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Morphological and intracellular alterations induced by cytotoxin VT2y produced by Escherichia coli isolated from chickens with swollen head syndrome.

Abstract Recently, a novel verocytotoxin named VT2y was described which belongs to the STx family and is produced by Escherichia coli isolated from domestic poultry with swollen head syndrome (SHS). The VT2y toxin induced apoptosis in Vero, HeLa, CHO, CEF (primary chicken embryo fibroblast) and PCK (primary chicken kidney) cell lines. Morphological evidence (nuclear shrinkage, chromatin condensation and blebbing of the plasma membrane) of apoptosis could be distinguished in 15 min and was maximal at 1 h after treatment with VT2y. This was confirmed by the terminal dUTP nick-end-labeling (TUNEL) method.

Introduction
Swollen head syndrome (SHS) in avian species was ¢rst described by Morley and Thomson [16] who attributed the disease to a mixed infection with Escherichia coli and coronavirus, paramyxovirus, or pneumovirus in which the initial viral infection caused acute rhinitis that permitted E. coli to invade the subcutaneous facial tissues [19]. This acute respiratory disease observed in domestic poultry is characterized by swelling of the peri-and infra-orbital sinuses, torcicollis, opisthotonus and lack of coordination [16,23]. Since the syndrome was described in South Africa, in 1984, there has been progressive spread to many other countries.
Verocytotoxin-producing E. coli (VTEC) are associated with infant diarrhea, hemorrhagic colitis and hemolyticû remic syndrome in humans, dysentery in calves, edema disease (ED) of pigs and, more recently, SHS in birds [6,18,20,26]. VTEC strains produce one or more VTs: VT1, and several variants of VT2. The VT2v is produced by E. coli isolated from pigs with ED [11]. Recently Par-reira and Yano [20] described a novel cytotoxin called VT2y and sequencing of a 1560-bp DNA fragment of VT2y showed that it contained the STx gene of Shigella dysenteriae, but the region 300 bp upstream of the start of the gene for the STxA subunit was identical to the corresponding sequence for E. coli O111:H-, except for 1 nucleotide, suggesting that VT2y is a member of the STx family [22].
The aim of this study is to illustrate the morphological and intracellular alterations induced in vitro by cytotoxin VT2y in Vero, HeLa, CHO, HEp-2, PCK and CEF cell lines.

Materials and methods
2.1. Bacterial strains E. coli SHS-8 strain isolated from chickens with SHS was used as a source of VT2y [21]. E. coli strain K12C600 was used as a negative control and strain J2 (O157 :H-VT2) was used as a source of VT2 (kindly donated by Yoshifumi Takeda, Japan) in these experiments. All E. coli strains were stored at 320³C in Luria^Bertani (LB) [14] broth to which 15% glycerol was added.

Puri¢cation of VT2y
The cytotoxin VT2y was puri¢ed [21], and protein content was assayed as described by Bradford [2].

Cytotoxicity assays
The cytopathic e¡ect (CPE) of puri¢ed VT2y was examined in Vero (African green monkey kidney), HeLa (human epitheloid cervical carcinoma), CHO (Chinese hamster ovary), HEp-2 (human larynx epidermoid carcinoma), PCK (primary chicken kidney) cell lines obtained from the Laboratory of Cell Culture, DMI, UNICAMP, Brazil; CEF (chicken embryo ¢broblast) cells were generously supplied by Fort Dodge Laboratories, Inc. Campinas, Brazil. All cell lines were maintained as described by Mac-Leod and Gyles [10]. The cells were grown at 37³C in Eagle's minimal essential medium (EMEM) with Earle's salts (Cultilab, Campinas, SP, Brazil), supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO, USA), penicillin (1000 U ml 31 ) and streptomycin (250 Wg ml 31 ). To determine cytotoxic activity [18], 100 Wl of medium and 2U10 5 cells per well were placed in 96-well plastic plates (Costar, Cambridge, MA, USA). After 24 h, culture medium was aspirated and 150 Wl of fresh EMEM without FBS, and 50 Wl (9 ng ml 31 ) of puri¢ed VT2y were added to each well. The plates were incubated at 37³C in a 5% CO 2 atmosphere for 24 h. Morphological changes were observed over the 24-h period and the test was scored as positive if more than 50% of the cells exhibited cytotoxic e¡ects. Control E. coli strains were J2 (O157:H-VT2) and K12C600.
The neutral red assay was performed in Vero, HeLa, CEF, PCK and CHO cells. Puri¢ed VT2y cytotoxin was applied to the cells and after 1 h, the medium was substituted with 0.2 ml of neutral red solution (50 Wg ml 31 ) per well and the plate incubated at 37³C in 5% CO 2 for 3 h, to allow for dye incorporation. Afterwards, the dye medium was removed and each well was rapidly washed with formol^calcium solution (10% formaldehyde and 10% calcium chloride) to remove unincorporated neutral red; 0.2 ml of solution containing 1 ml acetic acid with ethanol 50% was added to each well and the plate kept for 15 min at room temperature to extract the dye from the cells.
Controls consisted of toxin-treated and untreated cells but without addition of neutral red, (blank). Normal cells, without addition of cytotoxin, but with addition of neutral red, were utilized as controls of 100% with viable cells.
The color reaction was measured with an ELISA plate reader (Multiskan Bichromatic) at 540 nm.
The CD 50 , was de¢ned as the minimum amount of VT2y required to kill 50% of the cells (1 CD 50 ), and was determined from the toxin concentration resulting in 50% neutral red absorbance compared to control cells as 100%.

Determination of lactate dehydrogenase (LDH)
Leakage of the cytosolic enzyme LDH, was assessed [15] to investigate whether VT2y a¡ected the integrity of the plasma membrane of Vero cells. Medium from Vero cells treated with 3 CD 50 of VT2y or without VT2y was collected after 15, 30, 60, 90 min and immediately chilled on ice until measurement of LDH activity. A fraction of each sample was added to a cuvette containing sodium pyruvate in 1 ml freshly prepared potassium phosphate bu¡er (0.1 M, pH 7.4) and the mixture was equilibrated for 30 min at 37³C. The enzyme activity was determined spectrophotometrically and the rate of enzyme leakage min 31 was calculated and expressed as percentage of total LDH activity as compared to untreated control cells.

Morphological characterization of apoptosis induced by
VT2y in Vero, HeLa, CEF, PCK and CHO cells The Vero, HeLa, CEF, PCK and CHO cells were grown on coverslips in 24-well plates using 10 4 cells ml 31 per well in a volume of 1 ml. The plates were incubated at 37³C in 5% CO 2 . After 24 h, culture medium was aspirated and replaced with 1 ml of fresh medium. Then, 3 CD 50 VT2y was added to each well, and incubated for the same time intervals as in the LDH assay. After incubation, the coverslips were washed with PBS, ¢xed in 10% formaldehyde solution in PBS for 1 h, then washed with distilled water and stained with 0.025% toluidine blue in McIlvaine pH 4.0 bu¡er [13].
The coverslips were washed for 15 min with distilled water, air-dried, cleared in xylene and mounted using Entellan (Merck, Germany).

DNA fragmentation assay in Vero cells
DNA fragmentation in VT2y-treated and untreated Vero cells was investigated in situ by the terminal dUTP nick-end-labeling (TUNEL) method using an In situ cell death detection POD kit (Roche, Mannheim, Germany). Experiments were performed according to the manufacturer's protocol.

Cytotoxic activity
The VT2y was cytotoxic to Vero, HeLa, CEF, PCK and CHO cells, but not for HEp-2 cells. By light microscopy the cytotoxin-a¡ected cells appeared round and shrivelled, and the cells were dead within 1 h.

E¡ect of VT2y on cell viability
The neutral red assay showed that Vero cells were more sensitive than other cells tested since 50% cell death was observed in the presence of 0.1 ng ml 31 VT2y. A linear increase in the percentage of dead cells was found as protein concentration was increased within the range indicated in Fig. 1.

LDH leakage
After 15 min minimal (5%) LDH leakage was observed as compared to the control (Fig. 2), but by 60 min an increase to 46% above control LDH leakage had occurred.

