A chromosomally located traHIJKCLMN operon encoding a putative type IV secretion system is involved in the virulence of Yersinia ruckeri

Abstract

Nucleotide sequence analysis of the region surrounding the pIVET8 insertion site in Yersinia ruckeri 150RiviXII, 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 in sequence and organization to the tra operon cluster found in the virulence-related plasmid pADAP from Serratia entomophila. Interestingly, the tra cluster of Y. ruckeri is chromosomally encoded, and no similar tra cluster has been identified yet in the genomic analysis of human pathogenic yersiniae. A traI insertional mutant was 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 this bacterium. All of these results suggest the implication of the tra cluster in a virulence-related type IV secretion/transfer system. Reverse transcriptase PCR studies showed that this cluster is transcribed as an operon from a putative promoter located upstream of traH and that the mutation of traI had a polar effect. A traI::lacZY transcriptional fusion displayed higher expression levels at 18 degrees C, the temperature of occurrence of the disease, and under nutrient-limiting conditions. PCR detection analysis indicated that the tra cluster is present in 15 Y. ruckeri strains from different origins and with different plasmid profiles. The results obtained in the present study support the conclusion, already suggested by different authors, that Y. ruckeri is a very homogeneous species that is quite different from the other members of the genus Yersinia.

FIG. 1.

Chromosomal arrangement of the region containing the traHIJKCLMN genes in Y. ruckeri 150R. Arrows indicate the direction of the transcription. The organization of the transcriptional fusion between traI and the promoterless genes cat and lacZY in Y. ruckeri 150RiviXII is shown underneath, and the putative promoter (P) selected by IVET is indicated. blaseq and catseq2 oligonucleotides were used to sequence the adjacent fragments to the pIVET8 integration site. E, EcoRI sites; cat, chloramphenicol acetyltransferase gene (promoterless); lacZY, genes for lactose fermentation (promoterless); bla, ampicillin resistance gene.

FIG. 2.

Agarose gel electrophoresis of the RT-PCR amplification products showing transcriptional organization. (A) The positions of the 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-PCR using RNA from Y. ruckeri 150R (lane 2) and 150RtraI (lane 3) using the oF and qR primers as a control for traI expression. Lane 4, RT-PCR of Y. ruckeri 150RtraI using qF and rR (overlapping region traJ-traK) to prove that the mutation has a polar effect. Lanes 9 (B) and 5 (C), control reactions to assess DNA contamination in RNA preparations; lane 1 (B and C), the molecular weight marker corresponding to sizes ranging from 1,000 to 100 bp.

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 arrows indicating the direction of transcription. The loci are designated with the letter corresponding to each gene. The Y. ruckeri 150R gene organization lacks 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 150R and Y. ruckeri 150RiviXII. Plasmid (lane 1) and chromosomal (lane 2) DNAs of Y. ruckeri 150R (A) and Y. ruckeri 150RiviXII (C) were separated by 0.75% agarose gel electrophoresis. The relative sizes of the plasmids of Y. ruckeri 150RiviXII were determined by using lambda PstI-digested DNA fragments as molecular markers (left lane) and from the length of their migration compared to plasmids from Y. ruckeri 955 harboring a large plasmid of 75 MDa and a smaller one of 15.5 MDa (; data not shown). (B) Southern blot analysis of plasmid and chromosomal DNA 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 chromosomal DNA from Y. ruckeri 150RiviXII using an internal fragment of bla as a probe. Only one hybridization mark appeared in chromosomal DNA. Lanes 1 and 2 correspond to plasmid and chromosomal DNAs, respectively. Please note that traces of chromosomal DNA were left in the plasmid extraction of Y. ruckeri 150RiviXII (lane 1, C and D).

FIG. 5.

Construction of traI isogenic mutant by insertional mutagenesis. (A) Homologous recombination between the 317-bp traI internal fragment 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, β-galactosidase and galactoside permease genes; bla, β-lactamase gene. (B) Southern blot analysis of the Y. ruckeri 150R mutated strain. Genomic DNA from the parental strain Y. 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. ruckeri 150RtraI chromosome containing the traI truncated gene.

FIG. 6.

PCR detection of traC, traN, and traI 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 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, strain 13/86; 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; lane 16, negative control. Flanking lanes, DNA molecular size markers from 1,000 to 100 bp.