INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Rhodococcus equi is an important pulmonary pathogen of foals and is increasingly isolated from pneumonic infections and other infections in human immunodeficiency virus (HIV)-infected patients (19, 33). Isolates from foals possess a large virulence plasmid, varying in size from 80 to 90 kb (45, 47, 49). Isolates lacking the plasmid are avirulent to foals (16, 51). Little is known about the function of the plasmid apart from its encoding a virulence-associated surface protein (VapA) (45, 49), the presence of a family of four vap genes (5), and the origin of replication (53). Infection with R. equi bacteria carrying the virulence plasmid may lead to immunomodulation in foals by causing failure to mount an effective Th1-based cellular immune response, but the basis of this effect is undefined (17). The expression of VapA is thermoregulated (34°C) and pH regulated (41, 42), so that in this respect the plasmid has similarities to the virulence plasmids of pathogenic Yersinia species, such as Yersinia pestis, and of Shigella species (11, 22, 30). The plasmid is of significant interest, since it is associated with survival of the bacterium inside macrophages (16, 21, 33). Understanding its structure and function may therefore yield insights not only into the basis of virulence of this organism but also into the mechanisms of macrophage survival of other facultative intracellular pathogens, including Mycobacterium tuberculosis, a fellow member of the mycolata.

To better understand the structure and function of the virulence plasmid, we have completely sequenced plasmids from two isolates of R. equi that were recovered from pneumonic foals, ATCC 33701 and 103. These strains have been used previously for laboratory and experimental infection studies (16, 51), and both are of the 85-kb type I (29, 46). We sequenced both plasmids on the assumption that a comparative approach would lead to a better understanding of the structure and function of the plasmid and of individual genes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains plasmid isolation. The plasmids from R. equi ATCC 33701, a capsular serotype 1 prototype, and 103, a capsular serotype 6 isolate, were used; these are designated p33701 and p103. Escherichia coli strain XL1 Blue was used for plasmid or phage cloning.

DNA sequencing procedure. For p33701, shotgun cloning was carried out. Plasmid DNA was extracted from bacterial cultures as described previously (45). Plasmid DNA was recovered using CsCl-ethidium bromide gradients to eliminate chromosomal contamination and then partially digested with Sau3AI. Partially digested plasmid DNA was fractionated by 10 to 40% sucrose gradient centrifugation, and fractions containing 1- to 2-kbp fragments were collected. These were ligated into the BamHI site of pUC19 or pBluescript (Stratagene, La Jolla, Calif.). Then, E. coli DH5 was electrotransformed using GenePulserTM (Bio-Rad). Each clone was sequenced in both directions using dye-terminator-labeled fluorescent cycle sequencing prism reagents and ABI 373A automated sequencers (Applied Biosystem Division, Perkin-Elmer). The sequence data was analyzed using SEQUENCHER version 3 (Gene Codes). From a total of 1,078 sequence data, 23 unique fragments (contigs) were obtained. To determine the position of each contig on p33701, EcoRI fragments b to i and HindIII fragments b to d of p33701 were cloned in pUC19, and both ends of each were sequenced. The positions of some contigs were determined from partial sequences of the EcoRI and HindIII fragments. The sequences of gap regions between contigs were determined by primer walking using pUC19 containing EcoRI and HindIII fragments as templates. For p103, sequencing was initially of a library of the seven smallest EcoRI fragments (c to i) cloned in pBluescript. The 27.5-kb and 15.4-kb EcoRI a and b fragments were subcloned from the cosmid vector pLARF1 after isolation from agarose using the Lambda DASH II cloning kit (Stratagene). Further subcloning of the a fragment into pBluescript was done after digestion with EcoRI, BamHI, or HindIII, and DNA was sequenced from the three fragments produced. Sequencing was from either end of the fragments from the T3 and T7 primers in pBluescript or Lambda DASH II and, in the case of the a and b fragments, by primer walking using sequence data available from some individual contigs of p33701. p103 was sequenced in both directions using dye-terminator-labeled fluorescent cycle sequencing Prism reagents and the ABI 377 (Perkin-Elmer) automated sequencers (Guelph Molecular Supercentre, University of Guelph, Guelph, Canada). After all the fragments were sequenced, the sequence data were assembled and analyzed using GeneRunner3.04 (Hastings Software, Hudson, N.Y.). Other sequences were aligned and compared using the Wisconsin package 10.0 from the Genetics Computer Group (GCG). Sequence data from the two plasmids were compared after alignment using GeneRunner. Where there were missing or discrepant base pairs between the two plasmids, the sequence obtained in these areas was reexamined and the area was resequenced if required.

