A Novel cdsAB Operon Is Involved in the Uptake of l-Cysteine and Participates in the Pathogenesis of Yersinia ruckeri

Abstract

Application of in vivo expression technology (IVET) to Yersinia ruckeri, an important fish pathogen, allowed the identification of two adjacent genes that represent a novel bacterial system involved in the uptake and degradation of l-cysteine. Analysis of the translational products of both genes showed permease domains (open reading frame 1 [ORF1]) and amino acid position identities (ORF2) with the l-cysteine desulfidase from Methanocaldococcus jannaschii, a new type of enzyme involved in the breakdown of l-cysteine. The operon was named cdsAB (cysteine desulfidase) and is found widely in anaerobic and facultative bacteria. cdsAB promoter analysis using lacZY gene fusion showed highest induction in the presence of l-cysteine. Two cdsA and cdsB mutant strains were generated. The limited toxic effect and the low utilization of l-cysteine observed in the cdsA mutant, together with radiolabeled experiments, strongly suggested that CdsA is an l-cysteine permease. Fifty percent lethal dose (LD₅₀) and competence index experiments showed that both the cdsA and cdsB loci were involved in the pathogenesis of the bacteria. In conclusion, this study has shown for the first time in bacteria the existence of an l-cysteine uptake system that together with an additional l-cysteine desulfidase-encoding gene constitutes a novel operon involved in bacterial virulence.

The genus Yersinia is known mainly because it includes three important human pathogenic species, Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, which have been deeply studied in the last 20 years. However, the genus also includes other species, such as Yersinia ruckeri, involved in pathological processes in intensive aquaculture which cause important economic losses in this kind of industry. This bacterium is the etiological agent of the enteric red mouth disease of fish. This disease is spread throughout the world and affects mainly the aquaculture of salmonids. Most of the mechanisms involved in the virulence of Yersinia species that cause human diseases are very well known. In contrast, only a few pathogenic mechanisms of Y. ruckeri have been described (7, 12, 14, 29). The application to Y. ruckeri of in vivo expression technology (IVET) has allowed the identification of 14 genes specifically induced during the infection process in rainbow trout (11). Some of them have been proven to participate in virulence, such as the iron uptake mechanism via the catecholate siderophore ruckerbactin (11), the YhlA hemolysin (13), and the tra chromosomally located operon, a type IV secretion system (22).

Cysteine is an important amino acid, because as a sulfur-containing compound, it is the sole entrance for reduced sulfur into cell metabolism in most organisms. Cysteine is essential for the biogenesis of Fe-S clusters of some enzymes; it also plays a crucial role in protein folding through disulfide bond formation, and the sulfur component is needed for synthesis of essential compounds, such as methionine, thiamine, biotin, etc. This amino acid residue also represents the rate-limiting nutrient in glutathione biosynthesis (1, 34), the major redox buffer and detoxification molecule in the cell. In spite of these important roles of cysteine, an increased cysteine level has been shown to be toxic to cells (18, 19, 26). For this reason, the intracellular cysteine levels need to be tightly regulated, by controlling not only its biosynthesis and degradation but also the transport of this amino acid from the extracellular medium to the cell and vice versa. In contrast to eukaryotes, cysteine uptake has been poorly characterized in bacteria (10, 24, 32). These systems are rare in prokaryotes, in part because this amino acid is readily oxidized to the disulfide-linked cystine and taken up in this form. For this reason, cystine uptake has been further studied (5, 6, 17).

There are several types of cysteine-degrading enzymes in bacteria. These include l-cysteine desulfurase (EC 2.8.1.7) and d-cysteine desulfhydrase (EC 4.4.1.15), both pyridoxal phosphate-dependent enzymes. Recently, a new type of l-cysteine-degrading enzyme has been described in the archaeal Methanocaldococcus jannaschii (33). This was defined as l-cysteine desulfidase and uses the [4Fe-4S] center instead of pyridoxal phosphate to catalyze the hydrolysis of l-cysteine to sulfide, ammonia, and pyruvate. The enzyme was oxygen sensitive, and on the basis of sequence comparison it was found that this protein was widely present in anaerobic bacteria (33).

In this paper, the analysis of a previously selected iviX clone (11) showed the presence in Y. ruckeri of a novel two-component operon involved in the assimilation of l-cysteine. According to sequence analysis, the first gene corresponds to an amino acid permease and the second to an l-cysteine desulfidase. Regulation studies and uptake experiments confirm the involvement of this system in the cysteine metabolism of the bacterium. Interestingly, the operon is needed for full virulence of Y. ruckeri in fish.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions.

The bacterial strains and plasmids used in this work are listed in Table 1. Y. ruckeri strains were routinely cultured in nutrient broth (NB; Difco) and on nutrient agar (NB with 1.5% agar) (NA) and M9 medium supplemented with 0.5% glucose (Scharlau Chemie S.A.) and 0.2% Casamino Acids (Becton, Dickinson and Company) (M9GC) at 18°C or 28°C. Escherichia coli strains were cultured in 2× tryptone-yeast extract (TY) broth or agar. In order to avoid l-cysteine oxidation, the bacteria were incubated in 50-ml Falcon screw-top airtight tubes containing 20 ml of medium. Under this condition, l-cysteine was stable as determined by the ninhydrin quantification method (16) (see below).

