The five residues preceding the cleavage site for CSF are important for CSF production.
We sought to determine whether the conservation in Phr proteins of five residues preceding the cleavage site for release of the mature Phr pentapeptides was due to a requirement for these residues for directing the cleavage event. To this end, we constructed mutants with substitutions at positions −5 through −1 relative to the cleavage site in PhrC that releases CSF. Substitutions that moved a residue away from the identified consensus sequence were introduced (Fig. ). Site-directed mutagenesis of phrC
was performed, and the mutant phrC
genes were introduced into B. subtilis
under the control of the IPTG-inducible Pspachy
promoter in strains lacking endogenous phrC
. The strains were grown to mid-exponential phase in minimal media, and then PhrC expression was induced. After this the cultures were grown for ~2 cell doublings, and then culture supernatants were harvested. The levels of CSF that had accumulated in the culture supernatants were determined. Briefly, the CSF in the culture supernatants was partially purified using reverse-phase chromatography to separate CSF from other signaling peptides that affect ComA-controlled gene expression, and then it was quantified based on the ability of CSF to induce expression of a ComA-responsive reporter fusion, srfA-lacZ
. Importantly, a strain lacking CSF had levels of activity either below or at the limit of detection (Fig. ) (15
), indicating that any activity that is observed with other strains is CSF dependent.
FIG. 1. Substitution of the five residues preceding the CSF cleavage site affects CSF production. (A) Amino acid sequence of the C-terminal 15 residues of PhrC. The five residues preceding the cleavage site are indicated by bold italics, and the mature signaling (more ...)
The presence of charged or polar residues at the −5 and −3 positions was predicted based on the consensus sequence, and we substituted Ala for the Asp and His residues that occur at these positions in PhrC to remove the charge and polarity. These substitutions resulted in 40% (P = 0.006, n = 4) and 65% (P = 0.03, n = 3) decreases in CSF production for the Asp-to-Ala and His-to-Ala substitutions, respectively (Fig. ). These data support the notion that a charged or polar residue is important at the −5 and −3 positions relative to the cleavage site for CSF, although the −3 position appears to be more important. A Phe residue occurs at the −4 position of PhrC, and in the strain expressing PhrC with a Lys residue at this position there was a 95% (P = 0.0007, n = 3) reduction in CSF production (Fig. ). These data are consistent with the prediction based on the consensus sequence that a hydrophobic residue is required at this position. A Val residue is predicted to be important at the −2 position, and as we reasoned that other hydrophobic residues might be functional at this position, we changed the Val residue of PhrC to Glu. Interestingly, in the strain expressing the Glu variant there was only a 50% (P = 0.02, n = 3) reduction in CSF production (Fig. ). Thus, while Val appears to be more optimal for CSF production than Glu, CSF production was surprisingly tolerant of a change from Val to a charged residue. The findings for the −1 position of the consensus sequence indicate that an Ala residue is critical. Intriguingly, a Thr residue is at this position in PhrC. We replaced this Thr residue by Ala and found that the strains produced indistinguishable levels of CSF compared to the strains with wild-type PhrC (P = 0.8, n = 3) (Fig. ). The side chains of Ala and Thr are both small and at least partially hydrophobic. To test whether these side chains are important for CSF production, we replaced the Thr residue of PhrC with Lys, an amino acid with a large, charged side chain. CSF production was tolerant of such a radical change, and in strains having the Thr-to-Lys substitution there was only a 35% (P = 0.04, n = 3) decrease in CSF production (Fig. ). Although the effects of some of the amino acid substitutions were relatively small, together, the data obtained indicate that the five residues that precede the cleavage site in PhrC for CSF are required for normal CSF production.
The five residues preceding the CSF cleavage site are not required for a functional signal sequence.
A possible explanation for the decrease in CSF production caused by amino acid substitutions at positions −1 through −5 relative to the CSF cleavage site is that the substitutions affect an extended signal sequence necessary for secretion. To test this possibility, we created fusions of the mutant PhrC proteins to the E. coli alkaline phosphatase protein (PhoA), which lacked its own signal sequence. The PhrC-PhoA fusions were expressed in B. subtilis from the thrC locus under the control of the IPTG-inducible Pspachy promoter. PhrC-PhoA secretion was monitored by assaying the alkaline phosphatase activity in culture supernatants. As expected, a strain expressing PhoA lacking a signal sequence had no measurable alkaline phosphatase activity (Fig. ). All of the strains expressing mutant PhrC-PhoA fusion proteins had alkaline phosphatase activities comparable to that of a strain expressing the wild-type PhrC-PhoA fusion protein (P > 0.25, n = 3) (Fig. ). These data indicate that all of the mutant PhrC proteins contained a functional signal sequence and suggest that a secretion defect was not the cause of the reduced CSF production by strains expressing the mutant PhrC proteins.
