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Article No. mb982155 J. Mol. Biol. (1998) 284, 421±433

Molecular Analysis of the Trimethylamine N-oxide(TMAO) Reductase Respiratory System from aShewanella Species

Jean-Philippe Dos Santos, Chantal Iobbi-Nivol, Carole CouillaultGeÂrard Giordano and Vincent MeÂ jean*

Laboratoire de ChimieBacteÂrienne, Institut de BiologieStructurale et MicrobiologieCentre National de la RechercheScienti®que, 31, chemin JosephAiguier, BP 71, 13402 MarseilleCedex 20, France

E-mail address of the [email protected]

Abbreviations used: BV, benzyl vdimethylsulfoxide; LDAO, N,N-dimN-oxide; TMAO, trimethylamine N-trimethylamine; TorA, TMAO reductranscriptase; TorC, c-type cytochro

0022±2836/98/470421±13 $30.00/0

Trimethylamine N-oxide (TMAO) is an abundant compound of tissues ofmarine ®sh and invertebrates. During ®sh spoilage, certain marine bac-teria can reduce TMAO to nauseous trimethylamine (TMA). One suchbacterium has been isolated and identi®ed as a new Shewanella species,and called Shewanella massilia. The anaerobic growth of S. massilia isgreatly increased when TMAO is added, indicating that TMAO reductioninvolves a respiratory pathway. The TorA enzyme responsible for TMAOreduction is a molybdenum cofactor-containing protein of 90 kDa locatedin the periplasm. Whereas TorA is induced by both TMAO and dimethyl-sulfoxide (DMSO), this enzyme has a high substrate speci®city andappears to only ef®ciently reduce TMAO as a natural compound. Thestructural torA gene encoding the TMAO reductase (TorA) and its ¯ank-ing regions were ampli®ed using PCR techniques. The torA gene is thethird gene of a TMAO-inducible operon (torECAD) encoding the TMAOrespiratory components. The torC gene, located upstream from torAencodes a pentahemic c-type cytochrome, likely to be involved in electrontransfer to the TorA terminal reductase. TorC was shown to be anchoredto the membrane and, like TorA, is induced by TMAO. Except for theTorE protein, which is encoded by the ®rst gene of the torECAD operon,all the tor gene products are homologous to proteins found in theTMAO/DMSO reductase systems from Escherichia coli and Rhodobacterspecies. In addition, the genetic organization of these systems is similar.Although these bacteria are found in different ecological niches, their res-piratory systems appear to be phylogenetically related, suggesting thatthey come from a common ancestor.

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Keywords: TMAO reductase; respiratory system; c-type cytochrome;molybdoenzyme; anaerobiosis
*Corresponding author
Introduction

Trimethylamine N-oxide (TMAO) is a major lowmolecular mass constituent of marine ®sh andinvertebrates in which it probably acts as an osmo-protector (Barrett & Kwan, 1985). TMAO is anorganic osmolyte that has the useful biologicalfunction of protecting proteins against denaturat-

ing author:

iologen; DMSO,ethyldodecylamineoxide; TMA,tase; RT, reverse

me.

ing stresses such as high concentration of urea(Yancey et al., 1982). This counteracting osmolytehas been recently refered to as a ``chemical chaper-one'' due to its in¯uence on protein folding (Wang& Bolen, 1997).

Various bacteria grow anaerobically usingTMAO as an alternative terminal electron acceptorof a respiratory transport chain (Barrett & Kwan,1985). During this energy-yielding reaction, TMAOis reduced to off-odour volatile trimethylamine(TMA). The bacteria capable of reducing TMAO toTMA are found in three different ecological niches.Accordingly, TMAO-reducing activity has beenobserved in marine bacteria (Photobacterium, Shewa-nella and Vibrio species), in photosynthetic bacteria

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422 TMAO Reductase from Shewanella massilia

living in ponds (Rhodobacter species) and, moresurprising, in most enterobacteria (Unemoto et al.,1965; Barrett & Kwan, 1985; Clarke & Ward, 1988;McEwan, 1994; Gram & Huss, 1996).

The properties of TMAO reductases have beenstudied in several organisms. A common feature isthe presence of a molybdenum cofactor in all theknown terminal enzymes. Based on their substratespeci®city, these enzymes can be divided into twogroups: the ®rst corresponds to TMAO reductaseswhich have high substrate speci®city, and thesecond includes DMSO/TMAO reductases whichcan reduce a broad range of N and S-oxide sub-strates.

The TMAO reductases from Escherichia coli(TorA; Iobbi-Nivol et al., 1996), Shewanella putrefa-ciens (Clarke & Ward, 1988) and Roseobacter denitri-®cans (Arata et al., 1992) are unable to reduceS-oxide compounds such as DMSO and belong tothe ®rst group. In the second group, the constitu-tive DMSO reductases from E. coli (DmsA; Simala-Grant & Weiner, 1996) and Proteus vulgaris(Styrvold & Strom, 1984), and the DMSOreductases from Rhodobacter capsulatus or R. sphaer-oides (McEwan, 1994) can reduce TMAO as well asother N and S-oxides. Except the constitutiveDMSO reductases from E. coli and P. vulgariswhich are membrane bound, all these molybdoen-zymes are located in the periplasm and induced byTMAO.

