Veterinary Microbiology 94 (2003) 143–158

Partial genome sequencing of Rhodococcus equi ATCC 33701 M.T. Rahman a , L.L. Herron b , V. Kapur b , W.G. Meijer c , B.A. Byrne d , J. Ren a , V.M. Nicholson a , J.F. Prescott a,∗ b

a Department of Pathobiology, University of Guelph, Guelph, Ont., Canada N1G 2W1 Departments of Microbiology and Veterinary Pathobiology and Biomedical Genomics Center, University of Minnesota, Minneapolis, MN 55455, USA c Department of Industrial Microbiology, Conway Institute of Biomolecular and Biomedical Research and Dublin Molecular Medicine Center, University College Dublin, Belfield, Dublin 4, Ireland d Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN 47907, USA

Received 28 January 2003; accepted 20 March 2003

Abstract Preliminary analysis of a partial (30% coverage) genome sequence of Rhodococcus equi has revealed a number of important features. The most notable was the extent of the homology of genes identified with those of Mycobacterium tuberculosis. The similarities in the proportion of genes devoted to fatty acid degradation and to lipid biosynthesis was a striking but not surprising finding given the relatedness of these organisms and their success as intracellular pathogens. The rapid recent improvement in understanding of virulence in M. tuberculosis and other pathogenic mycobacteria has identified a large number of genes of putative or proven importance in virulence, homologs of many of which were also identified in R. equi. Although R. equi appears to have currently unique genes, and has important differences, its similarity to M. tuberculosis supports the need to understand the basis of virulence in this organism. The partial genome sequence will be a resource for workers interested in R. equi until such time as a full genome sequence has been characterized. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Rhodococcus equi; Genome sequence; Virulence

1. Introduction Rhodococcus equi is a Gram-positive, facultative intracellular pathogen that persists and multiplies within the macrophages of humans and animals. Largely soil saprophytes, the genus Rhodococcus is a member of the mycolata, bacteria with cell wall mycolic acids, ∗

Corresponding author. Tel.: +1-519-823-8800x4716; fax: +1-519-767-0809. E-mail address: [email protected] (J.F. Prescott). 0378-1135/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1135(03)00100-7

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that also include the genera Mycobacterium, Corynebacterium and Nocardia. R. equi is an important respiratory pathogen of foals (Prescott, 1991) and has emerged as a significant opportunistic pathogen in patients with AIDS (Harvey and Sunstrum, 1991). In both species the infection causes pyogranulomatous pneumonia. The genetic relatedness between R. equi and Mycobacterium tuberculosis is reflected in the similarities between the pathology of the disease. Understanding the detailed pathogenesis of R. equi may therefore contribute to understanding of the pathogenesis of tuberculosis. R. equi primarily infects alveolar macrophages of young foals and immunocompromised humans (Giguere et al., 1999). Following uptake by macrophages, virulent R. equi grow rapidly in the phagosome (Hondalus and Mosser, 1994). Virulence in foal and some human isolates is associated with the presence of 80–90 kb plasmids which encode a protein called VapA, whereas in pig and some human isolates, virulence is associated with 30–100 kb plasmids (reviewed by Takai, 1997) which encode a VapA homolog called VapB. The foal virulence plasmid is essential for virulence in mice and foals and for intracellular survival in murine and equine macrophages. Some isolates from immunocompromised humans and animals lack virulence-associated plasmids altogether, suggesting that virulence is not only plasmid associated (Takai et al., 1994). In isolates with virulence plasmids, virulence is likely to involve a complex interaction between plasmid and chromosomally encoded genes, including those controlling regulatory and metabolic pathways that allow the pathogen to thrive within the host. In recent years, the wealth of data provided by complete genome sequencing of important pathogenic microorganisms has generated unprecedented opportunities to understand microbial physiology, metabolism, nutrition, pathogenesis, and to reveal the genetic basis of microbial evolution and adaptation. The whole genome sequencing approach has proven to be the most efficient method available to date to understand the interaction of pathogen with its host and environment. We describe the results of a partial genome sequence of R. equi ATCC 33701, assessing the similarity to M. tuberculosis and identifying potential virulence genes and candidate immunogens that are of interest for further studies. The partial genome sequence will be an invaluable resource to workers interested in this organism and should stimulate efforts to obtain the complete sequence. 2. Materials and methods 2.1. Isolation of genomic DNA R. equi strain ATCC 33701 was used for sequencing. DNA was prepared by growth in trypticase soy broth (Difco, Detroit, MI, USA), sheared using the Hydroshear device (GeneMachines, San Carlos, CA, USA) and 1.8–2 kb fragments cloned into a SmaI digested pUC18 vector essentially as described (Herron et al., 2002). 2.2. Library analysis Plasmids were purified using the 96 Turbo Miniprep preparation, according to the protocol of the manufacturer (Qiagen, Valencia, CA, USA). Samples were transferred to new 96-well

