Transcriptional regulation of the virR operon of the intracellular pathogen Rhodococcus equi

Abstract

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.

FIG. 1.

Regulation of virR operon gene transcription by temperature and pH. mRNAs were isolated from Rhodococcus equi cells grown under noninducing (30°C, pH 8.0) or inducing (37°C, pH 6.5) growth conditions, followed by absolute quantification of the mRNA molecules using real-time RT-PCR. Transcription levels are indicated for each gene of the virR operon (virR, orf5, vapH, orf6, orf7, and orf8) and the divergently transcribed orf3 gene following growth under noninducing (gray bars) and inducing (black bars) conditions.

FIG. 2.

Determination of P_virR and P_orf5 promoter activities by real-time RT-PCR. (A) Schematic representation of inserts in the promoter probe vector pREV6. The hairpin structure marked with a “T” represents the transcriptional terminators. Genes and their direction of transcription are indicated by black arrows. The open white box represents the t tag. The transcription level of the t tag was measured by real-time RT-PCR. (B) mRNA transcript levels of the t tag in R. equi cells harboring pORF3PEX, containing both P_virR and P_orf5 (1); pVirRT, containing P_orf5 (2); p004 (3); or pREV6 (4). (C) mRNA transcript levels of the t tag in R. equi cells harboring pVirRT containing only P_orf5 grown at 37°C and pH 6.5 (1) or at 30°C and pH 8.0 (2) and in R. equi cells harboring pREV6 grown at 37°C and pH 6.5 (3) or 30°C and pH 8.0 (4).

FIG. 3.

Determination of transcriptional start sites of the virR operon of R. equi. Fluorescent primer extension was carried out with a Cy5-labeled primer and 5 μg of total cellular RNA extracted from R. equi cells grown under inducing conditions (37°C and pH 6.5). The upper panels show the D4-labeled primer extension products combined with DNA size standards and analyzed with a CEQ 8000 fragment analysis system. The lower panels show dideoxy sequencing reactions spiked with the D4-labeled primer extension products. The arrows indicate the transcriptional start sites where the D4-labeled cDNA and sequencing products overlapped. (A) Determination of the P_virR transcriptional start site, using D4-VIR5022. (B) Determination of the P_orf5 transcriptional start site, using D4-2IntPex5564. AU, arbitrary units.

FIG. 4.

Analysis of VirR DNA binding. Various concentrations of VirR were incubated with 2 ng of radiolabeled DNA containing (A) the P_virR promoter region in a 459-bp DNA fragment or (B) the P_orf5 promoter region in an 846-bp DNA fragment. The amount of protein added to each lane was as follows: lanes 1, radiolabeled DNA fragment only; lanes 2, 50 ng VirR-His; lanes 3, 100 ng VirR-His; lanes 4, 200 ng VirR-His; lanes 5, 300 ng VirR-His; and lanes 6, 400 ng VirR-His. The reaction volume was 20 μl. Protein-DNA complexes are indicated with black arrowheads. Nonbound DNA is indicated with a gray arrowhead. (C) HincII restriction protection assay. DNAs were incubated with VirR and HincII, followed by analysis of the restriction digest by gel electrophoresis. Lanes 1 to 3 contain a 459-bp DNA fragment with P_virR, and lanes 5 and 6 contain an 846-bp DNA fragment with P_orf5. Lanes 1 and 5, no VirR or HincII added; lanes 2 and 6, HincII added; lanes 3 and 7, HincII and VirR added. (D) Schematic representation of the 5′ end of the virR operon. Genes and the direction of transcription are indicated by black arrows. The HincII sites used in the restriction protection assay and the positions of P_virR and P_orf5 are indicated above the arrows. The nucleotide sequence of the sequence upstream of virR containing the P_virR promoter is shown. The P_virR transcriptional start site is indicated (+1).

FIG. 5.

Autoregulation of virR transcription. mRNAs were isolated from R. equi P⁻ harboring either pREV531 (orf3-virR′) or pREV5341 (orf3-virR-orf5′). virR and gyrB transcripts were subsequently detected by RT-PCR as 200-bp amplification products. The oligonucleotides used for this experiment (Table 2) amplified a region upstream of P_orf5. Lane 1, 100-bp marker; lane 2, R. equi(pREV5341) (virR); lane 3, R. equi(pREV531) (virR); lane 4, R. equi(pREV5341) (gyrB); lane 5, R. equi(pREV531) (gyrB); lanes 6 and 7, RT-PCR of gyrB genes of R. equi(pREV5341) and R. equi(pREV531), respectively, without RT.