Trimethylamine N-oxide Reductase Activity Research Articles

Cholera Toxin Production Induced upon Anaerobic Respiration is Suppressed by Glucose Fermentation in Vibrio cholerae.

The causative agent of pandemic cholera, Vibrio cholerae, infects the anaerobic environment of the human intestine. Production of cholera toxin (CT), a major virulence factor of V. cholerae, is highly induced during anaerobic respiration with trimethylamine N-oxide (TMAO) as an alternative electron acceptor. However, the molecular mechanism of TMAO-stimulated CT production is not fully understood. Herein, we reveal that CT production during anaerobic TMAO respiration is affected by glucose fermentation. When the seventh pandemic V. cholerae O1 strain N16961 was grown with TMAO and additional glucose, CT production was markedly reduced. Furthermore, an N16961 Δcrp mutant, devoid of cyclic AMP receptor protein (CRP), was defective in CT production during growth by anaerobic TMAO respiration, further suggesting a role of glucose metabolism in regulating TMAO-mediated CT production. TMAO reductase activity was noticeably decreased when grown together with glucose or by mutation of the crp gene. A CRP binding region was identified in the promoter region of the torD gene, which encodes a structural subunit of the TMAO reductase. Gel shift assays further confirmed the binding of purified CRP to the torD promoter sequence. Together, our results suggest that the bacterial ability to respire using TMAO is controlled by CRP, whose activity is dependent on glucose availability. Our results reveal a novel mechanism for the regulation of major virulence factor production by V. cholerae under anaerobic growth conditions.

Adaptation of anaerobic cultures of Escherichia coli K-12 in response to environmental trimethylamine-N-oxide.

SummarySystematic analyses of transcriptional and metabolic changes occurring when E scherichia coli K‐12 switches from fermentative growth to anaerobic respiratory growth with trimethylamine‐N‐oxide (TMAO) as the terminal electron acceptor revealed: (i) the induction of torCAD, but not genes encoding alternative TMAO reductases; (ii) transient expression of frmRAB, encoding formaldehyde dehydrogenase; and (iii) downregulation of copper resistance genes. Simultaneous inference of 167 transcription factor (TF) activities implied that transcriptional re‐programming was mediated by 20 TFs, including the transient inactivation of the two‐component system ArcBA; a prediction validated by direct measurement of phosphorylated ArcA. Induction of frmRAB, detection of dimethylamine in culture medium and formaldehyde production when cell‐free extracts were incubated with TMAO suggested the presence of TMAO demethylase activity. Accordingly, the viability of an frmRAB mutant was compromised upon exposure to TMAO. Downregulation of genes involved in copper resistance could be accounted for by TMAO inhibition of Cu(II) reduction. The simplest interpretation of the data is that during adaptation to the presence of environmental TMAO, anaerobic fermentative cultures of E . coli respond by activating the TorTSR regulatory system with consequent induction of TMAO reductase activity, resulting in net oxidation of menaquinone and inhibition of Cu(II) reduction, responses that are sensed by ArcBA and CusRS respectively.

The biogenesis of c-type cytochromes in Escherichia coli requires a membrane-bound protein, DipZ, with a protein disulphide isomerase-like domain.

A mutant of Escherichia coli K-12, JCB606, which lacks all five c-type cytochromes synthesized during anaerobic growth in the presence of nitrite or trimethylamine-N-oxide (TMAO), was totally defective in Nrf activity and also partially defective in TMAO reductase activity. The mutation in strain JCB606 was shown to affect expression of the tor operon, which contributes almost equally with the products of the dms operon to the rate of TMAO reduction by bacteria during anaerobic growth in the presence of TMAO. The mutation in strain JCB606, dipZ, was mapped by P1 transduction close to the mel operon at co-ordinate 4425 on the E. coli chromosome, the gene order being nrf-fdhF-mel-dipZ-ampC. Recombinant plasmids that restored Nrf activity to test-tube cultures of the mutant were isolated from a cosmid library. A 2.7 kb EcoRV-SmaI fragment (co-ordinates 4443 to 4446 kb on the physical map of the E. coli chromosome) was found potentially to encode three genes arranged in at least two operons. The second gene, dipZ, was sufficient to complement the JCB606 mutation. The translated DNA sequence predicts that DipZ is a 53 kDa integral membrane protein with a 37 kDa N-terminal domain including at least six membrane-spanning helices and a 16 kDa carboxy-terminal hydrophilic domain which includes a protein disulphide isomerase-like motif. It is suggested that DipZ is essential for maintaining cytochrome c apoproteins in the correct conformations for the covalent attachment of haem groups to the appropriate pairs of cysteine residues.