Kinetic study of the morphological alteration in Vero cells induced by VT2y
The cytotoxin VT2y caused morphological alterations in Vero cells, such as cytoplasm vacuolization, blebbing of the plasma membrane, chromatin condensation within the nucleus, nuclear shrinkage and apoptotic bodies within 15 min. The e¡ects became more pronounced with time to a¡ect the majority of cells. By 1 h, 100% cell death was observed (data not shown).

Detection of apoptosis in cells after treatment with verotoxin (VT2y)
When cells were cultured in the presence of VT2y, they showed condensed chromatin (Fig. 3B), cleaved nuclei (Fig. 3C) and cytoplasmic blebbing (Fig. 3D), ¢ndings typical of apoptosis. We also observed the presence of cytoplasmic vacuoles in conjunction with apoptotic fea-tures (Fig. 3C). However, none of these features occurred in control cells (Fig. 3A).
The cleavage of nuclear DNA, a major feature of apoptosis, occurred during VT-induced death of Vero cells, as con¢rmed by in situ detection by the TUNEL method (Fig. 4B). The same Vero cells clearly incorporated biotinylated nucleotides, indicating the occurrence of DNA fragmentation induced by VT2y, in contrast with control cells (Fig. 4A).

Discussion
Programmed cell death, or apoptosis, is de¢ned by the cell's ultrastructural morphology [7] and is characterized by cell shrinkage, membrane blebbing, and condensation of nuclear chromatin. These morphological changes are accompanied by DNA fragmentation, and this form of cell death is a natural process by which an organism removes damaged or unnecessary cells. It may also be triggered by external stimuli [12]. Recently, reports have claimed that VTs induce programmed cell death, apoptosis, in cell lines such as Vero, MDCK [5,25] and others.
In our studies, we showed evidence of morphological alterations characteristic of apoptosis (Fig. 3) in three mammalian cell (Vero, HeLa and CHO) and two avian cell lines (PCK and CEF), but not in HEp-2 cells. The most sensitive line to VT2y was Vero cells (Fig. 1), probably due to receptor speci¢city of the cells, as it is known that variants of VT2 are more cytotoxic to Vero cells than HeLa cells [4]; the receptor for VT1 and VT2 is globotriosyl ceramide (GB3) [9], whereas the preferred receptor for VT2v is globotetraosyl ceramide (GB4) [24]. It will be interesting to study the receptor for this toxin on the avian cells PCK and CEF, since VT2y is produced by avian E. coli strains. We also observed the presence of multiple vacuoles in the cytoplasm of these cell lines (Fig. 3C), that  simultaneously showed apoptotic features induced by VT2y cytotoxin. The microscopic nuclear fragmentation and incorporation of nucleotides shown by the TUNEL method occurred following VT-treatment of Vero cells (Fig. 4). By performing the LDH assay we veri¢ed a similar time-response using 3 CD 50 VT2y; marked apoptosis e¡ects were also observed when cells were assessed using a toluidine blue assay. Based on these data, we suggest that the VT2y-induced cell death occurs by the apoptotic pathway after plasma membrane damage.
Interestingly morphological evidence of apoptosis was observed within 15 min and was maximal within 1 h of toxin administration to Vero cells, which is considerably more rapid than previously described for apoptosis induced by VT2 in epithelial cells [8,27].
ED in pigs is caused by the toxin variant VT2v produced by E. coli [11]. This disease is characterized by anorexia, edema of the eye-lids and neurological abnormalities, such as lack of coordination and paralysis [3] ; these characteristics, are similar in some respects to those shown by chickens with SHS [17]. Here we observed that VT2v induced apoptosis in Vero cells, but in contrast to VT2y the morphological alterations of apoptosis were detected 3 h after VT-treatment (data not shown). From this evidence, a histopathological study of the action of VT2y in vivo is necessary to determine whether apoptosis as a VT-mediated mechanism of cell damage is a major feature in the pathogenesis of SHS.


Array of hope for gene technology.

A Washington-based bioinformatics company is developing sophisticated DNA microarrays that should help researchers measure and analyze gene expression faster, more economically, and with greater precision than ever before possible. The FlexJet system, as the microarray product is known, uses inkjet technology to propel microscopic strands of DNA nucleotides onto slides, "printing" arrays of DNA molecules in a process not unlike the manner in which a printer deposits ink onto paper, forming distinct patterns of characters and images. Microarray technology may revolutionize the field of toxicogenomics by helping scientists target new drugs, discover gene function, determine biologic pathways, and better understand diseases such as cancer, cystic fibrosis, and cardiovascular disease at the molecular level.

Background
An organism's ability to perceive, and respond to the challenges arising in its environment depends critically on the specific nature and design of its sensory system. Alterations in the molecular components of the transduction cascade can have profound consequences for sensory perception, offering the opportunity to better understand the evolution of these molecular pathways in the context of the environments in which an animal may reside. Sensory systems are therefore ideally suited for evolutionary investigations at the molecular level. For example, it has been shown that fish living at increasing depths have adapted to the changing light environment by blue-shifting their visual sensitivities via specific amino acid substitutions in one of the visual pigments that form the first step in the sensory visual pathway [1].
Visual systems are particularly amenable to molecular evolutionary analysis not only because of the diversity of light environments and visually-mediated tasks in which vision may be required, but also because the details of the primary phototransduction cascade have been fairly well characterized. In vertebrates, the first step in the visual cascade are the visual pigments, membrane-spanning protein complexes located in the outer segments of the rod and cone photoreceptor cells of the retina [2]. They are composed of two parts, an opsin protein component, which is a member of the G-protein coupled receptor superfamily, and a vitamin A-derived chromophore that is covalently bound to the opsin via a Schiff-base linkage. Absorbing a photon of light causes the chromophore to isomerize, triggering a conformational change in the opsin protein that allows it to activate the second messenger transducin, a heterotrimeric G-protein, which ultimately leads to a neural signal that light has been perceived [3,4]. Different visual pigments can be most sensitive to photons of differing wavelengths. Spectral tuning, the process by which a visual pigment's spectral sensitivity is modified, is usually achieved through amino acid substitutions that alter the environment of the chromophore within the opsin binding pocket [5,6]. Visual pigments in vertebrates are composed of five families of opsin genes [7]: the SWS1, SWS2, RH2, and LWS cone opsins that function under brightlight, or photopic conditions, and the RH1 rod opsins, or rhodopsins, that allow for vision under dim-light, or scotopic, conditions. This dichotomy in the vertebrate visual system is reflected in the fact that cone and rod photoreceptors generally have distinct morphologies and physiological functions related to their divergent roles in day vs. night vision, with cones tending to be less photosensitive but having faster response kinetics than rods [8]. At least some of these differences are thought to be mediated by differing components of the visual transduction pathways contained within each cell type. In addition to cone visual pigments, cone-specific transducins [9], cGMP phosphodiesterases [10], cGMP-gated ion channels [11], and arrestins [12] have been found. While many of the specifics of the molecular influences on photoreceptor response kinetics remain unknown, it seems likely that some of the differences in response are due to differences in G-protein activation, as cone visual pigments are known to be less efficient in activating transducin relative to rod pigments [13].
The study of vision in fish has long attracted the attention of comparative and evolutionary biologists due to the varied and often extreme lighting conditions present in the underwater environment [14,15]. In recent years, it has been discovered that many fishes have experienced several additional rounds of opsin duplication and divergence relative to other vertebrate groups. For example, where the typical vertebrate might possess one member of each of the LWS, SWS1, SWS2, and RH2 cone opsin families, the zebrafish (Danio rerio) possesses two LWS, one SWS1, one SWS2, and four RH2 opsins [16], while the distantly related medaka (Oryzias latipes) possesses two LWS opsins, one SWS1 opsin, two SWS2 opsins, and three RH2 opsins [17]. Phylogenetic relationships suggest that these additional opsin duplication events occurred independently in the lineages leading to these two fishes [17], though this pattern may also reflect concerted evolution, resulting in homogenized gene sequences within species [18]. Though it is tempting to suggest that these increased opsin numbers reflect one of the whole genome duplication events thought to have occurred in the fish lineage [19], this does not appear to be the case; evidence from whole genome sequencing projects and the sequencing of opsins from genomic libraries suggests that the SWS2 and LWS opsins are consistently arranged in one tandem array, while the multiple RH2 opsins are arranged in another (zebrafish D. rerio [16]; medaka O. latipes [17], Lake Malawi cichlids [20], cavefish Astyanax fasciatus [21], and the pufferfish Tetraodon nigroviridins [19]). This pattern is consistent with shared opsin duplication events occurring early in these fishes' evolution, but through local duplication events, not genome duplications. In many cases, the duplicated opsins display numerous amino acid substitutions and substantial differences in spectral sensitivity (e.g., the RH2 opsins of zebrafish, whose spectral sensitivity maxima are located from 467 nm to 505 nm; [16]), which is indicative of functional divergence following the duplication events.
In this paper we describe our investigation of the LWS opsins of the guppy, Poecilia reticulata. The guppy has long served as a model system for researchers interested in natural and sexual selection, and is notable among fish in that it displays extremely high levels of variation for a number of traits, both behavioural and morphological [22,23]. Both male colour patterns [24] and female mate preferences for these colour patterns [25], for instance, vary dramatically among individuals within and among populations. Furthermore, microspectrophotometry (MSP) analysis of the guppy's photoreceptor cells revealed visual system variation; guppies appears to possess three photoreceptors maximally sensitive to different wavelengths of long-wavelength light, but the retina of individual fish may have different numbers and combinations of these three photoreceptors [26,27]. It was suggested that these three different photoreceptors reflect the presence of two spectrally-distinct LWS visual pigments, expressed either independently or in conjunction [27]. We characterized the LWS opsins of the guppy using degenerate primers to amplify LWS opsin fragments from a cDNA library constructed from a single individual. Surprisingly, our results indicate that guppy LWS opsins have experienced several recent rounds of duplication and divergence, and reveal the presence of at least six expressed LWS opsin sequences in a single individual.

