Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, 31, chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
Correspondence
Chantal Iobbi-Nivol
iobbi{at}ibsm.cnrs-mrs.fr
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ABSTRACT |
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INTRODUCTION |
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In Escherichia coli, TorA is the terminal reductase of the Tor system, the main anaerobic respiratory pathway responsible for reduction of trimethylamine N-oxide (TMAO) to trimethylamine (Méjean et al., 1994). TorA is translocated into the periplasm by the Tat system (Weiner et al., 1998
; Santini et al., 1998
; Berks et al., 2000
) and it receives the electrons from the membrane anchored c-type cytochrome TorC (Gon et al., 2001
). The genes encoding TorC and TorA are part of the torCAD operon, which is induced in the presence of TMAO or related compounds by the two-component regulatory system TorS/TorR (Méjean et al., 1994
; Iobbi-Nivol et al., 1996
; Jourlin et al., 1997
). The reduction of TMAO in E. coli is also catalysed by the DMSO reductase complex (made up of three subunits: DmsA, DmsB and DmsC) synthesized in anaerobiosis (Weiner et al., 1992
). As described above, DmsA and TorA belong to type II and III, respectively, of the DMSO reductase family of molybdoenzymes (McDevitt et al., 2002
). They also differ in their substrate specificity, since DmsA reduces a wide range of S- and N-oxide compounds, whereas TorA only reduces TMAO (Simala-Grant & Weiner, 1996
; Iobbi-Nivol et al., 1996
).
TorD, the product of the last gene of the torCAD operon, does not participate in the electron transfer pathway but it is the specific chaperone of TorA (Pommier et al., 1998; Ilbert et al., 2003
). In vivo, the lack of TorD leads to a significant decrease of the final amount of TorA; however, specific activity and biochemical properties of the enzyme are not altered (Pommier et al., 1998
). Recently, we demonstrated, by an in vitro approach, the involvement of TorD during the first step of the maturation process of TorA (Ilbert et al., 2003
). When an appropriate molybdenum cofactor source is provided to the apoform of TorA, the addition of TorD increases fourfold the level of apoTorA maturation and leads to the maturation of most of the apoenzyme. We have also shown that TorD binds to apoTorA and probably modifies its conformation to a competent state, facilitating its maturation (Ilbert et al., 2003
).
Based on sequence similarity and on the results described below, DmsD seems to belong to the TorD specific chaperones, dedicated to the maturation of TMAO/DMSO reductases (Sargent et al., 2002; Tranier et al., 2003
). Indeed, recent work has shown that DmsA processing requires the presence of the DmsD chaperone (Oresnik et al., 2001
). The absence of DmsD leads to the loss of anaerobic growth of the bacteria on DMSO-containing medium, indicating that DmsD is required for the DMSO reductase biogenesis (Ray et al., 2003
). Furthermore, DmsD was described as binding to the signal peptide of preDmsA and preTorA, targeting these proteins to the Tat translocation system (Oresnik et al., 2001
). However, this point seems debatable since the fusion of the signal sequence of each enzyme to green fluorescent protein (GFP) indicated that DmsD was not required for the functioning of these signal peptides (Ray et al., 2003
). Although the role of DmsD is not yet clearly defined, a recent report indicates that, under certain conditions of bacterial growth, DmsD is associated with the inner membrane and that the TatB and TatC subunits could be important for this interaction (Papish et al., 2003
).
In a previous study, we showed that the TorD family comprised eleven proteins from various bacteria such as E. coli, Shewanella and Rhodobacter species, Vibrio cholerae, Salmonella typhimurium and Haemophilus influenzae (Tranier et al., 2003). The TorD family contains proteins of approximately 200 amino acids and shares sequence similarities with the E. coli TorD (TorDec) (Sargent et al., 2002
; Tranier et al., 2003
). The location of their genes is often found in operons also encoding molybdoenzymes of the DMSO reductase family. Among the members of the TorD family, a dimer of TorD from Shewanella massilia (TorDsm) was crystallized and its three-dimensional structure was solved at 2·4 Å resolution (Tranier et al., 2002
, 2003
). The X-ray structure of TorDsm revealed extensive domain swapping between the two subunits and an all-helical architecture of the globular domains, showing no similarity with other known protein structures (Tranier et al., 2003
).
This report presents an extension and classification of the TorD family members made possible by the increasing availability of sequenced bacterial genomes. Moreover, a structural and functional study of various members of the TorD family indicates that although they are structurally related, they seem to be highly specific. This analysis thus reveals a possible co-evolution of the molybdoenzymes of the DMSO reductase family and of their dedicated chaperones of the TorD family.