Dimethyl sulfoxide reductase is not required for trimethylamine N-oxide reduction in Escherichia chcoli

Deletion mutants of Escherichia coli lacking dimethyl sulfoxide (DMSO) reductase activity and consequently unable to utilize DMSO as an electron acceptor for anaerobic growth have been isolated. These mutants retained the ability to use trimethylamine N-oxide (TMAO) as an electron acceptor and the TMAO reductase activity was found to be unaltered. Heating the cell-free extract of the wild-type strain at 70 degrees C for 15 min selectively inactivated the DMSO reductase activity while the TMAO reductase activity remained unchanged for at least 1 h.

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, Mr=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.

Assimilatory reduction of trimethylamine N-oxide in the yeast Sporopachydermia cereana

Washed microsomal preparations (100 000 xg sediment) from the yeast Sporopachydermia cereana that had been grown on trimethylamine N-oxide as sole nitrogen source catalysed the NAD(P)H-dependent reduction of trimethylamine N-oxide to trimethylamine. Under anaerobic conditions, this was the sole reaction product, but under aerobic conditions only small amounts of trimethylamine accumulated, most being further metabolized to methylamine and formaldehyde (no detectable dimenthylamine accumulated due to its rapid turnover). In the absence of NAD(P)H, no formation of amines or formaldehyde from trimethylamine N-oxide was detected. The trimethylamine N-oxide reductase activity was inhibited by quinacrine, Cu2+ ions, triethylamine N-oxide (apparent Ki 0.43 mM) and dimethyl sulphoxide (Ki 0.94 mM). Chlorate and nitrate failed to inhibit the enzyme. The Km for trimethylamine N-oxide was 29 μM. Triethylamine N-oxide was also reduced by the microsomal preparation with the formation of acetaldehyde, and this reduction was sensitive to the same inhibitors as trimethylamine N-oxide, suggesting that both amine oxides are metabolized by the same enzyme(s). It is concluded that trimethylamine N-oxide is metabolized in this yeast via an NAD(P)H-dependent reductase.

Trimethylamine N-oxide (TMAO) reductases from Escherichia coli K-12

The TMAO reductase activity of Escherichia coli grown anaerobically in the presence or absence of TMAO was analysed on linear sucrose gradients and on non-denaturing polyacrylamide gels. The results, together with those obtained by analysis of some other properties of TMAO reductase, showed that there are significant differences between the enzyme synthesized in the absence of TMAO (“constitutive” enzyme) and that synthesized in its presence (“inducible” enzyme). A similar study of a tor mutant specifically altered in the structural gene for TMAO reductase, showed that the enzymes synthesized under the 2 growth conditions are probably 2 distinct enzymes encoded by different genes.

Escherichia colimutants defective in trimethylamineN-oxide reductase

Trimethylamine N-oxide (TMAO) is widespread in marine fish and invertebrates [1]. Enteric bacteria reduce TMAO to trimethylamine. Stimulation of anaerobic growth by TMAO has been observed in Escherichia coli [2], Salmonella typhimurium [3] and Proteus sp. strain NTHC153 [4]. According to the chemiosmotic theory, electron flow through the respiratory chain is accompanied by electrogenic extrusion of protons [5]. Proton extrusion and generation of membrane potential coupled to TMAO reduction is observed in anaerobically grown E. coli [6]. Thus, like nitrate reductase and fumarate reductase, TMAO reductase is a terminal electron acceptor for respiratory electron flow. Both TMAO reductase and nitrate reductase accept electrons from NADH, formate or viologen dyes. Defects of TMAO reductase activity in chlD mutants suggest that both TMAO reductase and nitrate reductase use the molybdenum cofactor in E. coli [6,7] and S. typhimurium [3]. However, TMAO reductase activity is found in chlC, chlE and chlG mutants, indicating that the enzyme does not share any polypeptides with nitrate reductase [6,7]. In this study, we isolated and characterized mutants of E. coli defective not in nitrate reductase but in TMAO reductase. We found that the mutation affecting TMAO reductase activity is located in a different gene from chl. 2.1. Bacterial strains and growth conditions

