Functional Analysis of the VirSR Phosphorelay from Clostridium perfringens

Jackie K. Cheung; Milena M. Awad; Sheena McGowan; Julian I. Rood

doi:10.1371/journal.pone.0005849

Abstract

Toxin production in Clostridium perfringens is controlled by the VirSR two-component signal transduction system, which comprises the VirS sensor histidine kinase and the VirR response regulator. Other studies have concentrated on the elucidation of the genes controlled by this network; there is little information regarding the phosphorelay cascade that is the hallmark of such regulatory systems. In this study, we have examined each step in this cascade, beginning with autophosphorylation of VirS, followed by phosphotransfer from VirS to VirR. We also have studied the effects of gene dosage and phosphorylation in vivo. We have used random and site-directed mutagenesis to identify residues in VirS that are important for its function and have identified a region in the putative sensory domain of VirS that appeared to be essential for function. In vitro phosphorylation studies showed that VirSc, a truncated VirS protein that lacked the N-terminal sensory domain, was capable of autophosphorylation and could subsequently act as a phosphodonor for its cognate response regulator, VirR. Conserved residues of both VirS and VirR, including the D57 residue of VirR, were shown to be essential for this process. By use of Targetron technology, we were able to introduce a single copy of virR or virR_D57N onto the chromosome of a virR mutant of C. perfringens. The results showed that in vivo, when virR was present in single copy, the production of wild-type levels of perfringolysin O was dependent on the presence of virS and an unaltered D57 residue in VirR. These results provide good evidence that phosphorylation is critical for VirR function.

Citation: Cheung JK, Awad MM, McGowan S, Rood JI (2009) Functional Analysis of the VirSR Phosphorelay from Clostridium perfringens. PLoS ONE 4(6): e5849. https://doi.org/10.1371/journal.pone.0005849

Editor: Holger Bruggemann, Max Planck Institute for Infection Biology, Germany

Received: April 7, 2009; Accepted: May 11, 2009; Published: June 9, 2009

Copyright: © 2009 Cheung et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research was supported by grants from the Australian National Health and Medical Research Council. S.M. was the recipient of an Australian Postgraduate Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Clostridium perfringens is a Gram-positive, endospore-forming, anaerobic pathogen that is the primary causative agent of gas gangrene or clostridial myonecrosis, and other human and animal diseases [1], [2]. The production of the extracellular toxins, α-toxin and perfringolysin O, which act synergistically in the gas gangrene disease process [3], [4], [5], [6], has been shown to be regulated by the VirS sensor histidine kinase and its cognate response regulator, VirR [1], [7], [8], [9]. VirR is a transcription activator that directly regulates the production of perfringolysin O, the cysteine protease α-clostripain, and the regulatory RNA molecules, VR-RNA, VirT and VirU [9], [10], [11], [12], [13]. It is through VR-RNA that the VirSR system indirectly regulates the production of α-toxin, collagenase (κ-toxin) [9], β2-toxin [14], a putative collagen adhesin [14], a cell-wall anchored DNase [15], and several housekeeping genes [9], [10], [16], [17].

The VirS sensor histidine kinase is predicted to contain six or seven transmembrane domains in its N-terminal region. Its C-terminal region contains several conserved motifs typical of histidine kinases, including the proposed site of autophosphorylation, H255, and the G box, which is thought to be involved in ATP binding [7], [18]. It is postulated that upon detection of an as yet unidentified signal by the N-terminal sensor region, VirS autophosphorylates at H255 [7]. The phosphoryl group is then transferred to a conserved aspartate residue, D57, that is located in the N-terminal region of VirR. This region also contains the E8, D9 and K105 residues, which in other response regulators are highly conserved and form a phosphoacceptor pocket [19]. After phosphorylation, activated VirR regulates the expression of its target genes by binding to specific DNA binding sites [20] via its C-terminal DNA binding domain [21]. We previously showed that VirR activates transcription of the perfringolysin O structural gene, pfoA, by binding independently to two imperfect 12-bp directly repeated sequences, called VirR boxes, located upstream of the pfoA promoter [20]. The maintenance of the integrity and spatial organisation of these VirR boxes is crucial for optimal perfringolysin O production [22]. In the three sequenced C. perfringens genomes [12], [23], VirR boxes have been identified upstream of several other genes, including genes encoding VR-RNA and α-clostripain. We have shown that these VirR boxes are functional and that VirR recognises and binds to each of these alternative binding sites [12], [22].

Although the role of the VirSR system in toxin production has been established, the N-terminal region(s) required for VirS function have not been determined and the role of phosphorylation in transcriptional activation has not been demonstrated. In this paper we identify a region located within one of the putative transmembrane domains that appears to be required for VirS function and show, using in vitro phosphorylation studies, that a truncated form of VirS is able to undergo autophosphorylation and subsequently act as the phosphodonor for VirR. Using perfringolysin O production as a reporter system for VirR function, we showed that activation of transcription was dependent on the presence of VirS and the D57 residue of VirR. These studies demonstrated that phosphotransfer from VirS to VirR and gene dosage were important factors in the regulation of perfringolysin O production by VirR.

Materials and Methods

Bacterial strains, plasmids and growth conditions

Bacterial strains are listed in Table 1, while plasmids are listed in Table S1. Escherichia coli strains were grown at 37°C in 2×YT or SOC media [24] supplemented with either 100 µg ml⁻¹ ampicillin or 30 µg ml⁻¹ chloramphenicol. C. perfringens strains were grown at 37°C in fluid thioglycollate medium (FTG) (Difco), trypticase-peptone-glucose (TPG) broth [25], Brain Heart Infusion (BHI) broth (Oxoid) or nutrient agar (NA) [26]. Where indicated, NA supplemented with 50 µg ml⁻¹ erythromycin (NAEm₅₀) or 30 µg ml⁻¹ chloramphenicol (NACm₃₀) was used for selection purposes. To screen for phospholipase C production, C. perfringens transformants were grown on egg yolk agar [27] supplemented with 30 µg ml⁻¹ chloramphenicol (EYACm₃₀), whilst perfringolysin O activity was visualised on horse blood agar (HBA) [7]. All C. perfringens agar cultures were incubated under anaerobic conditions in 10% (v/v) H₂, 10% (v/v) CO₂ in N₂.

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Table 1. Bacterial Strains and Plasmids.

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Perfringolysin O assay

The level of perfringolysin O activity in the culture supernatants was determined by a doubling dilution hemolysin assay using horse red blood cells, as previously described [3]. The titre was defined as the reciprocal of the last well that showed complete hemolysis.

Molecular techniques

Plasmid DNA was isolated from E. coli [28] and C. perfringens [29] cells as previously described. DNA for sequencing was prepared as per the PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Restriction endonucleases and other enzymes were used as specified by the manufacturer (Roche Diagnostics, New England Biolabs). All oligonucleotide primers are listed in Table S2

Competent E. coli [30] and C. perfringens [31] cells were prepared and transformed as described previously, unless otherwise indicated. C. perfringens genomic DNA was isolated from 5 ml FTG broth cultures as before [32]. PCR amplification was carried out as before [22]. PCR products were purified either directly using QIAquick® PCR Purification Kit (Qiagen) or were extracted from agarose gels using the QIAquick® Gel Extraction Kit (Qiagen), according to the manufacturer's instructions.

Random Mutagenesis

Random virS mutants were isolated after passage through the DNA repair deficient strain Epicurean Coli® XL1-Red (Stratagene). The target plasmid was pJIR884 [7], which contains an intact copy of the virS gene and the upstream virR promoter. Four independently derived pJIR884 DNA samples were obtained from transformed XL1-Red cells and used to transform C. perfringens strain JIR4000, a JIR325 derivative in which the virS gene has been insertionally inactivated by Tn916 [7]. To isolate random mutations by chemical mutagenesis, pJIR884 was incubated in 1 M hydroxylamine in 1 mM EDTA (pH 6) for 30 to 180 min at 70°C. Plasmid DNA was then used to transform strain JIR4000. Irrespective of the mutagenesis method, virS mutants were detected as non-hemolytic colonies on HBA supplemented with 50 µg ml⁻¹ erythromycin. Since perfringolysin O production is dependent upon the VirSR system [22], [33], an inability to complement the virS mutation in JIR4000, i.e. no hemolysis on HBA, was used as a direct means of detecting loss of VirS function. To facilitate analysis, plasmid DNA was recovered from all non-hemolytic strains of C. perfringens and used to transform E. coli. Sequence analysis was carried out on DNA isolated from the resultant E. coli strains to identify plasmids with mutations within the virS gene. Sequence analysis was used to identify plasmids with mutations within the virS gene. Selected plasmids derived from both methods of random mutagenesis were further analysed and are listed in Table 2.

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Table 2. The effect of virS mutations on perfringolysin O production.