The TMAO respiratory systems have been wellstudied at the molecular level in E. coli and Rhodo-bacter species. In E. coli, the TMAO reductase andthe DMSO reductase systems are encoded by thetorCAD and the dmsABC operons, respectively(Bilous et al., 1988; MeÂjean et al., 1994). The torCgene encodes a pentahemic c-type cytochrome(TorC) which is anchored to the inner membrane.TorC is likely to transfer electrons directly to theperiplasmic TorA terminal enzyme encoded by thetorA gene. We have recently shown that TorD is acytoplasmic protein. Interestingly, TorD is prob-ably a TorA speci®c chaperone as it displays agreat af®nity for the unfolded TorA protein(Pommier et al., 1998). The anaerobic expression ofthe torCAD operon is strictly controlled by the pre-sence of TMAO or related compounds through theTorS/TorR two component system (Jourlin et al.,1997). The E. coli dmsABC operon, which is consti-tutively expressed regardless of the substrateduring anaerobiosis, encodes the three subunits ofthe DMSO reductase complex: the molybdenum-containing catalytic subunit (DmsA), an electrontransfer subunit containing four 4Fe-4S clusters(DmsB) and a membrane anchor subunit (DmsC).

Although the DMSO reductase systems fromRhodobacter species reduce TMAO as well asDMSO, they appear closely related to the E. coliTor system. Firstly, the terminal enzymes arelocated in the periplasm (McEwan, 1994). Sec-ondly, the probable dorCBA operon in R. sphaer-oides 2.4.1T (also called dmsCBA in R. sphaeroides f.sp. denitri®cans) encodes three proteins, DorC,

DorB and DorA, which are, respectively, homolo-gous to the E. coli TorC, TorD and TorA proteins(Ujiiye et al., 1996; Mouncey et al., 1997). Thus,dorC encodes a c-type cytochrome anchored to themembrane and DorB could be a DorA speci®c cha-perone located in the cytoplasm. Thirdly, the dorC-BA operon is anaerobically expressed in thepresence of DMSO. The DorS/DorR proteinsresponsible for the DMSO control belong to thetwo component regulatory systems and shareextensive similarities with the E. coli TorS/TorRregulatory system (Mouncey et al., 1997; Ujiiyeet al., 1997).

Although TMAO is mainly found within themarine environment, no extensive molecular studyhas been performed for the TMAO respiratory sys-tem of marine bacteria. TMAO reduction by Vibriospecies has received very little attention, and muchmore is known about the TMAO reductase of She-wanella putrefaciens (previously called Alteromonasor Pseudomonas putrefaciens; Lee et al., 1977), aGram-negative bacterium frequently responsiblefor spoilage of protein-rich food such as marine®sh (Barrett & Kwan, 1985; Gram & Huss, 1996).The terminal enzyme in this bacterium contains amolybdenum cofactor and appears to be eitherperiplasmic (Easter et al., 1983) or loosely bound tothe outer part of the cytoplasmic membrane(Stenberg et al., 1984).

Since the studies on marine bacteria are poorlydocumented and sometimes controversial (Barrett& Kwan, 1985; Clarke & Ward, 1988), the aim ofour study was ®rst to isolate and identify a marinebacterium possessing a high level of TMAOreductase activity, and then to analyze the com-ponents involved in this process.

Results

Isolation and characterization ofShewanella massilia

A marine ®sh (Mullus surmuletus), freshlycaught, was placed in a sterile tube containing seawater. After 48 hours of incubation at room tem-perature without shaking, the ®sh decayed and thegrowing bacteria were isolated on solid rich med-ium. One of the bacteria accounts for up to 45% ofthe total culturable bacteria. Crude extract fromthis strain exhibits a high benzyl viologen (BV)-TMAO reductase activity (2.7 mmol/min per mg ofprotein) as compared to that of other strains and toTMAO reductase activities described in the litera-ture (Silvestro et al., 1989). This strain has beenidenti®ed as a new Shewanella species and calledShewanella massilia (see below).

We also studied the in vivo TMAO reducingcapability of S. massilia. During anaerobiosis with-out TMAO, S. massilia grows slowly in a rich med-ium at 30�C (doubling time: about ®ve hours).With TMAO, the growth rate increases signi®-cantly (doubling time: about 1.5 hours) and volatileoff-¯avoring TMA is simultaneously produced. In

TMAO Reductase from Shewanella massilia 423

contrast, when DMSO is added to the medium, thegrowth rate does not increase. Although DMSOreduction does take place, since volatile DMS isproduced, the effect of DMSO is clearly differentfrom that of TMAO for the growth of S. massilia.These results strongly suggest that the TMAOreduction is coupled to an energy-yielding reactionin S. massilia, whereas reduction of DMSO eitherlacks ef®ciency or is uncoupled to a respiratoryprocess.