DNA sequence analysis and annotation. Open reading frames (ORFs) encoding at least 50 amino acids were identified using GeneRunner or GeneMark (24) programs to display start codons, stop codons, and codon usage statistics for each reading frame. The start codon farthest upstream was used to annotate the ORF start. Comparisons of the predicted protein sequence with protein sequences in the available databases and pairwise protein alignments were done using the program BLAST at the server of the National Center for Biotechnology Information at the National Library of Medicine (1). Known genes and putative functions were assigned for individual ORFs by inspection of the search output. Homologies were considered to be significant when at least 25% of the amino acids were identical for at least 50% of the protein in the database. When a putative protein did not show significant homology with any known proteins, we analyzed the upstream DNA for known ribosomal binding sites. Where relevant, multiple protein sequences were aligned by DNASTAR (DNASTAR Inc., Madison, Wis.) using the algorithm developed by Lipman et al. (23). DNASTAR was also used to construct a phylogeny tree. Direct and indirect repeats were also identified using DNASTAR. Protein sequences were also analyzed for functionally important motifs using PROSITE (3) and for functional domains using Pfam (38) and BLOCKS (20), using Internet-based protein database programs. For Fam and BLOCK, e values of 0.01 were regarded as significant, and for PROSITE normalized scores of 9.0 were considered significant.

GenBank nucleotide sequence accession numbers. The annotated sequence of p33701 was submitted to the DNA Databank of Japan under the accession number AP001204, and the sequence of p103 was submitted to GenBank under accession number AF116907.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

General description. p33701 (80,610 bp) and p103 (80,609 bp) are virtually identical plasmids. We describe here the main features of p33701, noting the few differences from p103. A genetic map of p33701 is shown in Fig. 1. Table 1 lists designated ORFs and their putative functions based on homology with genes of known function identified by BLAST search and by protein domain or motif database searches. Homology given in Table 1 is the percent amino acid homology between the virulence plasmid ORF and the entire gene in GenBank to which it is compared. The minor differences between the plasmids are described below.

View larger version (35K): [in this window] [in a new window]   FIG. 1.   Map of the R. equi virulence plasmid p33701. The circle shows orientation of the ORFs, with the direction of reading denoted by shading. Genes with a known or suspected putative function based on homology with genes of a known function in DNA databases are named (see Table 1).

Pathogenicity island and virulence-associated protein genes. A 27,536-bp region bounded by two transposon resolvases (ORF61 and ORF22) has the characteristics of a pathogenicity island because it is bounded by insertion elements, contains a block of apparently foreign genes (GC content, 60.8%, compared to 66.6% for the rest of the plasmid), and contains a tRNA gene (a lysyl-tRNA synthetase [lsr2] gene) (18, 26). This region contains vapA and six other related virulence-associated protein genes, discussed below. It is apparent that a Rhodococcus plasmid has at some stage acquired a pathogenicity island from a source of as yet unknown origin and that this region of the plasmid may be the virulence region. Based on homology with genes or protein families of known function and given the presence of this pathogenicity island, the remainder of the plasmid appears to be concerned with conjugation, replication, and partition functions, as discussed below. The discovery of a pathogenicity island is important, in part since it opens the way to more ready sequence analysis of the virulence region in other R. equi plasmids, including that of the intermediately virulent isolates (40, 43, 44).

View larger version (98K): [in this window] [in a new window]   FIG. 2.   Virulence-associated protein amino acid alignment of the seven Vap (VapA and VapC to VapH) proteins on p33701 and of VapB identified in an intermediately virulent R. equi isolate. Solid shading shows the consensus of the majority of amino acids and indicates the extensive homology in the C-terminal halves of these proteins. VapF has extensive homology in its C-terminal half, but this is out of frame with the other Vap's. Dash, no amino acid in this Vap protein.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   Phylogenetic tree of Vap proteins from p33701 and of VapB identified in an intermediately virulent R. equi isolate.

Regulatory genes in the pathogenicity island. Two ORFs with homology to previously described regulatory genes are present upstream of six of the seven vap genes in the virulence region. ORF4 has extensive homology with members of the LysR group of positive regulatory proteins, one of the characteristics of which is negative autoregulation of their own transcription. In Salmonella, the plasmid virulence (spv) genes are activated by the LysR-like transcription factor SpvR and by the alternative sigma factor RpoS in response to signals received during intracellular growth or as the bacteria enter the stationary growth phase. Chromosomally encoded DNA-binding proteins contribute to the control of spv expression (25). Since LysR proteins can be positive regulators of a regulon (6), it will be of interest to determine whether expression of the vap genes is regulated by this protein. It will also be interesting to examine the role of both plasmid and chromosomal genes in regulating the function of ORF4. ORF8 is a gene with extensive homology to the two-component regulator gene resD or phoP of Bacillus subtilis (52) and to many similar two-component regulator genes. The unusual feature of this regulator gene is that although it is homologous with the response regulator component of a two-component regulatory gene, no gene homologous to the sensor kinase component was identified. This characteristic is shared with a homologous glutamine synthetase glnR gene response regulator in S. coelicolor (52). If the response regulator is phosphorylated by a sensor kinase, the gene for this may be located on the chromosome. In B. subtilis, ResD activates global regulation of aerobic and anaerobic respiration (39). The position of this response regulator upstream of the vapA-vapD gene cluster suggests an important role for this regulator in controlling expression of vap genes. It may also be significant that the promoter of the mtrA gene of M. tuberculosis, a gene with which it shares significant homology, is activated on entry into macrophages (50).