Results and Discussion
We amplified and sequenced, from cDNA obtained from a single guppy individual, six LWS opsin sequences corresponding to a 390 bp fragment of the LWS opsin (designated as 'guppy LWS1-6'; Genbank accession numbers DQ865167-DQ865172). This fragment of the opsin corresponds to LWS exons 4 and 5, and codes for the portion spanning from the beginning of the second extracellular loop to the middle of the seventh transmembrane domain (Fig. 1). Variation within this portion of the gene is known to have a disproportionately large effect on visual pigment phenotype [6]. Most notably, this fragment contains transmembrane domains 5 and 6, which include many of the known LWS spectral tuning sites, including residues 277 and 285 that account for much of the difference between human red and green visual pigments [6]. This fragment also contains the third cytoplasmic loop, which is thought to strongly influence the interaction between the activated visual pigment and the downstream G-protein [3,4].
In order to eliminate errors due to enzyme misincorporations, template switching and other PCR artifacts, multiple clones from separate PCR reactions were sequenced, and an error rate of ~0.2% was allowed among the clones (see Methods for error rate calculations, and number of clones sequenced). One of the opsins, LWS5, which may be expressed at low levels, was only isolated once from cDNA, but several clones were isolated and sequenced from genomic DNA. The other five LWS opsin sequences obtained from cDNA were confirmed by designing primers specific to each LWS sequence, and performing amplifications from genomic DNA as well. These genomic amplification products were then sequenced to confirm they were the same as those isolated from cDNA.
Among the six opsin sequences found, there were a total of 59 variable nucleotide sites and 20 variable amino acid sites (Fig. 2). Percentage sequence differences among the guppy LWS opsin sequences ranged from 1.04% to 14.3 % (distances computed with the method of Hasegawa et al. (HKY) [28]; Additional file 1). All guppy sequences had functionally critical residues known to be conserved among vertebrate opsins, such as the highly conserved lysine to which the chromophore is covalently bound via a Schiff-base linkage (Lys312; all numbering according to human LWS opsin), and a cysteine residue (Cys207) involved in a disulphide linkage with a residue in the third transmembrane helix, Cys126 [3,4,6]. These observations, along with the fact that the sequences were obtained from mRNA expressed in the head, suggest that the guppy LWS opsins are functional.
We aligned the guppy LWS opsins with a sample of teleost fish LWS opsin sequences (Fig. 2), including the medaka (Oryzias latipes) [17], two cichids (Dimidiochromis compressiceps and Oreochromis niloticus) [20], two pufferfish (Tetraodon nigroviridis and Takifugu rubripes) [29], and a close relative of the guppy, the bluefin killifish (Lucania goodei) [30]. LWS opsins from the more distantly related ayu smelt (Plecoglossus altivelis) [31], Atlantic salmon (Salmo salar) [32], and zebrafish (Danio rerio) [16] were also included as outgroups. Figure 3 presents the results of phylogenetic analyses performed on this alignment. Though some nodes are weakly supported, the relationships among the ingroup acanthopterygian fish are consistent with current phylogenetic hypotheses for this group [33,34]. Interestingly, the LWS opsins of the guppy and the killifish, which is currently known to have at least two LWS opsins, display trans-specific evolution; that is, guppy LWS6 is most closely related to L. goodei LWSB while guppy LWS1-5 are most closely related to L. goodei LWSA (Figs. 3,4). This pattern suggests that a LWS opsin duplication event (either involving the divergence of alleles within a locus, or the establishment of separate loci) pre-dated the guppy-killifish split, and that four more duplication events have occurred more recently within the guppy lineage. Whether or not all of the different guppy LWS opsin sequences represent products of true gene duplication events, or instead divergence among alleles within a single gene locus, is not currently known, but the results suggest that the guppy has at least three and possibly as many as six LWS opsin loci.
Observing this many distinct LWS opsin sequences in a single individual guppy is a highly surprising result. Not only is this number larger than the estimate derived from MSP analysis of guppy LWS photoreceptor cells [27], but it is, to our knowledge, greater than has been found for any other fish studied to date. Whole genome sequencing or screening and mapping of genomic libraries has confirmed single LWS opsins in two species of pufferfish [29], and two LWS opsins in both the zebrafish [16] and medaka [17], while screening and Southern blotting of a genomic library suggests the presence of two (or possibly three) LWS loci in the cavefish (Astyanax fasciatus) [21]. In humans, the LWS opsins are located tandemly along the X-chromosome; this tandem array expands and contracts as a result of non-homologous recombination events [6], and individuals can have from one to six X-linked LWS opsins per X chromosome [35,36]. The genomic organization of guppy LWS opsins is not currently known, but if guppy LWS opsins are arranged in a tandem array then it is possible that similar mechanisms may have generated the larger number of guppy LWS opsins.
Evolutionary patterns of selective constraint were investigated in our data set using codon-based likelihood phylogenetic models, as implemented in PAML [37,38]. Estimating the ratio of non-synonymous to synonymous rates (dN/dS or ω) in a single parameter model (M0) on the tree shown in Fig. 3 revealed a low overall dN/dS ratio of 0.08737 (Table 1). This is indicative of fairly strong purifying selection across sites, a result typical of most studies of proteins with highly conserved functional roles [39]. Models that allow ω to vary across sites were implemented in order to test for sites that may be under positive selection [40][41][42]; however, none of these tests were able Schematic diagram of an LWS opsin, with each circle representing an amino acid residue Figure 1 Schematic diagram of an LWS opsin, with each circle representing an amino acid residue. The portion of the guppy opsins isolated in this study is shaded grey. Sites found to be variable among the guppy opsins that were targeted in Franke et al.'s mutagenesis study of transducin activation capacity [48] are green with diagonal hatching. Those shown to be involved in spectral tuning [6,[45][46][47] are marked in red with vertical hatching. All other variable sites in the guppy opsins are shaded black. All amino acid numbering follows the human LWS opsins. The cytoplasmic (CM) and extracellular (EM) membranes, the transmembrane domain (TD), and the three extracellular (E1-E3) and four cytoplasmic (C1-C4) loops are indicated. Transmembrane boundaries were inferred by analogy to those of the bovine rhodopsin crystal structure [65].
to detect evidence of positive selection in our data set. Models incorporating site classes under positive selection were not found to be significantly better than neutral models in likelihood ratio tests, including M1a/M2a, and M7/M8 comparisons (Table 1). Only the M0/M3 comparison was significant, though this is not a strict test of positive selection. For the M3 model, the third site class was found to have a greatly elevated dN/dS ratio, though even in this case it remains below 1 (ω 2 = 0.64).
Branch [43] and branch-sites [42,44] models were also implemented in order to test for changes in selective constraint along lineages leading to the more divergent sequences, LWS5 and LWS6 (Fig. 3). Both lineages were found to have a statistically significant, or nearly significant, increase in dN/dS ratio (over 5-fold in both cases; Table 1). These ratios were found to be even more elevated in branch-sites models (LWS5, ω 2 = 16.5; LWS6, ω 2 = 1.0), although the addition of this site class was not found to be statistically significant in either lineage ( Table  1).
The significant changes in selective constraint in some of the LWS opsin lineages led us to examine in further detail sites that may be important in shifting opsin function in our data set. Several LWS opsin spectral tuning sites have been identified through comparative sequence analysis and site-directed mutagenesis studies [6,[45][46][47]. Among the guppy LWS opsins identified in this study, variation exists at several sites known to be functionally important ( Fig. 4; Table 2). Most notable are the differences at sites 277 and 285; guppy LWS5 possesses the same residues at those sites as the human green-LWS opsin (phenylalanine and alanine, respectively), while guppy LWS1-4,6 possess the same residues as the human red-LWS opsin (tyrosine and phenylalanine). These two substitutions are known, through mutagenesis, to cause a relatively large spectral shift of approximately 15-25 nm, and are the primary substitutions responsible for the functional difference between human red and green LWS opsins [6,[45][46][47]. This strongly suggests that the LWS5 may shifted toward shorter wavelengths (i.e., more toward the green) relative to the other guppy LWS opsins isolated in this study. This shift in function may be the reason for the significantly elevated dN/dS ratio found in codon-based models allowing for an additional ω parameter along this branch (Table 1). Moreover, branch-sites models identified both residues 277 and 285 as possible targets of positive selection, though inclusion of this class of positively selected sites was not found to be statistically significant (Table 1).
Sites 230 and 233, minor spectral tuning sites, are also variable in some of the sequences in our data set (Fig. 4, Table 2). At site 230, guppy LWS6 possesses a threonine, while the rest possess an isoleucine; this mutation was found to alter spectral sensitivity by a few nm in human LWS opsins [45]. At site 233, guppy LWS3-5 possess a glycine, while the rest possess an alanine; mutagenesis studies have shown that substituting a serine for an alanine at this site does have a small effect on spectral sensitivity in Amino acid alignment of guppy LWS opsins with a sample of fish LWS opsins Phylogeny showing the relationship between the LWS opsins of the guppy (Poecilia reticulata) and other teleost fishes Figure 3 Phylogeny showing the relationship between the LWS opsins of the guppy (Poecilia reticulata) and other teleost fishes. This topology represents one of ten most parsimonious trees found (see Methods for details). Branch lengths were estimated for this topology using maximum likelihood methods under the HKY+G model. Bootstrap support values are indicated above each node for parsimony (upper left), neighbour-joining (upper right), maximum likelihood (lower left) and Bayesian analysis (lower right). Except for some of the basal nodes for which there was little support, particularly in the likelihood and Bayesian analyses, this tree was congruent among all the different types of analyses used.