Phylogenetic analysis of S. massilia

From physiological and biochemical character-istics, S. massilia appears to be a Gram-negativemotile rod (data not shown). To unambiguouslyidentify this new isolate, the sequence of the 16 SrRNA gene of S. massilia was determined. The 16 SrDNA of S. massilia was ampli®ed by PCR, and thepuri®ed PCR product was directly sequenced (seeMaterials and Methods). Approximately 95% of the16 S rRNA gene sequence was determined; boththe 50 and 30 end sequences were incompletebecause internal primers were chosen to amplifythe target gene. The results, presented as a phylo-genetic tree (Figure 1), clearly indicate that S. mas-silia is related to the Shewanella group. The highest16 S rRNA gene similarity values are foundbetween S. massilia and S. alga Bry (97.8%) orS. putrefaciens (96.8%). In contrast, only 89.9% and87.7% of 16 S rRNA gene similarities were foundwith a Vibrio species (V. marinus) and E. coli,

Figure 1. Phylogenetic tree based on 16 S rRNA genesequence comparison between Shewanella massilia andstrains belonging to the g-subdivision of Proteobacteria.The tree was constructed as described in Materials andMethods. The root of the tree was determined by includ-ing the 16 S rRNA gene sequence of Escherichia coli(DSM30083T) in the analysis. The scale bar below thedendogram indicates 1 per 100 nucleotide substitution.The per cent of 16 S rRNA gene sequence similaritywith respect to the S. massilia 16 S rRNA gene sequenceis indicated in parentheses.

respectively. Based on this phylogenetic analysis,we consider that Shewanella massilia is a newspecies of the Shewanella group.

Induction and location of the TMAO reductaseterminal enzyme

Cells of S. massilia were anaerobically grown in arich medium with or without TMAO. In theabsence of TMAO, no signi®cant TMAO reductaseactivity was detected in the cell extracts, whereasin the presence of TMAO, TMAO reductaseactivity was found principally in the periplasmicfraction (Table 1). Consequently, TMAO is necess-ary for induction of the TMAO reductase enzymewhich is located in the periplasm. It is of interestthat the activity of the TMAO reductase found inthe periplasmic fraction (21 mmol/min per mg ofprotein) is very high, in comparison to that ofE. coli (5 mmol/min per mg of protein; Silvestroet al., 1989).

DMSO is also an ef®cient inducer for TMAOreductase since a high level of TMAO reductaseactivity was observed when DMSO was added tothe growth medium (Table 1). The fact that thisactivity is mainly found in the periplasmic fractionsuggests that the same periplasmic enzyme whichreduces TMAO can be induced by TMAO as wellas DMSO. To con®rm this hypothesis, we havecompared the periplasmic proteins synthesized inthe presence or absence of inducers (Figure 2). Theperiplasmic protein pattern, obtained after Coo-massie-blue staining of a SDS-polyacrylamide gel,is similar whatever the growth conditions, exceptthat an additional protein of about 90 kDa is pre-sent when the cells were grown in the presence ofeither TMAO or DMSO (Figure 2(a)). To show thatthis inducible protein can reduce TMAO, sampleswere loaded under non-denaturing conditions,revealling the TMAO reductase activity directly onthe gel (Pommier et al., 1998). Indeed, only a singleTMAO reductase active band of the expected sizewas revealed when the periplasmic fraction wasprepared from cells grown with added TMAO orDMSO (Figure 2(b)). This implies that the S. massi-lia TMAO reductase (TorA) is a single periplasmic

Table 1. Subcellular location of TMAO reductaseactivity

Specific activity of TMAO reductaseb

Subcellularfractionsa ± � TMAO � DMSO

Periplasm <0.2 21 (78%) 27 (83%)Cytoplasm <0.2 2.6 (21.6%) 2 (15 %)Membranes <0.2 <0.2 (0.4%) 0.3 (2%)

a Periplasmic, cytoplasmic and membrane fractions wereobtained from cells grown anaerobically at 30�C, in LB mediumsupplemented, when indicated, with 10 mM TMAO or 10 mMDMSO.

b TMAO reductase activity is expressed as mmol of TMAOreduced per minute per mg of protein. The distribution of theTMAO reductase activity is indicated as a percentage of thetotal TMAO reductase activity.

Figure 2. SDS-PAGE analysis of periplasmic proteinsfrom Shewanella massilia grown in different conditions.Periplasmic proteins (25 mg) from cells grown in anaero-biosis at 30�C in LB medium (lane 1), supplementedwith 10 mM TMAO (lane 2) or 10 mM DMSO (lane 3)were loaded on a SDS/7.5% polyacrylamide gel. Theposition of the TMAO reductase (TorA) is indicated byan arrow. (a) Samples were heated prior to loading andthe gel was stained with Coomassie blue; M, molecularweight markers. (b) Samples were not heated prior toloading and the gel was stained for TMAO reductaseactivity.


protein of about 90 kDa, induced by both TMAOand DMSO.