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plates, dried overnight, and resuspended with 6 pmol of primer, then sequenced on ABI 3700 automated DNA sequencers (ABI, Foster City, CA, USA). All sequence similarity searches were performed using NCBI’s BLAST software. BLASTx (amino acid) homology searches for R. equi sequences was done in the public databases (GenBank) against all the available sequences of different species (Altschul et al., 1997). Each sequence is described as a read. The following criteria were used in homology searching. An e-value ≥−15 (e.g., e − 16) was considered significant and results recorded. Occasionally, an e-value ≤−15 (e.g., e − 13) was regarded as significant if visual inspection showed that only a small part of the gene was present in the sequence available (“read”), even though BLAST searches account for such “edge” effects (Altschul et al., 1997). All the genes that were identified from the sequence homology search were tabulated into major functional categories, which are freely available with the raw sequence reads, from the corresponding author. The raw data can be BLAST searched on http://pathogenomics.umn. edu/rhodococcus index.htm.

3. Results and discussion 3.1. Overview A genome library of 1.8–2.0 kb genomic fragments of R. equi ATCC 33701 was inserted into pUC18 and transformed into E. coli. The average sequence length for readable sequence was about 950 bp and the total number of unique bases characterized was about 1.34 Mb, representing about 31% of the estimated 4.4 Mb genome. In the absence of a complete sequence of, and a map of the genome organization of, R. equi conclusions deduced from the available data have to be tentative; nevertheless, important preliminary conclusions can be made, as discussed below. Of 1716 sequences analyzed, 1138 sequences (66%) showed significant homology with known or hypothetical genes in the public database and 279 (16%) had no significant homology to known or hypothetical genes currently in the public database (Fig. 1). An e-value ≥−15 is a moderately stringent assessment of homology; use of a less stringent criterion would have reduced the “no homology” grouping. Interestingly, about 16% of the M. tuberculosis genome consists of genes with no homology to other known genes (Cole et al., 1998). The remaining 299 sequences (17%) were unreadable, likely because the high guanine and cytosine content of the DNA led to repeated runs of identical nucleotides. The R. equi genome consists of about 66% guanine and cytosine nucleotides. Among the 1417 readable sequences obtained, 53 (3.7%) were of fragments of the 80.6 kb R. equi virulence plasmid, which has recently been sequenced (Takai et al., 2000). The most striking finding was that 799 sequences (47%) of the total sequences with homology to known or hypothetical genes in the GenBank database demonstrated homology to genes in M. tuberculosis, of which 571 (71%) have known function and 234 (29%) are hypothetical open reading frames (ORFs) with unknown function. Fig. 2 shows the comparison of the e-values of the gene sequences most closely related to M. tuberculosis compared to genes with higher homology (e-values) from other bacterial genera and species but which were also homologs of genes of M. tuberculosis. Fig. 2 illustrates the high homology between R. equi and M. tuberculosis for many genes. A total of 159 sequences (10%) had

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Fig. 1. Overview of R. equi partial genome.

highest homology with Streptomyces spp. with none to M. tuberculosis, and 127 (7%) had highest homology to a wide variety of other bacterial genera and species but none to M. tuberculosis (Fig. 3). No insertion sequence elements were identified but seven bacteriophage sequences related to Corynebacterium, Mycobacterium, Pseudomonas, and Streptomyces phage were identified. An association between R. equi bacteriophages, beta-lactam antibiotic resistance, and virulence has been suggested earlier (Nordmann et al., 1994).

