Published Ahead of Print 16 December 2008. 10.1128/AEM.01377-08.

2009, 75(4):937. DOI:Appl. Environ. Microbiol. Pérez-Pascual, R. Navais and J. A. GuijarroJ. Méndez, L. Fernández, A. Menéndez, P. Reimundo, D. 

Yersinia ruckeriVirulence of Secretion System Is Involved in theOperon Encoding a Putative Type IV

traHIJKCLMNA Chromosomally Located

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2009, p. 937–945 Vol. 75, No. 40099-2240/09/$08.00�0 doi:10.1128/AEM.01377-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Chromosomally Located traHIJKCLMN Operon Encoding a PutativeType IV Secretion System Is Involved in the Virulence of

Yersinia ruckeri�

J. Mendez, L. Fernandez, A. Menendez, P. Reimundo, D. Perez-Pascual,R. Navais, and J. A. Guijarro*

Area de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo,33006 Oviedo, Spain

Received 19 June 2008/Accepted 8 December 2008

Nucleotide sequence analysis of the region surrounding the pIVET8 insertion site in Yersinia ruckeri150RiviXII, previously selected by in vivo expression technology (IVET), revealed the presence of eight genes(traHIJKCLMN [hereafter referred to collectively as the tra operon or tra cluster]), which are similar both insequence and organization to the tra operon cluster found in the virulence-related plasmid pADAP fromSerratia entomophila. Interestingly, the tra cluster of Y. ruckeri is chromosomally encoded, and no similar tracluster has been identified yet in the genomic analysis of human pathogenic yersiniae. A traI insertional mutantwas obtained by homologous recombination. Coinfection experiments with the mutant and the parental strain,as well as 50% lethal dose determinations, indicate that this operon is involved in the virulence of thisbacterium. All of these results suggest the implication of the tra cluster in a virulence-related type IVsecretion/transfer system. Reverse transcriptase PCR studies showed that this cluster is transcribed as anoperon from a putative promoter located upstream of traH and that the mutation of traI had a polar effect. AtraI::lacZY transcriptional fusion displayed higher expression levels at 18°C, the temperature of occurrence ofthe disease, and under nutrient-limiting conditions. PCR detection analysis indicated that the tra cluster ispresent in 15 Y. ruckeri strains from different origins and with different plasmid profiles. The results obtainedin the present study support the conclusion, already suggested by different authors, that Y. ruckeri is a veryhomogeneous species that is quite different from the other members of the genus Yersinia.

Yersinia ruckeri causes yersiniosis, a systemic infection affect-ing mainly salmonids in fish farms with important economicconsequences. The major source of infection is the shedding ofbacteria from the intestines of carrier fish, and transmission isalso favored by the ability of most strains to survive and remaininfective in the aquatic environment (46), sometimes formingbiofilms (11). Although fish are generally treated with inacti-vated whole-cell vaccines, outbreaks still occur under severestress or poor hygiene conditions. Moreover, commercial vac-cines do not confer protection against a new biogroup recentlyisolated in southern England (2), Spain (21), and the UnitedStates (1). Early approaches to the study of this bacteriumindicated that some extracellular products, including hemo-lysins, lipases, and proteases, could play an important role invirulence (36). Nevertheless, only recently have some of thepathogenic mechanisms involved in the development of theinfection been identified (47). The use of in vivo expressiontechnology (IVET) permitted the selection of a set of in vivo-induced (ivi) genetic loci, some of which have been proven toparticipate in virulence, such as the iron uptake mechanism viathe catecholate siderophore ruckerbactin (14) and YhlA he-molysin/cytolysin (16). Fernandez et al. (17) also demonstratedthe involvement in virulence of Yrp1, a serralysin-type pro-

tease. However, the pathogenic mechanisms of this bacteriumare still not very well understood.

Type IV secretion systems (T4SS) from gram-negative bac-teria are mainly involved in the spread of plasmids. T4SSconstitute complex conjugation machineries composed of apilus and a mating channel through which the DNA transferintermediate is translocated. The T4SS genes involved in thiskind of process are generally carried by conjugative plasmids,such as RP4 and pKM101 (7), which harbor antibiotic resis-tance genes that are disseminated among bacteria. Over thelast several years, the adaptation in some bacteria of T4SS fordelivering virulence factors into eukaryotic cells has been de-scribed (9, 42, 50). These T4SS are divided into two subgroupsaccording to their similarities to the Agrobacterium tumefaciensVirB system (type IVA) or to the Legionella pneumophila icm/dot system (type IVB). Functionally, the VirB system of A.tumefaciens is an example of a type IV exporter of DNA acrossbacterial membranes to be introduced into plant cells to inducetumor proliferation (52). Extracellular pathogens such as Hel-icobacter pylori (6) and Bordetella pertussis (12, 48) use T4SS todeliver the CagA protein or the pertussis toxin, respectively,into the extracellular milieu or the cytoplasm of eukaryoticcells. Finally, in the case of some intracellular pathogens, suchas L. pneumophila (41), Brucella sp. (33), and Bartonella sp.(40), T4SS participate in the transfer of different effector mol-ecules into the target cells in order to ensure their survivalwithin macrophages or red blood cells.