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Site-directed Mutagenesis

Site-directed mutagenesis of the virS gene carried on pJIR2056 was performed using a modification of the unique site elimination method [34] with the U.S.E. Mutagenesis Kit™ (Amersham Pharmacia Biotech), the GeneEditor™ in vitro Site-Directed Mutagenesis Kit (Promega) [21], or the PCR-based Quikchange™ XL Site-Directed Mutagenesis Kit (Stratagene). The virS genes and upstream promoter regions of the mutated plasmids were sequenced to confirm that only the desired mutation was present. To test the effect of the mutations on VirS function, a 3.8-kb XbaI/SalI fragment from each mutated plasmid was isolated, cloned into the XbaI/SalI site of the E. coli/C. perfringens shuttle vector pJIR751, and used to transform C. perfringens strain JIR4000. Site-directed mutagenesis of the virR gene and the cloning of the resultant mutated genes were carried out as described previously [21].

Construction of expression plasmids

To overexpress VirSc and its derivatives, VirSc_H255I and VirSc_G402D, a 0.69 kb PCR product was generated using the primers JRP1873 and JRP1133 (Table S2). These primers incorporated NdeI and XhoI sites at the 5′ and 3′ ends, respectively, which facilitated the insertion of the DNA fragment into the expression vector pET-22b(+) (Novagen) to construct pJIR2699, pJIR2792 and pJIR2825, respectively (Table S1). To overexpress N-terminal 6×His tagged VirR_D57N, a 0.9 kb XbaI/HindIII fragment from pJIR1732 (Table S1) was subcloned into pRSETA (Invitrogen) to produce pJIR1747.

Overexpression and purification of His-tagged proteins

To induce the expression of recombinant proteins, E. coli C43 (DE3) [35] derivatives were induced with 1 mM IPTG for 3 h at 37°C (VirSc,VirSc_H255I & VirSc_G402D) or 1 h at 37°C (VirR & VirR_D57N). Proteins were purified using Talon resin (Clonetech) and dialysed as described previously [33]. Protein concentration was determined with the bicinchoninic acid protein assay kit (Pierce).

Autophosphorylation of VirSc and Phosphotransfer from VirSc to VirR

VirSc was diluted in phosphorylation buffer (50 mM HEPES [pH 8.0], 50 mM KCl, 5 mM MgCl₂, 0.5 mM EDTA, 2 mM DTT) to a final concentration of 20 µM, and aliquoted for use in individual phosphorylation reactions. Phosphorylation was initiated by the addition of 0.1 volumes of a 10× reaction mixture containing either 2.5 µM [γ-³²P]ATP (111TBq/millimole) (Perkin Elmer Life Sciences) and 247.5 µM unlabelled ATP, or 247.5 µM unlabelled ATP alone. The latter reaction mixture was added to unlabelled reactions that were subject to Western blot analysis using a Penta-His antibody (Qiagen). To examine autophosphorylation specificity, reactions were pre-incubated with 50 mM EDTA or 2.5 mM unlabelled ATP at room temperature (approx 23°C) for 5 min before the addition of the 10× reaction mixture. All reactions were incubated at room temperature for 5 min, stopped by the addition of an equal volume of 2× gel loading buffer, and then subjected to SDS-PAGE [36]. The labelled gel was then vacuum dried and exposed to X-ray film (Fuji).

For phosphotransfer studies, VirSc (10 µM) was autophosphorylated as above, in a total volume of 10 µl, with the exception that reactions were carried out in phosphotransfer buffer (50 mM MOPS [pH 7.0], 50 mM KCl, 10 mM MgCl₂, 0.5 mM EDTA, 2 mM DTT) and incubated at room temperature for 1 h. VirR or VirR_D57N was then added to a final concentration of 0.9 µM, and reactions were incubated for a further 15 min at room temperature before the addition of 10× reaction mixture and incubation at room temperature for varying amounts of time. Proteins were separated by SDS-PAGE and gels were treated as before. The presence of proteins in these phosphorylation studies was verified by Western blot analysis using His-tag antibodies on duplicate reaction samples (data not shown).

Isolation of C. perfringens cell extracts

Cell pellets derived from 1 ml TPG broth cultures were resuspended in 350 µl of 1× PBS, approximately 100 µl of 150–212 micron glass beads (Sigma-Aldrich) was added and the cells were lysed by vigorous agitation, twice for 45 sec, in a FastPrep FP120 Cell Disrupter (Savant/Bio101). Cell debris was removed by centrifugation at 12,000 g for 10 min at 4°C, and the soluble whole cell extracts were collected. Following determination of the protein concentration, 20 µg of each extract was used in Western blot analysis with VirR-specific antisera.

Western blot analysis

Following SDS-PAGE, proteins were transferred to Protran nitrocellulose membrane (Schleicher and Schuell) using a mini Trans-Blot® Electrophoretic Transfer Cell (BioRad) for Western blot analysis. The nitrocellulose membrane was developed using Penta-His antibody (1∶2000 dilution) or VirR-specific antiserum (1∶1000 dilution) [21] and the Western Lightning Chemiluminescence Reagent (Perkin Elmer Life Sciences), in accordance with the manufacturer's instructions.

Introduction of virR and virR_D57N onto the chromosome by use of a Targetron

To construct the plasmids used to introduce virR and virR_D57N onto the chromosome, the genes were PCR amplified using primers JRP2812 and JRP2813 (Table S2). The oligonucleotides introduced MluI sites that facilitated cloning into the unique MluI site within the plc target region of pJIR750ai [37], such that the genes were transcribed in the same direction as the intron. The resultant plasmids, pJIR3326 (virR) and pJIR3243 (virR_D57N) were introduced into C. perfringens strain TS133 (Table 1) by electroporation as before [38]. Transformants were selected on NACm₃₀ and several transformants were subcultured into FTG broth. After overnight incubation at 37°C, 1 ml of each culture was used to inoculate 20 ml of BHI broth and grown at 37°C for 4 h. Cultures were then serially diluted and subcultured onto EYACm₃₀. After overnight incubation at 37°C, colonies that were not surrounded by a white zone of opalescence were selected for further analysis. These colonies represented cells in which the α-toxin gene, plc, had been disrupted by the Targetron. The insertion of the element was confirmed by PCR, using primers JRP2873 and JRP2874 (Table S2). To cure the strains of the replicating plasmid constructs, the strains were passaged twice a day in 20 ml of BHI broth for five days. Single colonies were isolated and those that were chloramphenicol sensitive were selected for further analysis. Southern blots were carried out as previously described [37] using DIG-labelled catP, virR and plc probes to confirm the insertion of the genes on the chromosome and the loss of the Targetron plasmid constructs (data not shown).

Isolation of RNA and Quantitative Real Time (QRT)-PCR

C. perfringens total RNA was isolated as described previously [18] and 2 µg of RNA converted to cDNA using AMV reverse transcriptase (Promega) in accordance with the manufacturer's instructions. The reaction products were diluted fivefold before use in QRT-PCR experiments, as described previously [32]. The QRT-PCR reactions were carried out on an ABI PRISM 7700 sequence detector, in a final volume of 25 µl with SYBR Green PCR master mix (Applied Biosystems), 2 µl of diluted RT reaction as the template and 120 nM of each primer (Table S2). To determine gene copy number, 0.2 ng of genomic DNA was used as the template. Total RNA or genomic DNA of each strain was isolated from three biological replicates, and each sample was assayed in triplicate. The values obtained were normalised to that of the rpoA gene for each strain, and the results expressed as a proportion of the wild-type.

Results

Identification of functional residues in VirS

VirS contains several residues and motifs that are conserved in C-terminal domains of sensor histidine kinases, including the putative site of autophosphorylation, H255, and the G (GxGL) motif (Fig. 1). In previous work, six or seven transmembrane regions were predicted in the N-terminal region of VirS. In this study, a new model of VirS was obtained using three independent transmembrane prediction algorithms: TMHMM (http://www.cbs.dtu.dk/services/TMHMM/), Phobius (http://phobius.cbr.su.se/) and Sosui (http://bp.nuap.nagoya-u.ac.jp/sosui/). These algorithms showed a consensus topology, where the extracellular N-terminus was followed by seven transmembrane domains, and the catalytic C-terminal region was cytoplasmically located (Fig. 1). Attempts at verifying this topology using PhoA/LacZ fusions were unsuccessful (data not shown).

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Figure 1. Model of the VirS sensor histidine kinase.

The predicted topology of VirS consists of an extracellular N-terminus, seven transmembrane domains (blue rectangles) and a cytoplasmic C-terminal region. The amino acids encompassing the transmembrane domains as predicted by the TMHMM, Phobius and Sosui algorithms are shown beside the predicted loop regions and are written in black, red and green, respectively. The consensus [39] H box (Fhxxh(S/T/A)H(D/E)h(R/K)TPLxxh), N box ((D/N)xxxhxxhhxNLhxNAh.(F/H/Y)(S/T)) and G box (GGxGLGLxhhxxhhxxxxGxhxhxxxxxxGxxFxhxh) are represented by the yellow squares, while the positions of the random mutations are as indicated.

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With this model in mind, random mutagenesis was used to identify VirS residues of functional importance, particularly within the N-terminal domain. Mutagenesis of the virS⁺ shuttle plasmid pJIR884 [7] led to the isolation of 134 non-hemolytic mutants, each containing plasmids that were indistinguishable from pJIR884 by restriction analysis. Eighteen of these plasmids were found to contain a single point mutation in the virS structural gene, 10 of which were unique (Fig. 1) and therefore were subjected to quantitative analysis. None of these mutants conferred any detectable perfringolysin O activity (Table 2), indicating that the mutations had eliminated all measurable VirS function. RT-PCR was used to confirm that the mutated virS genes were still transcribed in C. perfringens (data not shown).