Inhibition of Escherichia coli Trimethylamine-N-oxide Reductase by Food Preservatives

Trimethylamine-N-oxide (TMA-O) reductase activity of resting cells of Escherichia coli was inhibited by tetrasodium ethylenediaminetetraacetate (Na4EDTA), benzoic acid (BA and methylparaben (MP). The 50% inhibitory concentrations of Na4EDTA, BA and MP were 20.2, 1.2 and 32.4 mM, respectively. BA at pH 6.5 or below most effectively inhibited the TMA-O reductase. Sorbic acid (SA), up to 0.70 mM, had no effect on TMA-O reductase activity, but SA inhibited the growth and subsequent TMA production in E. coli at or above 0.3S mM.

Proton translocation coupled to trimethylamine N-oxide reduction in anaerobically grown Escherichia coli.

Proton translocation coupled to trimethylamine N-oxide reduction was studied in Escherichia coli grown anaerobically in the presence of trimethylamine N-oxide. Rapid acidification of the medium was observed when trimethylamine N-oxide was added to anaerobic cell suspensions of E. coli K-10. Acidification was sensitive to the proton conductor 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847). No pH change was shown in a strain deficient in trimethylamine N-oxide reductase activity. The apparent H+/trimethylamine N-oxide ratio in cells oxidizing endogenous substrates was 3 to 4 g-ions of H+ translocated per mol of trimethylamine N-oxide added. The addition of trimethylamine N-oxide and formate to ethylenediaminetetraacetic acid-treated cell suspension caused fluorescence quenching of 3,3'-dipropylthiacarbocyanine [diS-C3-(5)], indicating the generation of membrane potential. These results indicate that the reduction of trimethylamine N-oxide in E. coli is catalyzed by an anaerobic electron transfer system, resulting in formation of a proton motive force. Trimethylamine N-oxide reductase activity and proton extrusion were also examined in chlorate-resistant mutants. Reduction of trimethylamine N-oxide occurred in chlC, chlG, and chlE mutants, whereas chlA, chlB, and chlD mutants, which are deficient in the molybdenum cofactor, could not reduce it. Protons were extruded in chlC and chlG mutants, but not in chlA, chlB, and chlD mutants. Trimethylamine N-oxide reductase activity in a chlD mutant was restored to the wild-type level by the addition of 100 microM molybdate to the growth medium, indicating that the same molybdenum cofactor as used by nitrate reductase is required for the trimethylamine N-oxide reductase system.

Trimethylamine N-oxide (TMAO) reductase activity in chlorate-resistant or respiration-deficient mutants ofEscherichia coli

The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is induced when the organism is grown anaerobically in the presence of TMAO [1,2]. It is a membrane bound enzyme which can accept electrons from formate, NADH or reduced viologen dyes [2]. Similarly to the nitrate reductase system of E. coli, under anaerobic conditions, TMAO reductase appears to act as a terminal electron acceptor for respiratory electron flow [13]. Kim and Chang [4] report evidence suggesting a close similarity between nitrate and TMAO reductases from a chlorate-resistant mutant of Salmonella typhimurium which lacked both activities. In the present paper we make use of several mutants of E. coli to demonstrate that (i) membrane associated electron transport between NADH and TMAO requires quinones and cytochromes, and (ii) chlorate-resistant mutants of E. coli lacking nitrate reductase activity are not necessarily deficient in TMAO reductase activity, indicating that these enzymes are functionally and genetically distinct.