INTRODUCTION

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Escherichia coli can survive in various growth conditions owing to its ability to adapt in response to environmental changes. For example, in anaerobiosis and according to the exogenous electron acceptor present in the medium, this organism synthesizes the energetically more appropriate respiratory system (18). Sometimes, more than one respiratory system is produced for a given substrate. For instance, reduction of nitrate can be carried out by at least three respiratory systems (10). At high concentrations of nitrate, only the membranous NarG system is synthesized (46), whereas at very low concentrations, the periplasmic Nap system is produced (36). The operon encoding a third system (NarZ) is expressed during the early stationary phase under control of ^s, irrespective of the presence of nitrate (8). Accordingly, whatever the nitrate concentration and the growth phase, at least one of the nitrate reductases is synthesized in the cell (8, 36).

Trimethylamine N-oxide (TMAO) is reduced to the volatile compound trimethylamine (TMA) by at least two respiratory systems, the TorCAD and the DmsABC systems (5, 29). The torCAD operon, which encodes the periplasmic Tor system, is induced in the presence of TMAO by the TorS-TorR two-component regulatory system (25), whereas the membranous dimethyl sulfoxide (DMSO) reductase system, encoded by the dmsABC operon, is synthesized constitutively in anaerobiosis (5). The reason for the presence in the same host of several systems dedicated to a common substrate is still unclear, but one possibility is that they allow the cell to better adapt to changing environmental conditions during the different growth phases.

The terminal reductase of the inducible Tor pathway, TorA, encoded by the torA gene, is a periplasmic molybdoenzyme of 90 kDa (29) that is thought to receive electrons from the membrane pool of menaquinone through the TorC protein. TorC, which is encoded by the first gene of the torCAD operon, is a pentahemic c-type cytochrome anchored to the inner membrane by its N-terminal extremity (21, 29). The last gene of the tor operon, torD, encodes a cytoplasmic protein of 23 kDa, which is a private chaperone that is required for TorA assembly (35). TorD interacts with TorA at an early stage of TorA synthesis, probably before the insertion of the molybdenum cofactor. After folding and cofactor insertion, TorA is translocated across the inner membrane by the Tat (twin arginine translocation) system (40, 41, 52). Except for the c-type cytochromes (49), the metalloproteins located into the periplasm are transported by this pathway. The N-terminal signal sequences of the metalloenzymes translocated by the Tat mechanism are recognized by their length (>30 amino acids), the twin-arginine motif RRXFL, and their hydrophobicity (3, 11).

According to sequence homologies and to its biochemical properties, TorA belongs to the DMSO reductase family (20, 22). In this family of molybdoenzymes, three groups of enzymes can be defined, as follows: (i) the specific TMAO reductases encoded by torA of E. coli or Shewanella massilia, which do not have any S-oxide reductase activity and can only reduce TMAO as a natural substrate (13, 23); (ii) the TMAO- DMSO reductases like the DmsA subunit of the E. coli membranous DMSO reductase or the periplasmic DorA and DmsA enzymes of Rhodobacter capsulatus and Rhodobacter sphaeroides, respectively, which are able to reduce a wide range of N- and S-oxide compounds, including DMSO and TMAO (42, 45); and (iii) the biotin d-sulfoxide reductases like BisC from E. coli and the biotin sulfoxide (BSO) reductase from R. sphaeroides, which are cytoplasmic enzymes involved primarily in the recycling of biotin from BSO (33, 34). An in vitro study showed that the R. sphaeroides enzyme can also poorly reduce other N- and S-oxides like TMAO and DMSO (17, 34). Although these three groups of enzymes share sequence homologies, a major difference is that enzymes of the first two groups are involved in anaerobic respiratory processes while the cytoplasmic BSO reductase enzymes are not.

