The spatial organization of the VirR boxes is critical for VirR-mediated expression of the perfringolysin O gene, pfoA, from Clostridium perfringens

Abstract

The transcriptional regulation of toxin production in the gram-positive anaerobe Clostridium perfringens involves a two-component signal transduction system that comprises the VirS sensor histidine kinase and its cognate response regulator, VirR. Previous studies showed that VirR binds independently to a pair of imperfect direct repeats, now designated VirR box 1 and VirR box 2, located immediately upstream of the promoter of the pfoA gene, which encodes the cholesterol-dependent cytolysin, perfringolysin O. For this study, we introduced mutated VirR boxes into a C. perfringens pfoA mutant and found that both VirR boxes are essential for transcriptional activation. Furthermore, the spacing between the VirR boxes and the distance between the VirR boxes and the -35 region are shown to be critical for perfringolysin O production. Other VirR boxes that were previously identified from the strain 13 genome sequence were also analyzed, with perfringolysin O production used as a reporter system. The results showed that placement of the different VirR boxes at the same position upstream of the pfoA promoter yields different levels of perfringolysin O activity. In all of these constructs, VirR was still capable of binding to the target DNA, indicating that DNA binding alone is not sufficient for transcriptional activation. Finally, we show that the C. perfringens RNA polymerase binds more efficiently to the pfoA promoter in the presence of VirR, indicating that interactions must occur between these proteins. We propose that these interactions are required for VirR-mediated transcriptional activation.

FIG. 1.

Sequence of the VirR binding site upstream of the pfoA gene and analysis of mutated VirR boxes. (A) The VirR boxes, labeled VB1 and VB2, are enclosed in gray boxes, and their sequences are shown in bold. The −35 and −10 boxes of the pfoA promoter are underlined, the transcription start point is indicated by an asterisk, and the start of the pfoA gene is indicated by a solid arrow. The locations of the oligonucleotide primers (4565 and 5126) used to amplify the DNA targets for the gel mobility shift assays are indicated by dashed arrows. (B) Cloning of mutated VirR boxes into pJIR2266. Inserts containing the wild-type VirR boxes or the various VirR box mutations were amplified with primers 14347 and 14348. The resultant PCR products were then cloned into the unique NsiI site in pJIR2266. This vector contains the pfoA gene (hatched arrow) and the 3′ end of the pfoR gene (checked rectangle). Each of the inserts harbored the −10 and −35 boxes, which are represented by gray and black boxes, respectively. The VirR boxes are shown as rectangles with vertical or horizontal stripes. The black stars indicate the mutated boxes, and the resultant plasmids are indicated to the right of each insert. (C) Cloning of VirR box cassettes into pJIR2373. Annealed complementary oligonucleotides containing the wild-type or mutated VirR boxes were inserted into the unique EcoRV site of pJIR2373. This vector contains the pfoA gene (hatched arrow), the −10 box (gray box), the −35 box (black box), and a truncated pfoR gene (checked rectangle). The VirR box sequences are shown in bold, while the inserted nucleotides are underlined. The deletion in the intervening region is indicated by dots. The resultant plasmids are shown to the left of each insert. The 21 bp separating the centers of the VirR boxes are indicated by a brace.

FIG. 2.

Gel mobility shift analysis of altered VirR box regions. Each 183-bp DIG-labeled DNA target was incubated in the presence of various amounts of purified VirR, as indicated above each lane. Note that 1 μg of VirR represents a final concentration of 1.55 μM. The free DNA (F), CI, and CII bands are labeled. The wedges above the lanes are used to distinguish the separate DNA targets. (A) WT, +5-bp, +10-bp, and −5-bp indicate wild-type VirR boxes and VirR boxes containing a 5- or 10-bp insertion or a 5-bp deletion, respectively. (B) WT, VB2-5-P_pfoA, and VB2-10-P_pfoA indicate wild-type VirR boxes and DNA targets containing a 5- or 10-bp insertion between VirR box 2 and the promoter, respectively.

FIG. 3.

(A) Alignment of the various VirR boxes. The VirR boxes are labeled and enclosed in gray boxes. The open reading frames found downstream of the VirR boxes are indicated to the left of the VirR box sequences. The conserved nucleotides in each VirR box are shown in bold, while the consensus sequence is shown below the alignment. Modified from reference . (B) Gel mobility shift analysis of VirR boxes upstream of different open reading frames in the C. perfringens genome. The DIG-labeled DNA targets containing the various VirR boxes were incubated with various amounts of VirR, as indicated above each lane. Note that 1 μg represents a final VirR concentration of 1.55 μM. The wedges above the lanes indicate the different DNA targets. The free DNA (F), CI, and CII bands are labeled.

FIG. 4.

Gel mobility shift analysis of the pfoA (A) and plc (B) promoter regions. [γ-³²P]ATP-labeled DNA fragments were incubated with various combinations of 1.8 pmol of VirR, 0.01 U of CpRNAP, and 0.01 U of EcRNAP, as indicated above each lane. All reactions were incubated in RNA polymerase buffer (see Materials and Methods), with the exception of the first binding reaction containing VirR alone. This reaction was incubated in a previously described buffer (11). Unbound target DNAs are indicated by arrows.