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METHODS |
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For the growth kinetics studies, anaerobic growth was performed in full-capped cuvettes at 37 °C with a minimal salt medium (MSM), derived from that described by Gon et al. (2000). The MSM was supplemented with 0·5 % (v/v) glycerol, as a nonfermentable carbon source, and with DMSO (28 mM). The MSM was inoculated at a dilution of 1 % with cells grown overnight in LB medium supplemented with ampicillin (50 µl ml1) when necessary, centrifuged and resuspended in the same volume of MSM. Growth was monitored in the same full-capped cuvettes at 600 nm. Data are typical of at least three independent experiments.
Construction of mutant strains.
Construction of strain LCB514, with the entire dmsD gene deleted, was performed according to the method of Datsenko & Wanner (2000). The dmsD gene was replaced by a chloramphenicol-resistance gene, generated by PCR. The primers were 60 nts long and included 40-nt sequence extensions, corresponding to the 5' region upstream of the start codon of dmsD gene, or to the 3' extremity of the dmsD gene and 20 nt priming sequences that hybridized to the resistance gene flanking sequence of plasmid pKD3. The PCR mixture was treated with DpnI and used to transform strain MC4100 carrying the
Red helper plasmid pKD46. The mutation of strain LCB514 (
dmsD) was transferred to LCB641 (torD) by P1 transduction, resulting in strain LCB515 (torD
dmsD).
Construction of plasmids.
Plasmids pTorDsm and pTorDec, allowing the synthesis of His6-tagged-TorDsm and -TorDec, respectively, were constructed previously (Tranier et al., 2002; Ilbert et al., 2003
). Plasmids pDmsD, pYcdY and pTorDso, allowing the synthesis of His6-tagged-DmsD, -YcdY and -TorDso, respectively, were constructed by cloning DNA fragments encoding each tagged protein preceded by a consensus ShineDalgarno motif into the XbaI and HindIII sites of the expression vector pET28 under the control of T7 promoter. In each construction, the His6-tag coding sequence comes from the reverse primers. The PCR amplification of the coding sequences were performed with E. coli or Shewanella oneidensis chromosomal DNA, according to the origin of the protein of interest, and appropriate primers, available upon request. All PCR products and fusion sites were confirmed by sequencing. The recombinant vectors pDmsD, pYcdY and pTorDso were introduced into strain BL21(DE3) according to the method of Chung & Miller (1988)
.
Plasmids pJFTorD, pJFDmsD, pJFYcdY were constructed as described above except that the PCR products were cloned into the corresponding sites of expression vector pJF119EH containing the PTAC promoter.
Plasmid p'TorA was constructed as for pTorA (Ilbert et al., 2003) except that the coding sequence of torA was deleted from nucleotide 4 to117, encoding the signal sequence of the protein. This construction leads to the production of a TorA protein devoid of its signal sequence.
Purification of recombinant proteins.
Recombinant TorD-like proteins were purified by a HiTrap chelating Ni2+ column (Amersham Pharmacia Biotech) as described previously for TorDec and TorDsm (Tranier et al., 2002; Ilbert et al., 2003
). As often happens during overproduction, the proteins presented several oligomeric states, which were separated during the elution from the affinity column. In all the experiments carried out in this study, only the fractions containing mainly monomeric forms of the TorD-like proteins were used (see Fig. 2a
). After purification, TorDec, TorDsm, DmsD, YcdY and TorDso were loaded on a desalting PD-10 column (Amersham Biosciences) equilibrated with 20 mM phosphate buffer (pH 7). The proteins were purified near to homogeneity as checked by SDS/15 % PAGE and Coomassie staining of the gel (data not shown). His6-tagged apoTorA and apo'TorA were overproduced in a molybdenum-cofactor-deficient strain harbouring plasmid pTorA or p'TorA, respectively, and purified from the soluble fraction as described previously (Ilbert et al., 2003
).
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Analytical procedure.
The soluble and membrane extracts were prepared in Tris buffer (40 mM, pH 7·6). Membranes (4 mg ml1) were solubilized with Triton X-100 (4 %, 30 min at 4 °C) in the same buffer. TMAO reductase activity was measured spectrophotometrically at 37 °C by following the oxidation of reduced benzyl viologen at 600 nm, coupled to the reduction of TMAO. SDS-PAGE was performed using 10 or 15 % polyacrylamide gels as indicated in the legends. DMSO reductase activity in the polyacrylamide gel was revealed as described for TMAO reductase activity, except that methyl viologen and DMSO were used as electron donor and acceptor, respectively. The amount of TorA present in the extracts was determined by rocket immunoelectrophoresis using a polyclonal antiserum specific for TorA (Pommier et al., 1998).