Substrate specificity of the TorA enzyme fromS. massilia

The TorA protein was puri®ed (see Materialsand Methods) and the reductase activity of thisenzyme for several N and S-oxide compounds wasmeasured spectrophotometrically by following theoxidation of reduced BV at 600 nm in the presence

Table 2. Kinetic parameters with different electron acceptors

Compoundsa

TMAO4-Methylmorpholine-N-oxide2-Picoline-N-oxideN,N-dimethyldodecylamine N-oxide (LDAO)

DMSODL-MethioninesulfoxideDiphenylsulfoxideDibutylsulfoxide

a Assays were performed in 100 mM phosphate buffer (pH 6.8) wb Activity values obtained were too low for a Km calculation.

of the potential substrate. As shown in Table 2,only two substrates (TMAO and 4-methylmorpho-line N-oxide) can be reduced ef®ciently by theTorA enzyme. Even if the activity values (kcat) aresimilar for all the N-oxides tested, the af®nityvalues (Km) are much higher for LDAO (N,N-dimethyldodecylamine N-oxide) and 2-picolineN-oxide than for TMAO or 4-methylmorpholineN-oxide. As a result, LDAO and 2-picoline N-oxidecannot be considered as ef®cient substrates for theTorA enzyme. For all the S-oxides tested, theactivity values were too low to allow a Km determi-nation. TorA is thus unable to reduce any of theS-oxides tested. In conclusion, the puri®ed TorAenzyme reduces a restricted range of electronacceptors. As 4-methylmorpholine N-oxide is asynthetic compound, the TorA enzyme fromS. massilia is likely to only reduce TMAO in nature.

Amplification and sequence analysis of theS. massilia torA gene

To determine the DNA sequence of the TMAOreductase encoding gene (torA) from S. massilia, wedecided ®rst to PCR amplify an internal torA DNAfragment using degenerate oligonucleotide primersand chromosomal DNA from S. massilia as a tem-plate. The ®rst set of degenerate primers (A�) wasbased on the expressly obtained amino-terminalsequence of the TorA protein. As all the knownTMAO reductases contain a molybdenum cofactor,the second set of degenerate primers (Aÿ) wasdesigned from a highly conserved region of molyb-doenzymes (Figure 3). The PCR ampli®cation wasperformed with the two converging sets of oligo-nucleotides as described in Materials and Methods.A predominant ampli®cation product of about1 kb was obtained. This DNA fragment, the size ofwhich was compatible with that expected for atorA internal fragment, was cloned into the pUCvector, and then sequenced.

The second step of the torA gene analysisinvolved the ampli®cation by inverse PCR of theDNA regions ¯anking the torA internal fragment.The ampli®ed DNA fragments were then puri®edand directly sequenced on both strands. The DNAsequence shows an open reading frame coding fora polypeptide of 829 amino acid residues. A poten-tial ribosome binding site (GGGAG) was found

catalysed by Shewanella massilia TorA enzyme

kcat (sÿ1) km (mM)

119.8 1.7 10ÿ2

109.4 2.2 10ÿ2

78 0.61151 1

<4 ±b

<4 ±<4 ±<4 ±

ith a concentration of 10 mM for each compound tested.

Figure 3 (legend on page 426)

Figure 4. (a) Physical map of the torECAD operon region of S. massilia. The large arrows show the location and theorientation of the open reading frames. The nucleotide sequence of the putative regulatory region is indicated. Thefour direct repeats of a decameric nucleotide motif (boxes 1 to 4) are indicated by a line above. The putative ÿ10 pro-moter box is underlined whereas the torE start codon and the putative Shine-Dalgarno sequence are in bold type. Theprimers (a, b and c) used for the RT-PCR are also indicated. (b) Analysis of the tor genes transcription by RT-PCR fol-lowed by 1.5% agarose gel electrophoresis. The RT-PCR was carried out with primers a and c (lanes 1-3) or primersb and c (lanes 4-9). Lanes 1, 4 and 7, control PCR with genomic DNA; lanes 2, 5 and 8, RT-PCR experiments with1 mg of S. massilia total RNA prepared from cells grown in the presence of TMAO (lanes 2 and 8) or in the absence ofTMAO (lane 5); lanes 3, 6 and 9, same experiments as in lanes 2, 5 and 8 but without reverse transcriptase to checkthe absence of DNA traces in the RNA preparation.


eight bases upstream from the ATG start codon.The high degree of similarity observed betweenthe deduced amino acid sequence and the E. coliTorA sequence (50% of identity) or the RhodobacterDorA (DmsA) sequences (40±45% of identity)strongly suggests that the encoded protein corre-sponds to the S. massilia TorA protein (Figure 3).

The amino-terminal sequence of the puri®edperiplasmic TorA protein matches residues 32±61of the amino acid sequence deduced from the torAgene, providing evidence that the torA geneencodes the S. massilia TMAO reductase and thatthe cleaved signal peptide of TorA is 31 aminoacids long. Taking into account the molybdenumcofactor mass (1.541 kDa), the molecular mass of

Figure 3. Amino acid sequence alignment of Shewanelreductase from Escherichia coli (TorA/E.c., MeÂjean et al., 1994oides and Rhodobacter capsulatus (DmsA/R.s., Yamamoto et alcleavage site and the redox protein export conserved motifviously proposed to be involved in the binding of the molyspond to the regions chosen for the synthesis of theampli®cation of an internal torA gene DNA fragment fromreverse contrast in black boxes, and shaded when present in

the mature S. massilia TorA protein is 90.699 kDa.This calculated molecular mass con®rms thatwhich was previously estimated (90 kDa,Figure 2(a)).