TABLE 1.

Bacterial strains and plasmids^a

Strain or plasmid	Relevant properties	Source or reference
Strains
Yersinia ruckeri
150R	Rifr derivative of strain 150	14
150RiviX	Strain containing ivi fusion expressed only in the host	11
cdsA mutant	RifrcdsA::pJP5603	This work
cdsB mutant	RifrcdsB::pJP5603	This work
146, 147, 148, 149, and 150	Strains isolated from an outbreak in trout (Danish fish farm)	J. L. Larsen (Denmark)
955, 956, and 43/19	Strains isolated from an outbreak in trout (U.S. fish farms)	CECT (Spanish Type Culture Collection)
35/85 and 13/86	Strains isolated from outbreaks in trout (Danish and United Kingdom fish farms, respectively)	C. J. Rodgers, University of Tarragona, Spain
A100 and A102	Strains isolated from outbreak in trout (Spanish fish farm)	I. Márquez, Laboratory of Animal Health
150/05, 158/05, and 382/05	Strains isolated from outbreak in trout (Spanish fish farm)	Proaqua Nutrición S.A.
Escherichia coli
S17-1λpir	Smr λ(pir) hsdR pro thi RP4-2 Tc::Mu Km::Tn7	31
MT1694	Helper strain HB101 containing pRK2013	15
Plasmids
pIVET8	AproriR6K mob+ promotorless cat-lacZY	20
pJP5603	KanroriR6K mob+ Plac promoter	27
pCdsA	pJP5603::BamHI (cdsA), Kanr	This work
pCdsB	pJP5603::BamHI (cdsB), Kanr	This work

^aAll Y. ruckeri strains with the exception of strain 956 (which belongs to serotype 2) belong to serotype 1. Rif^r, rifampin resistance; Ap^r, ampicillin resistance; Sm^r, streptomycin resistance; Kan^r, kanamycin resistance.

For incubation under aerobic conditions, 250-ml Erlenmeyer flasks containing 20 ml of medium were inoculated with the bacteria and incubated in an orbital shaker at 250 rpm. If required, the following antibiotics were added to the medium: 100 μg/ml ampicillin, 50 μg/ml rifampin, 50 μg/ml streptomycin, and 50 μg/ml kanamycin, all of them from Sigma-Aldrich Co. Growth was monitored by determining the optical density at 600 nm (OD₆₀₀) of a culture with a Hitachi U-2900 spectrophotometer at different times during incubation at the appropriate temperature.

Genetic techniques.

Routine DNA manipulation was performed as described by Sambrook and Russell (30). Phage T4 DNA ligase and calf intestinal alkaline phosphatase were purchased from Roche Ltd., restriction enzymes were from Takara Bio Inc., and oligonucleotides were from Sigma-Aldrich Co.

DNA sequencing was performed by the dideoxy chain termination method with the BigDye Terminator version 3.1 (Applied Biosystems) according to the manufacturer's instructions in an ABI Prism 3130xl DNA sequencer at the Universidad de Oviedo. Sequences were compared to those in the databases with the BLAST (Basic Local Alignment Search Tool) program.

Plasmid DNA from clone iviX was recovered from the Y. ruckeri chromosome by triparental mating (11), and the DNA fragment situated upstream of the cat and bla genes was sequenced using the initial primer catseq-2 (5′-CGGTGGTATATCCAGTG-3′), corresponding to nucleotides 31 to 15 of the cat gene from the pIVET8 plasmid (11).

In vitro regulation studies.

Cells of Y. ruckeri 150RiviX containing a cdsB::pIVET8 transcriptional fusion were incubated in the presence of different amino acids, such as l-serine, l-leucine, l-threonine, l-methionine, l-tryptophan, l-tyrosine, l-cysteine, and l-cystine, at concentrations of 0.5 mM and 20 mM in screw-top airtight tubes and flasks containing M9GC medium supplemented with 100 μg/ml ampicillin up to an OD₆₀₀ of about 1.0. Cells were centrifuged at 12,000 × g for 5 min, and β-galactosidase activity was assayed in cells by the Miller method (23) using o-nitrophenyl-β-d-galactopyranoside (ONPG) as a substrate. For the analysis of the maximal l-cysteine concentration required for induction of the cdsAB operon, l-cysteine was included in the culture medium in concentrations of 10 μM, 25 μM, 50 μM, 100 μM, 250 μM, 0.5 mM, 1 mM, 2 mM, and 5 mM, and β-galactosidase activity was assayed when culture reached an OD₆₀₀ of 1.0. For time course induction experiments, 0.5 mM l-cysteine was added to the culture medium, and at different times of incubation β-galactosidase activity was determined.

Construction of cdsA and cdsB mutants.