It is well established that some components of T4SS involvedin conjugation share structural similarity, display considerable

* Corresponding author. Mailing address: Area de Microbiología,Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Astu-rias, Spain. Phone: 34985104218. Fax: 34985103148. E-mail: jaga@fq.uniovi.es.

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sequence homology, and consequently have identical proteinfunctions, with the T4SS related to pathogenicity (8, 19).For instance, traH, traI, traJ, and traK genes from thetraHIJKCLMN (hereafter referred to as the tra operon or tracluster) transfer loci, which are present among others in the traregion of plasmid R64 (28, 29), code for proteins similar tothose encoded by the dot/icm genes of the virulence-relatedtype IVB secretion system of L. pneumophila (29). The presenceof conjugative plasmids has been described in several Yersiniaspecies (24, 25, 45, 49). Recently, a type IVB secretion system hasbeen identified in the genomic analysis of Y. pseudotuberculosisIP31758, the causative agent of Far East scarlet-like fever (13).This system is encoded by plasmid pYpsIP31758.1, and it is phy-logenetically related to the type IVB dot/icm secretion system ofL. pneumophila (13). Until now, no other type IVB secretionsystem had been reported in the genus Yersinia.

Here, we report the in-depth analysis of the previously iso-lated Y. ruckeri iviXII clone (14) that allowed the identificationof a tra chromosomally located operon, which is structurallyrelated to the DNA transfer system present in plasmid pADAPof S. entomophila (26). A traI::lacZY transcriptional fusionshowed that the operon was regulated by nutritional conditionsand temperature. The fact that the identification of the clusterwas accomplished via IVET, together with in vivo competitionstudies and 50% lethal dose (LD50) experiments, indicatedthat the tra operon contributes to the virulence of Y. ruckeri. Inaddition, PCR analysis showed the presence of this operon inY. ruckeri strains from different origins.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions. The bacterial strains andplasmids used in the present study are listed in Table 1. Bacterial strains wereroutinely cultured in nutrient broth (NB) and nutrient agar (NA) from Difco at

18 or 28°C for Y. ruckeri and in 2�TY (1.6% tryptone, 1% yeast extract, and0.5% NaCl) broth (Difco) and agar at 37°C for Escherichia coli. When required,the following compounds were added to the media: 100 �g of ampicillin/ml, 50�g of rifampin/ml, and 50 �g of streptomycin/ml (all from Sigma-Aldrich Co.).Growth was monitored by determining the optical density at 600 nm (OD600) atdifferent times during incubation, and bacterial concentrations were determinedby serial dilutions and plate counts. Long-term storage of the strains was done in25% glycerol at �20°C and �70°C, and strains were lyophilized at �70°C.Experiments on the regulation of gene expression were performed with minimalmedium M9, prepared as described by Romalde et al. (37).

Genetic techniques. Routine DNA manipulation was performed as describedby Sambrook et al. (39). Phage T4 DNA ligase and calf intestinal alkalinephosphatase were purchased from Roche, Ltd.; restriction enzymes were fromAmersham Pharmacia Biotech, Ltd.; and oligonucleotides were from Sigma-Aldrich Co.

DNA sequencing was performed at the Universidad de Oviedo facility by thedideoxy chain termination method with a DR terminator kit (Applied Biosys-tems) according to the manufacturer’s instructions in an ABI Prism 310A auto-mated DNA sequencer from Perkin-Elmer. The sequences were compared usingthe computer programs BLASTx and BLASTp to those in the databases.

Plasmid DNA was recovered from the Y. ruckeri 150RiviXII chromosome bytriparental mating (34). Briefly, cultures of the Y. ruckeri fusion-containing strain150RiviXII, an E. coli strain plus the recipient (S17-1�pir), and an E. coli strainharboring a helper plasmid (pRK2013) that encodes Tra were mixed in equalamounts (100 �l of overnight cultures), pelleted, plated onto a 2�TY plate, andincubated at 28°C for 6 h. Transconjugants containing the transcriptional fusioninduced in vivo were selected in plates with both ampicillin and streptomycin atthe concentrations mentioned above. The DNA fragment situated upstream ofthe cat gene from plasmid pIVET8 was sequenced by using the initial primercatseq-2 (5�-CGGTGGTATATCCAGTG-3�), corresponding to nucleotides 31to 15 of the cat gene (Fig. 1). Thus were obtained the partial sequence of traI andthe complete sequence of traH. To analyze the fragment adjacent to the partialtraI sequence in clone iviXII, genomic DNA from Y. ruckeri 150RiviXII wasdigested with EcoRI (Fig. 1). The restriction fragments were religated and themixture was used to transform cells of E. coli S17-1�pir. Transformants wereselected on 2�TY agar medium containing ampicillin. The resulting plasmid,containing the rest of the cluster except for the end of gene traN, was sequencedwith the initial primer blaseq from the pIVET8 bla gene. The rest of gene traNwas obtained by inverse PCR. Briefly, genomic DNA from Y. ruckeri 150R wasdigested with ClaI, and the generated fragments were religated. The ligation

TABLE 1. Bacterial strains and plasmids

Strain or plasmida Characteristics and/or originb Source or reference

StrainsYersinia ruckeri

150R Rifr derivative of 150 17150RiviXII Strain containing ivi fusion expressed only in the host 14150RtraI Rifr traI::pIVET8 This study146, 147, 148, 149, and

150Strains 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. fishfarms)

CECT (Spanish Type Culture Collection)

35/85 and 13/86 Strains isolated from outbreaks in trout (Danish andUnited Kingdom fish farms, respectively)

C. J. Rodgers, University of Tarragona,Spain

A100 and A102 Strains isolated from an outbreak in trout (Spanishfish farm)

I. Marquez, Laboratory of Animal Health

150/05, 158/05, and 382/05 Strains isolated from outbreak in trout (Spanish fishfarm)

ProAqua Nutricon S.A.