Three of the VirS substitutions, L99F, T100A and E102K, were located in the N-terminal region, within a putative transmembrane domain (TMD 4). The other changes were all located within the cytoplasmic C-terminal domain. Four substitutions, H255Y, D256G, N259K and H260Y, were located either in or near H255, the putative site of autophosphorylation. One alteration, C335Y, was close to the conserved N box, a region predicted to form part of the catalytic domain that binds ATP. The G402D and G415R substitutions were located in or near the G box, which is also predicted to form part of the catalytic domain [39].

To define the functional region in this N-terminal portion of VirS, several leucine residues on either side of L99, and upstream of E102, were targeted for substitution. The sequence of this leucine rich region is ₈₈IMISLIFWLFMLTVEAL₁₀₄. The leucine residues adjacent to L99, L104 and L96, were altered to phenylalanine, on the basis that the random L99F mutation eliminated VirS function. The L104F substitution had no significant effect on perfringolysin O production (Table 2), suggesting that this residue is not essential for VirS function. By contrast, the L96F mutation had a dramatic effect on VirS function, eliminating VirS activity. Further mutagenesis of L96 to alanine or asparagine, had no significant effect on perfringolysin O activity, suggesting that the size of the side-chain was the critical factor at this position. To further delineate this putative motif, L92 and I88 were also altered to phenylalanine. These changes had little effect on perfringolysin O activity (Table 2), implying that these residues were not of functional significance.

The amino acid requirements at positions 99 and 100 were also determined by site-directed mutagenesis, the requirement for a glutamate residue at position 102 having been determined previously [18]. The substituted amino acids were chosen so that they had an altered size or charge compared to the original residue in that position. Alteration of L99 to either alanine or asparagine abolished VirS function (Table 2), implying that both the length and hydrophobicity of the side-chain at this position was important. At position 100 a hydroxyl group was required since T100V was inactive, T100C was partially functional and T100S (a conservative substitution) had almost wild-type activity. Based on these data we have designated this region of VirS as the L[T/S]×E motif, and postulate that due to its location it is either required for the conformational change that induces autophosphorylation, or for the structural integrity of VirS.

Residues in the C-terminal domain of VirS are essential for autophosphorylation

The next step in our systematic analysis of the VirSR phosphorelay was to determine whether VirS was able to undergo autophosphorylation. The VirS protein is postulated to contain multiple transmembrane domains in the N-terminal region, with the catalytic motifs being localised in the cytoplasmic C-terminal domain [7], [18]. Since attempts to purify full-length VirS were unsuccessful, a virS segment encoding the C-terminal domain (amino acids 215 to 440) was cloned into the pET-22b(+) expression vector, and a 27.7 kDa 6×-His tagged protein (VirSc) overexpressed. After purification on a Talon column, VirSc was used in autophosphorylation experiments using [γ−³²P]ATP or [α-³²P]ATP. A single band corresponding to the correct size of VirSc (27.7 kDa) was observed in the [γ−³²P]ATP reaction (Fig. 2A, lane 3), indicating that VirSc was able to undergo autophosphorylation. Moreover, labelling of the protein occurred through the specific transfer of the γ-phosphoryl group from ATP to VirSc, since the protein could be labelled with [γ−³²P]ATP, but not with [α-^32P]ATP (Fig. 2A, lane 4), indicating that labelling occurred through a specific phosphotransfer process. The autophosphorylation reaction was found to require divalent metal ions, since pre-incubation of VirSc with 50 mM EDTA inhibited autophosphorylation (Fig. 2A, lane 5). The addition of a 10-fold molar excess of unlabelled ATP competitor also abrogated autophosphorylation (Fig. 2A, lane 6).

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Figure 2. Autophosphorylation of VirSc and its derivatives.

(A) In the absence of purified VirSc, a labelled protein band was not observed in the presence of [γ-³²P]ATP (lane 1) or [α-³²P]ATP (lane 2). A labelled band of ∼27 kDa was observed when purified VirSc (20 µM) was incubated in phosphorylation buffer in the presence of [γ-³²P]ATP (lane 3). To show specificity of autophosphorylation, VirSc was incubated with [α-³²P]ATP (lane 4), or with [γ-³²P]ATP in the presence of 50 mM EDTA (lane 5) or 2.5 mM unlabelled ATP (lane 6). (B) Autophosphorylation of substituted derivatives of VirSc. VirSc was able to autophosphorylate (lane 1), while VirSc_H255I (lane 2), VirSc_C335Y (lane 3) and VirSc_G402D (lane 5) could not undergo autophosphorylation. VirSc_G400A (lane 4) showed severely reduced autophosphorylation. The band corresponding to phosphorylated VirSc (∼27 kDa) is indicated by the arrow. Molecular size markers (kDa) are as indicated.

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In a previous study, mutation of the conserved histidine residue (H255I), or the first glycine residue in the conserved G box (G400A), was shown to eliminate VirS function [18]. Similarly, the C335Y and G402D substitutions of VirS, which were identified through random mutagenesis in this study, also eliminated VirS function (Table 2). To examine what effects these changes had on autophosphorylation in vitro, VirSc proteins containing these modifications were purified and tested for their ability to autophosphorylate. The results showed that a phosphorylated protein band was not observed with VirSc_H255I (Fig. 2B, lane 2), indicating that this derivative was no longer able to carry out autophosphorylation and that this histidine residue was involved in this reaction. Analysis of the VirSc_C335Y derivative showed that this mutation also eliminated the ability of the protein to autophosphorylate (Fig. 2B, lane 3). Therefore, although C335 is located near to, but is not part of the actual N box, it still plays an important structural or functional role in the VirS autophosphorylation process. Significantly reduced VirS-specific autophosphorylation was detected with VirSc_G400A (Fig. 2B, lane 4), implying that although the first glycine residue in the G box is important for the autophosphorylation of VirS, very low level phosphorylation is possible when it is altered. However, alteration of the second glycine residue in this motif (G402D) completely eliminated autophosphorylation (Fig. 2B, lane 5). Taken together, these results provided experimental evidence that the motifs and residues predicted to be essential for ATP binding and kinase activity are required for autophosphorylation.

Phosphotransfer from VirSc to VirR in vitro

Using phosphorylated VirSc (VirSc-P) as the phosphodonor, we then tested the ability of purified VirR to act as a phosphoacceptor. The appearance of a labelled band corresponding to VirR was observed (Fig. 3A). This result provided evidence for the transfer of the phosphoryl group from VirSc-P to VirR, indicating that VirR was able to interact with the truncated VirS protein. A phosphorylated response regulator band was not observed when purified VirR containing a substitution at the putative D57 phosphoacceptor site (VirR_D57N) was tested under the same conditions (Fig. 3B). Furthermore, the intensity of this VirSc-P band did not appear to diminish over time. It was concluded that the VirR_D57N was not able to be phosphorylated and that D57 was most likely the site of phosphorylation in VirR.

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Figure 3. Phosphotransfer from VirSc to VirR.

VirSc (10 µM) was incubated in phosphotransfer buffer in the presence of [γ-³²P]ATP at room temperature before purified (A) VirR or (B) VirR_D57N was added. Reactions were incubated at room temperature for the time indicated above each well. The first lane of each gel contains autophosphorylated VirSc, while subsequent lanes show the reactions with both phosphorylated VirSc and VirR or VirR_D57N. The band corresponding to phosphorylated VirSc (∼27 kDa) or VirR (∼32 kDa) is indicated by the arrow or an asterisk, respectively. Molecular size markers (kDa) are as indicated.

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VirR proteins with mutations in conserved residues are still functional in vivo

To determine whether the VirR_D57N protein was functional in vivo, the virR_D57N gene was subcloned into the E. coli-C. perfringens shuttle vector, pJIR750 [40], and the resultant plasmid, pJIR1882, was introduced into C. perfringens virR mutant TS133 [8] (Table 1). Since complementation of the virR mutation in TS133 with a plasmid carrying a wild-type virR gene results in the restoration of perfringolysin O activity [8], perfringolysin O production was used to determine the effect of the mutations. Unexpectedly, when the shuttle plasmid carrying the virR_D57N gene was introduced into TS133, the resultant strain carrying the mutated gene produced less perfringolysin O than the wild-type, but still produced very significant levels of the toxin (Table 3).

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Table 3. Effect of virR genes on perfringolysin O production.

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Another derivative was subsequently constructed in which the putative essential lysine residue [7] was substituted (K105E). When this mutated virR gene was introduced into TS133, similar results were obtained (Table 3). Finally, in case alternate acidic residues in the phosphoacceptor site were being used as phosphoacceptors, a mutant was constructed in which D57 and the other residues in the acceptor pocket, E8 and D9, were substituted. This virR_{E8N D9N D57N} mutation, referred to as virR_NNN, was also capable of fully complementing the virR mutation in TS133 (Table 3). Confirmation that all the plasmids analysed in this strain had retained the expected mutations and had not undergone further virR changes was obtained after plasmid extraction and sequence analysis.