Internal fragments of 525 and 529 bp of the predicted cdsA and cdsB open reading frames (ORFs), respectively, were amplified by PCR with the following primers: forward primer cdsA-1 (5′-ATGCGGATCCTATTAGGCGCACTTTAT-3′), with nucleotides 347 to 363 of the cdsA gene in bold type, and reverse primer cdsA-2 (5′-ATGCGGATCCTTAACAGCTTCATCGTG-3′), with nucleotides 881 to 865 in bold type, to amplify the internal fragment of the cdsA gene; forward primer cdsB-1 (5′-ATGCAGATCTCGATACAGGAGAGTGAT-3′), with nucleotides 356 to 372 in bold type, and reverse primer cdsB-2 (5′-ATGCAGATCTAGATATTCGGCCACCAC-3′), with nucleotides 884 to 868 in bold type, to amplify the internal fragment of cdsB. Primers contained restriction sites for BamHI and BglII, in italics, and four additional bases at their 5′ end. The amplicons generated were digested with the corresponding enzymes and ligated into pJP5603 previously digested with BamHI and dephosphorylated. The ligation mixture was used to transform electrocompetent cells of E. coli S17-1λpir. Selected transformants, containing the plasmid with the insert, were used to conjugate with Y. ruckeri 150R to obtain the cdsA::pJP5603 and cdsB::pJP5603 mutants, as previously described (14). The mutations were confirmed by Southern blot analysis after digestion of genomic DNA with EcoRI of the parental strain and the cdsA and cdsB mutants. The previously amplified internal fragments from cdsA and cdsB were used as probes. Probe labeling, hybridization, and developing were performed with the DIG DNA labeling and detection kit from Roche by following the manufacturer's instructions. The stability of the mutants in the absence of kanamycin was analyzed by doing several passes on nonselective medium, followed by comparison of the number of cells able to grow on plates with or without antibiotic.

To complete the sequence of cdsB, genomic DNA from the Y. ruckeri cdsB mutant was digested with EcoRI, the only restriction site on the pJP5603 plasmid. The restriction fragments were religated, and the mixture was used to transform cells of E. coli S17-1λpir. Transformants were selected on 2× TY agar medium containing kanamycin. The plasmid containing the cdsB gene was sequenced with the initial primer RP (5′-CAGGAAACAGCTATGAC-3′) from the lacZ gene from the pJP5603 plasmid.

Growth studies and l-cysteine quantification.

To check if cdsA and cdsB mutations played any role in the growth of Y. ruckeri, wild-type and mutant strains were incubated in 50-ml Falcon screw-top airtight tubes containing M9GC and M9GC supplemented with 0.5 mM l-cysteine. Growth was monitored by determining the OD₆₀₀ at different times during incubation at 18°C. Growth curves were determined in triplicate and repeated three times. For l-cysteine quantification in the supernatant of cultures at different incubation times through the growth curve, the acid ninhydrin assay described by Gaitonde (16) was used. Briefly, the content of this amino acid was determined colorimetrically by monitoring the absorbance at 560 nm and reading from a standard curve (r² = 0.99). M9GC supplemented with 0.5 mM l-cysteine with no cells was used as the control of the total amount of the amino acid in the medium. The experiment was repeated three times, and the mean and standard deviation were computed. Cystine (0.5 mM) was used as a negative control.

Amino acid transport assay.

Y. ruckeri parental and cdsA strains were cultured in 50-ml Falcon screw-top airtight tubes containing M9GC medium supplemented with 0.5 mM l-cysteine at 18°C up to an OD₆₀₀ of 0.8. Thirty milliliters of each strain was harvested, washed twice with buffer A, containing 100 mM Tris-HCl (pH 7.3), 100 mM NaCl, and 0.5 mM MgCl₂, and resuspended in 9 ml of the same buffer. After 5 min in the presence of chloramphenicol (15 μg/ml) to inhibit protein synthesis, a solution containing 2 mM l-cysteine as a carrier, 20 μCi/ml of ³⁵S-labeled l-cysteine (specific activity, 1,075 Ci/mmol) (Perkin Elmer, Boston, MA), and 10 mM dithiothreitol was added (final concentrations of l-cysteine and dithiothreitol were 200 μM and 1 mM, respectively). Assays were performed at 18°C, and duplicated samples of 0.5 ml were withdrawn at intervals, diluted 20-fold in cold buffer A, and filtered through 25-mm-diameter GF/C Millipore nitrocellulose membrane filters (0.45-μm pore size). Filters were washed with 10 ml of buffer A, dried, and immersed in Filter-Count scintillation cocktail (Perkin Elmer, Boston, MA), and the radioactivity was measured in a liquid scintillation counter. l-Cysteine uptake activity was calculated in nmol/min/mg protein. Total protein was quantified by the Bradford (AppliChem, Germany) protein method.

For competition studies, the different amino acids were added in 10-fold excess in relation to cysteine, except cystine, which was insoluble in buffer A at this concentration, and a 1:1 ratio was used. The rate of l-cysteine uptake was measured in a 5-min reaction. The proton gradient inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to the cells 10 min before the labeled compound.