Fig. 2. Level of e-value in homology searches, showing genes that had highest homology to M. tuberculosis compared with M. tuberculosis homologs which had higher homology to other bacterial genera and species.

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Fig. 3. Homology of R. equi genes to different bacterial genera or species. Besides M. tuberculosis and S. coelicolor, homologs of genes in other Streptomyces spp., Pseudomonas aeruginosa, Mezorhizobium loti, Rhodococcus erythropolis, Corynebacterium glutamicum, Mycobacterium leprae, Caulobacter spp. occurred with decreasing frequency. There were also homologs of genes in 47 other bacterial genera and species.

When a random sample of 30 adjacent genes on the R. equi genome was examined, 21 were also found to be adjacent on the M. tuberculosis Rv37 genome. This analysis was too limited to make any firm statements about gene organization. 3.2. Functional classification of genes in comparison to M. tuberculosis genome All the genes that were identified from the sequence homology search were tabulated into major functional categories (Cole et al., 1998). A visual comparison of the relative proportion of a broad functional classification of R. equi genes sequenced to those in the M. tuberculosis genome is shown in Figs. 4 and 5. 3.2.1. Small molecule metabolism 3.2.1.1. Metabolic pathways. Degradation of carbon, amino acids, and fatty acids: Genes for degradation of carbon compounds, in amino acid and amine degradation, and in fatty acid degradation include genes with high homology to those found in M. tuberculosis. M. tuberculosis possesses many genes involved in lipid degradation; 38 homologous genes were found in the R. equi partial genome sequence, a similar proportion to those in the M. tuberculosis genome. These include acyl-CoA synthases, enoyl-CoA hydratases and isomerases, and acetyl-CoA C-acetyltransferases. Genes encoding enzymes of beta-oxidation like fadA and fadB series were also recorded. The catabolism of lipids, including those of macrophage membranes, by M. tuberculosis produces acetyl-CoA, which is subsequently used in the citric acid cycle and glyoxylate shunt. Glyoxylate pathway mutants of

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Fig. 4. Comparison of functional categories of R. equi and M. tuberculosis genome. CCD: carbon compound degradation; AAAD: amino acid and amine degradation; FAD: fatty acid degradation; EM: energy metabolism; OXD: oxidoreductase; CIM: central intermediate metabolism; AAB: amino acid biosynthesis; BCP: biosynthesis of cofactors and prosthetic groups; RET: respiration and electron transport; PPNN: purines, pyramidines, nucleosides and nucleotides; PE and PPE: PE and PPE family proteins; LB: lipid biosynthesis; PNRPS: polyketide and non ribosomal peptide synthesis; BRF: broad regulatory functions.

M. tuberculosis have markedly impaired virulence (McKinney et al., 2000). Lipids including those of macrophage membranes are likely also to be a major source of energy for R. equi. Interestingly, the macrophage-induced gene (mig) of Mycobacterium avium is a medium-chain acyl-CoA synthetase (Morsczeck et al., 2001), emphasizing the importance of fatty acids in the survival of this and likely related organisms in macrophages. Additionally, two homologs of fadB2 and fadD13, the fatty acid degradation M. tuberculosis genes induced under acid shock (Fisher et al., 2002), were identified in R. equi. A cholesterol oxidase gene, choE, of R. equi has been characterized by Navas et al. (2001); we identified an additional cholesterol oxidase, choD. Energy metabolism: Genes were identified for enzymes involved in energy metabolism through glycolysis, in pyruvate dehydrogenase activity, the pentose phosphate-, the TCA-and glyoxylate-cycles. The R. equi isocitrate lyase (aceA) gene which has been sequenced earlier is organized in an operon together with fadB2, an arrangement similar to that of M. tuberculosis (Kelly et al., 2002); we identified the next gene in the by-pass pathway, encoding malate synthetase (glcB in M. tuberculosis). We identified only 19 miscellaneous oxidoreductases, proportionately less than the 171 predicted in the M. tuberculosis genome; a complete genome sequence is required to confirm this paucity before attempting to assess its significance.