The gene dosage of virR is important for the normal regulation of perfringolysin O production

We postulated that this apparent phosphorylation independence occurred as a result of gene dosage effects. To test this hypothesis, we utilised an α-toxin (plc)-targeted Targetron vector [37] as a novel means of introducing virR genes back onto the chromosome in single copy. Using this method we constructed separate strains in which the wild-type virR gene or the virR_D57N gene was inserted into the plc gene. As a result of the genetic organisation of the virRS operon, the original insertional inactivation of virR in TS133 also eliminated the expression of virS [41]. Therefore, to reconstitute the VirSR regulatory system, pJIR884, which carries the wild-type virS gene [7], was introduced into each of these strains to provide virS in trans. Control strains carried the vector plasmid pJIR751.

QRT-PCR using genomic DNA showed that as in the wild-type strain there was one copy of virR or virR_D57N in the Targetron-derived chromosomal plc insertion derivatives although there were an average of 15 copies of virR and 18 copies of virR_D57N in the TS133 strains carrying these genes on plasmids. As an internal control, the copy number of the pfoA gene was determined to be one in all of these strains, as expected (Fig. 4A).

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Figure 4. Determination of virR copy number, virR expression levels and VirR levels in C. perfringens.

(A) Determination of virR copy number. Genomic DNA from each strain was isolated from three biological replicates and assayed in triplicate by QRT-PCR. The gene copy number of virR (black bars) and pfoA (white bars) in each strain relative to the wild-type (WT) copy number normalised to the rpoA gene is shown. The strains are indicated below each set of bars, and the chromosomal (c) or plasmid (p) location of the virR, virR_D57N and/or virS genes is shown beneath each strain. (B) Determination of virR expression using QRT-PCR. Total RNA from each strain was isolated from three biological replicates and assayed in triplicate. The expression levels of virR (black bars) in each strain relative to WT levels normalised to the rpoA gene (relative gene expression) are shown. The strains are indicated as for (A). In (A) and (B), the error bars represent the standard error of the mean, and the asterisks (*) represent a P-value of<0.05 calculated by two-tailed Student's t-test. (C) Immunoblot analysis of wild-type and TS133 derivatives. Western blots were carried out on whole cell extracts of the strains indicated above each lane. A brief description of each strain is given below the blot. The blots were probed with polyclonal rabbit VirR antiserum, followed by chemiluminescent detection. Molecular size markers (kDa) are as indicated.

https://doi.org/10.1371/journal.pone.0005849.g004

Perfringolysin O assays were carried out to determine the phenotypic effect of these different virR gene doses. Expression of both the virR and virR_D57N genes on multicopy plasmids in TS133 (strains JIR4508 and JIR4487, respectively) resulted in wild-type levels of perfringolysin O activity (Table 3), despite the absence of virS in these strains. By use of QRT-PCR analysis, the expression levels of the virR genes in these strains were shown to be significantly higher than in the wild-type strain (Fig. 4B). In addition, Western blotting using VirR-specific antiserum showed that there were higher levels of VirR protein in these cells (Fig. 4C). These results indicated that when VirR or VirR_D57N was present in the cell at higher concentrations, the response regulator no longer needed to be activated by phosphorylation to be functional. This observation is clearly illustrated by the results with VirR_D57N, which was not able to be phosphorylated in vitro, but was still able to stimulate high levels of perfringolysin O production when encoded on a multicopy plasmid. By contrast, when the virR_D57N gene was introduced onto the chromosome in single copy, as confirmed by genomic QRT-PCR analysis, the resultant strain was not able to produce perfringolysin O (Table 3). The expression levels of the single copy genes, including the wild-type strain, were significantly lower than the genes carried on the multicopy plasmids (Fig. 4B), and the respective protein concentrations were so low that they were not detected by the VirR-specific antisera in Western blots (Fig. 4C). These results suggest that the virR gene dosage is an important factor in the regulation of perfringolysin O production.

Analysis of chromosomal mutants reveals that phosphorylation of VirR is essential for optimal perfringolysin O production

The chromosomal derivatives were also used to assess the effect of the presence of virS, that is, the effect of phosphotransfer, on perfringolysin O production (Table 3). The results showed that despite the presence of a plasmid carrying the wild-type virS gene, no perfringolysin O activity could be detected in the virR mutant TS133, indicating that, as expected, overexpression of virS alone could not activate perfringolysin O production. By contrast, perfringolysin O activity was restored to wild-type levels when the virS plasmid, pJIR884, was introduced into TS133 complemented with the chromosomal virR gene. These results provided in vivo evidence that VirS, in the presence of VirR, is required to activate the expression of pfoA and were consistent with the in vitro phosphorylation data. Analysis of the data obtained with the chromosomal virR_D57N showed that no detectable perfringolysin O activity was observed even when virS was present. It was concluded that D57 was an essential residue for VirS-dependent VirR function in vivo.

QRT-PCR using total RNA was carried out to determine whether these results were due to variation in virR or virR_D57N expression levels. In each of the chromosomal complementation derivatives the relevant virR gene was expressed at levels similar to the wild-type (Fig. 4B). Therefore, even though the virR_D57N gene was being expressed at levels similar to wild-type virR, the resultant protein was not able to activate the production of perfringolysin O. Furthermore, the presence of a functional virS gene in multicopy did not increase the expression of the virR genes (Fig. 4B), nor the amount of VirR protein (Fig. 4C). These results suggest that the higher perfringolysin O activity in the chromosomal virR complementation derivative was a result of VirS modifying the VirR protein, a process that requires the D57 residue of VirR. Taken together the results provide evidence that in vivo phosphorylation is crucial for optimal perfringolysin O production.

Discussion

Although the VirSR two-component regulatory system is well studied [1], [13], [20], very little is known about the actual phosphorelay cascade. It has always been postulated, but never experimentally demonstrated, that activation of the VirSR system begins with the interaction of an unknown signal molecule with VirS and subsequent VirS autophosphorylation. In this study, we have identified an essential N-terminal VirS motif, L[T/S]×E, that may be involved in the activation of VirS after detection of the unknown signal molecule. Further studies would be required to test this hypothesis, once the signalling molecule has been identified. Early studies on toxin production [42], [43], [44] have led to the hypothesis that the VirSR system may potentially be activated by a quorum sensing mechanism involving a secreted molecule called substance A [1], [20]. Although, this elusive molecule has yet to be isolated, recent studies have suggested that it is a cyclic derivative of the quorum sensing peptide (TSACLWFT), which is the secreted product of the agrBD^CP genes [45].

Alternatively, the L[T/S]×E region may play an important role in transmembrane helix packing. Residues such as glutamate, serine and threonine have been found to be crucial in the tight helical packing of membrane bound proteins, which in turn contributes to the stability, folding and subsequently, the function of the protein [46]. Therefore, the introduced changes may have caused a disruption in protein structure, leading to either misfolding or instability of VirS. At present, however, reliable VirS antisera is not available to examine whether protein stability is affected by the mutations. Nevertheless, the observation that not all the residues surrounding the L[T/S]×E motif were required for VirS function implies that the residues in this motif are of functional significance. We have also shown that a truncated VirS protein, VirSc, is able to autophosphorylate, in the absence of the N-terminal sensory domain. Autophosphorylation requires divalent cations and is ATP-dependent, with the γ-phosphoryl moiety of ATP being transferred to VirSc. Furthermore, we have shown that the conserved C-terminal motifs postulated to be required for autophosphorylation are indeed functional.

The conserved histidine residue in sensor kinases is the site of autophosphorylation [19], [47], where it is suggested to function as the nucleophile that attacks the ATP γ-phosphate [48], [49]. In this study, it was shown that a VirSc_H255I protein could not be phosphorylated in vitro, which was consistent with the in vivo results obtained previously [18], where a virS_H255I gene was not able to complement a chromosomal virS mutation. Taken together these results provide good evidence that H255 is essential for VirS function. Although H255 has been postulated to be the site of autophosphorylation, the finding that another histidine residue, H260, also affected protein function suggests that either histidine could potentially act as the site of phosphorylation. However, determination of the actual site was beyond the scope of this study. The glycine residues of the G box were also found to be important. In particular, the second glycine, G402, was shown to be essential, since mutation of this residue resulted in elimination of autophosphorylation. A non-functional virSc_C335Y mutant was isolated in the random mutagenesis experiments and the resultant VirSc_C335Y protein was not labelled in the presence of [γ-³²P]ATP, suggesting that C335 is required for autophosphorylation. The C335Y substitution was located about 10 amino acids N-terminal to the proposed N box, which in other systems is important in ATP binding and hence, autophosphorylation [50], [51], [52]. Residues located outside of the conserved motifs have been found to be involved in interactions with the bound nucleotide [53]. Therefore, it is possible that although C335 is not part of the N-box, it may associate with the bound ATP molecule. Alternatively, the effect of the cysteine to tyrosine change could be structural. Note that it was not possible to test the effect of the substitutions in the L[T/S]×E motif on autophosphorylation because we were unable to purify full-length VirS and the truncated VirSc protein did not include these residues.