Analysis of the S. massilia tor locus

To determine the sequence of the genes neartorA, we have again employed the inverse PCRtechnique as a chromosomal walking method. Thesequence analysis of the ampli®ed productsrevealed the presence in the tor locus of four genes(torA, torC, torD and torE) close together and tran-scribed in the same direction (Figure 4). The geneorder is torE, torC, torA and torD and the distance

la massilia TMAO reductase (TorA/S.m.) with TMAO) and TMAO/DMSO reductases from Rhodobacter sphaer-

., 1995; DorA/R.c., Shaw et al., 1996). The signal sequence(R-R-x-F-L) are indicated. The ®ve conserved regions pre-bdenum cofactor are underlined. The large arrows corre-two degenerate primer sets (A � /Aÿ) that allowed

S. massilia. Residues identical in all sequences are inat least three out of the four sequences.

Figure 5 (legend on page 428)


Figure 6. Detection of c-type cytochromes by TMBZstaining. Membranous proteins (30 mg) from cells grownanaerobically at 30�C in LB medium (lane 1), sup-plemented with 10 mM TMAO (lane 2) or 10 mMDMSO (lane 3) were loaded on a SDS/12.5% polyacryl-amide gel and stained for heme with 3,30,5,50-tetra-methylbenzidine (TMBZ) after electrophoresis. Thearrow corresponds to the cytochrome (TorC) induced byTMAO and DMSO.


between these genes is 43, 15 and 94 bp, respect-ively. Each gene contains a potential ribosomebinding site 7±9 bp upstream from the ATG startcodon (GGAG for torE, GAGGA for torC andtorD). The putative genes preceding or followingthe torECAD cluster are transcribed divergently,and a non-coding region of about 0.5 kb is foundupstream from the ®rst gene (Figure 4(a)). Todetermine whether the torE, torC, torA and torDgenes are organized in an operon, RT(reverse tran-scriptase)-PCR were carried out with appropriateoligonucleotides that hybridize to the four torgenes and to the torE upstream region (Figure 4(b),and data not shown). These experiments show thatthe four genes are in a single transcription unit,and that the transcription start site of the torECADoperon is located within 50 bases of the translationstart point of torE. Moreover, signi®cant ampli®ca-tion products were only obtained when TMAOwas added to the cell culture (compare lanes 5 and8 of Figure 4(b)). This strongly suggests thatexpression of the torECAD operon is induced byTMAO.

The torE gene is predicted to encode a smallmembrane protein homologous to the NapE pro-tein of Thiosphaera pantotropha (Figure 5(a)). TheNapE protein belongs to the nitrate reductase sys-tem but its role is still unknown (Berks et al., 1995).Neither the E. coli tor operon nor the Rhodobacterdor operons contain such a gene.

The deduced torC gene product of 392 aminoacid residues contains ®ve C-X-X-C-H motifscharacteristic of c-type heme-binding sites(Figure 5(b)). Four of the heme-binding sites areclustered in the ®rst half of the protein, the ®fthbeing located near the carboxy-terminal end of theprotein. Although the overall TorC protein exhibitsa hydropathy pro®le characteristic of soluble pro-tein, a potential transmembrane hydrophobicsequence is present in its amino-terminal region,from residue 18 to 38. TorC could be, like its E. coliTorC and R. sphaeroides DorC homologs, a pentahe-mic c-type cytochrome anchored to the cytoplasmicmembrane of the cell (MeÂjean et al., 1994; Ujiiyeet al., 1996; Mouncey et al., 1997).

To con®rm the presence of such a c-type cyto-chrome in S. massilia, we have performed heme-detection experiments. Samples from subcellularfractions of S. massilia bacteria, anaerobicallygrown with or without TMAO, were heme-stainedafter being subjected to SDS-PAGE (Figure 6, and

Figure 5. (a) Amino acid comparison of the deduced aminThiosphaera pantotropha napE gene product (NapE/T.p., Berkslined. Identical residues are in black boxes. (b) Similarities btorC gene (TorC/S.m.) with its homologs from Escherichia co(DmsC/R.s., Ujiiye et al., 1996) and Rhodobacter capsulatus (Dterminal hydrophobic segment is underlined and the ®ve c-tboxes correspond to identical residues in all sequences. Shafour sequences. (c) Similarities between S. massilia torD genecoli (TorD/E.c., MeÂjean et al., 1994), Rhodobacter sphaeroides (D(DorD/R.c., accession number U49506).

data not shown). Only one c-type cytochromeappeared to be synthesized speci®cally in the pre-sence of TMAO, whereas several others wererevealed even without TMAO. Furthermore, thisinduced c-type cytochrome is found in the mem-brane fraction, and its estimated molecular mass(44 kDa) corresponds with that calculated for thetorC gene product (44.5 kDa). Together, theseresults strongly suggest that this hemoprotein isthe product of the torC gene.