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Fig. 5. Comparison of functional categories of R. equi and M. tuberculosis genome. RPS: ribosomal peptide synthesis; ATR: aminoacyl t-RNA synthesis; DRRR: DNA replication, repair and recombination; RSM: RNA synthesis and modification; DM: degradation of macromolecules; LP: lipoproteins; OMP: other membrane proteins; TBP: transport and binding proteins; HSP: heat shock proteins; CD: cell division; AAC: adaptations and atypical conditions; VIR: virulence; APR: antibiotic production and resistance; HP: hypothetical proteins.

Like M. tuberculosis, R. equi possesses genes involved in both aerobic and anaerobic respiration. Under aerobic growth conditions, R. equi generates ATP through oxidative phosphorylation through genes such as ctaC, ctaD, ctaE, nuoC, nuoD, and nuoF that were identified in the current study. Although R. equi is an obligate aerobe, its genome contains components of anaerobic phosphorylative electron transport chains homologous to those of M. tuberculosis, including genes we identified for nitrate reductase (narG) and nitrite reductase (nirB). It has been hypothesized that mycobacterial granulomas are low in oxygen. In addition, and perhaps more importantly, the phagosomes of activated macrophages have lower oxygen tension than those of unstimulated macrophages (James et al., 1995). A narG mutant of Mycobacterium bovis BCG was markedly less virulent in immunodeficient SCID mice than the parent strain, possibly because the mutant was unable to exploit nitrate as a source of oxygen (Weber et al., 2000). A further nitroreductase gene has been described in M. tuberculosis, the expression of which is co-regulated by the heat shock protein gene acr (hspX) which is produced under low oxygen conditions and in standing versus shaking cultures (Purkayastha et al., 2002). Only four R. equi proteins (bacterioferritin, bfpB, the phosphodiesterase glpQ1, Rv1739c, Rv3908) were homologs of the 47 proteins of M. tuberculosis H37Rv expressed under low oxygen conditions (Sherman et al., 2001). The paucity of these genes in R. equi in comparison to M. tuberculosis (many of which are hypothetical genes) may suggest that surviving hypoxia is less important in rhodococcal infections than in tuberculosis. However, this is speculative since only a partial sequence was determined. In addition, the virulence plasmid possesses a homolog of a two-component regulatory gene,