Recently, it has been proposed that a newly characterized gene, bisZ, encodes a second BSO reductase in E. coli (12). Genetic and biochemical evidence showed that the bisZ product was responsible for the 4% background BSO reductase activity observed in a bisC mutant. Moreover, the bisZ gene encodes a protein that exhibits 62% sequence identity with the bisC product. We demonstrate in this paper that, in contrast to BisC, the bisZ gene product is periplasmically located. Furthermore, this enzyme has an extended substrate specificity which includes, in addition to BSO, other N- and S-oxides, such as TMAO. Significantly, it exhibits greater catalytic activity with TMAO than with BSO. Accordingly, we propose to rename bisZ as torZ. We also show that torZ (bisZ) is the second gene of the torYZ (yecK bisZ) operon and that torY encodes a c-type cytochrome homologous to TorC. Finally, TorY and TorZ constitute a respiratory system.

	MATERIALS AND METHODS

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Chemicals. The N- and S-oxide compounds were purchased from Sigma or Aldrich except for d-biotin d-sulfoxide, which was synthesized from biotin as described by Melville (28).

Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this work are listed in Table 1. To maintain selection for plasmids or to select for transductant strains, we used antibiotics as follows: ampicillin, 50 µg/ml; chloramphenicol, 10 µg/ml; kanamycin, 50 µg/ml; and spectinomycin, 25 µg/ml. For the biochemical study, cells were grown anaerobically at 37°C on Luria-Bertani (LB) media. The concentration of arabinose or of glucose, added in the growth medium, is detailed for each experiment in Results. Otherwise, growth of E. coli was performed under anaerobic conditions in 3-ml full-cap cuvettes at 37°C with a minimal salt medium (MSM) derived from that described by Bilous and Weiner (4). It contained K₂HPO₄ (3.5%); KH₂PO₄ (1%), (NH₄)₂SO₄ (0.5%), MgSO₄ (0.05%), CaCl₂ (0.015%), Na citrate (0.3%), casein acid hydrolysate (0.15%; Difco), and thiamine hydrochloride (0.002%, pH 7). The MSM was supplemented with 0.5% glycerol as a nonfermentable carbon source and with 1 mM isopropyl--D-thiogalactopyranoside (IPTG) when necessary. Electron acceptors were used at a concentration of 50 mM. The MSM was inoculated at a dilution of 1% with cells grown overnight in LB broth (supplemented with ampicillin when necessary), centrifuged, and resuspended in the same volume of MSM. Growth was monitored in the same full-capped cuvettes at 600 nm. Values (about 0.15 after 24 h) obtained from a control curve with cells in MSM supplemented with glycerol but without electron acceptor were subtracted from the experimental values.

DNA manipulations. DNA was prepared with the High Pure DNA Isolation kit from Boehringer Mannheim. Plasmid preparation, restriction endonuclease digestions, and DNA purification and ligation were carried out as described by Sambrook et al. (39). Transformations were performed according to the method of Chung and Miller (9). PCR amplification was carried out using standard procedures according to the supplier's instructions. Reverse transcriptase-PCR (RT-PCR) was performed with the Promega Access system. The oligonucleotides used were as follows: a, 5'-ATTAGAGGAATCTATGCGAGGGAAAAAACG-3'; b, 5'-GTGGACAGTTCCCTGATATTCCTC- 3'; c, 5'-CGCTATGGCAATACGCTGAAAAACTTG-3'; d, 5'-TAACAATTGACCATGATCAGGGAGGAAGTTATGACATTAAC-3'; and e, 5'-CCGCCGCCGTAGACTGTAAGGAAT-3'. One microgram of total RNA prepared with the High Pure RNA Isolation kit (Boehringer Mannheim) was denatured at 94°C for 2 min in the presence of either primers a and b, primers c and e, or primers d and e. Immediately afterward, reverse transcription and 35 cycles of PCR amplification were carried out according to the supplier's protocol.