Experimental animal studies.

To determine the role in virulence of cdsA and cdsB mutations, 50% lethal dose (LD₅₀) experiments with the parental and the mutant strains were carried out as described by Fernández et al. (14). Rainbow trout (Oncorhynchus mykiss) weighing between 8 and 10 g were kept in 60-liter tanks at 18 ± 1°C in continually flowing dechlorinated water. Groups of 10 fish were challenged by intraperitoneal injection of 0.1 ml serial 10-fold dilutions of an exponential-phase culture of bacteria in phosphate-buffered saline (PBS) over a range of 10² to 10⁸ cells, and the mortalities were followed up for 7 days. Control fishes were injected with 0.1 ml PBS. Two different experiments were carried out, and the LD₅₀ determinations were calculated by the method of Reed and Muench (28).

For in vivo competition assays, parental and mutant strains were grown separately in NB at 18°C in orbital shakers at 250 rpm up to an OD₆₀₀ of 0.5 (approximately 10⁸ cells/ml). Two and a half milliliters of parental and mutant strains were mixed, and 10-fold dilutions of this suspension were plated onto NA (to measure total CFU) and NA containing kanamycin (to determine mutant CFU). From this, the exact input ratio of mutant to wild-type CFU was calculated. A sample of 0.1 ml of the 10⁻² dilution of both mixes (parental/cdsA or parental/cdsB mutant strains; approximately 10⁶ cells/ml of each strain) was used to infect rainbow trout weighing from 8 to 10 g by intraperitoneal injection. After 72 h, spleen, liver, and intestine from each infected fish were homogenized together in NB with a stomacher. Aliquots of the suspensions were plated onto NA and NA containing kanamycin as a selective medium to determine the output ratio of mutant to parental cells. In each experiment, three fish were used, and the competence index (CI) was determined as the mean of data of the three fish. The experiment was repeated twice, and the mean and standard deviation were determined. The competence index is defined as the output ratio (mutant/parental) divided by the input ratio (mutant/parental). Animal experiments were performed in accordance with the European legislation governing animal welfare, and they were authorized and supervised by the Animal Experimentation Ethics Committee of Universidad de Oviedo.

PCR detection of cdsA and cdsB in different Y. ruckeri strains.

Fifteen Y. ruckeri strains were studied in regard to the presence of cdsA and cdsB genes. PCR was performed using the following primers: DetC-1 (5′-ATGCGGATCCTTTCATTATGTTAGTTA-3′) and DetC-2 (5′-CCATTATTCCATTTTGA-3′) to check the presence of the cdsA gene, and cdsB-1 and cdsB-2 (described previously) to check the presence of the cdsB gene. All PCR components (DNA polymerase, reaction buffer, and deoxynucleoside triphosphates) were provided by Biotools. The amplification reactions were performed in a Perkin-Elmer 9700 GeneAmp thermocycler with an initial denaturation cycle at 94°C for 5 min, followed by 25 cycles of amplification (denaturation at 94°C for 30 s, annealing at 38°C for cdsA and 44°C for cdsB for 60 s, and extension at 72°C for 1 min), and a final 7-min elongation step at 72°C. The reaction products corresponding to the two groups were mixed, and 1.5% agarose gel electrophoresis was used to separate the generated PCR amplicons.

Nucleotide sequence accession number.

The GenBank/EMBL/DDBJ accession number for the sequences reported in this paper is HM769941.

RESULTS

CdsB shows conserved amino acid positions with respect to l-cysteine desulfidase enzyme.

Selection of specific in vivo-induced genes in Y. ruckeri was previously carried out by IVET (11). One out of 14 isolated clones, the iviX, contained an in vivo-induced transcriptional fusion (Fig. 1) with a partial ORF encoding a protein which showed a significant similarity with hypothetical transmembrane proteins from different bacteria (11). Further sequencing and analysis of the adjacent region of this genetic locus revealed the presence of an additional ORF separated by 49 bp and proceeded by a consensus −10 (TTTAAT) and −35 (CAGATA) promoter sequence and a ribosome binding site (RBS) (AGGCGA). The upstream gene consists of 1,326 bp and encodes a protein of 442 amino acids which shares a high identity with hypothetical proteins of Y. ruckeri ATCC 29473 (442/442 [100%]), Y. enterocolitica (418/442 [94%]), Salmonella enterica serovar Typhimurium LT2 (389/445 [87%]), and Citrobacter koseri (387/445 [86%]) and with the protein YhaO of Escherichia coli B (162/175 [92%]). The translational compound encoded by the first ORF shows 11 transmembrane helixes (putative membrane localization) and a conserved domain related to transmembrane amino acid transporter proteins. The second ORF of 1,323 bp encodes a protein of 441 amino acids which shares high identity with hypothetical proteins of Y. ruckeri ATCC 29473 (441/441 [100%]), Y. enterocolitica (329/439 [74%]), Escherichia albertii TWO7627 (300/440 [68%]), and Salmonella enterica CT-02021853 (296/440 [67%]) and the protein YhaM CFTO73 of Escherichia coli B (297/441 [67%]). It encodes a protein which presents two conserved domains: COG3681 (l-cysteine desulfidase) and cI12120 (l-serine dehydratase, iron-sulfur-dependent, alpha subunit). Similar domains exist in the l-cysteine desulfidase of M. jannaschii, which catalyzes the breakdown of l-cysteine into pyruvate, ammonia, and sulfide (33). In fact, the protein encoded by the second ORF in Y. ruckeri presents 28 out of 29 amino acids conserved in specific positions of the l-cysteine desulfidase of M. jannaschii, including the four cysteines (C27, C327, C366, and C373) described by Tchong et al. as possible ligands for the [3Fe-4S] center (33) (Fig. 2). The differential amino acid position corresponds to P346 in Y. ruckeri, and it also differs from M. jannaschii in other orthologous loci found in Y. enterocolitica, Klebsiella pneumoniae, and Proteus mirabilis, among others (Fig. 2). On the basis of these amino acid identities and domains as well as other results shown below, from now on the two ORFs identified from the analysis of the iviX clone are designated cdsB (cysteine desulfidase) and cdsA, for ORF2 and ORF1, respectively.