Escherichia coliS17-1�pir Smr �(pir) hsdR pro thi RP4-2 Tc::Mu Km::Tn7 43MT1694 Helper strain HB101 containing pRK2013 18

PlasmidspIVET8 Apr oriR6K mob� promoterless cat-lacZY 30pTRAI pIVET8::BglII (traI), Apr This study

a All Y. ruckeri strains with the exception of strain 956 (which belongs to serotype 2) belong to serotype 1.b Rifr, rifampin resistance; Apr, ampicillin resistance; Smr, streptomycin resistance.

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mixture was used as template DNA for a PCR, using a long amplification kit(Biotools) and oligonucleotides corresponding to the known DNA sequence.The reaction was performed in a Perkin-Elmer 9700 GeneAmp thermocycler.Sequences were compared to the GenBank NR database using BLASTx andBLASTp.

Localization of the tra genes in the Y. ruckeri genome. Total DNA was isolatedfrom 1-ml portions of overnight cultures of Y. ruckeri 150R and Y. ruckeri150RiviXII strains by using a Gene Elute genomic bacterial DNA kit (Sigma).Plasmid DNA was obtained by using the protocol described by Kado and Liu(27). Chromosomal and plasmid DNA were separated by electrophoresis in0.75% agarose gels, transferred to a nylon membrane, and hybridized withprobes corresponding to internal fragments of traI and bla. Probe labeling,hybridization, and development were performed with a DIG DNA labeling anddetection kit (Roche) according to the manufacturer’s instructions.

In vitro regulation analysis. For promoter expression studies, 20 ml of culturemedium supplemented with ampicillin was inoculated with 200 �l of Y. ruckeri150RiviXII overnight cultures, followed by incubation in orbital shakers at 250rpm. To determine the influence of temperature, cells were incubated in minimalmedium M9 at 18 or 28°C. To determine the effect of the presence of differentsalts, cells were incubated in M9 supplemented with NaCl (250 mM), MgCl2 (25mM), or CaCl2 (1.25 mM). Samples from stationary-phase cultures were col-lected and stored at �20°C. The �-galactosidase activity of the traI::pIVET8transcriptional fusion was measured as described by Miller (32), using ONPG(o-nitrophenyl-�-D-galactopyranoside) as a substrate.

Construction of traI insertion mutant. A 317-bp internal fragment of traI wasamplified by PCR with the primers traI-1 (5�-ATGCAGATCTGTTTTTGGGGCGGACGC-3�, nucleotides 480 to 464 are in boldface type) and traI-2 (5�-ATGCAGATCTCCGTGGGGTTTCAGGGA-3�, nucleotides 164 to 180 are in bold-face type). Both primers contained restriction sites for BglII (indicated in italics)and four additional bases at their 5� ends. The generated amplicon was digestedwith BglII and ligated into pIVET8, previously digested with the same enzymeand dephosphorylated. The ligation mixture was used to transform electrocom-petent cells of E. coli S17-1�pir. Selected transformants, containing the plasmidwith insert, were used to conjugate with Y. ruckeri 150R to obtain the traI mutant.The mutation was confirmed by Southern blot analysis. Genomic DNA isolatedfrom Y. ruckeri 150R and the mutant strain 150RtraI was digested with EcoRIand separated in a 0.75% (wt/vol) agarose gel. DNA was transferred to a nylonmembrane (Amersham), fixed by UV irradiation, and hybridized with the 317-bpPCR-generated internal fragment from the traI. Probe labeling, hybridization,and development were performed with the DIG DNA labeling and detection kitfrom Roche according to the manufacturer’s instructions.

Salt tolerance of the traI mutant. The parental (Y. ruckeri 150R) and mutant(Y. ruckeri 150RtraI) strains were grown up to an OD600 of 0.5 (�108 cells/ml).Five 10-fold serial dilutions were prepared, and 100-�l portions of 10�4 and 10�5

dilutions were plated onto NA and NA supplemented with 250 mM NaCl. Thenumbers of bacteria on both types of media were determined after 48 h. Threeindependent experiments were carried out, and in each one plating was carriedout in triplicate.

LD50 studies with the traI mutant strain. To determine the role in virulence ofthe traI mutation, LD50 experiments with the parental and mutant strains werecarried out as described by Fernandez et al. (17). Rainbow trout (Oncorhynchusmykiss) weighing between 8 and 10 g were kept in 60 l tanks at 18 1°C indechlorinated water. Groups of 10 fish were challenged by intraperitoneal injec-tion of 0.1-ml serial 10-fold dilutions of exponential-phase cultures of the strainsY. ruckeri 150R and Y. ruckeri 150RtraI corresponding to a range of 102 to 108

cells, and mortalities were monitored for up for 7 days. The microorganisms werepreviously washed and resuspended in phosphate-buffered saline. Control fishwere injected with 0.1 ml of phosphate-buffered saline. In parallel, 100-�l ali-quots of dilutions 10�4 and 10�5 of the cultures corresponding to both strainswere plated in duplicate on NA to estimate the numbers of cells injected into thefish. Two independent experiments were carried out, and LD50 determinationswere calculated by the method of Reed and Muench (35). All of the animalexperiments were conducted under the European legislation governing animalwelfare and were authorized and supervised by the Animal ExperimentationEthics Committee from the Oviedo University.