The C-terminal region also appears to be capable of protein-protein interactions with the cognate response regulator, VirR, since phosphotransfer from phosphorylated VirSc to VirR was observed. This result represents the first time phosphotransfer has been demonstrated in this system. The response regulator superfamily is defined by conservation of N-terminal residues that are involved in phosphorylation [54]. The key aspartate and lysine residues are invariant in these phosphorylation-dependent regulators. The remaining conserved residues, an acidic pair, a hydroxyl side-chain and an aromatic residue are proposed to have functional roles in the coordination of the essential divalent cation and phosphoryl group. Alignment of VirR with other response regulators suggested that the central aspartate was D57 [7]. In this study, the results provide evidence that D57 is highly likely to be the site of phosphorylation, since phosphotransfer was not observed with VirR_D57N under in vitro phosphotransfer conditions in which the wild-type VirR protein acted as a phosphoacceptor. In addition, D57 was shown to be essential for VirR function in vivo; when the virR_D57N gene was present on the chromosome in single copy, perfringolysin O production was not activated. The results also indicated that other residues in the phosphorylation pocket or elsewhere in the protein were not phosphorylated instead of D57. It is possible that the D57N mutation caused the misfolding of the resulting protein and the associated loss of activity, but the observation that the mutated gene, when present in multicopy, was able to complement the virR mutation of the strain argues against this possibility. Unexpectedly, when the wild-type gene was re-introduced back onto the chromosome of TS133, the resultant VirR protein could stimulate a low level of perfringolysin O production in the absence of VirS. This result may be due to phosphorylation of the VirR protein by small molecular weight phosphodonors. This phenomenon has been observed in other two-component systems [55], [56] and involves an alternative phosphoryl moiety, such as acetyl phosphate or carbamoyl phosphate, donating its phosphate group to a response regulator. Phosphorylation by this process is not as efficient as that involving the cognate sensor histidine kinase [57], [58], which may explain why perfringolysin O production was only partially restored.

In some response regulators phosphorylation alters the protein conformation so that steric inhibition is relieved, and consequently, dimerization and DNA binding can occur [59], [60], [61], [62]. In others, phosphorylation is required for interaction with RNA polymerase [63], [64], [65], [66]. We do not know the precise role of phosphorylation in the VirR-dependent expression of the pfoA gene, but previous work has shown that phosphorylation is not essential for DNA binding [22], [33]. We postulate that phosphorylation is required to optimize protein-protein interactions between VirR monomers, VirR and RNA polymerase, or both reactions, since binding to DNA alone is not sufficient to activate perfringolysin O production [22].

In conclusion, based on the data presented in this paper, we propose that the regulation of perfringolysin O production in C. perfringens is dependent on three factors. The first is the autophosphorylation of VirS following the detection of an unknown signal. This process may involve the L[T/S]×E motif. The second is a requirement for VirS-dependent VirR phosphorylation, a process that involves conserved motifs in both proteins. The third is the virR gene dosage in the cell, since overexpression of wild-type and mutated virR genes resulted in VirS-independent activation. It appears that the VirSR system is not limited to C. perfringens, with genes encoding orthologs of VirR and/or VirS identified in Clostridium difficile [32], Clostridium tetani [67] and Clostridium botulinum [68]. In addition, VirR binding sites have been identified in C. difficile, and its VirR ortholog, RgaR, has been shown to recognise and bind specifically to the C. perfringens VirR boxes [32]. Therefore, our studies clearly have relevance to the analysis of two component signal transduction system in other pathogenic clostridial species.

Supporting Information

Table S1.

https://doi.org/10.1371/journal.pone.0005849.s001

(0.07 MB DOC)

Table S2.

https://doi.org/10.1371/journal.pone.0005849.s002

(0.06 MB DOC)

Acknowledgments

We thank Melissa Bateman and Hayley Newton for the initial construction of several mutants and Dr Ji Yang (University of Melbourne) for helpful discussions.

Author Contributions

Conceived and designed the experiments: JC MMA SM JIR. Performed the experiments: JC MMA SM. Analyzed the data: JC MMA SM JIR. Wrote the paper: JC MMA SM JIR.