Construction of plasmids. To create plasmids ptorZ and ptorYZ, we used PCR to generate DNA fragments corresponding to the torZ and the torYZ coding sequences with, in both cases, an upstream MfeI site and a downstream SmaI site. After enzymatic hydrolysis, the PCR products were cloned into the compatible EcoRI and SmaI sites of pJF119EH (16), yielding plasmids ptorZ and ptorYZ. In these plasmids, torZ and torYZ are under the control of the P_tac promoter. To create the pBtorYZ, the same torYZ PCR product was cloned into the EcoRI and SmaI sites of pBAD24 (19). In this construct, the torYZ genes are under the control of the arabinose promoter. To create plasmid ptorY, the torZ gene was partly deleted from plasmid ptorYZ after an EcoRI digestion, followed by an intramolecular ligation. To create plasmid ptorCAD, we used PCR to generate a promoterless torCAD DNA fragment. This purified PCR product was then cloned into the pPCR-script vector (Stratagene), according to the supplier's protocol, to yield pPStorCAD. The PstI-SacI fragment from pPStorCAD was cloned into the same sites on pJF119EH, resulting in plasmid ptorCAD, in which the entire tor operon is under the control of the P_tac promoter. To create plasmid pPTorYZ, we amplified the torY promoter region by PCR (from position 283 to position +170 relative to the first nucleotide of the initiation codon of torY gene). The DNA fragment was blunted using the blunting kit from Takara and introduced into plasmid pGE593 (14), previously linearized by SmaI, thus placing the lacZ gene under the control of the putative torY promoter. All the PCR products and fusion sites were confirmed by sequencing, except for the torCAD coding region of ptorCAD which was subjected to a PCR amplification of only 13 cycles in order to minimize the number of possible mutations.

Construction of strain LCB504. The mutation of strain DSS401 (dms Km^r) was transferred to LCB502 by P1 transduction, resulting in a torC dms strain (LCB504). Integration of the Km^r gene at the correct position on the chromosome was verified by PCR.

-galactosidase assays. Strain LCB504 carrying pPTorYZ was grown anaerobically at 37°C in LB medium alone or supplemented with 50 mM TMAO, DMSO, or BSO. -galactosidase activities were measured according to the method of Miller (30) from culture samples that were taken during the exponential- and the stationary-growth phases.

Preparation of subcellular fractions. Crude extracts were prepared by disrupting the cells in a French press as described by Iobbi-Nivol et al (24). The periplasmic fractions were prepared according to the sucrose-lysosyme-EDTA procedure described by Osborn et al. (32). Membranous and cytoplasmic fractions were obtained from the spheroplasts after disruption in a French press and ultracentrifugation as detailed by Silvestro et al. (44).

Enzyme purification. TorZ (BisZ) was purified from the periplasm fraction obtained from 4 g of LCB620/pBtorYZ cells grown anaerobically in the presence of 0.1% arabinose and ampicillin. The periplasm was dialyzed to remove the sucrose and applied to an ion-exchange Q Sepharose, HiLoad 16/10 column (Pharmacia-Biotech). The fractions obtained from a 0 to 0.5 M NaCl linear gradient elution were assayed for TMAO reductase activity, and active fractions were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (26). Two fractions, possessing also the highest specific activity, presented only two Coomassie blue-stained bands each. The major one, corresponding to more than 80% of the stained proteins, had a calculated molecular mass of about 90 kDa, whereas the minor band was slightly smaller. These fractions were then tested for TMAO and DMSO reductase activities on SDS gels according to the procedure described by Pommier et al. (35). The single active band obtained for both the TMAO and DMSO activities corresponded to the major Coomassie blue band. The two active fractions were pooled and used for analytical experiments. A total quantity of 1.76 mg of protein was obtained with a specific activity of 709 µmol of TMAO reduced per min per mg of protein.

Analytical procedures. TMAO reductase activity was measured spectrophotometrically at 37°C by following the oxydation of reduced benzyl viologen at 600 nm coupled to the reduction of TMAO. Each of the compounds tested (see Table 4 for a list) was prepared at a final concentration of 1 or 2 M and used at least at eight different concentrations with three to four duplicates. Kinetic parameters were determined as previously described (23).