The torD gene, the last gene of the tor operon,encodes a 209 amino acid polypeptide similar tothe E. coli TorD and the Rhodobacter DorB (orDmsB) proteins (Figure 5(c)). At ®rst glance, theS. massilia TorD protein might have been a mem-branous protein, as believed for its E. coli and Rho-dobacter homologs, as it likewise contains twohydrophobic segments at the amino and carboxyends, respectively. However, the membranouslocation previously proposed for the E. coli TorDprotein has been recently discarded. In fact, E. coliTorD proved to be a cytoplasmic protein involvedin the folding process of the TorA precursor pro-

o acid sequence of S. massilia torE gene (TorE/S.m.) withet al., 1995). The conserved hydrophobic region is under-etween the deduced amino acid sequence of S. massilia

li (TorC/E.c., MeÂjean et al., 1994), Rhodobacter sphaeroidesorC/R.c., accession number U49506). The shared amino-ype heme binding sites are indicated (C-x-x-C-H). Blackded amino acids are identical in at least two out of theproduct (TorD/S.m.) and its homologs from EscherichiamsB/R.s., Ujiiye et al., 1996) and Rhodobacter capsulatus


tein (Pommier et al., 1998). As S. massilia TorDshows 27% identity with the E. coli TorD protein,its cellular location and role remain to be de®ned.

Discussion

Gram-negative bacteria belonging to the Shewa-nella genus are widespread in nature and havemainly been isolated from the aquatic environmentbut also from sediments, oil ®elds and even humaninfections (Lee et al., 1977; Vogel et al., 1997). Anessential property of the Shewanella species is itsability to reduce TMAO to TMA during an anaero-bic respiratory process. That S. massilia belongs tosuch a genus is not surprising. This strain, whichhas been isolated from a marine environment, isable to ef®ciently use TMAO as a terminal electronacceptor in anaerobiosis. S. massilia accounts foralmost half of the culturable bacteria found during®sh spoilage, probably because anaerobic respir-ation with TMAO, an abundant ®sh compound,confers a competitive advantage to it over strainsunable to reduce TMAO when oxygen becomesdepleted (Gram & Huss, 1996).

Based on whole-cell protein pro®les, ribotyping,phenotypic characterization and 16 S rRNA genesequence analysis, Vogel et al. (1997) have recentlyproposed that the Shewanella genus can be dividedinto four main related groups: S. alga, S. benthica,S. hanedai and S. putrefaciens. In their classi®cation,S. alga (Bry) belongs to the S. putrefaciens grouprather than to the S. alga group. S. massilia is clo-sely related to S. alga (Bry) and could therefore alsobelong to the same group. In any case, the 16 SrRNA gene sequence analysis (Figure 1) clearlyshowed that S. massilia is a new species of the She-wanella genus.

We have shown that the main enzyme (TorA)responsible for TMAO reduction in S. massilia is alarge molybdoprotein of 90 kDa located in theperiplasm (Figure 2). The TorA primary sequenceshares high similarities with both the E. coli TorAand Rhodobacter DorA (DmsA) periplasmic pro-teins. These enzymes are thus probably phylogen-etically related even if the bacteria containing themare distant from each other. Nevertheless, thereduction capacities of the TorA and DorAenzymes are somewhat different, since the formerspeci®cally reduces TMAO (and the 4-methylmor-pholine N-oxide synthetic compound), whereas thelatter reduces a broader range of substrates includ-ing TMAO and DMSO (Table 2; Iobbi-Nivol et al.,1996). This raises interesting questions concerningthe active site structure of the TorA enzymes com-pared to that of the DorA enzymes (Schindelinet al., 1996; Schneider et al., 1996; Czjzek et al.,1998).

The amino-terminal end of the mature S. massiliaTorA enzyme, obtained by Edman degradation,coincides with the torA gene product from residue32 to residue 61. Thus, the ®rst 31 amino acids ofTorA constitute an unusually long signal sequence

with a classic A-x-A cleavage site and a motif (R-R-x-F-L, Figure 3) found in several exported metal-loproteins (Berks, 1996; Shaw et al., 1996). Thiskind of signal sequence could be involved in a newtype of membrane crossing mechanism. Indeed,the E. coli TorA enzyme crosses the inner mem-brane as a folded molybdo-containing protein by asec-independant mechanism (Santini et al., 1998;Weiner et al., 1998).

The presence of a speci®c cytochrome for TMAOreduction in the Shewanella species remains contro-versial. For example, Morris et al. (1990) did not®nd any speci®c cytochrome in S. putrefaciensNCMB 400. Our biochemical analysis clearlyshows that a c-type cytochrome (TorC), found inthe membrane fraction, is speci®cally induced byTMAO in S. massilia (Figure 6). Moreover, theS. massilia torC gene, located upstream from thetorA gene, encodes a c-type cytochrome which islikely to correspond to the TorC protein (Figures 4and 5). This protein could be anchored to the innermembrane by its amino-terminal hydrophobic seg-ment and involved in the electron transfer to theterminal periplasmic reductase.

Two additional genes, torE and torD, encodingproteins possibly involved in the TMAO reductaserespiratory pathway, have been found in theS. massilia tor operon (Figure 4). The fact that bothTorA and TorC are induced in the presence ofTMAO (Figures 2 and 6) and that the tor mRNA isdetected only from TMAO-induced bacteria(Figure 4), strongly suggests that the expression ofthe tor operon is controlled by TMAO. The E. colitorCAD and Rhodobacter putative DorCBA operonsare also only expressed when the substrate is pre-sent in the medium (MeÂjean et al., 1994; Ujiiye et al.,1996; Mouncey et al., 1997).