FIG. 1.

Chromosomal arrangement of the region containing cdsA and cdsB genes in Y. ruckeri 150R. Arrows indicate the direction of the transcription. The organization of the transcriptional fusion between cdsB and the promoterless genes cat and lacZY in Y. ruckeri 150RiviX is shown underneath, and the putative promoter (P) selected by IVET is indicated. Catseq-2 oligonucleotide was used to sequence the fragments adjacent to the pIVET8 integration site. cat, chloramphenicol acetyltransferase gene (promoterless); lacZY, genes for lactose fermentation (promoterless); bla, ampicillin resistance gene.

FIG. 2.

Conserved residues among the orthologs of CdsB. GenBank data accession numbers correspond to M. jannaschii (MJ1025), Y. enterocolitica (YE0448), K. pneumoniae (KPN_04419), P. mirabilis (PMI0145), and D. nodosus (DNO_0106). Note that the amino acid variations correspond with position 346 of the CdsB protein. An additional variation is found in the 311 position of D. nodosus (DNO_0106). The four cysteines C27, C327, C366, and C373 described by Tchong et al. (33) as possible ligands for the [3Fe-4S] center are underlined.

cdsA and cdsB orthologs are present in several bacterial groups.

Comparative nucleotide sequence analysis using the BLAST program showed that similar cdsAB clusters were present in C. koseri ATCC BAA-895 (CKO_04511 and CKO_04510), S. enterica subsp. enterica serovar Typhimurium strain LT2 (yhaO and yhaN), Shigella sonnei Ss046 (yhaO and yhaNM), Escherichia coli B strain REL606 (yhaO and yhaM), Dichelobacter nodosus VCS1703A (DNO_0107 and DNO_0106), Chromobacterium violaceum ATCC 12472 (CV_1823 and CV_1824), and Y. enterocolitica subsp. enterocolitica 8081 (YE0447 and YE0448) (Fig. 3). However, the cluster was absent from species closely related to Y. ruckeri and Y. enterocolitica, such as Y. pseudotuberculosis IP 32953 and Y. pestis CO92. These two species present similar cdsA loci (YPTB0334 and YPO0277, respectively) in their genomes, but these are followed by the metC gene, which encodes a cystathionine β-lyase. Interestingly, this enzyme also catalyzes the degradation of l-cysteine to pyruvate, ammonia, and sulfide in Escherichia coli (3). A second group of bacteria, including M. jannaschii, was found in which the cdsB locus was not preceded by the corresponding cdsA locus. A phylogenetic analysis based on CdsB protein sequence showed the relationship between Y. ruckeri and its other relatives (Fig. 3).

FIG. 3.

Phylogenetic tree based on the CdsB protein sequence, showing the relationships between Y. ruckeri and its other relatives. GenBank data accession numbers are indicated: Y. enterocolitica (YE0448), Escherichia coli (YHAM), Shigella sonnei Ss046 (YP_312078), Salmonella enterica (NP_462152), Citrobacter koseri (YP_001456001), Chromobacterium violaceum (CV_1824), Dichelobacter nodosus (DNO_0106), Proteus mirabilis (PMI0145), Treponema denticola (TDE2144), Aeromonas hydrophila (YP858502), Lactobacillus sakei (YP_394765), Actinobacillus pleuropneumoniae (ZP_00133782), Methanocaldococcus jannaschii (MJ1025), Methanococcus maripaludis (MMP1468), Fusobacterium nucleatum (FN1147), Thermoanaerobacter tengcongensis (TTE0269), Clostridium perfringens (CPE0806), Geobacter sulfurreducens (GSU1527), Desulfitobacterium hafniense (DSY1109), Vibrio parahaemolyticus (VP2173), Porphyromonas gingivalis (PG0909), Klebsiella pneumoniae (YP_001338041), Bacteroides thetaiotaomicron (BT2080), Desulfovibrio desulfuricans (Dde_0807), Shewanella oneidensis (SO_1403), and Clostridium tetani (CTC02309). Note that the species grouped in the first division from CdsB of Y. ruckeri to DNO_0106 of Dichelobacter nodosus presented a cdsAB orthologous operon in their genome. The large black box groups the species close to Y. ruckeri which harbor the cdsAB cluster.