	RESULTS

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Gene organization and putative products of the bisZ locus. The torZ (bisZ) gene is located at kb 1955 on the chromosome of E. coli. The analysis of the DNA sequences surrounding this gene revealed the presence of a 1-kb open reading frame that we propose to call torY (formerly designated yecK), located immediately upstream from torZ (bisZ) and transcribed in the same orientation (Fig. 1). The distance between torY (yecK) and torZ (bisZ) is only 24 bp, while a noncoding region of 387 bp separates torY (yecK) from the upstream cutC gene which is transcribed in the same orientation and ends with a probable transcriptional terminator. The next gene downstream from torZ (bisZ) (yecP) is transcribed divergently. To determine whether the torY (yecK) and torZ (bisZ) genes are organized in a single transcription unit, RT-PCR was performed using RNA extracted from a strain grown anaerobically and appropriate oligonucleotide pairs that hybridize to regions in the beginning or within torY (yecK) and within torZ (bisZ), as shown in Fig. 1. The PCR synthesis of a DNA fragment that overlaps the region between the end of torY (yecK) and the beginning of torZ (bisZ) strongly suggests that these genes are organized in an operon. Amplification of DNA fragments that cover the beginning of torY (yecK) indicates that the transcriptional start of this operon is, as expected, upstream from the ATG start codon of torY (yecK). The intergenic region located between the cutC and the torY (yecK) genes was cloned upstream from the promoterless lacZ gene of the multicopy plasmid pGE593 to yield plasmid pPTorYZ. Unfortunately, the -galactosidase activity measured from strain MC4100 carrying pPTorYZ was very low (10 to 20 Miller units) under all the growth conditions tested (see Materials and Methods). This result suggests either that the torYZ (yecK bisZ) operon is always expressed at a very low level or that the conditions for inducing this operon have not been discovered.

View larger version (47K): [in this window] [in a new window]   FIG. 1.   (a) Physical map of the torYZ operon region of E. coli. The large arrows show the locations and the orientations of the reading frames. The primers (a, b, c, d, and e) used for the RT-PCR are indicated. (b) Analysis of tor gene transcription by RT-PCR was followed by 2% agarose gel electrophoresis. The RT-PCR was carried out with primers c and e (lanes 1 to 4), primers d and e (lanes 5 to 8), or primers a and b (lanes 9 to 12). Lanes 1, 5, and 9, RT-PCR experiments with 1 µg of E. coli total RNA; lanes 2, 6, and 10, same experiments as described for lanes 1, 5, and 9 but without reverse transcriptase to check the absence of DNA traces in the RNA preparation; lanes 3, 7, and 11, control PCR with genomic DNA; lanes 4, 8, and 12, the same experiments as described for lanes 1, 5, and 9 but without RNA (negative control); lane M, DNA ladder corresponding to the 1-kb ladder of Gibco BRL.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Amino acid sequence alignment of the N-terminal part of the DMSO-TMAO reductases of R. sphaeroides (RsDmsA) (53) and R. capsulatus (RcDorA) (43), the TMAO reductases of S. massilia (SmTorA) (13) and E. coli (EcTorA) (29), the product of the H. influenzae bisZ gene (HibisZ) (47), and the product of the E. coli torZ (bisZ) gene (EctorZ). The signal sequence cleavage site and the redox protein export conserved motif are in italic and in bold type, respectively.