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Cladogram showing the relationships among guppy (Poecilia reticulata) and bluefin killifish (Lucania goodei) LWS opsins, with sub-stitutions of likely functional importance mapped on the tree Figure 4 Cladogram showing the relationships among guppy (Poecilia reticulata) and bluefin killifish (Lucania goodei) LWS opsins, with substitutions of likely functional importance mapped on the tree. These substitutions were the result of most parsimonious ancestral reconstructions inferred using MacClade v4.0 [62]. The branch leading to guppy LWS5, marked with an X, and the branch leading to guppy LWS6 and L. goodei LWSB, marked with a Y, were analyzed in PAML using branch-specific and branch-site models.
human LWS opsin [45]. The effects of such substitutions on spectral sensitivity are small enough that they are often undetected by methods such as MSP, which could explain why MSP studies of guppy photoreceptor cells suggested a relatively low number of visual pigments (2) sensitive to long-wavelength light [26,27] relative to the higher number of LWS opsin sequences (6) found in this study. There are also additional substitutions observed among guppy LWS opsin sequences that have not yet been explored by mutagenesis methods which may influence spectral sensitivity as well (for example, site 234, which is immediately adjacent to the minor spectral tuning site 233). Such mutations will require experimental evalua-tion through site-directed mutagenesis and spectrophotometric analysis of expressed visual pigments [45].
Variation also exists among guppy LWS opsin sequences at sites that likely influence other, non-spectral properties of the visual pigment (Fig. 4, Table 2). The most notable differences occur within the third cytoplasmic loop, a portion of the visual pigment known to be critically important for binding and activating transducin following the absorption of a photon of light [48,49]. Guppy LWS6 differs from the rest at seven AA sites within this loop. The effect of several of these sites on transducin activation rates was experimentally tested in mutated bovine rho-   dopsin expressed in vitro [48] ( Table 2). The effect of most substitutions was to reduce transducin activation in the mutants relative to wildtype bovine rhodopsin. The physiochemical properties of residues at sites 247, 248, and 264, guppy LWS6 are more similar to the wildtype bovine rhodopsin, suggesting that guppy LWS6 may have an enhanced transducin activating ability as compared with other guppy LWS opsins. However, the residues possessed by guppy LWS6 at sites 256, 261, and 263 appear to be unique among vertebrate opsins, with all other vertebrate opsins being either invariant or possessing residues with fairly different physiochemical properties.
The resemblance of the residues in guppy LWS6 at key sites with rhodopsin, combined with the known importance of the third cytoplasmic loop in G-protein binding and activation [49,50], raise the interesting possibility that the guppy LWS6 opsin may have divergent function with regards to transducin activation relative to other LWS opsins. Many of these same substitutions in the third cytoplasmic loop are present in L. goodei LWSB, the killifish opsin most closely related to guppy LWS6, including the unique substitutions at site 256, 261, and 263 (Fig. 3, Table 2), suggesting that variation in transducin activating capacity is being selectively maintained. This is consistent with our codon-based analyses using branch-specific models, which revealed a nearly statistically significant, 5.4-fold increase in ω along the branch leading to guppy LWS6 and L. goodei LWSB (Fig. 4), even if the branch-site analysis of this lineage did not provide any evidence of positive selection (Table 1). Interestingly, Bayes Empirical Bayes [42] analysis did identify five residues (including sites with known influence on transducin activation [48]), all within the third cytoplasmic loop (residues 248, 249, 256, 261 and 263).
The evolutionary mechanisms that have promoted and maintained this diversity of LWS opsins in the guppy remain to be investigated. Guppies are known to have highly variable colour patterns and mate preferences [22]. The chroma of orange patches on male guppies varies among individuals, and it has been suggested that these differences may reflect differences in male foraging ability or parasite load [22]. By having multiple LWS opsins with slight variation in spectral sensitivity, female guppies may be able to better discriminate among differently coloured males [51,52] or the brightly-coloured fruits that occasionally fall into the guppy's streams [53]. Also, it has been observed in the cichlid Metriaclima zebra that a region of the cichlid genome that influences colour pattern is closely linked to the SWS2-LWS opsin tandem array [54]; selection acting to maintain colour pattern variation could potentially lead to the maintenance of variation in physically linked opsin genes. Both directional [22,25] and balancing [55,56] selection have been shown to oper-ate on variation in male colour patterns in the guppy, and could therefore function to influence visual variation if a similar linkage exists in the guppy. In future work, we plan to investigate the relationship between behavioural ecology and opsin molecular biology in the guppy by studying the influence of opsin genes on individual behaviour and fitness.

Conclusion
Our results demonstrated the presence of at least six expressed LWS opsin sequences within a single guppy individual. This number is substantially higher than the number found in any other fish to date, and higher than the minimum number previously estimated for the guppy by MSP. Phylogenetic analysis reveals that these guppy LWS opsins fall into two groups, one group of relatively closely related sequences, and another that appears to have descended from a duplication event pre-dating the guppy-killifish split. Many of the amino acid substitutions that distinguish these guppy LWS opsins appear at sites thought to be functionally important, either for spectral tuning or transducin activation. This is consistent with codon-based analyses, which indicate changes in selective constraint along two of the more divergent lineages of guppy LWS opsins.