The cdsAB operon is induced specifically by l-cysteine.

The strain Y. ruckeri 150RiviX, obtained by IVET, contains a transcriptional fusion between the cdsAB promoter and lacZY (Fig. 1). This construction was used for the analysis of the expression of these genes in response to different culture conditions. The results obtained by β-galactosidase activity determination in cultures grown in 50-ml Falcon screw-top airtight tubes containing M9GC medium showed that l-cysteine and l-cystine were the only promoter inductors. Specifically, β-galactosidase activity was increased approximately 4-fold in the presence of 0.5 mM concentrations of these amino acids. This increase was detected in both exponential- and stationary-phase cultures. The catabolism of these amino acids produced the generation of abundant H₂S, as was determined by its characteristic unpleasant odor and the production of a black precipitate after culture centrifugation when an iron salt was included in the culture medium. It should be noticed that during incubation under these conditions and in the presence of a reducing agent such as H₂S, which changed the redox potential of cultures, l-cystine turned into l-cysteine, as was determined by the ninhydrin method (data not shown). This, together with the limited induction (1.7-fold) observed in the presence of l-cysteine when incubation was carried out under oxygenation conditions, in which l-cysteine is turned into l-cystine, strongly suggests that l-cysteine is the unique operon inductor, and a progressive increase in β-galactosidase activity was found until a maximum at a 1 mM concentration of the amino acid. Other amino acids, such as leucine, serine, threonine, tryptophan, tyrosine, and methionine, in both low (0.5 mM) and high (20 mM) concentrations, did not significantly induce the promoter.

The cdsA mutant but not the cdsB mutant is more resistant to l-cysteine than the parental strain due to an alteration in l-cysteine uptake.

To investigate the relative roles of the cdsAB operon in bacterial physiology, two mutants with independent mutations in cdsA and cdsB were constructed by insertional mutagenesis as described by Fernández et al. (11). PCR and Southern blot analysis showed that the genes cdsA and cdsB were inactivated in the respective mutants, and both were genetically stable under nonselective antibiotic pressure (data not shown). No differences in growth were detected between the parental and the mutant strains (cdsA and cdsB mutants) when they were grown in screw-top airtight tubes containing M9GC or in M9GC supplemented with specific amino acids, such as threonine, serine, tryptophan, or tyrosine. A similar result was obtained when the complex medium NB was used. However, the parental and the cdsB mutant strains showed a significant growth limitation in medium containing 0.5 mM cysteine, while the cdsA mutant partially overcame this growth limitation (Fig. 4).

FIG. 4.

l-Cysteine effect on the growth of Y. ruckeri parental and cdsA mutant strains. Strains were grown under simulated anaerobic conditions in M9GC (black symbols) and M9GC supplemented with 0.5 mM l-cysteine (white symbols). ▴ and ▵, parental strain; ▪ and □, cdsA mutant. Growth curves were determined by three independent experiments, and the media were represented. The standard deviations were lower than ±0.06.

To determine if the partial resistance of the cdsA mutant to cysteine was a consequence of a limited consuming capacity of this amino acid, the cysteine present in the medium was quantified during the growth curves of Y. ruckeri parental and mutant strains in M9GC medium plus 0.5 mM cysteine. As can be observed in Table 2, the cysteine remaining in the culture medium after 48 h of incubation was considerably higher in the cdsA mutant than in the parental and cdsB mutant strains. This difference was observed along the whole growth curve. Therefore, the limited toxic effect that cysteine has in the growth of the cdsA mutant, together with its limited capacity for cysteine consumption, suggests that the cdsA gene encodes a cysteine permease.

TABLE 2.

l-Cysteine remaining in the supernatant of cell cultures of Y. ruckeri parental and cdsA and cdsB mutant strains after 48 h of incubation in M9GC supplemented with 0.5 mM l-cysteine

Strain	l-Cysteine in culture supernatant (mg/ml)	Utilization of l-cysteine (mg/mg protein)a
No cells	0.065 ± 0.001
Y. ruckeri parental	0.005 ± 0.002	1.02 ± 0.09
Y. ruckeri cdsA mutant	0.038 ± 0.001	0.27 ± 0.08
Y. ruckeri cdsB mutant	0.006 ± 0.003	0.89 ± 0.08

^aThe utilization of l-cysteine was inferred per milligram of protein, considering the amount of this amino acid present at the end of the incubation period in the culture medium without cells. The data represent the average of results from three independent experiments.