torYZ (yecK bisZ) operon encodes a respiratory system. To test our hypothesis that the TorY and TorZ proteins are involved in an electron transfer pathway similar to that of the TorCAD system, a strain that was unable to reduce TMAO was constructed. This strain, LCB504, carries an interposon in the beginning of the first gene of the torCAD operon and a deletion of the entire dmsABC operon (Table 1). As expected, strain LCB504, containing the pJF119 expression vector, grew at an extremely slow rate under anaerobic conditions with TMAO as the only exogenous electron acceptor (Fig. 3). When plasmid ptorCAD, carrying the torCAD operon under the control of the P_tac promoter, was introduced into strain LCB504, the recombinant strain exhibited a high growth rate in the presence of IPTG. Introduction of plasmid ptorYZ, carrying the torYZ (yecK bisZ) operon under the control of the P_tac promoter, into strain LCB504 also yielded a recombinant strain with similar IPTG-dependent growth rate (Fig. 3). In the absence of IPTG, no significant growth was observed with either recombinant strain (Fig. 3). It is noteworthy that production of the characteristic odor of volatile TMA occurred under condition of rapid growth, indicating that TMAO was reduced to TMA in both experiments. Therefore, when expressed to a certain level, the torYZ (yecK bisZ) operon, like torCAD, allows the strain to use TMAO as a substrate for anaerobic respiration.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Anaerobic growth profiles of E. coli strain LCB504 carrying plasmid ptorYZ or plasmid ptorCAD. The LCB504 recombinant strains were grown in MSM in the presence of 1 mM IPTG (unless otherwise indicated) and of a 50 mM concentration of either TMAO (strains carrying ptorCAD [], ptorYZ [], ptorYZ but without IPTG [], pJF119EH [], ptorY [], or ptorZ []), DMSO (strain carrying ptorYZ []), or BSO (strain carrying ptorYZ []). Growth was monitored at 600 nm as described in Materials and Methods. Data are typical of those obtained from at least three independent experiments. OD, optical density.

TorYZ respiratory system is made up of a membrane-anchored cytochrome and a periplasmic reductase. For the production of both the torY (yecK) and torZ (bisZ) products, the genes were cloned under the strict control of the P_bad promoter into the pBAD24 vector. The resulting plasmid, pBtorYZ, was then introduced into strain LCB620 and grown in the presence of either arabinose or glucose for the induction or repression, respectively, of torYZ expression. First, to confirm the predictions deduced from the sequence analysis about the cellular location of TorY as well as the presence of heme in it, various fractions of the strain, carrying both plasmids pBtorYZ and pEC86, grown in presence of 0.2% arabinose or 0.2% glucose were tested by SDS-PAGE followed by heme staining. In the membrane fraction of cells grown in the presence of arabinose, a cytochrome of about 40 kDa was observed (Fig. 4). It is distinguishable from TorC by its slightly smaller size, and it is absent when glucose is added to the growth media or in the soluble extracts of the bacteria (Fig. 4 and data not shown). Only five c-type cytochromes have been described previously for E. coli: two of them are involved in the periplasmic nitrate respiratory system (Nap), two others are involved in the nitrite reduction pathway (Nrf), and one, TorC, belongs to the TMAO respiration system (21). This study shows that the E. coli genome contains a gene, torY, able to encode a sixth c-type cytochrome. According to the results obtained from a search in the Colibri databank using the heme binding motif as a pattern, no other multihemic cytochrome seems to be encoded by the E. coli genome.

View larger version (51K): [in this window] [in a new window]   FIG. 4.   Detection of c-type cytochromes by TMBZ staining. Membranous proteins (50 µg) from cells carrying pBtorYZ grown anaerobically at 37°C in LB medium supplemented with 0.2% glucose (lane 3) or supplemented with 0.2% arabinose (lane 4) were loaded on an SDS-10% polyacrylamide gel. As a control, purified TorC (2.5 µg) was also loaded onto the gel (lane 1). Sigma Wide Range color markers were also used (lane 2). Samples were heated prior to loading, and after electrophoresis, the gel was stained for heme with TMBZ. The arrow indicates the position of the torY gene product.

Catalytic properties of TorZ (BisZ). The kinetic study was performed on the purified product of torZ (bisZ) as described in the Materials and Methods. The compounds tested were structurally related to TMAO or DMSO and have been previously used to determine the specificity of TorA and the membranous DMSO reductase of E. coli (23, 45).