Methods
Total RNA was extracted from the head of a single male guppy sampled from a tributary of the Paria River, Trinidad. The fish was anesthetized in MS-222 solution, its head dissected and ground on dry ice, and the RNA isolated using an RNeasy Mini Kit (Qiagen . In order to confirm the existence of the different LWS opsin sequences found in cDNA, portions of the opsin that distinguished the guppy LWS sequences from each other were also amplified from genomic DNA using specific primers, and sequenced. For the LWS5 opsin, although only one cDNA clone was isolated, multiple clones (7) were sequenced from genomic DNA amplifications.
Vertebrate LWS opsin sequence alignment was carried out via ClustalW [57]. Amino acid sequences were aligned, and from this the nucleotide alignment was inferred. Preliminary rounds of phylogenetic analysis (Neighbourjoining method), carried out separately on the upstream and downstream halves, revealed a small number of clones as likely instances of recombination among clones during amplification; these clones were excluded from further analysis. The low amount of statistical power afforded by the small size of the dataset precluded the use of more refined methods for detecting instances of template switching. Several different methods of phylogenetic analyses were performed on the alignment shown in Fig Hierarchical likelihood ratio tests, implemented in Modeltest 3.6 [58], identified the HKY+G model as the best fitting model. Neighbour-joining, maximum parsimony, and maximum likelihood analyses were carried out in PAUP* 4.0b10 [59]. Parsimony heuristic searches were implemented with 1000 random addition replicates. Node support was assessed using bootstrap analysis [60], as implemented in PAUP* 4.0b10 [59], for the neighborjoining (1000 replicates, HKY+G model), parsimony (1000 replicates), and maximum likelihood analyses (100 replicates, HKY+G model). Bayesian analyses were carried out in Mr. Bayes 3.1.1 [61]. Four simultaneous chains were run for 10 7 generations, with tree sampling every 100 generations (for a total of 100000 trees). The first 5000 trees were considered 'burn-in' and discarded. Most parsimonious ancestral reconstructions, and mapping of amino acid substitutions onto the phylogeny were carried out using MacClade v4.0 [62].
Evolutionary patterns of selective constraint were assessed using codon-based likelihood analyses as implemented in PAML v3.15 [38], using the aligned nucleotide dataset and the tree topology shown in Fig. 3. PAML uses a maximum likelihood framework to estimate dN/dS (ω) for the entire dataset and tree (M0 model), for particular lineages (branch models), for particular codons (sites models), or for particular codons within particular lineages (branch-site models) (reviewed in [37]; also see [40][41][42][43][44] for specificities on the various models employed). The ω value is thought to reflect the degree of evolutionary constraint, or selection pressure operating on the site class and/or lineage of interest, with 0 < ω < 1 indicating purifying selection, ω = 1 neutral evolution, and ω > 1 positive selection [63]. Nested models are compared via likelihood ratio tests, with the test statistic following a χ 2 distribution with degrees of freedom equal to the difference in the number of parameters between the two models [60,64]. Likelihood ratio tests were carried out using the program chi2, which is bundled with the PAML software package [37].
Bayes Empirical Bayes analysis, as implemented in PAML, was used to identify particular residues within the dataset as likely targets of positive selection [42].

List of abbreviations
LWS = long-wavelength sensitive, SWS1 = type 1 shortwavelength sensitive, SWS2 = type 2 short-wavelength sensitive, RH1 = rod, RH2 = rod-like (or medium-wavelength sensitive), MSP = microspectrophotometry ing, Jo Wolfe for assistance in data management, Scott Salsbury for help with implementing the PAML software on the workstation, and David Guttman for his suggestions and comments on the manuscript. This study was funded by an NSERC Grant (BSWC), and an NSERC Post Graduate Scholarship (CJW).
This article has been published as part of BMC Evolutionary Biology Volume 7, Supplement 1, 2007: First International Conference on Phylogenomics. The full contents of the supplement are available online at http:// www.biomedcentral.com/bmcevolbiol/7?issue=S1.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Chemistry

text:
Electron Transfer and Binding of the c-Type Cytochrome TorC to the Trimethylamine N-Oxide Reductase in Escherichia coli *

Reduction of trimethylamine N-oxide (E′0(TMAO/TMA) = +130 mV) in Escherichia coli is carried out by the Tor system, an electron transfer chain encoded by the torCAD operon and made up of the periplasmic terminal reductase TorA and the membrane-anchored pentahemicc-type cytochrome TorC. Although the role of TorA in the reduction of trimethylamine N-oxide (TMAO) has been clearly established, no direct evidence for TorC involvement has been presented. TorC belongs to the NirT/NapC c-type cytochrome family based on homologies of its N-terminal tetrahemic domain (TorCN) to the cytochromes of this family, but TorC contains a C-terminal extension (TorCC) with an additional heme-binding site. In this study, we show that both domains are required for the anaerobic bacterial growth with TMAO. The intact TorC protein and its two domains, TorCN and TorCC, were produced independently and purified for a biochemical characterization. The reduced form of TorC exhibited visible absorption maxima at 552, 523, and 417 nm. Mediated redox potentiometry of the heme centers of the purified components identified two negative midpoint potentials (−177 and −98 mV) localized in the tetrahemic TorCN and one positive midpoint potential (+120 mV) in the monohemic TorCC. In agreement with these values, thein vitro reconstitution of electron transfer between TorC, TorCN, or TorCC and TorA showed that only TorC and TorCC were capable of electron transfer to TorA. Surprisingly, interaction studies revealed that only TorC and TorCN strongly bind TorA. Therefore, TorCCdirectly transfers electrons to TorA, whereas TorCN, which probably receives electrons from the menaquinone pool, is involved in both the electron transfer to TorCC and the binding to TorA.

expressed at a low level (6). Each of these systems comprises a periplasmic terminal reductase and a membrane-anchored ctype cytochrome. The membranous Me 2 SO reductase, which is constitutively expressed in anaerobiosis, is also capable of TMAO reduction (7).
The TorCAD system is encoded by the torCAD operon, and its expression is under the control of the TorS/TorR two-component regulatory system, which mediates the response to the presence of TMAO in the medium (8). The torA gene encodes the periplasmic molybdoreductase TorA (5). According to sequence homologies, TorA belongs to the Me 2 SO reductase family, a group of periplasmic enzymes that contain a molybdenum cofactor as a single prosthetic group and are capable of utilizing TMAO and/or Me 2 SO as electron acceptors (9). In the past few years, structures have been published for several members of this family (10 -13). They are organized in four domains surrounding a bismolybdopterin guanine dinucleotide cofactor that is consequently buried in a deep depression of the protein surface. The insertion of the molybdenum cofactor into the TorA apoprotein is a cytoplasmic event (14) that probably involves the TorD cytoplasmic chaperone (15).
It was previously shown that the first gene of the tor operon, torC, encodes protein TorC, a 46-kDa pentahemic c-type cytochrome (5,16). TorC is anchored to the inner membrane by a sequence of about 20 hydrophobic residues, while a globular domain containing five hemes faces the periplasm. Heme binding to the five consensus CXXCH motifs takes place in the periplasm by a mechanism involving the c-type cytochrome maturation machinery encoded by the ccm genes (17,18). From sequence comparisons, TorC has been included in the NirT/ NapC class of membrane-anchored multiheme c-type cytochromes. This family originally was composed of tetrahemic c-type cytochromes of about 20 kDa that were involved in periplasmic nitrite and nitrate reduction (19). The N-terminal region of TorC, which is homologous to NirT and NapC, possesses the four heme binding sites, whereas the C-terminal part, which is present specifically in the Me 2 SO/TMAO respiratory systems, carries the fifth heme motif (5). Recently, two members of the NirT/NapC family, NapC and the TorC homologue DorC, have been characterized, and the redox potential values determined for the four hemes of NapC and the five hemes of DorC were all negative (20,21).
In TMAO and Me 2 SO reduction systems, pentahemic cytochromes proved to be essential for the electron transfer and thus appeared to be the intermediate components between the membrane quinone pool and the terminal reductase (6,22). Since it has been shown in E. coli that TMAO reduction involves menaquinones (23), TorC should receive the electrons from the menaquinone pool (EЈ 0(menaquinol/menaquinone) ϭ Ϫ74 mV) and transfer them to the active site of TorA, where TMAO reduction (EЈ 0(TMAO/TMA) ϭ ϩ130 mV) occurs.
Interestingly, a recent genetic study showed that TorC in its unprocessed form is a negative regulator of the torCAD operon (18). Indeed, apo-TorC seems to inhibit the kinase activity of the TMAO sensor TorS by an unknown mechanism. This negative autoregulation probably means that the maturation of TorC is the limiting step for the Tor system biogenesis.
This paper provides in vivo and in vitro evidence that the mature form of TorC is directly involved in the electron transfer to the terminal reductase, TorA. We also show that TorC exhibits four negative redox potentials and a positive one corresponding to the fifth heme. The latter group is responsible for the direct electron transfer to the catalytic site of TorA. In addition, TorC binds TorA, and this interaction involves the N-terminal domain of TorC.

