The virulence of the intracellular pathogen Rhodococcus equi in foals is dependent on the presence of an 81-kb virulence plasmid encoding the virulence protein VapA. Expression of this protein is induced by exposure to oxidative stress, high temperatures, and low pHs, which reflect the conditions encountered by R. equi when it enters the host environment. The aim of this study was to determine whether the LysR-type transcriptional regulator VirR, which is encoded by the virulence plasmid, is required for the expression of vapA. It was shown that the virR gene is cotranscribed with four downstream genes, one of which encodes a two-component response regulator. The expression of VapA, as monitored by Western blotting, was completely dependent on the presence of virR. Maximal expression was observed when vapA was present together with the complete virR operon, suggesting that at least one of the virR operon genes, in addition to virR, is required for the expression of vapA to wild-type levels. The transcriptional start site of vapA was determined to be a cytidine located 226 bp upstream from the vapA initiation codon. His-tagged VirR protein was expressed in Escherichia coli and purified by nickel affinity chromatography. DNA binding studies showed that purified VirR binds to a DNA fragment containing the vapA promoter. We therefore conclude that VirR is required for the activation of vapA transcription.The gram-positive bacterium Rhodococcus equi is a facultative intracellular pathogen of alveolar macrophages. Although young foals are the primary host of this pathogen, the incidence of R. equi infection in immunocompromised humans has increased markedly over the past 15 years (9,23,46). Infection with R. equi leads to life-threatening pyogranulomatous pneumonia accompanied by gross lesions such as macroabscesses and cavitation (32). The virulence of R. equi in foals is dependent on an indigenous plasmid, which varies in size between 80 and 85 kb (40,42). Plasmid-cured strains are unable to proliferate in macrophages (12,17). A recent analysis of the nucleotide sequences of two virulence plasmids revealed the presence of a 27.5-kb DNA fragment characterized by a significantly lower GϩC content than the remainder of the virulence plasmid (39). The expression of genes located within this region of the virulence plasmid is upregulated following the internalization of R. equi by macrophages, suggesting that this part of the plasmid is a pathogenicity island (33

The virR operon, located on the virulence plasmid of the intracellular pathogen Rhodococcus equi, contains five genes, two of which (virR and orf8) encode transcriptional regulators. The first gene of the operon (virR), encoding a LysR-type transcriptional regulator, is transcribed at a constitutive low level, whereas the four downstream genes are induced by low pH and high growth temperature. Differential regulation of the virR operon genes could not be explained by differential mRNA stability, as there were no major differences in mRNA half-lives of the transcripts representing each of the five genes within the virR operon. Transcription of virR is driven by the P virR promoter, with a transcription start site 53 bp upstream of the virR initiation codon. The four genes downstream of virR are transcribed from P virR and from a second promoter, P orf5 , located 585 bp downstream of the virR initiation codon. VirR binds to a site overlapping the initiation codon of virR, resulting in negative autoregulation of the virR gene, explaining its low constitutive transcription level. The P orf5 promoter is induced by high temperature and low pH, thus explaining the observed differential gene expression of the virR operon. VirR has a positive effect on P orf5 activity, whereas the response regulator encoded by orf8 is not involved in regulating transcription of the virR operon. The P virR promoter is strikingly similar to those recognized by the principal sigma factors of Streptomyces and Mycobacterium, whereas the P orf5 promoter does not share sequence similarity with P virR . This suggests that P orf5 is recognized by an alternative sigma factor.

The gene encoding virulence associated protein A (VapA) is clustered with three vapA homologues (vapICD) within the pathogenicity island of the virulence plasmid of Rhodococcus equi. Northern blot analysis showed a vapA transcript of c. 700 nucleotides (nt) suggesting that vapA is a monocistronic transcript. However, using the more sensitive RT-PCR, it was shown that vapA is cotranscribed with the downstream vapICD genes forming a 2.3-kb operon. This initial transcript is subsequently processed to give rise to a 700 nt vapA transcript with a half-life of 7.5 min. In contrast, the vapI, vapC and vapD transcripts have an average half-life of 1.8 min, identical to that of the five cistronic virR operon located upstream of the vapA operon. It is speculated that the need for differential gene expression arises from the different localisation of the Vap proteins. VapA is tethered to the surface of the cell wall, whereas VapC and VapD are secreted, diffusable proteins. The intercistronic region between vapC and vapD harbours two short ORFs (OrfA, OrfB). These ORFs are translationally coupled to vapC and vapD in which the start codon overlaps the stop codon of the preceding gene.

Rhodococcus equi is an intracellular pathogen of macrophages, causing disease in young foals, humans, and sporadically other animals. Although R. equi is easy to grow and manipulate, the analysis of virulence is hampered by a lack of molecular tools. This paper describes the development of a number of versatile plasmids for use in R. equi. Plasmids pREV2 and pREV5 use origins of replication derived from the Mycobacterium fortuitum plasmids pAL5000 and pMF1. These plasmids and their derivatives are compatible in R. equi, allowing their use for analysis of gene function in trans. The stability of these plasmids in R. equi in the absence of selection for the plasmid borne antibiotic resistance markers, and their integrity following passage through Escherichia coli and R. equi was determined.