References

1. Rood JI (1998) Virulence genes of Clostridium perfringens. Annu Rev Microbiol 52: 333–360.
- View Article
- Google Scholar
2. Songer JG (2005) Clostridial diseases in domestic animals. In: Dürre P, editor. Handbook on Clostridia. Boca Raton: CRC Press. pp. 527–542.
3. Awad MM, Bryant AE, Stevens DL, Rood JI (1995) Virulence studies on chromosomal α-toxin and θ-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens-mediated gas gangrene. Mol Microbiol 15: 191–202.
- View Article
- Google Scholar
4. Awad MM, Ellemor DM, Boyd RL, Emmins JJ, Rood JI (2001) Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens-mediated gas gangrene. Infect Immun 69: 7904–7910.
- View Article
- Google Scholar
5. Ellemor DM, Baird RN, Awad MM, Boyd RL, Rood JI, et al. (1999) Use of genetically manipulated strains of Clostridium perfringens reveals both alpha-toxin and theta-toxin are required for vascular leukostasis to occur in experimental gas gangrene. Infect Immun 67: 4902–4907.
- View Article
- Google Scholar
6. Stevens DL, Tweten RK, Awad MM, Rood JI, Bryant AE (1997) Clostridial gas gangrene : Evidence that α and θ toxins differentially modulate the immune response and induce acute tissue necrosis. J Infect Dis 176: 189–195.
- View Article
- Google Scholar
7. Lyristis M, Bryant AE, Sloan J, Awad MM, Nisbet IT, et al. (1994) Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens. Mol Microbiol 12: 761–777.
- View Article
- Google Scholar
8. Shimizu T, Ba-Thein W, Tamaki M, Hayashi H (1994) The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens. J Bacteriol 176: 1616–1623.
- View Article
- Google Scholar
9. Shimizu T, Yaguchi H, Ohtani K, Banu S, Hayashi H (2002) Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins. Mol Microbiol 43: 257–265.
- View Article
- Google Scholar
10. Banu S, Ohtani K, Yaguchi H, Swe T, Cole ST, et al. (2000) Identification of novel VirR/VirS-regulated genes in Clostridium perfringens. Mol Microbiol 35: 854–864.
- View Article
- Google Scholar
11. Shimizu T, Shima K, Yoshino K, Yonezawa K, Shimizu T, et al. (2002) Proteome and transcriptome analysis of the virulence genes regulated by the VirR/VirS system in Clostridium perfringens. J Bacteriol 184: 2587–2594.
- View Article
- Google Scholar
12. Myers GSA, Rasko DA, Cheung JK, Ravel J, Seshadri R, et al. (2006) Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens. Genome Res 16: 1031–1040.
- View Article
- Google Scholar
13. Okumura K, Ohtani K, Hayashi H, Shimizu T (2008) Characterization of genes regulated directly by the VirR/VirS system in Clostridium perfringens. J Bacteriol 190: 7719–7727.
- View Article
- Google Scholar
14. Ohtani K, Kawsar HI, Okumura K, Hayashi H, Shimizu T (2003) The VirR/VirS regulatory cascade affects transcription of plasmid-encoded putative virulence genes in Clostridium perfringens strain 13. FEMS Microbiol Lett 222: 137–141.
- View Article
- Google Scholar
15. Okumura K, Kawsar HI, Shimizu T, Ohta T, Hayashi H, et al. (2005) Identification and characterization of a cell-wall anchored DNase gene in Clostridium perfringens. FEMS Microbiol Lett 242: 281–285.
- View Article
- Google Scholar
16. Ohtani K, Takamura H, Yaguchi H, Hayashi H, Shimizu T (2000) Genetic analysis of the ycgJ-metB-cysK-ygaG operon negatively regulated by the VirR/VirS system in Clostridium perfringens. Microbiol Immunol 44: 525–528.
- View Article
- Google Scholar
17. Kawsar HI, Ohtani K, Okumura K, Hayashi H, Shimizu T (2004) Organization and transcriptional regulation of myo-inositol operon in Clostridium perfringens. FEMS Microbiol Lett 235: 289–295.
- View Article
- Google Scholar
18. Cheung JK, Rood JI (2000) Glutamate residues in the putative transmembrane region are required for the function of the VirS sensor histidine kinase from Clostridium perfringens. Microbiol-SGM 146: 517–525.
- View Article
- Google Scholar
19. Stock AM, Robinson VL, Goudreau PN (2000) Two-component signal transduction. Annu Rev Biochem 69: 183–215.
- View Article
- Google Scholar
20. Cheung JK, McGowan S, Rood JI (2005) Two-component signal transduction systems in clostridia. In: Dürre P, editor. Handbook on Clostridia. Boca Raton: CRC Press Taylor & Frances Group. pp. 545–560.
21. McGowan S, Lucet IS, Cheung JK, Awad MM, Whisstock JC, et al. (2002) The FxRxHrS motif: a conserved region essential for DNA binding of the VirR response regualtor from Clostridium perfringens. J Mol Biol 322: 997–1011.
- View Article
- Google Scholar
22. Cheung JK, Dupuy B, Deveson DS, Rood JI (2004) The spatial organization of the VirR boxes is critical for VirR-mediated expression of the perfringolysin O gene, pfoA, from Clostridium perfringens. J Bacteriol 186: 3321–3330.
- View Article
- Google Scholar
23. Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, et al. (2002) Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci USA 99: 996–1001.
- View Article
- Google Scholar
24. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
25. Rood JI, Maher EA, Somer EB, Campos E, Duncan CL (1978) Isolation and characterization of multiple antibiotic-resitant Clostridium perfringens strains from porcine feces. Antimicrob Agents and Chemother 13: 871–880.
- View Article
- Google Scholar
26. Rood JI (1983) Transferable tetracycline resistance in Clostridium perfringens strains of porcine origin. Can J Microbiol 29: 1241–1246.
- View Article
- Google Scholar
27. Sloan J, Warner TA, Scott PT, Bannam TL, Berryman DI, et al. (1992) Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid. Plasmid 27: 207–219.
- View Article
- Google Scholar
28. Morelle G (1989) A plasmid extraction procedure on a miniprep scale. Focus 11: 7–8.
- View Article
- Google Scholar
29. Roberts I, Holmes WM, Hylemon PB (1986) Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum. Appl Environ Microbiol 52: 197–199.
- View Article
- Google Scholar
30. Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96: 23–28.
- View Article
- Google Scholar
31. Scott PT, Rood JI (1989) Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens. Gene 82: 327–333.
- View Article
- Google Scholar
32. O'Connor JR, Lyras D, Farrow KA, Adams V, Powell DR, et al. (2006) Construction and analysis of chromosomal Clostridium difficile mutants. Mol Microbiol 61: 1335–1351.
- View Article
- Google Scholar
33. Cheung JK, Rood JI (2000) The VirR response regulator from Clostridium perfringens binds independently to two imperfect direct repeats located upstream of the pfoA promoter. J Bacteriol 182: 57–66.
- View Article
- Google Scholar
34. Deng WP, Nickoloff JA (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem 200: 81–88.
- View Article
- Google Scholar
35. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260: 289–298.
- View Article
- Google Scholar
36. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680–685.
- View Article
- Google Scholar
37. Chen Y, McClane BA, Fisher DJ, Rood JI, Gupta P (2005) Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron. Appl Environ Microbiol 71: 7542–7547.
- View Article
- Google Scholar
38. Czeczulin JR, Collie RE, McClane BA (1996) Regulated expression of Clostridium perfringens enterotoxin in naturally cpe-negative type A, B, and C isolates of C. perfringens. Infect Immun 64: 3301–3309.
- View Article
- Google Scholar
39. Wolanin PM, Thomason PA, Stock JB (2002) Histidine protein kinases: key signal transducers outside the animal kingdom. Genome biology 3: REVIEWS3013.
- View Article
- Google Scholar
40. Bannam TL, Rood JI (1993) Clostridium perfringens-Escherichia coli shuttle vectors that carry single antibiotic resistance determinants. Plasmid 29: 223–235.
- View Article
- Google Scholar
41. Ba-Thein W, Lyristis M, Ohtani K, Nisbet IT, Hayashi H, et al. (1996) The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens. J Bacteriol 178: 2514–2520.
- View Article
- Google Scholar
42. Higashi T, Chazono M, Inoue K, Yanagase Y, Amano T, et al. (1973) Complementation of θ-toxinogenicity between mutants of two groups of Clostridium perfringens. Biken J 16: 1–9.
- View Article
- Google Scholar
43. Imagawa T, Higashi Y (1992) An activity which restores theta toxin activity in some theta toxin-deficient mutants of Clostridium perfringens. Microbiol Immunol 36: 523–527.
- View Article
- Google Scholar
44. Imagawa T, Tatsuki T, Higashi Y, Amano T (1981) Complementation characteristics of newly isolated mutants from two groups of strains of Clostridium perfringens. Biken J 24: 13–21.
- View Article
- Google Scholar
45. Ohtani K, Yuan Y, Hassan S, Wang R, Wang Y, et al. (2009) Virulence gene regulation by the agr system in Clostridium perfringens. J Bacteriol Apr 10: [Epub ahead of print].
- View Article
- Google Scholar
46. Eilers M, Shekar SC, Shieh T, Smith SO, Fleming PJ (2000) Internal packing of helical membrane proteins. Proc Natl Acad Sci USA 97: 5796–5801.
- View Article
- Google Scholar
47. Parkinson JS, Kofoid EC (1992) Communication modules in bacterial signaling proteins. Annu Rev Genet 26: 71–112.
- View Article
- Google Scholar
48. Bilwes AM, Alex LA, Crane BR, Simon MI (1999) Structure of CheA, a signal-transducing histidine kinase. Cell 96: 131–141.
- View Article
- Google Scholar
49. Dutta R, Inouye M (2000) GHKL, an emergent ATPase/kinase superfamily. TIBS 25: 24–28.
- View Article
- Google Scholar
50. Dutta R, Inouye M (1996) Reverse phosphotransfer from OmpR to EnvZ in a kinase⁻/phosphatase⁺ mutant of EnvZ (EnvZ.N347D), a bifunctional signal transduction transducer of Escherichia coli. J Biol Chem 271: 1424–1429.
- View Article
- Google Scholar
51. Hsing W, Russo FD, Bernd KK, Silhavy TJ (1998) Mutations that alter the kinase and phosphatase activities of the two-component sensor EnvZ. J Bacteriol 180: 4538–4546.
- View Article
- Google Scholar
52. Hirschman A, Boukhvalova M, VanBruggen R, Wolfe AJ, Stewart RC (2001) Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli. Biochem 40: 13876–13887.
- View Article
- Google Scholar
53. Marina A, Mott C, Auyzenberg A, Hendrickson WA, Waldburger CD (2001) Structural and mutational analysis of the PhoQ histidine kinase catalytic domain. J Biol Chem 276: 41182–41190.
- View Article
- Google Scholar
54. West AH, Stock AM (2001) Histidine kinases and response regulator proteins in two-component signaling systems. TIBS 26: 369–376.
- View Article
- Google Scholar
55. Cho HS, Pelton JG, Yan D, Kustu S, Wemmer DE (2001) Phosphoaspartates in bacterial signal transduction. Curr Opin Struct Biol 11: 679–684.
- View Article
- Google Scholar
56. Kim S-B, Shin B-S, Choi S-K, Kim C-K, Park S-H (2001) Involvement of acetyl phosphate in the in vivo activation of the response regulator ComA in Bacillus subtilis. FEMS Microbiol Lett 195: 179–183.
- View Article
- Google Scholar
57. Da Re SS, Deville-Bonne D, Tolstykh T, Véron M, Stock JB (1999) Kinetics of CheY phosphorylation by small molecule phosphodonors. FEBS Letts 457: 323–326.
- View Article
- Google Scholar
58. Mayover TL, Halkides CJ, Stewart RC (1999) Kinetic characterization of CheY phosphorylation reactions: comparison of P-CheA and small-molecule phosphodonors. Biochem 38: 2259–2271.
- View Article
- Google Scholar
59. Fiedler U, Weiss V (1995) A common switch in activation of the response regulators NtrC and PhoB: phosphorylation induces dimerization of the receiver modules. EMBO J 14: 3696–3705.
- View Article
- Google Scholar
60. Zhang JH, Xiao G, Gunsalus RP, Hubbell WL (2003) Phosphorylation triggers domain separation in the DNA binding response regulator NarL. Biochem 42: 2552–2559.
- View Article
- Google Scholar
61. Da Re S, Schumacher J, Rousseau P, Fourment J, Ebel C, et al. (1999) Phosphorylation-induced dimerization of the FixJ receiver domain. Mol Microbiol 34: 504–511.
- View Article
- Google Scholar
62. Da Re S, Bertagnoli S, Fourment J, Reyrat J-M, Kahn D (1994) Intramolecular signal transduction within the FixJ transcriptional activator: in vitro evidence for the inhibitory effect of the phosphorylatable regulatory domain. Nuc Acids Res 22: 1555–1561.
- View Article
- Google Scholar
63. Boucher PE, Stibitz S (1995) Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis. J Bacteriol 177: 6486–6491.
- View Article
- Google Scholar
64. Boucher PE, Menozzi FD, Locht C (1994) The modular architecture of bacterial response regulators. Insights into the activation mechanism of the BvgA transactivator of Bordetella pertussis. J Mol Biol 241: 363–377.
- View Article
- Google Scholar
65. Makino K, Amemura M, Kawamoto T, Kimura S, Shinagawa H, et al. (1996) DNA binding of PhoB and its interaction with RNA polymerase. J Mol Biol 259: 15–26.
- View Article
- Google Scholar
66. Qi Y, Hulett FM (1998) PhoP∼P and RNA polymerase σ^A holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP∼P activator sites within the coding region stimulate transcription in vitro. Mol Microbiol 28: 1187–1197.
- View Article
- Google Scholar
67. Brüggemann H, Bäumer S, Fricke WF, Wiezer A, Liesegang H, et al. (2003) The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc Natl Acad Sci USA 100: 1316–1321.
- View Article
- Google Scholar
68. Sebaihia M, Peck MW, Minton NP, Thomson NR, Holden MT, et al. (2007) Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes. Genome Res 17: 1082–1092.
- View Article
- Google Scholar

[ref1] 1. Rood JI (1998) Virulence genes of Clostridium perfringens. Annu Rev Microbiol 52: 333–360.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Songer JG (2005) Clostridial diseases in domestic animals. In: Dürre P, editor. Handbook on Clostridia. Boca Raton: CRC Press. pp. 527–542.