In order to confirm the role of CdsA in cysteine uptake, studies using [³⁵S]l-cysteine were carried out. Cells of the cdsA mutant and parental strains were grown in the presence of 0.5 mM cysteine, and at mid-logarithmic-growth phase (OD₆₀₀ = 0.7 to 0.8) cysteine uptake was measured. When the kinetics of l-cysteine uptake were determined in the presence of a 200 μM concentration of this amino acid, the results showed a significant delay in the uptake of radiolabeled cysteine in the cdsA mutant compared to that in the parental strain (Fig. 5).

FIG. 5.

Uptake of [³⁵S]l-cysteine by Y. ruckeri parental and cdsA mutant strains. l-Cysteine taken up by cells of parental (•) and cdsA mutant (▪) strains during a 5-min period. The data points represent the mean values from three separate experiments.

To define the substrate specificity of CdsA, competitive transport studies were carried out in the presence of a 10-fold excess of unlabeled competing ligand. Among the various amino acids used, only l-methionine, l-cystine, and to a lesser extent l-glutamic acid compete with the cysteine uptake (Table 3). The other tested amino acids (tryptophan, leucine, serine, and threonine) had no effect on the cysteine uptake. The presence of the uncoupler of phosphorylation CCCP resulted in a considerable loss in the cysteine uptake, which implies that CdsA-mediated cysteine transport is an energy-dependent process (Table 3).

TABLE 3.

Effect of different compounds on l-cysteine transport by cells of Y. ruckeri

Compound	% uptake ± SD
No inhibitor	100
l-Cysteine	43 ± 12
l-Threonine	98 ± 9
l-Serine	109 ± 3
l-Leucine	75 ± 16
l-Methionine	39 ± 10
l-Tryptophan	86 ± 8
l-Glutamic acid	63 ± 14
l-Cystinea	51 ± 14
CCCPb	36 ± 1
DMSO	95 ± 12

^a[³⁵S]l-cysteine was used at a concentration of 200 μM. All competitors were added to a final concentration of 2 mM, a 10-fold excess over the labeled substrate, except l-cystine, added in a final concentration of 200 μM.

^bCells were preincubated with the metabolic inhibitor CCCP for 10 min before addition of the [³⁵S]l-cysteine. CCCP was dissolved in dimethyl sulfoxide (DMSO), and hence this compound was analyzed for its effect on l-cysteine uptake. The data represent the mean values from three independent experiments. When [³⁵S]l-cysteine was used at a concentration of 20 μM and all the competitors were added to a final concentration of 200 μM, the results were similar.

The cdsAB operon is needed for full virulence of Y. ruckeri, and it is conserved in different isolates.

The fact that the Y. ruckeri 150RiviX strain was obtained as an IVET clone suggested that the cdsAB operon could have a role during the infection process. To go further into this, in vivo competition assays and LD₅₀ experiments were carried out using the parental and the cdsA and cdsB strains. The competence indexes (CI) obtained for in vivo experiments were 0.03 ± 0.01 and 0.24 ± 0.14 for the cdsA and cdsB mutants, respectively, which indicated a significant lower recovery of cells of the mutant strains than that of the parental strain. The results obtained in LD₅₀ experiments showed that the mutant strains showed significantly higher values than those of the parental strain. Thus, the means of LD₅₀ values were 5.65 × 10⁴ CFU/fish for the parental strain and 1.18 × 10⁶ and 3.55 × 10⁵ CFU/fish for the cdsA and cdsB mutants, respectively. Therefore, gene IVET selection, together with the results obtained from in vivo CI and LD₅₀ experiments, confirmed that the cdsAB operon is necessary for full virulence and thus for the development of the infection process.

The presence of the cdsAB operon was analyzed by PCR in 15 different Y. ruckeri strains from different sources and origins. All of them showed the amplification of two bands of 756 bp and 549 bp, corresponding to internal fragments of cdsA and cdsB, respectively (Fig. 6). This result indicates intraspecies genetic homogeneity, given that the genes are present in serotype I and II as well as recently isolated new biotype strains from outbreaks in Spain.

FIG. 6.

PCR detection of cdsA and cdsB genes from different Y. ruckeri strains. Independent PCRs were carried out for each gene. The amplicons obtained were then mixed and separated in a 1.5% agarose gel. The sizes of the amplicons generated were 756 bp and 549 bp for cdsA and cdsB, respectively. Lanes: 2, strain 146; 3, strain 147; 4, strain 148; 5, strain 149; 6, strain 150; 7, strain 955; 8, strain 956; 9, strain 35/85; 10, strain 13/86; 11, strain 43/19; 12, strain A100; 13, strain A102; 14, strain 150/05; 15, strain 158/05; 16, strain 382/05; 17, negative control. Lanes 1 and 18, DNA molecular size markers from 1,000 to 100 bp.

DISCUSSION

Once more, IVET has allowed the in-depth investigation of the pathogenic mechanisms of Y. ruckeri. In this case, analysis of the sequences surrounding the iviX clone (11) revealed the presence of two ORFs. Comparative analysis and functional predictions indicated that they could represent a new system involved in the uptake and further degradation of the amino acid cysteine. Major clues were the presence in the CdsB protein of 28 out of 29 conserved amino acids of the l-cysteine desulfidase of M. jannaschii (33) and the presence in CdsA of a clear permease domain related to amino acid transporter proteins.