Bacterial Strains, Plasmids, Media, and Growth Conditions
The bacterial strains and plasmids used in this work are listed in Table I. To maintain selection for plasmids, antibiotics were added at the following concentrations: ampicillin, 100 g/ml; chloramphenicol, 10 g/ml. For the biochemical study, cells were grown anaerobically at 37°C on Luria broth medium (24). The concentration of arabinose or glucose, added to the growth medium, is detailed for each experiment under "Results." The growth kinetics studies were performed as described by Gon et al. (6).

Constructions Leading to TorC, TorC N , and TorC C Production
To synthesize the membrane His tag TorC, the complete torC coding sequence was amplified from MC4100 chromosomal DNA by polymerase chain reaction, using an oligonucleotide (C5Hc) that corresponds to an EcoRI site followed by the 5Ј coding sequence of torC and a primer (Cct) that corresponds to a SmaI site followed by a sequence encoding a C-terminal His 6 tag and the complementary sequence of the 3Ј-end of torC (Fig. 1A).
To synthesize the N-terminal domain of TorC (TorC N , positions 1-194 relative to TorC) that includes the first four heme binding sites of TorC (Fig. 1B), the corresponding coding sequence was amplified by polymerase chain reaction using oligonucleotide C5Hc and a primer that corresponds to a SmaI site followed by a His 6 tag coding sequence and the appropriate torC internal sequence.
To synthesize the periplasmic C-terminal domain of TorC (TorC C , from position 198 to 390) that contains the fifth heme of TorC (Fig. 1C), the corresponding torC coding sequence was amplified by polymerase chain reaction using an oligonucleotide that contains an EcoRI site followed by the TorT signal peptide (18 codons) coding sequence (28) and the appropriate torC sequence and primer Cct. Detailed information on the primer sequences is available on request.
The purified polymerase chain reaction products were digested by the appropriate restriction enzymes and ligated into the corresponding cloning sites of the expression vector pBAD24 (27) downstream from the arabinose-inducible P BAD promoter to give pBC, pBC N , and pBC C ( Table  I). All of the polymerase chain reaction products and the fusion sites were confirmed by sequencing. DNA preparations were carried out with high pure DNA isolation kits from Roche Diagnostics. Transformations were performed according to the method of Chung and Miller (29). Plasmids pBC, pBC N , and pBC C were digested with NheI and HindIII, and the resulting DNA fragments encoding the modified His tag TorC proteins were cloned just downstream of torD, into the compatible sites XbaI and HindIII of plasmid pTorAD (15), leading to plasmids pTorADC, pTorADC N , and pTorADC C , respectively.

Purification of TorA, TorC, TorC N , and TorC C
The purification of the TMAO reductase (TorA) was performed from the periplasmic fraction of cells DSS401/pEC86. TorA protein enrichment was achieved as follows. The periplasmic fraction of strain DSS401/pEC86, grown anaerobically in the presence of TMAO (30 mM), was prepared by the lysozyme-EDTA procedure (30) and precipitated with ammonium sulfate 60% (w/v). The pellet was resuspended into Tris-HCl 40 mM buffer (pH 7.6) and then loaded on a Mono Q HR 5/5. The TMAO reductase active fractions, obtained from a 50 -500 mM NaCl gradient were then dialyzed, pooled, concentrated by 60% (w/v) ammonium sulfate precipitation, and loaded on a Superdex G200 HR 10/30 column. At this stage, the active fractions contained 3 mg of protein, and the purity of the preparation was checked on Coomassie Bluestained gel.
Purification of TorC and TorC N was performed from the membrane fraction of cells MC4100, containing both plasmids pEC86 and either pBC or pBC N , grown anaerobically in the presence of 0.0005% arabinose. Membrane fraction, prepared as described by Silvestro et al. (4), was incubated for 1 h at 4°C in 20 mM phosphate buffer (pH 7.4) with 500 mM NaCl containing 2% Triton X-100. After centrifugation at 14,000 rpm for 30 min at 4°C, the fraction containing the solubilized cytochrome c, TorC or TorC N , was diluted two times with the same buffer containing 0.2% Triton X-100 and 2 mM imidazole and loaded onto a HiTrap TM chelating Ni 2ϩ column (Amersham Pharmacia Biotech). The proteins were eluted with a step gradient of imidazole from 20 to 500 mM. Both TorC and TorC N cytochromes were eluted with 150 mM imidazole.
TorC C , the soluble C-terminal domain of TorC, was purified from the periplasmic fraction of cells MC4100 containing both pEC86 and pBC C grown under anaerobic conditions in the presence of 0.1% arabinose. The periplasm was diluted two times into 20 mM phosphate buffer (pH 7.4) with 500 mM NaCl, 2 mM imidazole, and the purification onto a HiTrap TM chelating Ni 2ϩ column was performed as described for TorC and TorC N .

Analytical Procedure
Protein analysis was carried out using 12.5 or 10% SDS-PAGE. After electrophoresis, the presence of hemes within TorC and derivatives was revealed by staining the gel for peroxidase activity using 3,3Ј,5,5Јtetramethylbenzidine as described by Thomas et al. (31); then Coomassie Blue staining of total proteins was performed (32). Protein concentrations were estimated using the technique of Lowry et al. (33).

Kinetics of Electron Transfer
The kinetic assays were achieved using anaerobic cuvettes, typically filled with phosphate buffer (20 mM, pH 7.4), TMAO (50 mM), and either TorC C (10 M), TorC N (5 M), or TorC (5 M) previously reduced by dithionite. Before adding TMAO reductase (350 units⅐ml Ϫ1 ), the cuvette mixture was flushed with argon (10 min). The oxidation rate of the cytochrome was determined from the absorption band at 552 nm.

Interaction Experiments
Interaction Studies on PAGE under Native Conditions-TorA (5 M) and either TorC (5 M), TorC N (5 M), or TorC C (10 M) were incubated for 30 min at room temperature in 20 mM phosphate buffer (pH 7.4), Triton X-100 0.05%. The interactions were analyzed by 10 or 12.5% PAGE under native conditions. For identification, the proteins were either stained for heme with 3,3Ј,5,5Ј-tetramethylbenzidine or were transferred to a Hybond TM ECL TM nitrocellulose membrane. The protein complexes were immunodetected with anti-His antibodies (Invitrogen). The ECL-Western blotting system was used as recommended by the supplier (Amersham Pharmacia Biotech).
Interaction Studies by Biosensor Experiments-The BIAcore apparatus (Amersham Pharmacia Biotech) was used to analyze, in real time, the binding between TorA (M r 94,000) and TorC (M r 46,000), TorC C (M r 23,000), or TorC N (M r 24,500), by using surface plasmon resonance. All experiments were carried out at 25°C. TorA, in 10 mM sodium acetate buffer (pH 5), was immobilized on a CM5 sensor chip (BIAcore) through amine coupling (3000 resonance units). TorC, TorC N , and TorC C were diluted in 20 mM phosphate buffer (pH 7.4), 0.05% Triton X-100 and injected using a constant flow rate of 10 l/min. The resulting sensorgrams were evaluated using the biomolecular interaction analysis evaluation software (BIAcore) to calculate the kinetic constants of the complex formation.