In vivo and in vitro competition assays. For in vivo competition assays, themutant and parental strains were grown separately in NB at 18°C in orbitalshakers at 250 rpm up to an OD600 of 0.5 (�108 cells/ml). Portions (2.5 ml) ofeach strain were mixed, and 10-fold dilutions of this suspension were plated ontoNA and NA with ampicillin to count the number of total CFU and mutant CFU,respectively. Based on these values, the exact input ratio of mutant to parentalstrains was calculated. A sample of 0.1 ml at dilution 10�2 (�106 cells of eachstrain/ml) was used to infect rainbow trout weighing from 8 to 10 g by intraper-itoneal injection. After 72 h, the fish were euthanized and, afterward, the spleens,livers, and intestines were recovered and homogenized in NB with a stomacher.Tenfold serial dilutions of the suspensions were plated onto selective media todetermine the output ratio of mutant to parental cells. The media used were NAand NA supplemented with ampicillin to count the number of total CFU andmutant CFU, respectively. From this, the output ratio of mutant to parental wascalculated. The competitive index (CI) is defined as the output ratio (mutant toparental) divided by the input ratio (mutant to parental).

For in vitro competition assays, 20 ml of NB was inoculated with 0.2 ml of thedilution 100 (108 cells of each strain/ml). The cultures were grown at 18°C up toan OD600 of 1.2. The input and output ratios of mutant strain to wild-type strainwere determined by selective plating as described for the in vivo competitionassay.

RT-PCR. Total RNA was obtained from 3-ml late-exponential-phase culturesof parental strain 150R and mutant 150RtraI grown in M9 at 18°C. RNA wasisolated by using an RNeasy minikit (Qiagen) and was treated with RNase-freeDNase (Ambion) to eliminate traces of DNA. Reverse transcription-PCR (RT-PCR) analyses were performed by using Superscript One-Step with Platinum Taq(Invitrogen Life Technologies); 40 ng of RNA was used in each reaction. ControlPCRs using Platinum Taq polymerase (Invitrogen Life Technologies) were per-

TABLE 2. Primers used for RT-PCRs

Oligonucleotide Sequence (5�–3�) Position(nt)a Gene

XII-oF CCCAGCTAGCCCGTCAA 247–264 traHXII-pR TGTGCCATATCGGTCGC 323–307 traIXII-pF GCGACCGATATGGCACA 307–323 traIXII-qR CGCAGACGTTCGCCTCG 320–304 traJXII-qF CGAGGCGAACGTCTGCG 304–320 traJXII-rR CACGACCGGAATACCCA 69–53 traKXII-rF TGGGTATTCCGGTCGTG 53–69 traKXII-sR CCAGCAGGTCGGCCATC 419–403 traCXII-tF GCACTGGCGGGAATGGC 3047–3063 traCXII-xR CCAATGACGCCGAAACG 178–162 traLXII-xF CGTTTCGGCGTCATTGG 162–178 traLXII-yR GAAGTGGGGTGTTGCTC 293–277 traMXII-yF GAGCAACACCCCACTTC 277–293 traMXII-zR GGAACAATATCGGCGGG 335–319 traN

a nt, nucleotides.

FIG. 1. Chromosomal arrangement of the region containing thetraHIJKCLMN genes in Y. ruckeri 150R. Arrows indicate the directionof the transcription. The organization of the transcriptional fusionbetween traI and the promoterless genes cat and lacZY in Y. ruckeri150RiviXII is shown underneath, and the putative promoter (P) se-lected by IVET is indicated. blaseq and catseq2 oligonucleotides wereused to sequence the adjacent fragments to the pIVET8 integrationsite. E, EcoRI sites; cat, chloramphenicol acetyltransferase gene (pro-moterless); lacZY, genes for lactose fermentation (promoterless); bla,ampicillin resistance gene.

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formed to determine whether RNA was free of contaminant DNA. The primersused are listed in Table 2.

PCR detection of traI, traC, and traN in different Y. ruckeri strains. Fifteen Y.ruckeri strains were studied in regard to the presence of traI, traC, and traN genes.PCR was performed using the primers traI-1 and traI-2 (previously described),the primers traC-1 (5�-CCACCGGCTCACATACC-3�) and traC-2 (5�-CCGGTTCGCTGCTGTTC-3�), and the primers traN-1 (5�-TGACCGGCTGGCATTTT-3�) and traN-2 (5�-CCTCAAAACGAACCTGT-3�). All PCR reagents wereprovided by Biotools. The amplification reactions were performed in a Perkin-Elmer 9700 GeneAmp thermocycler with an initial denaturation cycle at 94°C for5 min, followed by 25 cycles of amplification (denaturation at 94°C for 30 s,annealing at 48°C for 60 s, and extension at 72°C for 1 min), and a final 7-minelongation step at 72°C. The reaction products corresponding to the three groupswere mixed, and 1.5% agarose gel electrophoresis was used to separate thegenerated amplicons.

