobic growth of halophilic archaeobacteria by reduction of dimethysulfoxide and trimethylamine N-oxide

Abstract Most representatives of the halophilic arachaeobacterial genera Halobacterium, Haloarcula and Haloferax tested were able to reduce dimethylsulfoxide (DMSO) to dimethylsulfide (DMS) and trimethylamine N -oxide (TMAO) to trimethylamine (TMA) under (semi)anaerobic conditions. In most cases the reduction of DMSO and TMAO was accompanied by an increase in cell yield. The ability to reduce DMSO or TMAO was not correlated to reduced DMSO or TMAO was not correlated with the ability to reduce nitrate to nitrite. Anaerobic respiration with DMSO and TMAO as electron acceptor supplies the halophilic archeobacteria with an additional mode of energy generation in the absence of molecular oxygen.     

DMSO respiration by the anaerobic rumen bacterium Wolinella succinogenes

The anaerobic rumen bacterium Wolinella succinogenes was able to grow by respiration with dimethylsulphoxide (DMSO) as electron acceptor and formate or H₂ as electron donors. The growth yield amounted to 6.7 g and 6.4 g dry cells/mol DMSO with formate or H₂ as the donors, respectively. This suggested an ATP yield of about 0.7 mol ATP/mol DMSO. Cell homogenates and the membrane fraction contained DMSO reductase activity with a high K _m (43 mM) for DMSO. The electron transport from H₂ to DMSO in the membranes was inhibited by 2-(heptyl)-4-hydroxyquinoline N-oxide, indicating the participation of menaquinone. Formation of DMSO reductase activity occurred only during growth on DMSO, presence of other electron acceptors (fumarate, nitrate, nitrite, N₂O, and sulphur) repressed the DMSO reductase activity. DMSO can therefore be used by W. succinogenes as an acceptor for phosphorylative electron transport, but other electron acceptors are used preferentially.Abbreviations DMN 2,3-Dimethyl-1,4-naphthoquinone - DMNH ₂ Reduced DMN - DMS Dimethylsulphide (CH₃)₂S - DMSO Dimethylsulphoxide (CH₃)₂SO - HQNO 2-(Heptyl)-4-hydroxyquinoline-N-oxide - TMAO Trimethylamine-N-oxide - Y _s Growth yield for substrate S     

Characterization and multiple molecular forms of TorD from Shewanella massilia,the putative chaperone of the molybdoenzyme TorA

Several bacteria use trimethylamine N-oxyde (TMAO) as an exogenous electron acceptor for anaerobic respiration. This metabolic pathway involves expression of the tor operon that codes for a periplasmic molybdopterin-containing reductase of the DMSO/TMAO family, a pentahemic c-type cytochrome, and the TorD cytoplasmic chaperone, possibly required for acquisition of the molybdenum cofactor and translocation of the reductase by the twin-arginine translocation system. In this report, we show that the TorD chaperone from Shewanella massilia forms multiple and stable oligomeric species. The monomeric, dimeric, and trimeric forms were purified to homogeneity and characterized by analytical ultracentrifugation. Small-angle X-ray scattering (SAXS) and preliminary diffraction data indicated that the TorD dimer is made of identical protein modules of similar size to the monomeric species. Interconversion of the native oligomeric forms occurred at acidic pH value. In this condition, ANS fluorescence indicates a non-native conformation of the polypeptide chain in which, according to the circular dichroism spectra, the alpha-helical content is similar to that of the native species. Surface plasmon resonance showed that both the monomeric and dimeric species bind the mature TorA enzyme, but that the dimer binds its target protein more efficiently. The possible biologic significance of these oligomers is discussed in relation to the chaperone activity of TorD, and to the ability of another member of the TorD family to bind the Twin Arginine leader sequences of the precursor of DMSO/TMAO reductases.     

Differentiation of the multiple S- and N-oxide-reducing activities ofEscherichia coli

InEscherichia coli, several terminal reductases catalyze the reduction of S- and N-oxide compounds. We have used mutants missing either the constitutive dimethylsulfoxide (DMSO) reductase,dmsABC, and/or the inducible trimethylamine N-oxide (TMAO) reductase,torA, to define the roles of each reductase. These studies indicated that the constitutive DMSO reductase can sustain growth on DMSO, TMAO, methionine sulfoxide (MetSO), and other N-oxide compounds. Only one inducible TMAO reductase is expressed inE. coli, and this enzyme sustains growth on TMAO but not DMSO or MetSO. Characterization of atorA ^–, dms^–double mutant revealed that adenosine N-oxide (ANO) reductase is specifically required for anaerobic respiration on ANO in this mutant.     

The specific functions of menaquinone and demethylmenaquinone in anaerobic respiration with fumarate,dimethylsulfoxide, trimethylamine N-oxide and nitrate by Escherichia coli

The respiratory activities of E. coli with H₂ as donor and with nitrate, fumarate, dimethylsulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as acceptor were measured using the membrane fraction of quinone deficient strains. The specific activities of the membrane fraction lacking naphthoquinones with fumarate, DMSO or TMAO amounted to 2% of those measured with the membrane fraction of the wild-type strain. After incorporation of vitamin K₁ [instead of menaquinone (MK)] into the membrane fraction deficient of naphthoquinones, the activities with fumarate or DMSO were 92% or 17%, respectively, of the activities which could be theoretically achieved. Incorporation of demethylmenaquinone (DMK) did not lead to a stimulation of the activities of the mutant. In contrast, the electron transport activity with TMAO was stimulated by the incorporation of either vitamin K₁ or DMK. Nitrate respiration was fully active in membrane fractions lacking either naphthoquinones or Q, but was 3% of the wild-type activity, when all quinones were missing. Nitrate respiration was stimulated on the incorporation of either vitamin K₁ or Q into the membrane fraction lacking quinones, while the incorporation of DMK was without effect. These results suggest that MK is specifically involved in the electron transport chains catalyzing the reduction of fumarate or DMSO, while either MK or DMK serve as mediators in TMAO reduction. Nitrate respiration requires either Q or MK.Abbreviations DMK demethylmenaquinone - MK menaquinone - Q ubiquinone - DMSO dimethylsulfoxide - TMAO trimethylamine N-oxide - DMS dimethylsulfide - TMA trimethylamine - BV benzylviologen     