[ref3] 3. Awad MM, Bryant AE, Stevens DL, Rood JI (1995) Virulence studies on chromosomal α-toxin and θ-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens-mediated gas gangrene. Mol Microbiol 15: 191–202.
View Article
Google Scholar

[6] View Article

[7] Google Scholar

[ref4] 4. Awad MM, Ellemor DM, Boyd RL, Emmins JJ, Rood JI (2001) Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens-mediated gas gangrene. Infect Immun 69: 7904–7910.
View Article
Google Scholar

[9] View Article

[10] Google Scholar

[ref5] 5. Ellemor DM, Baird RN, Awad MM, Boyd RL, Rood JI, et al. (1999) Use of genetically manipulated strains of Clostridium perfringens reveals both alpha-toxin and theta-toxin are required for vascular leukostasis to occur in experimental gas gangrene. Infect Immun 67: 4902–4907.
View Article
Google Scholar

[12] View Article

[13] Google Scholar

[ref6] 6. Stevens DL, Tweten RK, Awad MM, Rood JI, Bryant AE (1997) Clostridial gas gangrene : Evidence that α and θ toxins differentially modulate the immune response and induce acute tissue necrosis. J Infect Dis 176: 189–195.
View Article
Google Scholar

[15] View Article

[16] Google Scholar

[ref7] 7. Lyristis M, Bryant AE, Sloan J, Awad MM, Nisbet IT, et al. (1994) Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens. Mol Microbiol 12: 761–777.
View Article
Google Scholar

[18] View Article

[19] Google Scholar

[ref8] 8. Shimizu T, Ba-Thein W, Tamaki M, Hayashi H (1994) The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens. J Bacteriol 176: 1616–1623.
View Article
Google Scholar

[21] View Article

[22] Google Scholar

[ref9] 9. Shimizu T, Yaguchi H, Ohtani K, Banu S, Hayashi H (2002) Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins. Mol Microbiol 43: 257–265.
View Article
Google Scholar

[24] View Article

[25] Google Scholar

[ref10] 10. Banu S, Ohtani K, Yaguchi H, Swe T, Cole ST, et al. (2000) Identification of novel VirR/VirS-regulated genes in Clostridium perfringens. Mol Microbiol 35: 854–864.
View Article
Google Scholar

[27] View Article

[28] Google Scholar

[ref11] 11. Shimizu T, Shima K, Yoshino K, Yonezawa K, Shimizu T, et al. (2002) Proteome and transcriptome analysis of the virulence genes regulated by the VirR/VirS system in Clostridium perfringens. J Bacteriol 184: 2587–2594.
View Article
Google Scholar

[30] View Article

[31] Google Scholar

[ref12] 12. Myers GSA, Rasko DA, Cheung JK, Ravel J, Seshadri R, et al. (2006) Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens. Genome Res 16: 1031–1040.
View Article
Google Scholar

[33] View Article

[34] Google Scholar

[ref13] 13. Okumura K, Ohtani K, Hayashi H, Shimizu T (2008) Characterization of genes regulated directly by the VirR/VirS system in Clostridium perfringens. J Bacteriol 190: 7719–7727.
View Article
Google Scholar

[36] View Article

[37] Google Scholar

[ref14] 14. Ohtani K, Kawsar HI, Okumura K, Hayashi H, Shimizu T (2003) The VirR/VirS regulatory cascade affects transcription of plasmid-encoded putative virulence genes in Clostridium perfringens strain 13. FEMS Microbiol Lett 222: 137–141.
View Article
Google Scholar

[39] View Article

[40] Google Scholar

[ref15] 15. Okumura K, Kawsar HI, Shimizu T, Ohta T, Hayashi H, et al. (2005) Identification and characterization of a cell-wall anchored DNase gene in Clostridium perfringens. FEMS Microbiol Lett 242: 281–285.
View Article
Google Scholar

[42] View Article

[43] Google Scholar

[ref16] 16. Ohtani K, Takamura H, Yaguchi H, Hayashi H, Shimizu T (2000) Genetic analysis of the ycgJ-metB-cysK-ygaG operon negatively regulated by the VirR/VirS system in Clostridium perfringens. Microbiol Immunol 44: 525–528.
View Article
Google Scholar

[45] View Article

[46] Google Scholar

[ref17] 17. Kawsar HI, Ohtani K, Okumura K, Hayashi H, Shimizu T (2004) Organization and transcriptional regulation of myo-inositol operon in Clostridium perfringens. FEMS Microbiol Lett 235: 289–295.
View Article
Google Scholar

[48] View Article

[49] Google Scholar

[ref18] 18. Cheung JK, Rood JI (2000) Glutamate residues in the putative transmembrane region are required for the function of the VirS sensor histidine kinase from Clostridium perfringens. Microbiol-SGM 146: 517–525.
View Article
Google Scholar

[51] View Article

[52] Google Scholar

[ref19] 19. Stock AM, Robinson VL, Goudreau PN (2000) Two-component signal transduction. Annu Rev Biochem 69: 183–215.
View Article
Google Scholar

[54] View Article

[55] Google Scholar

[ref20] 20. Cheung JK, McGowan S, Rood JI (2005) Two-component signal transduction systems in clostridia. In: Dürre P, editor. Handbook on Clostridia. Boca Raton: CRC Press Taylor & Frances Group. pp. 545–560.

[ref21] 21. McGowan S, Lucet IS, Cheung JK, Awad MM, Whisstock JC, et al. (2002) The FxRxHrS motif: a conserved region essential for DNA binding of the VirR response regualtor from Clostridium perfringens. J Mol Biol 322: 997–1011.
View Article
Google Scholar

[58] View Article

[59] Google Scholar

[ref22] 22. Cheung JK, Dupuy B, Deveson DS, Rood JI (2004) The spatial organization of the VirR boxes is critical for VirR-mediated expression of the perfringolysin O gene, pfoA, from Clostridium perfringens. J Bacteriol 186: 3321–3330.
View Article
Google Scholar

[61] View Article

[62] Google Scholar

[ref23] 23. Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, et al. (2002) Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci USA 99: 996–1001.
View Article
Google Scholar

[64] View Article

[65] Google Scholar

[ref24] 24. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

[ref25] 25. Rood JI, Maher EA, Somer EB, Campos E, Duncan CL (1978) Isolation and characterization of multiple antibiotic-resitant Clostridium perfringens strains from porcine feces. Antimicrob Agents and Chemother 13: 871–880.
View Article
Google Scholar

[68] View Article

[69] Google Scholar

[ref26] 26. Rood JI (1983) Transferable tetracycline resistance in Clostridium perfringens strains of porcine origin. Can J Microbiol 29: 1241–1246.
View Article
Google Scholar

[71] View Article

[72] Google Scholar

[ref27] 27. Sloan J, Warner TA, Scott PT, Bannam TL, Berryman DI, et al. (1992) Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid. Plasmid 27: 207–219.
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref28] 28. Morelle G (1989) A plasmid extraction procedure on a miniprep scale. Focus 11: 7–8.
View Article
Google Scholar

[77] View Article

[78] Google Scholar

[ref29] 29. Roberts I, Holmes WM, Hylemon PB (1986) Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum. Appl Environ Microbiol 52: 197–199.
View Article
Google Scholar

[80] View Article

[81] Google Scholar

[ref30] 30. Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96: 23–28.
View Article
Google Scholar

[83] View Article

[84] Google Scholar

[ref31] 31. Scott PT, Rood JI (1989) Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens. Gene 82: 327–333.
View Article
Google Scholar

[86] View Article

[87] Google Scholar

[ref32] 32. O'Connor JR, Lyras D, Farrow KA, Adams V, Powell DR, et al. (2006) Construction and analysis of chromosomal Clostridium difficile mutants. Mol Microbiol 61: 1335–1351.
View Article
Google Scholar

[89] View Article

[90] Google Scholar

[ref33] 33. Cheung JK, Rood JI (2000) The VirR response regulator from Clostridium perfringens binds independently to two imperfect direct repeats located upstream of the pfoA promoter. J Bacteriol 182: 57–66.
View Article
Google Scholar

[92] View Article

[93] Google Scholar

[ref34] 34. Deng WP, Nickoloff JA (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem 200: 81–88.
View Article
Google Scholar

[95] View Article

[96] Google Scholar

[ref35] 35. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260: 289–298.
View Article
Google Scholar

[98] View Article

[99] Google Scholar

[ref36] 36. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680–685.
View Article
Google Scholar

[101] View Article

[102] Google Scholar

[ref37] 37. Chen Y, McClane BA, Fisher DJ, Rood JI, Gupta P (2005) Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron. Appl Environ Microbiol 71: 7542–7547.
View Article
Google Scholar

[104] View Article

[105] Google Scholar

[ref38] 38. Czeczulin JR, Collie RE, McClane BA (1996) Regulated expression of Clostridium perfringens enterotoxin in naturally cpe-negative type A, B, and C isolates of C. perfringens. Infect Immun 64: 3301–3309.
View Article
Google Scholar

[107] View Article

[108] Google Scholar

[ref39] 39. Wolanin PM, Thomason PA, Stock JB (2002) Histidine protein kinases: key signal transducers outside the animal kingdom. Genome biology 3: REVIEWS3013.
View Article
Google Scholar