Nucleotide sequence accession number. The sequence accession number forthe genes in the GenBank database is EU828793.

RESULTS

Sequence analysis of the Y. ruckeri tra cluster. The applica-tion of IVET to Y. ruckeri allowed the selection of 14 clones,carrying in vivo-induced transcriptional fusions between Y. ruckeripromoters and the promoterless cat and lacZY genes (14). Se-quence analysis of the region surrounding the pIVET8 insertionsite in one of these ivi clones, iviXII, surprisingly revealed thepresence of a partial open reading frame (ORF) encoding aprotein with identity to TraI (58%), one of the components of thesystem Tra encoded by the S. entomophila virulence plasmidpADAP (26). In order to examine more closely the function ofthis system in Y. ruckeri, the region adjacent to this genetic locuswas sequenced. An 8,234-bp DNA segment containing eight ad-jacent ORFs oriented in the same direction was found. The de-duced amino acid sequences of these ORFs showed identities tothe proteins encoded by the tra region involved in the transfer ofsome plasmids, such as pADAP of S. entomophila (ranging from

35 to 58%), pDC3000A of Pseudomonas syringae (ranging from41 to 62%), pEL60 of Erwinia amylovora (ranging from 26 to53%), pCTX-M3 of Citrobacter freundii (ranging from 27 to 53%),ColIb-P9 of Shigella sonnei (ranging from 30 to 50%), and thechromosomal icm/dot genes of L. pneumophila (ranging from 27to 50%) or Coxiella burnetii (ranging from 23 to 34%). By analogywith the genes tra from S. entomophila, the eight ORFs from Y.ruckeri were designated traH-I-J-K-C-L-M-N, referred to heresimply as the tra operon or cluster (Fig. 1). The predicted bio-chemical properties of the tra operon products are similar to theones described for the same genes found in R64 plasmid de-scribed by Komano et al. (29).

The small size of the intergenic spaces and the absence ofputative promoter sequences within the tra cluster suggested thatit might be transcribed as a single unit. This is in agreement withthe presence of putative promoter, with �35 (5�-TAGTTA-3�),�10 (5�-TATATT-3�), and RBS (5�-AGGCGA-3�) sequencesupstream of traH and a stem-loop palindromic sequence locatedat the end of traN (data not shown). RT-PCR analysis confirmedthe prediction that the tra cluster genes form an operon. Theresults obtained with this analysis are shown in Fig. 2B. Fragmentscorresponding to the overlapping regions of the eight genes wereamplified when RNA from the parental strain was used, confirm-ing that all of the genes are cotranscribed. However, when RNAfrom the mutant Y. ruckeri 150RtraI strain was used, no mRNAcorresponding to the overlapping region traJ-traK was found (Fig.2C). This result confirms that the traI mutation has a polar effect.

The overall G�C content of this cluster, 53.6%, is slightlyhigher than the 47 to 48% reported for Y. ruckeri, indicating apossible horizontal gene transfer of this region. The geneticorganization of the tra genes of Y. ruckeri was almost identicalto the tra cluster of the S. entomophila pADAP plasmid(pADAP_08/pADAP_01) and similar to the ones present in C.freundii (pCTX-M3_053/pCTX-M3_061), Erwinia amylovora(pEL60p43/pEL60p50), and Pseudomonas syringae (PSPTOA0043/PSPTO A0052) (Fig. 3). The comparison of the flankingregions of the tra cluster of Y. ruckeri to those from S. ento-mophila, C. freundii, E. amylovora, and P. syringae hints at theirlikely evolutionary origin through horizontal gene transferamong these bacteria. Since the tra genes of S. entomophilapresented the same genetic organization as the cluster tra of Y.ruckeri and because of the significant level of similarity be-tween them, it is quite possible that the origin of the locus traof Y. ruckeri is related to that of the tra cluster of pADAP (Fig.3). In fact, a deletion of the traO-traG region in this plasmid,would explain the organization of the tra genes in Y. ruckeri(Fig. 3). The information collected from the data banks indi-cated that the cluster described here is not present in any of thethree human pathogenic Yersinia species.

Chromosomal localization of the tra operon in Y. ruckeri. Inorder to determine whether the tra cluster in Y. ruckeri 150Rwas located in the chromosome or a plasmid, both DNA typesof this strain were separated in agarose gels (Fig. 4A). Y.ruckeri 150R carried a large plasmid of 75 MDa and a smallerone of 12.7 MDa, as described for most Y. ruckeri strainsbelonging to serotype O1 (23). When a traI internal fragmentwas used as a probe for Southern blot analysis, there werehybridization marks in both chromosomal DNA and 75-MDaplasmid (Fig. 4B). It is important to emphasize that the tragenes are part of conjugal transfer systems and, therefore,

FIG. 2. Agarose gel electrophoresis of the RT-PCR amplificationproducts showing transcriptional organization. (A) The positions ofthe primers used within the tra genes of Y. ruckeri are indicated.(B) RT-PCR of Y. ruckeri 150R using the primers oF and pR (lane 2),pF and qR (lane 3), qF and rR (lane 4), rF and sR (lane 5), tF and xR(lane 6), xF and yR (lane 7), and yF and zR (lane 8). (C) RT-PCRusing RNA from Y. ruckeri 150R (lane 2) and 150RtraI (lane 3) usingthe oF and qR primers as a control for traI expression. Lane 4, RT-PCR of Y. ruckeri 150RtraI using qF and rR (overlapping regiontraJ-traK) to prove that the mutation has a polar effect. Lanes 9 (B) and5 (C), control reactions to assess DNA contamination in RNA prep-arations; lane 1 (B and C), the molecular weight marker correspondingto sizes ranging from 1,000 to 100 bp.