Although a ÿ10 promoter box (TATAGT) exhi-biting signi®cant homology with the E. coli ÿ10consensus sequence is present in the 50 untrans-lated torE region (Lisser & Margalit, 1993), no puta-tive ÿ35 box matching the E. coli consensussequence could be found 16 to 18 bp upstreamfrom the TATA box (Figure 4). This is not surpris-ing, since the absence of a consensus ÿ35 box is ageneral feature of promoters that are positivelyregulated (Busby & Ebright, 1994). In E. coli andR. sphaeroides, the TorR and DorR activators bindto speci®c direct repeats located in the promoterregion (Simon et al., 1995; Ujiiye et al., 1997). If theS. massilia tor promoter is the target for an activatorresponsible for TMAO induction related to theE. coli TorR or Rhodobacter DorR response regula-tors, then direct repeats of a consensus areexpected in this regulatory region for the bindingof the hypothetical response regulator. This isindeed the case, since four homologous sequences,which have six to ten matches of the consensusdecameric sequence CCATTCATAT, are presentupstream from the torE gene (Figure 4). It is worth-while noting that the consensus sequence is closeto that (CTGTTCATAT) of the E. coli TorR bindingboxes (Simon et al., 1995). More striking, the four


direct repeats are found at exactly the same dis-tances from the ÿ10 promoter box in E. coli andS. massilia. Therefore, the direct repeats present inthe tor promoter region of S. massilia are probablythe binding sites for a regulatory protein related tothe E. coli TorR response regulator (Simon et al.,1994). Experiments are in progress to con®rm thishypothesis.

This study, together with previous ones, sup-ports a close phylogenetic relationship betweenthose TMAO/DMSO reductase respiratory systemswith a periplasmic terminal enzyme. Firstly, all theknown periplasmic TMAO/DMSO reductases arehomologous molybdoenzymes which lack anyadditional prosthetic group (Yamamoto et al., 1995;KnaÈblein et al., 1996; Schindelin et al., 1996;Schneider et al., 1996). In this context, the overallstructure of the TMAO reductase (TorA) fromS. massilia is highly similar to that of the DMSOreductases (DorA) from Rhodobacter species (Czjzeket al., 1998). Secondly, the TMAO/DMSO respirat-ory systems contain a related c-type cytochromeand a terminal reductase chaperone (MeÂjean et al.,1994; Ujiiye et al., 1996; Mouncey et al., 1997;Pommier et al., 1998). Finally, the genes encodingthe respiratory components are always organizedin an operon. All these systems are induced byboth TMAO and DMSO whatever their substratespeci®city (the TorA enzymes reduce speci®callyTMAO whereas the DorA enzymes reduce bothTMAO and DMSO). An attractive hypothesis isthat these systems have evolved from a commonancestor capable of reducing a broad range of sub-strates and thus are induced by these variouspotential substrates. Since bacterial species contain-ing these related respiratory systems are distantfrom each other, it remains probable that such bac-teria have acquired the TMAO/DMSO reductasefunction by horizontal gene transfer.

Materials and Methods

Strains, media and growth conditions

The ®sh (Mullus surmuletus) used for bacterial strainisolation was from the Mediterranean sea (Marseillesarea). Sea water was sterilized by ®ltration through a0.45 mm Millipore ®lter. Shewanella massilia was grown at30�C in Luria Broth (LB) medium. For TMAO reductaseinduction, the medium was supplemented with 10 mMTMAO or DMSO as indicated. E. coli TG1 strain (�(lac-pro) supE thi �(hsdM-mcrB)5/F0 traD36 proA�B� lacIq

�(lacZ)M15) was grown aerobically at 37�C in LBmedium. Ampicillin was used at the concentration of50 mg/ml.

Preparation of subcellular fractions

Crude extracts of S. massillia were performed as pre-viously described for E. coli (Silvestro et al., 1989). Theperiplasmic fraction was prepared following the Easteret al. (1983) procedure, except that 1 mM of benzamidinewas added to the 10 mM Tris-HCl (pH 8) buffer. Mem-branous and cytoplasmic fractions were prepared fromthe spheroplasts as described by Silvestro et al. (1989).

Enzyme purification

Puri®cation of the S. massilia TMAO reductase wasperformed from the periplasmic fraction obtained from30 g cells grown anaerobically in the presence of 10 mMTMAO. The periplasmic fraction was applied to a DE 52ion exchange column and the proteins were eluted witha linear 0±0.4 M NaCl gradient. The TMAO reductaseactive fractions, recovered at a salt concentration ofabout 0.15 M, were pooled and desalted by dialysisbefore being loaded on a Mono Q HR 16/10 column.The active fractions, obtained from a 0.1±0.2 M NaCl lin-ear gradient elution, were pooled and concentrated witha DE 52 column. The last step of the puri®cation was car-ried out with a preparative electrophoresis (model 491Prep Cell, BioRad) using a 4.5% polyacrylamide gel.A quantity of 1.5 mg pure TMAO reductase wasobtained with a speci®c activity of 300 mmol TMAOreduced per minute per mg protein. The puri®edenzyme appears as a single polypeptide of about 90 kDaon a SDS-PAGE (Laemmli, 1970).

Analytical procedures

TMAO reductase activity was either measured spec-trophotometrically or visualized directly after SDS-PAGEby an activity staining method (Pommier et al., 1998).The determination of kinetic parameters was performedas previously described (Iobbi-Nivol et al., 1996).