Electron donors and the quinone involved in dimethyl sulfoxide reduction inEscherichia coli

Escherichia coli can use dimethyl sulfoxide (DMSO) as an electron acceptor during anaerobic growth on the oxidizable substrate, glycerol. During growth, the DMSO is reduced to dimethyl sulfide (DMS). For the reduction of DMSO, NADH, formate, lactate, reduced benzyl viologen, reduced methyl viologen, and dithionite can serve as electron donors. The terminal reductase and the dehydrogenases linking the various electron donors to the electron transport chain were found to be membrane bound. Chlorate-resistant mutants (chl) were unable to grow and reduce DMSO. However, in the case of thechlD mutant, growth and DMSO reduction can be restored by growth in the presence of high concentrations of molybdate. Mutants ofE. coli blocked in menaquinone (vitamin K₂) biosynthesis—menB, menC, andmenD—were unable to grow with DMSO as an electron acceptor, even though the terminal reductase is present in these mutants. Both growth and DMSO reduction could be restored in these mutants by growth in the presence of the menaquinone intermediates,o-succinylbenzoate and 1,4-dihydroxy-2-naphthoate, depending on the metabolic block of the mutant. Thus menaquinone is involved in electron transport during DMSO reduction.     

A Single enzyme is Responsible for Both Dimethylsulfoxide and Trimethylamine-N-oxide Respirations as the Terminal Reductase in a Photodenitrifier: Rhodobacter sphaeroides f.s. denitrificans

Polyacrylamide gel electrophoresis of the periplasmic fractionfrom Rhodobacter sphaeroides f.s. denitrificans grown with dimethylsulfoxide(DMSO) and trimethylamine-Af-oxide (TMAO) showed that only onepolypeptide was stained for both DMSO and TMAO reductase activities,and it was the same as the purified DMSO redutase. Determinationof DMSO and TMAO reductase activities of intact cells grownwith DMSO or TMAO showed that each reagent induced both DMSOand TMAO activities and that DMSO showed much higher inductionactivity than TMAO. These results indicate that a single enzymeis responsible for both DMSO and TMAO respirations as the terminalreductase. (Received July 15, 1987; Accepted November 24, 1987)     

Redox mechanisms in “oxidant-dependent” hexose fermentation by Rhodopseudomonas capsulata

Trimethylamine N-oxide (TMAO) can function as an electron acceptor in the anaerobic metabolism of both Rhodopseudomonas capsulata and Escherichia coli. In both bacteria, anaerobic growth in the presence of TMAO induces a system that can reduce TMAO to trimethylamine (TMA). Comparative studies, however, show that TMAO reduction serves different purposes in the organisms noted. In E. coli, anaerobic growth on sugars does not require the presence of TMAO, but in cells induced for TMAO reductase, TMAO can act as the terminal electron acceptor for membrane-associated oxidative phosphorylation. Anaerobic dark growth of R. capsulata is dependent on the presence of TMAO (or an analog) and in this organism a soluble system catalyzes anaerobic oxidation of NADH with TMAO. The mechanism, in R. capsulata, appears to involve a flavoprotein of the flavodoxin type and presumably represents a system for maintenance of redox balance during anaerobic dark fermentation of hexoses and related compounds.     

Growth of Campylobacter jejuni supported by respiration of fumarate,nitrate, nitrite,trimethylamine-N-oxide,or dimethyl sulfoxide requires oxygen

The human gastrointestinal pathogen Campylobacter jejuni is a microaerophilic bacterium with a respiratory metabolism. The genome sequence of C. jejuni strain 11168 reveals the presence of genes that encode terminal reductases that are predicted to allow the use of a wide range of alternative electron acceptors to oxygen, including fumarate, nitrate, nitrite, and N- or S-oxides. All of these reductase activities were present in cells of strain 11168, and the molybdoenzyme encoded by Cj0264c was shown by mutagenesis to be responsible for both trimethylamine-N-oxide (TMAO) and dimethyl sulfoxide (DMSO) reduction. Nevertheless, growth of C. jejuni under strictly anaerobic conditions (with hydrogen or formate as electron donor) in the presence of any of the electron acceptors tested was insignificant. However, when fumarate, nitrate, nitrite, TMAO, or DMSO was added to microaerobic cultures in which the rate of oxygen transfer was severely restricted, clear increases in both the growth rate and final cell density compared to what was seen with the control were obtained, indicative of electron acceptor-dependent energy conservation. The C. jejuni genome encodes a single class I-type ribonucleotide reductase (RNR) which requires oxygen to generate a tyrosyl radical for catalysis. Electron microscopy of cells that had been incubated under strictly anaerobic conditions with an electron acceptor showed filamentation due to an inhibition of cell division similar to that induced by the RNR inhibitor hydroxyurea. An oxygen requirement for DNA synthesis can thus explain the lack of anaerobic growth of C. jejuni. The results indicate that strict anaerobiosis is a stress condition for C. jejuni but that alternative respiratory pathways can contribute significantly to energy conservation under oxygen-limited conditions, as might be found in vivo.