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orf 8, with homology to ResD of Bacillus subtilis, which regulates expression of a series of genes induced under conditions of low oxygen (Nakano et al., 1996; Takai et al., 2000). Other metabolic pathways: Forty-four genes associated with amino acid biosynthesis were identified, and 27 associated with purine, pyrimidine, nucleoside and nucleotide biosynthesis and metabolism. A glutamine synthase gene, glnA1, with high homology to that of M. tuberculosis was identified. In M. tuberculosis, this gene is involved in resistance to killing by human macrophages (Miller and Shinnick, 2000). The gene in M. tuberculosis catalyzes the extracellular synthesis of poly-l-glutamine, an important cell wall component produced and released in large amounts into the extracellular environment (together with superoxide dismutase) though a bacterial leakage process (Tullius et al., 2001). It may also be involved in altering the ammonia level (and pH) of the phagosome (Tullius et al., 2001). The glnA gene has been identified as a target of novel antimicrobial therapy for M. tuberculosis (Harth and Horwitz, 1999; Harth et al., 2000). Among the genes involved in biosynthesis of cofactors, prosthetic groups and carriers, genes homologous to M. tuberculosis included a number involved in biotin, folic acid, pantothenate, pyridoxine, thiamine, riboflavin, thioredoxin, menaquinone, heme and cobalamine synthesis. Lipid biosynthesis: R. equi possesses the unique lipid-rich cell envelope of the mycolata (Sutcliffe, 1997); the lipoarabinomannam of R. equi has recently been characterized (Garton et al., 2002). As expected, genes involved in fatty acid and mycolic synthesis and modification (n = 17) were identified, in slightly lower proportion than in the M. tuberculosis genome (Fig. 4). An fbpB gene homolog of the mycolyltransferase gene, antigen 85, of M. tuberculosis was identified. The antigen 85 complex in M. tuberculosis are exported immunogenic proteins involved in fibronectin binding (Armitige et al., 2000). A marked increase in expression of fbpB occurs in M. tuberculosis early after infection in human monocytes that correlates with TNF␣ expression (Wilkinson et al., 2001) although, unlike fbpA, the gene is not important for intra-macrophage growth of the M. tuberculosis (Armitige et al., 2001). Polyketides: Several genes homologous to those involved in mycobactin synthesis in M. tuberculosis were identified; these are discussed below under iron acquisition. Only one gene homolog of a M. tuberculosis polyketide synthase gene was recognized. Drug resistance: Like M. tuberculosis, the hydrophobic cell envelope makes R. equi resistant to the entry of many antibiotics. No beta-lactamase enzymes or aminoglycoside acetyl transferase genes were identified, but potential drug-efflux systems included 12 efflux protein genes and at least 25 ABC transporter proteins, some of which might be involved in drug resistance, genes among the approximately 40 membrane protein genes identified. Broad regulatory functions: A wide variety of regulatory components were detected in the genome, among them partial or complete genes for 64 repressor/activators, 14 two-component systems and a serine/threonine protein kinase. Like M. tuberculosis, the presence of these regulatory proteins governs the ability of R. equi to adapt to the very different environments in soil and inside different host macrophages. R. equi may therefore have more two-component regulatory systems than the 11 complete pairs found in M. tuberculosis (Cole et al., 1998), but this remains to be shown. The two-component regulatory genes identified are discussed below under virulence. Sigma factors are discussed below.