The amino-terminal sequence of the TMAO reductaseof S. massilia (KAGINEDEWLTTGSHFGAFKMKRKNG-VIAE) was determined by Edman degradation (AppliedBiosystems, model 470A) after electroblotting of TorAonto a polyvinylidene di¯uoride membrane (Towbinet al., 1979).

The presence of covalently bound hemes in c-typecytochromes was revealed by staining for peroxidaseactivity using 3,30,5,50 tetramethylbenzidine (TMBZ;Thomas et al., 1976).

The protein concentration was estimated by theLowry et al. (1951) technique.

DNA manipulations

Plasmid preparation, restriction endonuclease diges-tions, DNA puri®cation and ligation were carried out asdescribed by Sambrook et al. (1989). Chromosomal DNAwas prepared as previously described (MeÂjean et al.,1994), except that the cells were lysed by the addition of0.5% SDS and that phenol was replaced by chloroform/isoamyl alcohol (24:1) for the ®rst extraction step. Stan-dard PCR ampli®cations were performed as describedby MeÂjean et al. (1994). E. coli transformations were per-formed according to the Chung & Miller (1988) method.

To amplify an internal torA DNA fragment of S. massi-lia, two sets of degenerate oligonucleotide primers weredesigned. The ®rst one (A � : 50-GGNATHAAYGAR-GAYGARTGG-30) was based on the amino-terminalsequence of the TorA protein (GINEDEW). The secondset of degenerate primers (A ÿ : 50-CCRAANCCNCC-NCCNGG-30) was designed by using a highly conservedregion of molybdoenzymes (PGGGFG; Figure 4). PCRreactions were performed as described by Jourlin et al.(1995). The predominant ampli®cation product (about 1kb) was recovered from agarose gel (1%) and puri®edwith the GeneClean kit (Bio 101). The puri®ed PCR pro-duct was blunted and ligated into a pUC vector linear-ized with the SmaI restriction enzyme. The ligation


mixture was used to transform E. coli TG1. The clonedDNA fragment was sequenced using the dideoxy chain-termination method.

Inverse PCR experiments were performed as pre-viously described (Simon et al., 1994). ChromosomalDNA of S. massilia was digested by the following restric-tion endonucleases SacII, RsaI, Sau3A or XhoI. DNA frag-ments produced by the restriction enzymes werepuri®ed with the GeneClean kit and ligated underdiluted conditions (®nal DNA concentration <2mg/ml).The resulting ligated DNA was used as a template forPCR ampli®cation with pairs of primers homologous tothe 50 or 30-end of the previously sequenced DNA frag-ments. The resulting products were puri®ed and directlysequenced on both strands.

RT-PCR was performed with the Promega Accesssystem. The oligonucleotides used are: a, 50-CATATCTC-CCCTCTATAGTGC-30; b, 50-CTACATCAGAGAAATC-CACGC-30; and c, 50-CATTTGTAAGCGTTTGGCTTC-30.One microgram of total RNA prepared with the highpure RNA isolation kit (Boehringer Mannheim) wasdenatured at 94�C for two minutes in the presence ofeither primers a and c or primers b and c. Immediatelyafter, reverse transcription and PCR ampli®cation werecarried out according to the supplier's protocol.

Molecular identification

The 16 S rRNA gene sequence analysis of Shewanellamassilia was determined by direct sequencing of PCR-ampli®ed 16 S rDNA. DNA manipulation and PCRampli®cation were performed as previously described(Rainey et al., 1996). Puri®ed PCR products weresequenced and the resulting 16 S rRNA sequence datawere put into the alignment editor ae2 (Maidak et al.,1996), aligned manually and compared with representa-tive 16 S rRNA gene sequences of organisms belongingto the g-subdivision of Proteobacteria. For comparison,16 S rRNA sequences were obtained from the EMBLdata base or RDP (Ribosomal Database Project). The 16 SrRNA gene similarity values were calculated by pairwisecomparison of the sequences within the alignment. Forconstruction of the phylogenetic dendogram, operationsof the Phylogeny Inference Package (version 3.5.1) wereused. Pairwise evolutionary distances were computedfrom percent similarities by the correction of Jukes &Cantor (1969). Based on the evolutionary distance values,the phylogenetic tree was constructed by the neighbor-joining method (Saitou & Nei, 1987).

Accession numbers

The accession numbers of the S. massilia DNAsequences are AJ006084 for the 16 S rRNA gene andAJ006085 for the tor gene cluster.

Acknowledgements

We thank Cathrin SproÈer from D.S.M.Z. for assistancein the phylogenetic analysis and the Centre de SeÂquencË-age of the I.B.S.M. for assistance in the DNA sequencing.We are grateful to Corinne Appia-Ayme, ViolaineBonnefoy and Janine Pommier for helpful advice. We areindebted to Susan Wells for the critical reading of themanuscript. This work was supported by grants fromthe C.N.R.S. and the MinisteÁre de l'Agriculture et de la

PeÃche (R 96/02). J.P. D-S. was supported by a grant fromthe Conseil ReÂgional PACA (Provence-Alpes-CoÃ ted'Azur).

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Edited by R. Huber

(Received 19 May 1998; received in revised form 7 August 1998; accepted 7 August 1998)