Early studies of the partner-switching mechanism focused on the general stress response and sporulation pathways of B. subtilis
). More recently, it has become apparent that partner-switching modules are also involved in the regulation of virulence in several gram-positive pathogens, including S. aureus
), L. monocytogenes
), and M. tuberculosis
). Since the σB
-regulated stress response is highly conserved in gram-positive bacteria, these effects are likely due to a compromised ability to withstand environmental stresses encountered during infection. Interestingly, perhaps the most illustrative example of a role for partner switcher orthologs in the regulation of a system that is entirely dedicated to virulence can be found in the gram-negative respiratory pathogen B. bronchiseptica
, which has adapted a variation of the partner-switching paradigm to control type III secretion.
Our previous studies demonstrated that nonpolar, in-frame deletions in btrU
, or btrV
eliminate TTS in Bordetella
). In this report, we have demonstrated that BtrW binds and phosphorylates BtrV at the conserved serine residue S55. In addition, we have shown that BtrW is capable of homotypic interactions. These data suggest that the BtrV-BtrW complex may be similar to the B. subtilis
RsbV-RsbW complex, in which RsbW forms a homodimer, with each RsbW monomer interacting with and phosphorylating one molecule of RsbV (9
Characterization of BtrV and BtrW mutants carrying substitutions at conserved residues gave further insight into the biochemical properties of these partner switcher orthologs. By analogy to BtrV orthologs described in B. subtilis
), it was predicted that alanine substitution at S55 would prevent phosphorylation of BtrV by BtrW and stabilize the BtrW-BtrV(S55A) complex. On the other hand, mutation of S55 in BtrV to a negatively charged aspartate was predicted to mimic phosphorylated serine and prevent BtrW binding. To our surprise, the high levels of β-galactosidase activity produced by the reporter strains carrying BtrV(S55A)-BtrW and BtrV(S55D)-BtrW fusions indicated that BtrW formed stable complexes with both BtrV(S55A) and BtrV(S55D). The ability of Bordetella
BtrV(S55D) to interact with BtrW distinguishes this mutant from the B. subtilis
RsbV(S56D), RsbS(S59D), and SpoIIAA(S58D) orthologs, which do not bind their cognate anti-sigma factor/serine kinase partners (23
). Moreover, mutation of BtrV S55 to a negatively charged glutamic acid did not prevent BtrW binding (data not shown). We propose that the structures of Bordetella
BtrW and/or BtrV differ from their B. subtilis
orthologs such that replacement of the phosphorylatable serine in BtrV with negatively charged residues does not create sufficient electrostatic or steric clashes to disrupt interactions with BtrW. Instead, BtrW binds these mutant proteins with increased stability, presumably because phosphorylation does not occur. To our knowledge, this is the first report of a STAS domain-containing protein that preserves its ability to bind an anti-sigma factor upon mutation of the phosphorylated serine residue to a negatively charged amino acid.
Mutagenesis of the conserved asparagine residue in the BtrW N box eliminated the ability to phosphorylate BtrV. In addition, the bacterial two-hybrid analysis indicated that the reporter strain carrying BtrW(N51A) and wild-type BtrV fusion pairs produced higher levels of β-galactosidase activity than the reporter strain carrying wild-type BtrW and BtrV fusions. These observations are consistent with those obtained with the BtrV(S55A) and BtrV(S55D) mutants. Together, they suggest that some level of phosphorylation occurs in the E. coli
two-hybrid system and that phosphorylation reduces the stability of the BtrV-BtrW complex. The properties of BtrW(N51A) support the prediction that BtrW forms a Bergerat ATP-binding fold, similar to the one in SpoIIAB (5
), with the conserved asparagine residue at position 51 playing an essential role in serine kinase activity.