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3.2.2. Macromolecule metabolism Among identified genes involved in synthesis and modification of macromolecules, genes involved in ribosomal protein synthesis and modification, aminoacyl tRNA synthesis, DNA replication, repair and recombination, protein translation and modification, RNA synthesis, modification and DNA transcription were identified in similar proportion to those in M. tuberculosis (Fig. 5). Genes involved in degradation of macromolecules were also in similar proportion to those in M. tuberculosis. Five putative sigma factors, including the alternative sigma factor sigE, were identified. In M. tuberculosis, sigE has been shown to be important in the ability of this organism to survive oxidative stresses and to grow in macrophages (Manganelli et al., 2001). In addition, in M. tuberculosis it controls the expression of 41 genes, of which 9 homologs were identified in R. equi (cspA, ctaE, fadD, fadD29, fbpB, qcrA, qcrB, sodA, Rv2927c) (Manganelli et al., 2001). In M. tuberculosis, sigE, as well as the heat shock genes dnaK and clpB (see below) are regulated by the alternative sigma factor SigH (Raman et al., 2001). Only three cell envelope lipoprotein homologs were identified, a strikingly lower proportion than expected compared to the 65 identified in M. tuberculosis. A complete sequence is required before attempting to determine the significance of this observation. 3.2.3. Cell processes Genes for transport or binding proteins were found in similar proportion to those in the M. tuberculosis genome. Six homologs of chaperonins and heat shock proteins (clpB, dnaJ, groEL1, groEL2, hspR, htrA) were identified. Expression of clpB is regulated by SigH (Raman et al., 2001). In Salmonella typhimurium, sigE regulates the expression of the shock-induced serine protease, htrA (Humphreys et al., 1999) a homolog of which was identified. In M. tuberculosis, htrA may also be part of an operon with sigE (Jensen-Cain and Quinn, 2001). This protein has been shown to be essential for replication in macrophages by Brucella abortus (Elzer et al., 1996), Legionella pneumophila (Pedersen et al., 2001), and S. typhimurium (Baumler et al., 1994). HtrA assists in the degradation in the periplasm of denatured or degraded proteins produced when bacteria encounter toxic environments. HtrA mutants have acted as vaccines in S. typhimurium and Yersinia enterocolitica (Pallen and Wren, 1997). In Brucella suis, clpB is induced under high temperature stress conditions and mutants are more sensitive to high temperature, ethanol and acid pH (Ekaza et al., 2001). clpA and clpB double mutants of B. suis were more susceptible than the parent strain to hydrogen peroxide killing (Ekaza et al., 2001). Notable detoxification genes with high homology to the same gene in M. tuberculosis were two superoxide dismutase genes, sodA and sodC. SodA is a ubiquitous metalloenzyme in aerobic bacteria that detoxifies superoxide anions and contributes to the virulence of a number of intracellular pathogens. M. tuberculosis is unusual in secreting large quantities of the iron-cofactored SodA (Tullius et al., 2001). SodA attenuated strains of M. tuberculosis were more susceptible than the parent to hydrogen peroxide killing and were markedly attenuated in virulence for mice (Edwards et al., 2001). It appears to prevent the early elimination of the organism by innate immune defenses. In enteric bacteria, SOD is part of the SoxRS regulon, which does not appear to be active in mycobacteria (Shiloh and Nathan, 2000); a putative soxR-like transcriptional regulator homolog was however identified in R. equi.

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3.2.4. Miscellaneous other findings A homolog of a M. tuberculosis hypothetical gene (Rv0365c) identified as involved in mycobacterial resistance to killing by human macrophages (Miller and Shinnick, 2001) was identified; unusually, this homolog occurred three times. A homolog of a hypothetical protein of M. tuberculosis (Rv2466c) regulated by SigH was identified (Raman et al., 2001). In M. tuberculosis, SigH also regulates expression of thioredoxin, trxC, a homolog of which was found in R. equi; thioredoxin is involved in resistance of M. tuberculosis and other to oxidative stress (Raman et al., 2001). In E. coli, trxC is also part of the OxyR regulon (Ritz et al., 2000). A purC homolog was identified; this gene is essential for virulence in M. tuberculosis and mutants have been shown to have potential as vaccines (Jackson et al., 1999). One phenolphthiocerol gene homolog (ppsE) was identified; these surface-exposed unusual lipids were thought to be unique to slow-growing pathogenic mycobacteria (Azad et al., 1997). 3.2.5. Differences from the M. tuberculosis genome Despite similarities, there were differences apparent between the genome of M. tuberculosis and R. equi. Most notably, genes homologous to two large unrelated families of acidic, glycine-rich proteins, the PE family (genes with a highly homologous sequence and a ProGlu [PE] signature amino acid sequence near the amino terminus) and PPE family (genes with highly homologous sequence and almost invariable presence of Pro-Pro-Glu [PPE] signature motif at the amino acid terminal), were not found. In M. tuberculosis 167 genes, about 10% of the coding capacity of the genome, belong to the PE and PPE family. Among other cell envelope proteins, no homolog to the 17 mmpL series of conserved M. tuberculosis membrane proteins was found. These results may represent a true absence or under-representation of these genes in R. equi as compared to M. tuberculosis, or may have been missed by chance alone because of the relatively low (∼0.3-fold) coverage of the R. equi genome. 3.3. Streptomyces spp. related genes One hundred and fifty nine R. equi genes were most homologous with genes and hypothetical genes of Streptomyces spp. in the public databases, with no homology to M. tuberculosis. Like Streptomyces spp., R. equi is a soil saprophyte and member of the order Actinomycetales. One hundred and seven gene sequences had highest homology to S. coelicolor and the remaining were homologs of other Streptomyces spp. Most were unnamed but had putative functions; 39 were hypothetical genes. Among the genes identified, there was a miscellany of genes involved in small molecule and macromolecule metabolism. The most interesting finding was in genes involved in polyketide and non-ribosomally synthesized peptide production. The synthesis of this class of compounds is directed by large multifunctional enzymes termed polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) and the corresponding biosynthetic genes have been isolated from a variety of fungi and bacteria, including actinomycetes (Stachelhaus et al., 1996; Stein and Vater, 1996). Ten genes were identified with these functions. One homolog of papA was identified, which is synthesized by S. pristinaespiralis and previously shown to encode one of the pristinamycin I (PI) synthetases (Blanc et al., 1997).