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there can be similar genes in conjugative plasmids. To confirmthe localization of the in vivo-induced tra cluster in Y. ruckeri150R, the chromosomal and plasmid DNA of the Y. ruckeri150RiviXII strain were separated by agarose gel electrophore-sis (Fig. 4C). Southern blotting was carried out with the blagene from pIVET8 as a probe and, in this case, only a hybrid-ization mark in the chromosomal DNA was obtained (Fig.4D). Since clone iviXII contains pIVET8 integrated inside thetraI coding sequence, this result showed that the tra genesdescribed here and selected by IVET are located in the chro-mosome.

Medium composition and temperature regulation of the traIpromoter. The Y. ruckeri 150RiviXII strain contains a transcrip-tional fusion between the promoter that controls the expres-sion of traI and the promoterless lacZY genes (Fig. 1). Thisclone was used to carry out studies on the regulation of theexpression of traI in response to different environmental con-

ditions. The �-galactosidase activity produced by the bacteriagrown in M9 at 18°C was considered to be 100%. When theincubation was carried out at 28°C, corresponding to the op-timal growth temperature, the �-galactosidase activity de-creased by 36% 1.92%. In addition, the influence of themedium composition on traI expression was also tested. In thiscase, the expression decreased by 35.5% 1.32% when themicroorganism was grown in the complex medium NB. Highconcentrations of NaCl (250 mM), MgCl2 (25 mM), or CaCl2(1.25 mM) did not influence the expression of traI (data notshown).

TraI mutation reduces Y. ruckeri virulence. A traI isogenicmutant strain (traI::pIVET8) was obtained by insertionalmutagenesis (Fig. 5A). The mutation was confirmed bySouthern blot analysis. After EcoRI digestion of totalgenomic DNA from the parental and mutant strains, thefragments obtained were hybridized with an internal frag-ment of traI gene. Whereas a single hybridization band of�15 kb appeared in the parental strain, two bands of ca. 11and 9.2 kb could be seen in the traI mutant (Fig. 5B). Sincethe plasmid pIVET8 has internal EcoRI sites (Fig. 5A),these patterns of hybridization show that a disruption of thetraI gene was obtained by insertion of this vector within thegenomic DNA. In conclusion, Southern blotting showed thatthe integration of the suicide vector containing the traIinternal fragment into the chromosome had occurred by asingle crossover event. The stability of the mutation in theabsence of ampicillin was checked by doing several passeson nonselective medium, followed by comparison of thenumber of cells able to grow with or without the antibiotic.This same experiment was carried out after every LD50

experiment in order to confirm that the bacteria recoveredfrom the fish had the mutation. In all cases, no significantreversion rate was observed. In addition, the ratio found inthe in vitro CI experiments when both strains (parental andmutant) were grown together was approximately 1, indicat-ing that growth of the mutant without antibiotic is similar tothat of the parental strain and also that no major reversionoccurred when that mutant strain was grown in the absenceof antibiotic.

As previously indicated, TraI of Y. ruckeri displays 30%identity to the DotC protein of L. pneumophila. Phenotypi-cally, the dotC mutant is described as salt resistant compared tothe parental strain (38). A similar result was found in the

FIG. 3. Genomic organization of the region surrounding the tra operon of Y. ruckeri150R and comparative analysis to that of S. entomophila(pADAP), C. freundii (pCTX-M3), E. amylovora (pEL60), and P. syringae pv. tomato (pDC3000B). Groups of genes are indicated by arrowsindicating the direction of transcription. The loci are designated with the letter corresponding to each gene. The Y. ruckeri 150R gene organizationlacks the traOPQRTUWXY, excB, trbABC, and traG loci present in S. entomophila, suggesting a deletion event. orf1, PSPTOA0046; orf2,PSPTOA0047, orf3, PSPTOA0061; orf4, PSPTOA0065.

FIG. 4. Analysis of the location of the tra operon in Y. ruckeri 150Rand Y. ruckeri 150RiviXII. Plasmid (lane 1) and chromosomal (lane 2)DNAs of Y. ruckeri 150R (A) and Y. ruckeri 150RiviXII (C) were sepa-rated by 0.75% agarose gel electrophoresis. The relative sizes of theplasmids of Y. ruckeri 150RiviXII were determined by using lambda PstI-digested DNA fragments as molecular markers (left lane) and from thelength of their migration compared to plasmids from Y. ruckeri 955 har-boring a large plasmid of 75 MDa and a smaller one of 15.5 MDa (23;data not shown). (B) Southern blot analysis of plasmid and chromosomalDNA from Y. ruckeri 150R using an internal fragment of traI as a probe.Hybridization marks appeared in both chromosomal DNA and the 75-MDa plasmid. (D) Southern blot analysis of plasmid and chromosomalDNA from Y. ruckeri 150RiviXII using an internal fragment of bla as aprobe. Only one hybridization mark appeared in chromosomal DNA.Lanes 1 and 2 correspond to plasmid and chromosomal DNAs, respec-tively. Please note that traces of chromosomal DNA were left in theplasmid extraction of Y. ruckeri 150RiviXII (lane 1, C and D).