Conjugation region. A second region of the plasmid appears to be concerned with conjugation functions, a complex process which involves many genes. For example, the conjugation regions of plasmids pGO1 of Staphylococcus aureus and pMRC01 of Lactococcus lactis consist of 14 and 16 genes, respectively (12, 27). By analogy, we suggest that the conjugation regions of p33701 and p103 include ORFs 23 to 40 (23106 to 54412), thus including ORF39, which has homology to a Streptomyces integral membrane protein which would probably be involved in conjugation, and ORF40, which has homology to a gene in the transfer gene complex of plasmid RP4.

Replication and partition functions. The remainder of the plasmid (ORF41 to ORF60; 55,914 to 75,827) appears to be devoted to replication and partition (Table 1). It contains the putative ori, a region of two 15-bp direct repeats and a 32-base pyrimidine run preceded by a 9-bp purine run, present within ORF56, which was identified during the development of an R. equi-E. coli plasmid shuttle vector (53). The origin of replication of the R. equi virulence plasmid appears to be a novel type, since with one exception there is no homology with genes involved in replication. The exception is a gene (ORF50) most homologous with the trbA gene of a Rhodococcus sp. plasmid, pSOX (10). The Rhodococcus sp. trbA gene is homologous to the broad-host-range plasmid RK2 trbA gene, which produces a protein which represses both plasmid replication and conjugative transfer. ORFs with homology to parA and parB genes are found in this replication and partition region, although parB is unexpectedly present inside the pathogenicity island. ORF56 was identified by protein domain analysis as a helix-turn-helix regulatory protein. ORF60 has minor (5%) amino acid homology with mycobacterial dnaB, a DnaB-like helicase function supported by protein domain analysis. It seems likely that the other, unknown ORFs in this large region are therefore also concerned with replication and partitioning functions, but further work will be required to confirm this supposition.

Insertion and bacteriophage elements. ORF53, which has significant homology to the mycobacteriophage TM4 gp82 protein, a protein of unknown function with homology to the putative excisionase protein gp 34.1 of mycobacteriophage D29 (13, 14), is present immediately adjacent to the origin of replication. An area with extensive homology to the mycobacteriophage TM4 gp17 protein (13, 14) occurs from position 51303 to 51671 within ORF36, an ORF homologous to an S. coelicolor putative peptidase and which, as described earlier, protein domain analysis identified as a protease with Gly-Gly endopeptidase activity. The only other bacteriophage element identified is a gene fragment with homology to bacteriophage T7 gene 7.7, present at 38,249 to 38,494. Apart from the pathogenicity island, the relative lack of transposable and bacteriophage elements in this plasmid is in contrast to the markedly mosaic nature of virulence plasmids in Y. pestis (22, 30).

Other elements. A series of two oligonucleotides of 16 and 21 bp at 76,493 and 76,517 is repeated at 76,975 and 76,999, immediately after ORF61.

Differences between the two plasmids. The product of ORF32 in p33701 was 825 amino acids in length, whereas in p103 it was 477 amino acids in length; both ORFs had the same start site. The shorter ORF32 in p103 more closely approximated the size of the homologous DNA topoisomerase of Salmonella enterica serovar Typhi. In addition to this difference, there are differences of seven base pair substitutions and six substitutions or deletions of 1 to 4 bp among the plasmids which do not affect the ORFs identified.

Conclusion. The nucleotide sequences of two virulence plasmids isolated from foal virulent R. equi provide important new information about the development of virulence functions in this plasmid and potential insight into virulence of this organism for foals. It will be important to understand the role of the series of Vap proteins in virulence and the factors which control their expression. The latter include the role of DNA supercoiling and of histone-like molecules, such as H-NS of chromosomal origin, in thermoregulation (2) and pH regulation of the vap genes. Foal virulent isolates contain virulence plasmids which can be classified into five closely related types based on restriction digestion patterns (29, 46). The two plasmids sequenced in this study belong to the 85-kb type 1 group. The basis of the additional DNA present in the 87- and 90-kb types remains to be determined; it may be the result of insertion of mobile genetic elements. Comparison of the virulence region of the plasmid recovered from foal isolates with that of the intermediately virulent, vapB-containing plasmid isolated from pigs and some AIDS patients will be of interest, since this may explain host-adapted virulence.