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The presence of these genes indicates that R. equiproduces a pristinamycin-related antibiotic. Parts of 5 two-component system and 15 response regulators homologs of those of S. coelicolor were identified. Six S. coelicolor homologs related with iron utilization, of which four genes are associated with siderophore transport, were also identified. 3.4. Other bacterial genera One hundred and twenty seven R. equi genes were most homologous with genes and hypothetical genes of a wide variety of other (usually) bacterial genera and species other than M. tuberculosis and Streptomyces spp, indicating that these were acquired through lateral gene transfer (Fig. 3). These genes had a wide miscellany of assigned functions. An additional cholesterol oxidase homolog most closely related to a gene of Pimelobacter simplex was identified. Thirteen genes were homologs to repressors or activators, 15 were ABC transporter homologs, and 10 were involved in iron utilization. 3.5. Virulence 3.5.1. Iron acquisition The R. equi genome has a large number of genes homologous to those involved in iron acquisition in other pathogens, including ideR (Boland and Meijer, 2000). We have identified furA, four mycobactin gene homologs (mbtA, mbtE, mbtF, mtbH), eight putative iron-siderophore ABC transporter protein genes, three other presumed iron transport protein genes, and four homologs of genes involved in siderophore formation in non-mycobacterial genera. Expression of selected genes on the pathogenicity island of the R. equi virulence plasmid are partially controlled by iron restriction (Ren and Prescott, 2003). Fur-like proteins are transcriptional repressors that regulate genes involved in iron metabolism through iron-dependent binding to DNA. Iron metabolism and oxidative stress are closely linked. In Escherichia coli, regulators involved in oxidative stress, OxyR and SoxRS, activate Fur, which is the global repressor for ferric iron uptake (Milano et al., 2001). In M. tuberculosis, furA is adjacent to katG, a catalase-peroxidase and major virulence factor that also activates the drug isoniazid (Cole et al., 1998). FurA regulates katG in M. tuberculosis as well as other genes involved in pathogenesis (Pym et al., 2001). In M. tuberculosis, the oxyR gene, the equivalent of the main regulator of the E. coli oxidative response stress response, is inactivated (Pagan-Ramos et al., 1998). Since M. tuberculosis lacks the oxidative response regulator OxyR, FurA may be a major regulator of the ability of the organism to survive under conditions of oxidative stress (Zahrt et al., 2001). The furA–katG linkage and arrangement, ubiquitous in mycobacteria, is reproduced in R. equi since there is a catalase gene, most homologous to one in B. abortus, immediately downstream of the furA homolog. The resistance of M. tuberculosis to oxidative killing is a function of activity of both a catalase-peroxidase (KatG) and an alkyl hydroperoxide reductase (AhpA) (Manca et al., 1999). In M. tuberculosis, katG is induced under iron restriction independently of IdeR; other genes so induced include a variety of NADH dehydrogenases (Rodriguez et al., 2002). We identified two homologs of these M. tuberculosis genes (nuoC, nuoD). In M. tuberculosis, furA and katG are co-transcribed from the furA promoter that is induced, along