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traI::pIVET8 strain of Y. ruckeri. Thus, ca. 10% more cells ofthe mutant strain than cells of the parental strain were recov-ered in NB plates supplemented with 250 mM NaCl.

The virulence of the traI::pIVET8 strain was first assessed inan in vivo competition assay in a rainbow trout model. A CI of0.2 0.07 was obtained after the coinoculation of 105 cells ofeach strain (parental and traI::pIVET8) in fish weighing 10 to15 g. This result shows that the mutant strain has significantlygreater difficulty growing inside the fish than did the parentalstrain. However, in vitro competition assays showed that themutant has no growth defect under optimal laboratory condi-tions, with an in vitro CI of 1.04 0.18. The means of the LD50

values obtained for the parental strain and mutant traI after 7days were 4.07 � 104 and 2.69 � 105 CFU per fish, respectively.Therefore, a slight but clear attenuation was found in themutant strain Y. ruckeri 150RtraI.

The tra genes are present in Y. ruckeri strains from differentorigins. PCR detection of traI, traC, and traN genes was carriedout with 15 Y. ruckeri strains from different origins. The resultsshowed, as can be observed in Fig. 6, that the three genes werepresent in all of the strains tested, which suggests that thiscluster is conserved in this species. This is especially significantbecause the 15 strains represented three different plasmid pro-files, and this fact thus strengthened the hypothesis of a chro-mosomal location.

DISCUSSION

In the present study, a cluster of eight genes involved invirulence was identified by analysis of a previously isolated

iviXII clone, the product of the application of IVET to Y.ruckeri (14). Nucleotide sequencing, the identities of the trans-lated products, and the genetic organization established thatthe tra cluster loci of Y. ruckeri are similar to the ones foundin plasmid pADAP of S. entomophila (26), pCTX-M3 of C.freundii (31), and pEL60 of E. amylovora (20). In fact, a colinear-ity and conservation of the sequence of the region surroundingthe tra cluster provided some hints about its possible evolution.

FIG. 5. Construction of traI isogenic mutant by insertional mutagenesis. (A) Homologous recombination between the 317-bp traI internalfragment from plasmid pTRA1 and the traI gene from the Y. ruckeri 150R chromosome resulted in insertional mutation of traI. Open rectangles,target traI gene; solid rectangles, interrupted traI gene. cat, chloramphenicol acetyltransferase gene; lacZY, �-galactosidase and galactosidepermease genes; bla, �-lactamase gene. (B) Southern blot analysis of the Y. ruckeri 150R mutated strain. Genomic DNA from the parental strainY. ruckeri 150R (lane 1) and mutant strain 150RtraI (lane 2) was digested with EcoRI and hybridized with the 317-bp internal traI fragment,previously labeled with digoxigenin as described in Materials and Methods. The hybridizing fragments are indicated with arrows as follows: A, Y.ruckeri 150R 15-kb EcoRI chromosome fragment flanking the traI gene; B and C, 11- and 9.2-kb EcoRI fragments, respectively, from the Y. ruckeri150RtraI chromosome containing the traI truncated gene.

FIG. 6. PCR detection of traC, traN, and traI genes from differentY. 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 as follows: traC(930 bp), traN (584 bp), and traI (317 bp). Lane 1, strain 146; lane 2,lane strain 147; lane 3, strain 148; lane 4, strain 149; lane 5, strain 150;lane 6, strain 955; lane 7, strain 956; lane 8, strain 35/85; lane 9, strain13/86; lane 10, strain 43/19; lane 11, strain A100; lane 12, strain A102;lane 13, strain 150/05; lane 14, strain 158/05; lane 15, strain 382/05; lane16, negative control. Flanking lanes, DNA molecular size markersfrom 1,000 to 100 bp.

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A deletion could explain the rearrangement undergone bythese regions from the pADAP plasmid to Y. ruckeri. Recently,the pYR1 plasmid, which contained a type IV conjugativetransfer system, has been described in Y. ruckeri (49). However,the conjugation genes contained in plasmid pYR1 did not showsignificant identity with the tra genes described here, and theirgenetic organization is completely different. A Y. enterocoliticaself-transmissible plasmid has been characterized (24), andgenomic analysis of Y. pestis (44) also revealed the existence oftype IV transfer systems in this species. In the case of Y.pseudotuberculosis IP31758, the etiological agent of Far Eastscarlet-like fever, Eppinger et al. (13) described a plasmid-borne putative type IVB secretion system related to the icm/dot system of L. pneumophila. However, the involvement invirulence of any of these T4SS systems has not been demon-strated thus far. In addition, they do not share a significantsimilarity with the in vivo-induced tra cluster from Y. ruckeri.These results stress again the uncertain taxonomic position ofY. ruckeri inside the genus Yersinia, as discussed by Bottone etal. (4).