Circular dichroism (CD) spectra.
Far UV CD spectra of purified monomeric forms of proteins were monitored from 190 to 260 nm. The spectra were recorded on a Jasco J-715 instrument equipped with a Peltier-type temperature control system (model PTC-348WI) with a 2 mm pathlength and a scan rate of 5 nm min1 at 20 °C. Each spectrum was a mean of three scans. Baselines were corrected by subtracting the buffer spectrum.
Phylogenetic analysis.
Amino acid sequences of the TorD homologues were retrieved via the NCBI server (http://www.ncbi.nlm.nih.gov/). Multiple alignments were performed using CLUSTALX and the phylogenetic relationships were calculated using the neighbour-joining approach (CLUSTALX) (Altschul et al., 1990).
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RESULTS AND DISCUSSION |
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Specificity of the TorD homologues
It was recently shown by an in vitro assay that TorDec plays a key role during the maturation of apoTorA, its presence leading to a fourfold increase in the apoenzyme maturation (Ilbert et al., 2003). This activation test was used to determine whether other types of TorD homologues, and in particular those from E. coli, could substitute for TorDec. In the assay, apoTorA and a source of molybdenum cofactor were mixed either with DmsD or YcdY. None of the proteins increased significantly the level of TMAO reductase activity recovered in the assay after 180 min incubation, indicating that neither DmsD nor YcdY could substitute for TorDec during apoTorA maturation (Fig. 3
). One possibility is that DmsD and YcdY are unable to replace TorDec because they do not belong to the same subfamily, and thus are quite distinct from TorDec. A similar test was therefore performed with TorDsm, a type III TorD homologue sharing 34 % identity with TorDec. Again, no complementation was observed, showing that TorDsm cannot replace TorDec either (Fig. 3
). From these experiments, it seems clear that TorDec is specifically devoted to apoTorA and cannot be replaced by any of the other proteins tested.
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The effect of TorDec is independent of the signal sequence of TorA
To determine if the signal sequence of apoTorA plays a role during the maturation process of the apoenzyme mediated by TorDec, we constructed a plasmid allowing the production of 'TorA (or apo'TorA in a mo mutant strain), a TorA form devoid of the signal sequence. The N-terminal extremity of purified apo'TorA (AQAAT) was checked by sequencing. The activation of apo'TorA in the presence of TorDec and of the molybdenum cofactor source clearly shows that the absence of the signal sequence does not hamper the action of TorDec during TorA maturation. Indeed, no significant difference was measured in the level of activation of apoTorA and apo'TorA (Fig. 3). DmsD was reported to bind to the signal sequence of TorA to target it to the Tat machinery (Oresnick et al., 2001
; Ray et al. 2003
). To test whether DmsD still interferes during the in vitro maturation of apo'TorA in the presence of TorDec, we performed an assay containing apo'TorA, the molybdenum cofactor source, TorDec (2·1 µM) and DmsD (5 µM). The maturation level was lower in the presence of DmsD than in its absence, showing that the inhibitory effect of DmsD is independent of the TorA signal sequence. The same conclusion could be drawn when the experiment was performed with YcdY or TorDsm.
TorDec cannot substitute for DmsD
To determine whether the high level of specificity between TorDec and TorA was a common feature of the TorD family proteins toward their cognate enzymes, we tested whether TorDec could replace DmsD during the maturation of DmsA. It was recently shown that a dmsD mutant strain cannot grow anaerobically on glycerol minimal medium supplemented with DMSO (Ray et al., 2003). As expected, the LCB514 strain (
dmsD) was unable to grow on DMSO-containing medium (Fig. 5
a). Moreover, the presence of the dmsD gene in trans (LCB514/pJFDmsD) restored the wild-type phenotype. In contrast, when TorDec was produced in this strain (LCB514/pJFTorD), no growth increase was observed compared to the mutant strain (LCB514) (Fig. 5a
). The absence of complementation by TorDec was confirmed using extracts of the same strains grown anaerobically on rich medium. After electrophoresis of the samples, the gel was stained for DMSO reductase activity; this was observed only in the lanes of DmsD-producing extracts (Fig. 5b
). This point indicates that the action of DmsD toward the DMSO reductase complex cannot be carried out by TorDec.
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ACKNOWLEDGEMENTS |
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Received 13 November 2004;
revised 7 January 2004;
accepted 7 January 2004.
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