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with a separate katG promoter, by exposure to hydrogen peroxide (Master et al., 2001). Interestingly, hydrogen peroxide triggers induction of virulence-associated proteins on the virulence plasmid of R. equi (Benoit et al., 2002). Mycobactin siderophores are essential for growth of M. tuberculosis in macrophages, since mutation in mbtB impairs growth (De Voss et al., 2000). Both mbtA, a M. tuberculosis homolog we identified, and mbtB have promoter regions controlled by IdeR (Gold et al., 2001). Other mycobactin homolog genes we identified (mbtE, mbtF, mtbH) also appear to be IdeR controlled in M. tuberculosis (Rodriguez et al., 2002). An exochelin and a 2,3-dihydroxybenzoate siderophore gene (dhbF) homolog were also identified. Several genes involved in heme metabolism were identified, including hemZ, a ferrochelatase. A ferrochelatase, hemH, was shown to be critical for the intracellular survival and virulence of B. abortus (Almiron et al., 2001). 3.5.2. Two-component regulatory systems and virulence PhoP–PhoQ: Expression of selected genes on the pathogenicity island of the R. equi virulence plasmid are partially controlled by magnesium restriction (Ren and Prescott, 2003). In M. tuberculosis, growth in a defined medium was restricted by acid pH and magnesium levels, with growth of the organism even in a mildly acid environment being dependent on the presence of sufficient Mg2+ (Piddington et al., 2000). PhoP–PhoQ is a two-component system that mediates the adaptation of bacteria to Mg2+ -limiting environments and regulates virulence in a number of gram-negative intracellular pathogens (Groisman, 2001). It also has an essential role in virulence in M. tuberculosis since a mutant showed reduced multiplication inside macrophages and impaired growth in a mouse infection model (Perez et al., 2001). A gene with high homology to the M. tuberculosis phoP was identified in the R. equi partial genome sequence. Other two-component regulatory systems: M. tuberculosis has at least 12 two-component regulatory systems, but their function and the signals sensed by them are not known. We identified a SenX3–RegX3, KdpD sensor kinase, and six sensor histidine kinases homologs of M. tuberculosis genes, as well as four putative two-component genes from Streptomyces spp. KdpP was detected using the selective capture of transcribed sequences (SCOTS) approach as up-regulated in M. avium growing in macrophages (Hou et al., 2002). 3.5.3. Acid and virulence Acid pH has been shown to induce expression of genes on the pathogenicity island of the R. equi virulence plasmid (Takai et al., 1996; Benoit et al., 2001; Ren and Prescott, 2003). Microarray analysis of the transcriptional response of M. tuberculosis genes to acidic pH identified 24 genes that were induced by short exposure to acid (Fisher et al., 2002). Homologs to four of these genes were identified in the partial R. equi genome sequence (fadB2, fadD13, Rv1245c, Rv3083). R. equi, like S. typhimurium, shows acid tolerance since it can adapt to acid conditions (Benoit et al., 2001). Genes involved in the acid tolerance response of S. typhimurium include fur and phoPQ (Foster, 1995). 3.5.4. Other virulence genes Four genes homologous to those in the M. tuberculosis mammalian cell entry (mce) operon (mce1, mce3, mce4) were identified; the M. tuberculosis mce operon confers on

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non-pathogenic E. coli the ability to invade and survive within macrophages and Hela cells (Arruda et al., 1993). Fifty nine genes expressed in macrophages by M. avium were detected by the SCOTS technique (Hou et al., 2002); the R. equi partial genome sequence contains six of these (homologs of Rv1292, Rv2714, kdpP, mbtE, mbtF, nirB, as well as related mce operon genes).

4. Conclusions Obtaining a complete genome sequence of this organism would be invaluable to support research on R. equi and related organisms. In the meantime, a large number of genes have been identified which can be investigated for their role in the virulence of this organism.

Acknowledgements This work was supported by Natural Sciences and Engineering Research Council of Canada and by the Ontario Ministry of Agriculture and Food.

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