Conjugative plasmids such as pCTX-M3 and pADAP canpromote horizontal gene transfer between bacteria. The dif-ference in G�C content between a particular gene clusterand the core genome can be a clue of this phenomenon. Thisseems to be the case of the Y. ruckeri tra operon, whoseG�C content is 53.6%, a value closer to the G�C content ofthe plasmid pADAP from S. entomophila (50%) andpCTX-M3 from C. freundii (51%) than to the 47% G�Ccontent of the Y. ruckeri genome. This, together with thesimilarities in genomic organization, suggests that Y. ruckeri,whose ecological niche is close to that of Serratia species,could have acquired this cluster from one of these speciesand eventually integrate it into the chromosome. This inte-gration event might be a necessary step toward its stabiliza-tion in the genome and could be the consequence of thepositive selection of virulence-related functions in thepathogen. For instance, in facultative intracellular bacteriasuch as Y. ruckeri this selection could be necessary for afunctional T4SS during the intracellular stage. Thus, muta-tions in the T4SS chromosomal genes in L. pneumophila,such as dotD, dotC, dotB, or icmT genes (51), counterpartsof the traH, traI, traJ, and traK genes, respectively, of Y.ruckeri, result in a defective intracellular growth. It shouldbe emphasized that the presence of other tra clusters har-bored by the plasmids present in Y. ruckeri 150 could not beexcluded. In fact, when Southern blot hybridization wascarried out with an internal fragment of traI as a probe, aband in the 75-MDa plasmid was found. However, all of theresults obtained in the present study only refer to the traoperon present in the chromosome.

The expression of the tra operon is transcriptionally reg-ulated by temperature and nutrient availability. Interest-ingly, as occurred in the case of other Y. ruckeri virulencefactors (15, 16), the tra operon is upregulated at 18°C, acondition that resembles that present in water when thebacterial cells are in contact with the fish and might initiatethe infection process. Although the differences found in thetra operon expression were not as high as in YhlA hemolysin(16), this result, together with those obtained for the pro-tease Yrp1 (17) and ruckerbactin production (14), clearly

establishes that temperature is an important environmentalfactor regulating the virulence of Y. ruckeri. tra cluster ex-pression was higher in minimal medium than in complexmedium, indicating that nutritional stress is a positive factorin its regulation. This result is reminiscent of the findings inother intracellular pathogenic bacteria such as Brucella suis(3) and L. pneumophila (5), in which nutritional limitationcould induce the secretion of effector molecules by T4SS tolyse the host cells. When these bacteria are in a rich intra-cellular environment, replication is the response. Althoughthe basis for the sodium resistance of dot mutants of L.pneumophila is still unknown, some researchers have sug-gested that the Dot-Icm protein complex may allow thediffusion of sodium into the cytoplasm of the L. pneumo-phila cells, resulting in a sodium-sensitive phenotype (22,38). The sodium-resistant phenotype of the traI mutant issimilar to the one described for L. pneumophila dot mutants.Therefore, the proteins from both microorganisms mighthave an identical functional effect. Nevertheless, NaCl didnot have any influence on the expression of the tra cluster,suggesting that the sodium resistance mechanism might notbe directly related to these loci but rather be an indirecteffect of the mutation.

Experiments carried out to determine the stability of the traIinsertional mutation showed that, under the assayed conditionsand with this particular construction, no reversion of the mu-tation occurred. These data provide enough support to con-clude that the results obtained with the mutant strain are validin terms of virulence and phenotypic properties. Taking thisinto account, it can be concluded that the tra operon is a newvirulence factor of this bacterium. First, it was initially identi-fied as an ivi gene. Second, a traI mutant strain was approxi-mately 10 times more attenuated than the parental strain and,finally, in vivo competition experiments showed a significantlylower recovery of the traI mutant strain. According to all of thecharacteristics of the chromosomal tra operon, it is tempting tospeculate that it could form part of a T4SS involved in thetransfer of virulence factors. Y. pseudotuberculosis harbors achromosomal pilLMNOPQRSUVW gene cluster involved inthe synthesis of type IV pilus that is regulated by environmen-tal factors and contributes to pathogenicity (10). Sequenceanalysis of the region flanking the tra cluster in Y. ruckeri,particularly upstream of the pilV gene, could help to locate thegenes coding for the synthesis of the pilus structure.

The tra operon is present in Y. ruckeri strains isolated fromdifferent outbreaks and locations, implying that these genes arelikely to play an important role in the biology and pathogenesisof this bacterium. This confirms that Y. ruckeri is a highlyhomogeneous species at the genetic level, as was previouslyindicated by different studies (4, 15).

In conclusion, a component of a T4SS, absent from humanpathogenic yersiniae, contributes to the virulence of Y. ruckeriand this, together with previously published data (4, 15),strongly supports the idea that the taxonomy of Y. ruckerishould be reconsidered, perhaps as a new genus within Entero-bacteriaceae. Further analyses are necessary to identify othercomponents of this T4SS to clarify its role in the environment,and especially during the infection process. In particular, thestudy of the intracellular stage of Y. ruckeri infection seems tobe more interesting in the light of this finding.

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ACKNOWLEDGMENTS

This study was supported by a grant from the MEC of Spain(AGL2006-07562). J.M. was the recipient of a grant from OviedoUniversity. A.M., P.R., and D.P.-P. were the recipients of grants fromthe Spanish Ministerio de Educacion y Ciencia.

We thank A. F. Brana for his help in keeping the laboratory equip-ment and facilities and ProAqua for providing some of the strains usedfor this study.

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