RESULTS
The Intact TorC Protein Is Required for TMAO Respiration-To establish that the c-type cytochrome TorC is required for electron transfer to the terminal enzyme TorA, we cloned the torAD genes alone (15) or together with torC under the control of the P TAC promoter of the plasmid pJF119. The resulting pTorAD and pTorADC plasmids were introduced into strain LCB504, which is unable to reduce TMAO. Indeed, this strain carries an interposon in the beginning of the first gene of the torCAD operon and a deletion of the entire dmsABC operon (6). The strains carrying either pJF119 or pTorAD grew very slowly and at the same rate under anaerobic conditions in the presence of TMAO (Fig. 2). In contrast, when plasmid pTorADC was introduced into strain LCB504, the recombinant strain exhibited a significant growth rate in the presence of TMAO (Fig. 2) and produced the characteristic odor of volatile TMA, which results from reduction of TMAO. These results confirmed the hypothesis that TorC is essential for TMAO reduction.
The TorC protein belongs to the NapC/NirT family of c-type cytochrome because its tetrahemic N-terminal domain (hereafter called TorC N ; Fig. 1B) is homologous to the NapC protein and contains four heme-binding sites. In addition, TorC contains a C-terminal domain (hereafter called TorC C ; Fig. 1C) with a single heme-binding motif. The C-terminal extension is found exclusively in TorC/DorC systems (35). To determine whether one or both of the two domains are required for the TMAO reduction process, we cloned DNA fragments encoding either one of these domains behind torAD. The resulting plas-mids pTorADC N and pTorADC C were introduced into strain LCB504, and the growth rate was monitored. The recombinant strains containing pTorADC N did not grow significantly in the presence of TMAO (Fig. 2), and the cells containing pTorADC C grew very slowly only after 36 h of incubation. These results strongly suggest that both domains of the TorC protein are required for the TMAO respiratory process.
Overproduction and Purification of TorC and of Its N-and C-terminal Domains-To study the biochemical characteristics of the TorC protein, we constructed recombinant plasmids that encode either TorC; TorC N , which corresponds to the N-terminal domain of TorC; or TorC C , which corresponds to the second half of the TorC protein (Fig. 1). DNA fragments encoding TorC, TorC N , and TorC C were cloned into the pBAD24 vector, giving rise to plasmids pBC, pBC N , and pBC C with His tags added at the C-terminal extremity of each protein. Moreover, since the heme incorporation occurs in the periplasm, we introduced the TorT signal peptide in front of TorC C to assure its periplasmic localization.
Overproduction of mature c-type cytochrome, and especially multiheme c-type cytochromes, is difficult in E. coli, due to the limited capacity for synthesis of the c-type cytochrome of this organism. To overcome this problem, we used a strain containing the plasmid pEC86 that carries the c-type cytochrome maturation ccm genes and thus improves production of c-type cytochromes (17). Nevertheless, the amount of mature cytochrome produced is rather low even in the presence of this plasmid, and the expression of cytochrome-encoding genes has to be controlled to prevent saturation of the maturation machinery. Accordingly, the TorC genes were cloned into the pBAD24 vector under the control of the P BAD promoter for controlled expression of TorC proteins. Plasmids pBC, pBC N , and pBC C were then introduced into strain MC4100 carrying the compatible plasmid pEC86. TorC and TorC N expression was induced with 0.0005% arabinose because concentrations higher than 0.001% apparently overloaded the heme delivery machinery and led to degraded proteins. Higher levels of the mature TorC C protein were obtained by using 0.1% arabinose, probably because TorC C is a monoheme protein. Heme-staining proteins of 47 and 24 kDa were present in membrane extracts of the strains containing pBC and pBC N , respectively, indicating that TorC and TorC N were matured (data not shown). Since these proteins were exclusively located in the membrane fraction (data not shown), detergent Triton X-100 was used to solubilize them. A 23-kDa heme-staining protein was detected in the periplasmic extract of the strain containing pBC C (data not shown). The detergent-solubilized fractions containing TorC or TorC N or the periplasmic fraction containing TorC C were loaded onto Ni 2ϩ columns, and the His-tagged proteins were eluted from the column at about 150 mM imidazole. Analysis by SDS-PAGE revealed the presence of TorC, TorC N , and TorC C as a single major band on gels stained for heme-dependent peroxidase activity (Fig. 3A). No other c-type cytochrome copurified with the proteins and no apparent degradation occurred. The same gels stained with Coomassie Blue (Fig. 3B) revealed one major band; moreover, this band cross-reacted with anti-His antibodies in Western blots (data not shown), confirming that His-tagged derivatives of TorC had been purified.