In the present work, it is shown that l-cysteine desulfidase orthologs were present in many facultative bacteria apart from E. coli (33), such as C. koseri, Y. ruckeri, K. pneumoniae, and Y. enterocolitica. Interestingly, unlike the l-cysteine desulfidase of M. jannaschii, the CdsB protein of these species is preceded in the genome by the permease CdsA. In fact, phylogenetic analysis based on the CdsB protein showed that these bacteria and others which also harbor the cdsAB loci are grouped in the same cluster, suggesting a common evolutionary origin. In other species, such as Y. pestis and Y. pseudotuberculosis, downstream from a locus similar to cdsA is located metC, a gene involved in the degradation of l-cysteine to pyruvate, ammonia, and sulfide in E. coli (3). The fact that cdsA appears adjacent to two different enzymes which use l-cysteine as a substrate reinforces the role of this protein as a cysteine permease.

The relation of these two genes with the uptake and further metabolism of l-cysteine was also supported by promoter induction experiments, comparative toxic effect, and cysteine consumption throughout the growth of parental and cdsA mutant strains and also by the differential cell uptake of labeled cysteine observed between the two strains.

In fact, promoter induction took place only in the presence of cysteine. The induction detected when cystine was in the culture medium seems to be a consequence of the conversion of this amino acid to cysteine. This was supported by the ninhydrin method, which detected high levels of cysteine in culture supernatants when cystine was the only amino acid included in the medium. The production of H₂S as a metabolite derived from cysteine and cystine utilization helped to keep a reductive state, as was confirmed by measuring the redox potential. On the contrary, under aerobic conditions, only limited induction was observed, probably due to the rapid conversion of cysteine to cystine (10). The fact that in aerobic conditions neither cysteine nor cystine produced significant promoter induction strongly supports the idea that this operon is induced specifically by cysteine. It should be emphasized that the genes are found widely in anaerobic and facultative bacteria but not in aerobic bacteria.

The analysis of mutations in cdsA and cdsB genes also supported the involvement of the cdsA gene in the uptake of cysteine. The cysteine toxic effect defined by Kari et al. (18) was lower in the cdsA mutant strain than in the parental and csdB mutant strains, probably due to the lesser utilization of cysteine by the cdsA mutant in the course of growth, just as was shown by the ninhydrin method.

Finally, radiotracer studies confirmed the involvement of cdsA in the uptake of l-cysteine in Y. ruckeri. Time course results as well as l-cysteine consumption during the growth curve suggested the existence of at least one additional cysteine uptake system given the residual cysteine uptake in the cdsA mutant. However, the comparative analyses of kinetic data in different biological systems are complex and subject to multiple factors. These, together with the special characteristics of this amino acid (in oxidizing conditions, cystine is the compound present in the environment), make extremely difficult the estimate and comparison of specific kinetic parameters between strains. These are the reasons information concerning cysteine uptake in bacteria is sparse and few examples of cysteine transporters have been found. In Campylobacter jejuni, CjaA is a receptor protein with a high affinity for l-cysteine (24). In Legionella pneumophila as well as in Cyanobacterium synechocystis, two cysteine transporters were identified (10, 32). In Saccharomyces cerevisiae, up to seven different permeases were described as taking up cysteine, and the uptake of this amino acid was found to be a nonsaturable process under various conditions (9).

The presence of cysteine desulfhydrases has been described in different microorganisms. In Bacillus subtilis, four different genes encoding this kind of enzyme have been found (2). Interestingly, in E. coli, a quintet mutant strain in five different cysteine desulfhydrases still showed some cysteine desulfhydrase activity (4). According to all the data, it seems that the cdsAB system remained undiscovered because specific culture conditions are needed for induction. It could represent one of the systems responsible for the residual cysteine degradation activity detected in this E. coli quintet mutant strain, given that E. coli possesses an orthologous cdsAB operon.

It should be indicated that local reducing environments caused by H₂S generation could be present in the gut tract of rainbow trout, one of the main locations of Y. ruckeri (14), as a consequence of bacterial metabolism. H₂S is also present in other tissues (25), since this gas is considered to be a gasotransmitter able to trigger cell signaling in vertebrate animals, including rainbow trout, with physiological levels of H₂S in the blood in the range from 10 to 300 μM (8, 21). It is possible that H₂S reduces cystine to cysteine under these conditions, and genes related to the transport and metabolism of this amino acid might be induced. This CdsAB system might be involved in the generation of iron-sulfur centers for proteins, as was proposed for the l-cysteine desulfidase of M. jannaschii (33), and also in glutathione accumulation, the major redox buffer and detoxification molecule in the cell. In any case, the absence of the CdsA and CdsB proteins led to bacterial attenuation as well as a significantly restricting growth in the fish, showing that this system is related to the progression of the bacteria inside the animal during the infection process. According to the presence of the cdsAB operon in different pathogenic species, it is probable that its involvement in the infection process is not exclusive to Y. ruckeri.