[110] View Article

[111] Google Scholar

[ref40] 40. Bannam TL, Rood JI (1993) Clostridium perfringens-Escherichia coli shuttle vectors that carry single antibiotic resistance determinants. Plasmid 29: 223–235.
View Article
Google Scholar

[113] View Article

[114] Google Scholar

[ref41] 41. Ba-Thein W, Lyristis M, Ohtani K, Nisbet IT, Hayashi H, et al. (1996) The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens. J Bacteriol 178: 2514–2520.
View Article
Google Scholar

[116] View Article

[117] Google Scholar

[ref42] 42. Higashi T, Chazono M, Inoue K, Yanagase Y, Amano T, et al. (1973) Complementation of θ-toxinogenicity between mutants of two groups of Clostridium perfringens. Biken J 16: 1–9.
View Article
Google Scholar

[119] View Article

[120] Google Scholar

[ref43] 43. Imagawa T, Higashi Y (1992) An activity which restores theta toxin activity in some theta toxin-deficient mutants of Clostridium perfringens. Microbiol Immunol 36: 523–527.
View Article
Google Scholar

[122] View Article

[123] Google Scholar

[ref44] 44. Imagawa T, Tatsuki T, Higashi Y, Amano T (1981) Complementation characteristics of newly isolated mutants from two groups of strains of Clostridium perfringens. Biken J 24: 13–21.
View Article
Google Scholar

[125] View Article

[126] Google Scholar

[ref45] 45. Ohtani K, Yuan Y, Hassan S, Wang R, Wang Y, et al. (2009) Virulence gene regulation by the agr system in Clostridium perfringens. J Bacteriol Apr 10: [Epub ahead of print].
View Article
Google Scholar

[128] View Article

[129] Google Scholar

[ref46] 46. Eilers M, Shekar SC, Shieh T, Smith SO, Fleming PJ (2000) Internal packing of helical membrane proteins. Proc Natl Acad Sci USA 97: 5796–5801.
View Article
Google Scholar

[131] View Article

[132] Google Scholar

[ref47] 47. Parkinson JS, Kofoid EC (1992) Communication modules in bacterial signaling proteins. Annu Rev Genet 26: 71–112.
View Article
Google Scholar

[134] View Article

[135] Google Scholar

[ref48] 48. Bilwes AM, Alex LA, Crane BR, Simon MI (1999) Structure of CheA, a signal-transducing histidine kinase. Cell 96: 131–141.
View Article
Google Scholar

[137] View Article

[138] Google Scholar

[ref49] 49. Dutta R, Inouye M (2000) GHKL, an emergent ATPase/kinase superfamily. TIBS 25: 24–28.
View Article
Google Scholar

[140] View Article

[141] Google Scholar

[ref50] 50. Dutta R, Inouye M (1996) Reverse phosphotransfer from OmpR to EnvZ in a kinase⁻/phosphatase⁺ mutant of EnvZ (EnvZ.N347D), a bifunctional signal transduction transducer of Escherichia coli. J Biol Chem 271: 1424–1429.
View Article
Google Scholar

[143] View Article

[144] Google Scholar

[ref51] 51. Hsing W, Russo FD, Bernd KK, Silhavy TJ (1998) Mutations that alter the kinase and phosphatase activities of the two-component sensor EnvZ. J Bacteriol 180: 4538–4546.
View Article
Google Scholar

[146] View Article

[147] Google Scholar

[ref52] 52. Hirschman A, Boukhvalova M, VanBruggen R, Wolfe AJ, Stewart RC (2001) Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli. Biochem 40: 13876–13887.
View Article
Google Scholar

[149] View Article

[150] Google Scholar

[ref53] 53. Marina A, Mott C, Auyzenberg A, Hendrickson WA, Waldburger CD (2001) Structural and mutational analysis of the PhoQ histidine kinase catalytic domain. J Biol Chem 276: 41182–41190.
View Article
Google Scholar

[152] View Article

[153] Google Scholar

[ref54] 54. West AH, Stock AM (2001) Histidine kinases and response regulator proteins in two-component signaling systems. TIBS 26: 369–376.
View Article
Google Scholar

[155] View Article

[156] Google Scholar

[ref55] 55. Cho HS, Pelton JG, Yan D, Kustu S, Wemmer DE (2001) Phosphoaspartates in bacterial signal transduction. Curr Opin Struct Biol 11: 679–684.
View Article
Google Scholar

[158] View Article

[159] Google Scholar

[ref56] 56. Kim S-B, Shin B-S, Choi S-K, Kim C-K, Park S-H (2001) Involvement of acetyl phosphate in the in vivo activation of the response regulator ComA in Bacillus subtilis. FEMS Microbiol Lett 195: 179–183.
View Article
Google Scholar

[161] View Article

[162] Google Scholar

[ref57] 57. Da Re SS, Deville-Bonne D, Tolstykh T, Véron M, Stock JB (1999) Kinetics of CheY phosphorylation by small molecule phosphodonors. FEBS Letts 457: 323–326.
View Article
Google Scholar

[164] View Article

[165] Google Scholar

[ref58] 58. Mayover TL, Halkides CJ, Stewart RC (1999) Kinetic characterization of CheY phosphorylation reactions: comparison of P-CheA and small-molecule phosphodonors. Biochem 38: 2259–2271.
View Article
Google Scholar

[167] View Article

[168] Google Scholar

[ref59] 59. Fiedler U, Weiss V (1995) A common switch in activation of the response regulators NtrC and PhoB: phosphorylation induces dimerization of the receiver modules. EMBO J 14: 3696–3705.
View Article
Google Scholar

[170] View Article

[171] Google Scholar

[ref60] 60. Zhang JH, Xiao G, Gunsalus RP, Hubbell WL (2003) Phosphorylation triggers domain separation in the DNA binding response regulator NarL. Biochem 42: 2552–2559.
View Article
Google Scholar

[173] View Article

[174] Google Scholar

[ref61] 61. Da Re S, Schumacher J, Rousseau P, Fourment J, Ebel C, et al. (1999) Phosphorylation-induced dimerization of the FixJ receiver domain. Mol Microbiol 34: 504–511.
View Article
Google Scholar

[176] View Article

[177] Google Scholar

[ref62] 62. Da Re S, Bertagnoli S, Fourment J, Reyrat J-M, Kahn D (1994) Intramolecular signal transduction within the FixJ transcriptional activator: in vitro evidence for the inhibitory effect of the phosphorylatable regulatory domain. Nuc Acids Res 22: 1555–1561.
View Article
Google Scholar

[179] View Article

[180] Google Scholar

[ref63] 63. Boucher PE, Stibitz S (1995) Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis. J Bacteriol 177: 6486–6491.
View Article
Google Scholar

[182] View Article

[183] Google Scholar

[ref64] 64. Boucher PE, Menozzi FD, Locht C (1994) The modular architecture of bacterial response regulators. Insights into the activation mechanism of the BvgA transactivator of Bordetella pertussis. J Mol Biol 241: 363–377.
View Article
Google Scholar

[185] View Article

[186] Google Scholar

[ref65] 65. Makino K, Amemura M, Kawamoto T, Kimura S, Shinagawa H, et al. (1996) DNA binding of PhoB and its interaction with RNA polymerase. J Mol Biol 259: 15–26.
View Article
Google Scholar

[188] View Article

[189] Google Scholar

[ref66] 66. Qi Y, Hulett FM (1998) PhoP∼P and RNA polymerase σ^A holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP∼P activator sites within the coding region stimulate transcription in vitro. Mol Microbiol 28: 1187–1197.
View Article
Google Scholar

[191] View Article

[192] Google Scholar

[ref67] 67. Brüggemann H, Bäumer S, Fricke WF, Wiezer A, Liesegang H, et al. (2003) The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc Natl Acad Sci USA 100: 1316–1321.
View Article
Google Scholar

[194] View Article

[195] Google Scholar

[ref68] 68. Sebaihia M, Peck MW, Minton NP, Thomson NR, Holden MT, et al. (2007) Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes. Genome Res 17: 1082–1092.
View Article
Google Scholar

[197] View Article

[198] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Bacterial strains, plasmids and growth conditions

Perfringolysin O assay

Molecular techniques

Random Mutagenesis

Site-directed Mutagenesis

Construction of expression plasmids

Overexpression and purification of His-tagged proteins

Autophosphorylation of VirSc and Phosphotransfer from VirSc to VirR

Isolation of C. perfringens cell extracts

Western blot analysis

Introduction of virR and virRD57N onto the chromosome by use of a Targetron

Isolation of RNA and Quantitative Real Time (QRT)-PCR

Results

Identification of functional residues in VirS

Residues in the C-terminal domain of VirS are essential for autophosphorylation

Phosphotransfer from VirSc to VirR in vitro

VirR proteins with mutations in conserved residues are still functional in vivo

The gene dosage of virR is important for the normal regulation of perfringolysin O production

Analysis of chromosomal mutants reveals that phosphorylation of VirR is essential for optimal perfringolysin O production

Discussion

Supporting Information

Table S1.

Table S2.

Acknowledgments

Author Contributions

References

Introduction of virR and virR_D57N onto the chromosome by use of a Targetron