Characterization of TorC by Optical Spectroscopy Shows That TorC Exhibited Positive and Negative Heme Redox Potentials-
The UV-visible spectrum of purified TorC shows a Soret peak at 411 nm in the oxidized state and peaks at 417 (Soret), 523, and 552 nm in the dithionite reduced form (Fig. 4A). These peaks are characteristic of c-type cytochromes and confirm that TorC is a c-type cytochrome as predicted by the presence of c-type heme binding sites (CXXCH) in the amino acid sequence. TorC was totally reduced by dithionite, but it was only partially reduced by ascorbate (Fig. 4A). These results indicate that at least one of the TorC hemes exhibits a positive redox potential, whereas the remaining hemes display more negative redox potentials. Similar UV-visible spectra were obtained with membrane extracts from strain DSS401 containing native TorC after reduction with ascorbate or dithionite (Fig. 4B), indicating that the purified His tag TorC cytochrome has spectral properties similar to those of the unmodified native TorC protein.
To determine which domain of TorC contains a positive redox potential center, we carried out UV-visible spectra with the purified TorC N and TorC C domains (Fig. 4, C and D). Fig. 4C shows that the hemes of TorC N are totally reduced by dithionite but not by ascorbate. In contrast, Fig. 4D shows that the unique heme of TorC C is totally reduced by both ascorbate and dithionite. These findings strongly suggest that the fifth heme of TorC has a positive potential, whereas the first four hemes exhibit negative potentials.
Determination of the Redox Potentials of the TorC Protein-To define more precisely the heme potentials, a mediated redox potentiometry was carried out on the purified TorC protein. This analysis revealed three distinct redox type centers, one at Ϫ177 mV (two hemes), one at Ϫ98 mV (two hemes), and one at ϩ114 mV (one heme) (Table II). These potentials are in agreement with the previous spectral evidence for one positive heme potential in the TorC C domain and four negative heme potentials in the TorC N domain. Mediated redox potentiometry of TorC C (Table II) yielded a value of ϩ120 mV, in complete agreement with the spectral evidence for a positive heme in the TorC C domain of TorC (Fig. 4D) and with the heme potential values obtained with the intact TorC protein.
Transfer of Electrons from TorC to the TMAO Reductase TorA-In the model of the Tor electron transfer pathway, TorC transfers electrons from the menaquinones, which are embedded in the cytoplasmic membrane, to the periplasmic TMAO reductase TorA. To demonstrate directly that TorC can act as an electron shuttle from the inner membrane to TorA, we studied electron transfer between purified TorC and TorA. TorC was previously reduced by dithionite under anaerobic conditions (Fig. 4A). Oxidation of TorC, followed at 552 nm ( Fig. 5) was specifically dependent upon TMAO reductase, since no oxidation occurred before the addition of TorA (data not shown).
Based on the redox potentials of TorC N (Ϫ177 and Ϫ98 mV) and TorC C (ϩ120 mV) and on EЈ 0(TMAO/TMA) (ϩ130 mV), we hypothesized that electrons are transferred from TorC N to TorC C and from TorC C to TorA. In this model, TorC C is the direct electron donor to TorA. Purified TorC N and TorC C were tested as electron donors for TMAO reductase. The monoheme TorC C domain was oxidized by the addition of TorA, whereas the tetrahemic TorC N domain was not (Fig. 5). Although oxidation of TorC C was slower than that of TorC, these results indicate that the electron donor for TorA is most probably located in the C-terminal part of TorC. When a mixture of purified TorC N and TorC C was tested, the electron transfer was slightly higher than that of TorC C alone but slower than that of TorC. These results indicate that TorC N also plays a role in the electron transfer pathway.
Binding of TorC to TorA Mainly Involves the TorC N-terminal Domain-The above results with purified components imply a direct interaction between these proteins. To characterize the binding between TorC and TorA, the formation of a TorC-TorA complex was studied under nondenaturing conditions by native PAGE and by BIAcore experiments. TorA and TorC were mixed together and loaded in the same conditions onto two polyacrylamide gels. After electrophoresis under native conditions, one gel was stained for heme (Fig. 6A, lanes 1 and  2), and a Western blot with anti-His antibodies was performed with the second gel (Fig. 6A, lanes 3 and 4). Significantly, the migration of TorC was retarded in the presence of TorA (Fig.  6A, compare lanes 1 and 3 with lanes 2 and 4, respectively). This result suggested that TorC can bind to TorA. To confirm this result, we studied the interaction between TorA and TorC by using the BIAcore technique (surface plasmon resonance). For this purpose, purified TorA protein was coupled to the dextran matrix of a sensor chip, and TorC was injected into the TorA-containing sensor chip. The sensorgram reflected the association and dissociation of two proteins, indicating that TorC directly interacts with TorA (Fig. 7A) in agreement with the above result.
The interaction of the individual domains with TorA was also studied by the same two approaches. Surprisingly, no binding between TorC C and TorA was detected on a native gel stained for heme or by Western blot with anti-His antibodies. Indeed, whatever the conditions used, TorC C migrated at exactly the same position in the presence and in the absence of TorA (Fig.  6B, compare lanes 1 and 3 with lanes 2 and 4, respectively). Similarly, no binding between TorC C and TorA was detected by the BIAcore procedure (Fig. 7B). As a control, we coupled TorC C to the dextran matrix and injected TorA, and again, no formation of a complex corresponding to the association of TorC C and TorA was detected (data not shown). When TorC N and TorA were mixed and loaded onto a polyacrylamide gel under nondenaturing conditions, TorC N migration was retarded by the presence of TorA (Fig. 6C), suggesting that the N-terminal domain of TorC is sufficient for TorA binding. The fact that TorC N can bind to TorA was confirmed by using the BIAcore technique. Indeed, an increase in the amount of recovered resonance units was observed when the TorC N protein was   injected (Fig. 7C). These results clearly show that the TorC N domain interacts with TorA.
The association rate constants (k on ) and the dissociation rate constant (k off ) of TorC and TorC N to TorA were determined at four different concentrations between 2.5 and 10 M (data not shown). TorC N exhibited a K d value of 4.5⅐10 Ϫ8 M (Table III). Significantly, the sensorgram of intact TorC binding to TorA exhibits at least two steps leading to the determination of two constants, K d1 ϭ 1.7⅐10 Ϫ8 M and K d2 ϭ 3.0⅐10 Ϫ6 M (Table III). These two steps could correspond to the fixation of TorC N and TorC C , but TorC N is mainly responsible for the binding of intact TorC to TorA because TorC C alone does not bind TorA. The low affinity between TorC C and TorA could also explain the poor catalytic efficiency in electron transfer to TorA exhibited by TorC C compared with that of the intact TorC protein (Fig. 5). DISCUSSION This study has revealed that TorC is made up of two domains of similar size, corresponding to its N-and C-terminal half (TorC N and TorC C , respectively) ( Figs. 1 and 3). As the other members of the NirT/NapC family with which it shares sequence homologies, TorC N contains a membrane anchor segment followed by four heme-binding sites (5,20). Production of the isolated TorC N domain resulted in a stable mature protein.
Unfortunately, and in contrast to what was described previously for NapC (20), the removal of the membrane anchor of TorC and TorC N led to an unstable protein (data not shown). The second domain, TorC C contains the fifth heme-binding motif of TorC (Fig. 1C) and presents sequence homologies with the corresponding region of pentahemic cytochromes specifically involved in TMAO/Me 2 SO respiration of various bacteria (6,35). Although the fifth heme binding site is located at the C-terminal extremity (positions 329 -333 relative to TorC), we were unable to produce a stable protein starting from position 285 of TorC, i.e. just upstream from the fifth heme binding site (data not shown). The region located between the fourth and the fifth heme binding sites probably plays an essential role in the stability and/or the folding of the TorC C domain.
The comparison between the midpoint redox potential values obtained for the soluble form of the Paracoccus denitrificans NapC (n ϭ 1, Ϫ235, Ϫ207, Ϫ181, and Ϫ56 mV) (20) and TorC N , the N-terminal domain of TorC (n ϭ 2, Ϫ177 and Ϫ98 mV) ( Table II), highlights that they are all negative and in the same range. This result confirms that NapC and TorC N are highly related. It is also striking that, for TorC N , each of the midpoint redox potentials corresponds to two heme centers. This feature could be representative of a symmetry in the N-terminal domain of TorC. This hypothesis agrees with the previous proposal of Roldan et al. (20), that the tetrahemic domain of NirT/NapC c-type cytochromes comes from a gene duplication leading to two related diheme subdomains. If true, then the redox potential of the first and third hemes of TorC or that of the second and the fourth ones should be very similar.
The midpoint redox potential described by Shaw et al. (21) for purified DorC, a TorC pentahemic homologue, is in the same range of values (Ϫ276, Ϫ185, Ϫ184, Ϫ128 mV, and Ϫ34 mV) as those detailed above for NapC. Surprisingly, all the potentials exhibited by DorC are negative, while a positive one has been detected for TorC. Indeed, we have clearly shown that the redox potential of the fifth heme is about ϩ120 mV, and the presence of a positive heme center has been observed in both the isolated TorC C domain and the intact TorC protein (Table  II). Moreover, this result has been confirmed by a study performed directly on native TorC using membrane extracts (Fig.  4B). These results are also in agreement with the fact that ascorbate reduced partially TorC and TorC C completely (Fig.  4). Positive midpoint redox potentials are often associated to His-Met-ligated hemes (36). The comparison of the sequences surrounding the fifth heme-binding site of the pentahemic cytochrome family reveals the presence of one highly conserved residue of methionine (position 353 in the TorC amino acid sequence) (35). This residue is a good candidate for the axial ligand, although a conserved histidine residue (position 340) might also play such a role. As shown by the in vitro electron transfer experiments, the FIG. 8. Model of binding and electron transfer between TorC and TorA. In our model, in a first step, TorC N , the N-terminal membrane-anchored domain of TorC, binds the periplasmic TorA protein (1). Then TorC C is correctly positioned to transfer electrons directly to the catalytic site of TorA, allowing the reduction of TMAO in TMA (2). The proposed electron transfer pathway is thus from menaquinones to TorC N , then from TorC N to TorC C , and finally to TorA.  TorC C domain donates the electrons directly to TorA (Fig. 5). This finding fits well with the positive midpoint redox potential exhibited by the TorC C heme (EЈ 0 ϭ ϩ120 mV) and that of the reduction reaction of TMAO (EЈ 0(TMAO/TMA) ϭ ϩ 130 mV). The rate of electron transfer is slightly but significantly enhanced by the presence of the tetrahemic domain TorC N in the reconstitution system. The enhanced rate in the presence of TorC N probably means that TorC N transfers the electrons to TorC C , since TorC N alone cannot directly feed TorA with electrons (Fig. 5). This result, together with the fact that the presence of both TorC N and TorC C domains are required for bacterial growth with TMAO as a sole exogenous electron acceptor (Fig.  2), support a model in which TorC N receives the electrons from the menaquinone pool and then transfers them to TorC C , which gives them to TorA. In the Nir and Nap respiratory systems, the TorC N homologues (NirT and NapC) and their associated diheme cytochromes (NirB and NapB, respectively) constitute distinct proteins (19,37). In the case of TorC, the TorC N tetrahemic domain is fused to the TorC C monohemic domain, and it is clear from our experiments that the intact TorC protein is more efficient than a mixture of the two isolated domains.
Although the simplest model of interaction between TorC and TorA would have been that TorC C binds TorA, since it transfers the electrons to TorA, we have shown by two different approaches that TorC C does not significantly bind TorA, whereas TorC N binds TorA efficiently (Figs. 6 and 7). The apparent equilibrium dissociation constant obtained by the analysis of the BIAcore data is similar for TorC N and TorC, although a second K d with a higher value can also be calculated for TorC (Table III). This latter might correspond to a weak interaction between TorC C and TorA that takes place after the TorC N -TorA binding, allowing the electron transfer. Based on these findings, we proposed a model in which the tetrahemic domain of TorC transfers the electrons to TorC C and binds TorA in such a way that TorC C is correctly positioned to transfer them to TorA (Fig. 8). This may also explain why TorC N and TorC C are fused. Finally, the binding of the TorC N domain to TorA raises the question of a possible binding of DorC and also of the tetrahemic homologues of TorC to their associated terminal reductase.