1

	DISCUSSION

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During this study, we have demonstrated that the torYZ (yecK bisZ) operon of E. coli encodes a new respiratory system. This system is made up of a membranous pentahemic c-type cytochrome and a periplasmic molybdoreductase, and it is closely related to the Tor and Dor respiratory systems of E. coli and Rhodobacter species (29, 31, 51). The association of a pentahemic membrane-bound cytochrome with a periplasmic enzyme seems to be the hallmark of respiratory systems capable of reducing either specifically TMAO (the Tor system of E. coli and of S. massilia) or TMAO and related N- and S-oxide compounds (the Dor system of Rhodobacter species). Accordingly, the best substrate among those tested for the TorYZ is TMAO. However, our in vitro and in vivo studies revealed that the specificity of TorZ is different from that of either TorA or DorA. In contrast to TorA, TorZ can reduce other N- and S-oxide compounds such as BSO, and in contrast to DorA, the best substrate for TorZ is not a sulfoxide compound but TMAO.

To avoid any confusion, we proposed to rename BisZ as TorZ because this enzyme is a better TMAO reductase than a BSO reductase and because it is located in the periplasm, whereas BisC (the higher TorZ [BisZ] homologue) is a cytoplasmic protein which is not involved in a respiratory process. Our data are in agreement with those of del Campillo-Campbell and Campbell (12), showing that TorZ can reduce BSO quite efficiently, but, from our results, it is probable that reduction of BSO takes place mainly in the periplasm rather than in the cytoplasm. It would be interesting to test whether TorZ can also reduce BSO when located in the cytoplasm. If this happens, the electron donor could be different from TorY which, most probably, faces the periplasm.

In E. coli, two homologous nitrate respiratory systems, nitrate reductase G and nitrate reductase Z, have been described as having issued from a duplication of ancestral genes (6, 24). For a long time, the narZYWV operon encoding nitrate reductase Z was supposed to be expressed constitutively at a very low level. However, a recent study showed that expression of this operon is induced in the early stage of the stationary phase of cell growth in a RpoS-dependent manner (8), while the nitrate reductase G is synthesized in anaerobiosis when a high concentration of nitrate is available (10, 36, 46). The torYZ operon originates probably from a duplication of the torCAD genes. In the case of the TorYZ system, no obvious regulation has been highlighted so far. In particular, the expression of the torYZ operon was very low whatever the growth phase and did not increase in the presence of TMAO, DMSO, or BSO. One can imagine that either the torYZ operon is constitutively expressed at a very low level or the induction conditions of its expression are still unknown. This raises the question of the existence of an unknown inducer for the torYZ operon which might be the best natural substrate of this respiratory system. The fact that the specificity of the TorYZ system is not exactly that of the TorCAD or the DmsABC system of E. coli suggests that the TorYZ system has evolved from the TorCAD system to play a specific role in E. coli respiration. It would be interesting to see whether related bacteria, such as Salmonella species, contain a TorYZ homologue and, if present, how this system is expressed. In this line of thought, a gene homologous to torZ (bisZ) was found in the chromosome of Haemophilus influenzae (47). This putative gene has been called bisZ, but the presence of a potential signal sequence in the N-terminal extremity of its deduced amino acid sequence indicates that it encodes a periplasmic protein (Fig. 2). Moreover, the presence before it of a gene encoding a pentahemic cytochrome homologous to TorC and to TorY raises the question of a possible respiratory role of this bisZ product.

An obvious difference between the torYZ and the torCAD operons is that a torD gene homologue is not found in the former. TorD is a private chaperone for the terminal reductase TorA, and its absence in a torD strain led to a significant decrease in the quantity of the TorA protein compared to that observed with a wild-type strain, but even in such a mutant strain, 30% of the active TorA protein is present in the periplasm (35). Our result indicates that TorZ is synthesized and folded in a torCAD strain. Thus, TorD is not required for TorZ maturation. Therefore, TorZ folding involves either no chaperone or a TorD homologue whose gene is located elsewhere on the chromosome. Interestingly, we have found several genes which could encode TorD homologues on the E. coli chromosome (unpublished results). We are investigating whether one of these TorD homologues plays the role of a TorZ chaperone.