In contrast to the expected behavior of the BtrW N-box mutant, the dual alanine replacements of the conserved aspartate and glycine residues in the G1 box of BtrW resulted in an unexpected phenotype. Based on similarity to GHKL superfamily proteins (14
), it was predicted that mutation of both D82 and G84 would eliminate the kinase activity of BtrW. Surprisingly, BtrW(D82AG84A) retained detectable phosphorylation activity upon incubation with wild-type BtrV, despite the inability to form stable complexes with BtrV, as indicated by a negligible level of β-galactosidase activity in the two-hybrid analysis. The ability of the S55A mutation in BtrV to partially rescue the binding defect observed with the D82AG84A mutations in BtrW may explain this apparent inconsistency. We propose that BtrW(D82AG84A) interacts with BtrV at a level that is sufficient for detectable phosphorylation but is below the minimum threshold of stability required for detection in the two-hybrid system. Substitution of a nonphosphorylatable substrate, BtrV(S55A), decreases the dissociation rate of the BtrV(S55A)-BtrW(D82AG84A) complex to a level that allows detection by the two-hybrid analysis. Interestingly, alanine substitutions for the conserved aspartate and two surrounding residues in the G1 box of the B. subtilis
ortholog RsbT also disrupted binding of the RsbT mutant with its antagonist RsbS (37
). Although the conserved aspartate and glycine residues in the G1 box of BtrW play an important role in formation of the BtrV-BtrW complex, they do not seem to be involved in intramolecular interactions, since alanine substitutions did not affect binding to another molecule of BtrW, nor did they distort the secondary or tertiary structure of the mutant protein as determined by circular dichroism and fluorescence emission spectroscopy.
On the basis of results obtained with in-frame deletion mutations, we initially proposed that formation of a stable BtrV-BtrW complex is required for TTS in Bordetella
and that phosphorylation plays a negative role by disrupting the BtrV-BtrW interaction (24
). Although this model agrees with the observed lack of TTS in strains carrying ΔbtrW
, or ΔbtrU
alleles, it is inconsistent with our current observations. Most notably, although BtrV(S55A), BtrV(S55D), and BtrW(N51A) are incapable of undergoing phosphorylation and consequently form stable complexes, they fail to support TTS. Our cumulative results, which are summarized in Table , suggest a revised model for partner switcher-mediated regulation of Bordetella
TTS. We propose that the ability of BtrW to phosphorylate BtrV is required for the activation of TTS. Upon phosphorylation, the BtrV-BtrW complex is expected to dissociate, releasing BtrW and BtrV~P. Free BtrW may then associate with an alternative partner, which could be a part of the TTS machinery, a chaperone, or another regulatory molecule. We predict that this association is essential for TTS.
Compilation of data obtained from genetic and biochemical analyses of BtrW and BtrV mutantsa
The suggestion that BtrW interacts with an alternative binding partner to activate TTS is supported by several lines of evidence. Deletion of btrW
eliminates TTS, and all BtrW orthologs that have been sufficiently characterized have alternative binding partners in addition to their BtrV-like antagonists. Furthermore, the phenotype associated with the btrW
) allele is consistent with this hypothesis. In vitro analyses indicated that the BtrW G1-box mutant protein is defective in the ability to form a stable complex with BtrV. BtrW orthologs, such as SpoIIAB and RsbT, use overlapping sites for interacting with both of their alternative binding partners (17
), and it is therefore possible that the same domain of BtrW is involved in binding to both BtrV and its competing partner. If so, the G1-box mutation might not only weaken binding of BtrW to BtrV but also disrupt the interactions between BtrW and one or more alternative binding partners that are required for TTS.
Although the prediction that activation of Bordetella TTS requires the ability of BtrW to phosphorylate BtrV and disrupt the BtrV-BtrW complex is consistent with phenotypes obtained with the btrV(S55A), btrV(S55D), btrW(N51A), btrW(D82AG84A), and ΔbtrW alleles, it is also necessary to account for the observation that deletion of btrU or btrV eliminates TTS. In the model described above, both BtrU and BtrV are expected to negatively regulate TTS, since BtrU-mediated dephosphorylation of BtrV~P would promote formation of a BtrV-BtrW complex, which presumably inactivates BtrW. To accommodate the fact that TTS is inactive in ΔbtrU and ΔbtrV mutants, we propose that BtrU and BtrV play positive roles in addition to their negative roles in partner switcher-mediated regulation of TTS. Although the exact nature of these additional functions remains to be determined, BtrU and BtrV could form part of the TTS apparatus and/or stabilize binding partners required for secretion.
It is apparent that the Bordetella BtrW, BtrV, and BtrU proteins participate in a complex regulatory cascade that represents a variation of the partner-switching paradigm. Analysis of the biochemical properties of BtrW and BtrV suggests that uncharacterized interactions with components of the TTS apparatus and other regulatory factors are likely to play a key role in governing secretion. The identification of additional proteins that associate with BtrW and perhaps BtrV~P will be essential for understanding the regulatory mechanisms that control TTS.