Electrochemical Trimethylamine N-Oxide Biosensor with Enzyme-Based Oxygen-Scavenging Membrane for Long-Term Operation under Ambient Air

An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O2 scavenging membrane. The TMAO sensor operates at a potential of −0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 µM and 15 mM, with a sensitivity of 2.75 ± 1.7 µA/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 µM TMAO.

Genetic Analysis of Trimethylamine N-Oxide Reductases in the Light Organ Symbiont Vibrio fischeri ES114

ABSTRACT Trimethylamine N-oxide (TMAO) reductases are widespread in bacteria and often function in anaerobic respiration. The regulation and expression of TMAO reductase operons have been well studied in model genera such as Escherichia, Shewanella, and Rhodobacter, although TMAO reductases are present in many other bacteria, including the marine Vibrio species. The genome sequence of Vibrio fischeri revealed three putative TMAO reductase operons, and a previous report identified TMAO reductase activity in symbiotic V. fischeri isolates associated with the light organs of adult Hawaiian bobtail squid, Euprymna scolopes. We examined the roles and regulation of these three operons using mutational analyses and promoter-reporter fusions. We found that the torECA promoter, and to a lesser extent the torYZ and dmsABC promoters, were active during symbiotic colonization of juvenile E. scolopes; however, a V. fischeri strain lacking TMAO reductase activity displays no discernible colonization defect over the first 48 h. Our studies also revealed that torECA has the most active promoter of the putative TMAO reductase operons, and TorECA is the major contributor to TMAO-dependent growth in V. fischeri under the conditions tested. Interestingly, the transcriptional regulation of TMAO reductase operons in V. fischeri appears to differ from that in previously studied organisms, such as Escherichia coli, which may reflect differences in gene arrangement and bacterial habitat. This study lays the foundation for using V. fischeri as a model system for studying TMAO reductases in the Vibrionaceae.

Functional and structural analysis of members of the TorD family, a large chaperone family dedicated to molybdoproteins

The trimethylamine N-oxide (TMAO) reductase TorA, a DMSO reductase family member, is a periplasmic molybdoenzyme of Escherichia coli. The cytoplasmic protein TorD acts as a chaperone for TorA, allowing the efficient insertion of the molybdenum cofactor into the apoform of the enzyme prior to its secretion. This paper demonstrates that TorD is a member of a large family of prokaryotic proteins that are structurally related. Moreover, their genes generally belong to operons also encoding molybdoenzymes of the DMSO reductase family. Both the TorD and the DMSO reductase families present a similar phylogenetic organization, suggesting a co-evolution of these two families of proteins. This hypothesis is also supported by the fact that the TorD and DmsD chaperones cannot replace each other and thus appear dedicated to specific molybdopartners. Interestingly, it was found that the positive effect of TorD on TorA maturation, and the partial inhibitory effect of DmsD and homologues, are independent of the TorA signal sequence.

Effects of ISSo2 Insertions in Structural and Regulatory Genes of the Trimethylamine Oxide Reductase of Shewanella oneidensis

ABSTRACT We have isolated three Shewanella oneidensis mutants specifically impaired in trimethylamine oxide (TMAO) respiration. The mutations arose from insertions of an ISSo2 element into torA, torR, and torS, encoding, respectively, the TMAO reductase TorA, the response regulator TorR, and the sensor TorS. Although TorA is not the sole enzyme reducing TMAO in S. oneidensis, growth analysis showed that it is the main respiratory TMAO reductase. Use of a plasmid-borne torE′-lacZ fusion confirmed that the TorS-TorR phosphorelay mediates TMAO induction of the torECAD operon.

Reconstitution of the Trimethylamine Oxide Reductase Regulatory Elements of Shewanella oneidensis in Escherichia coli

ABSTRACT Several bacteria can grow by using small organic compounds such as trimethylamine oxide (TMAO) as electron acceptors. In Shewanella species, the TMAO reductase respiratory system is encoded by the torECAD operon. We showed that production of the TMAO reductase of S. oneidensis was induced by TMAO and repressed by oxygen, and we noticed that a three-gene cluster (torSTR) encoding a complex two-component regulatory system was present downstream of the torECAD operon. We introduced the torSTR gene cluster into Escherichia coli and showed that this regulatory gene cluster is involved in TMAO induction of the torE promoter but plays no role in the oxygen control. The TorR response regulator was purified, and gel shift and footprinting experiments revealed that TorR binds to a single region located about 70 bases upstream of the transcription start site of the tor structural operon. By deletion analysis, we confirmed that the TorR operator site is required for induction of the tor structural promoter. As the TMAO regulatory system of S. oneidensis is homologous to that of E. coli, we investigated a possible complementation between the TMAO regulatory components of the two bacteria. Interestingly, TorSec, the TMAO sensor of E. coli, was able to transphosphorylate TorRso, the TMAO response regulator of S. oneidensis.

The torYZ (yecK bisZ) Operon Encodes a Third Respiratory Trimethylamine N-Oxide Reductase inEscherichia coli

ABSTRACT The bisZ gene of Escherichia coli was previously described as encoding a minor biotin sulfoxide (BSO) reductase in addition to the main cytoplasmic BSO reductase, BisC. In this study, bisZ has been renamed torZ based on the findings that (i) the torZ gene product, TorZ, is able to reduce trimethylamine N-oxide (TMAO) more efficiently than BSO; (ii) although TorZ is more homologous to BisC than to the TMAO reductase TorA (63 and 42% identity, respectively), it is located mainly in the periplasm as is TorA; (iii)torZ belongs to the torYZ operon, and the first gene, torY (formerly yecK), encodes a pentahemic c-type cytochrome homologous to the TorC cytochrome of the TorCAD respiratory system. Furthermore, the torYZ operon encodes a third TMAO respiratory system, with catalytic properties that are clearly different from those of the TorCAD and the DmsABC systems. ThetorYZ and the torCAD operons may have diverged from a common ancestor, but, surprisingly, notorD homologue is found in the sequences aroundtorYZ. Moreover, the torYZ operon is expressed at very low levels under the conditions tested, and, in contrast to torCAD, it is not induced by TMAO or dimethyl sulfoxide.