FIG. 2. Mutant PhrC proteins are secreted at the same level as wild-type PhrC as measured by alkaline phosphatase activity for strains carrying PhrC-PhoA fusions. PhoA activity was measured using culture supernatants of strains BAL2199 (Δss-PhoA), BAL2200 (more ...) Synthetic pro-CSF peptides with amino acid substitutions are cleaved less efficiently to CSF.
The data described above indicated that strains expressing PhrC mutant proteins with amino acid substitutions at positions −5 through −1 relative to the CSF cleavage site produce less CSF and that the reduction in CSF production was not due to a reduction in secretion of PhrC. We reasoned that this reduction could have been due to reduced PhrC protein expression or reduced protease recognition and/or cleavage of PhrC to CSF. We measured the levels of phrC mRNA for the three mutant strains with the lowest levels of CSF production (i.e., the mutants with substitutions at the −2 to −4 positions) and observed no significant differences in the levels of phrC mRNA (see Fig. S1 in the supplemental material). There is no method to directly test the levels of PhrC inside cells at this time, and attempts to indirectly measure PhrC levels using a C-terminal epitope tag have been unsuccessful, possibly due to proteolytic removal of the tag extracellularly (data not shown). Given that the mutant PhrC-PhoA fusion proteins were expressed at levels similar to the levels of the wild-type fusion protein, it seemed unlikely that the defect in CSF production was due to a defect in PhrC expression.
To test directly the possibility that the mutant PhrC proteins were cleaved less efficiently to CSF, we synthesized peptides that corresponded to the portion of PhrC predicted to be C terminal to the signal sequence for secretion. The sequence of one peptide, pro-CSF-WT, was identical to the sequence of the C-terminal 15 residues of PhrC, and this peptide has been used previously in studies of CSF proteolytic processing (15
). We also synthesized peptides that individually had the three amino acid substitutions that resulted in the greatest defects in CSF production, pro-CSF-F32K, pro-CSF-H33A, and pro-CSF-V34E. These peptides differed at the −2, −3, or −4 position relative to the CSF cleavage site (Fig. ).
FIG. 3. Synthetic pro-CSF peptides with amino acid substitutions at the −4, −3, and −2 positions are cleaved less efficiently to mature CSF. (A) Sequence of synthetic pro-CSF peptides. (B) Synthetic pro-CSF peptides were incubated with (more ...)
To test for cleavage of the synthetic pro-CSF peptides to CSF, the peptides were incubated with washed whole cells of B. subtilis
strain BAL950, which lacks phrC
and thus cannot produce any CSF. BAL950 also lacks the oligopeptide permease responsible for uptake of CSF from media and comQ
, which encodes a protein needed to produce a signaling peptide with activity similar to that of CSF. All three of these mutations were included to increase the sensitivity of the assay for detection of CSF. Approximately 108
cells were incubated with pro-CSF for 40 min, a time sufficient for nearly complete cleavage of 100 pmol of pro-CSF-WT to CSF (15
). After incubation, the cells were removed, and the culture medium was fractionated on a reverse-phase C18
column to separate pro-CSF from CSF. The amount of CSF that eluted from the column was then determined by determining the level of β-galactosidase specific activity after treatment of cells containing the ComA-controlled reporter fusion srfA-lacZ
with dilutions of the eluates.
When the cells were incubated with pro-CSF-WT, CSF production was observed, but CSF production was not observed when the cells and pro-CSF-WT were incubated separately (Fig. and data not shown). Compared to pro-CSF-WT, each of the mutant pro-CSF peptides produced less CSF when it was incubated with cells (Fig. ). Incubation with the pro-CSF-F32K peptide, with a substitution at the −4 position, yielded only 10% of the CSF produced with the pro-CSF-WT peptide (P = 0.01, n = 3), similar to the results obtained with the same amino acid substitution in the in vivo context (compare Fig. and ). Incubation with the pro-CSF-V34E and pro-CSF-H33A peptides yielded 25% (P = 0.004, n = 3) and 60% (P = 0.02, n = 3) of the CSF produced with the pro-CSF-WT peptide, respectively. The magnitude of the defect caused by these substitutions was different than the magnitude observed when the same substitutions were encoded by phrC (compare Fig. and ). This may have been because there was a slightly different profile of proteases able to cleave pro-CSF to CSF after cells were washed. Nevertheless, the defect in CSF production caused by the amino acid substitutions at positions −2 to −4 when they were part of an exogenously added peptide supports the hypothesis that these amino acid substitutions decreased the efficiency of cleavage of PhrC to CSF.
To confirm that the mutant peptides were cleaved less efficiently to the CSF peptide sequence ERGMT, we measured the amounts of CSF produced after incubation of the pro-CSF peptides using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for CSF. This assay recorded the intensity of the transition of the doubly charged parent ion (m/z
297.2) to the singly charged fragment ion (m/z
386.5) under collisionally activated dissociation, multiple-reaction-monitoring (MRM) conditions, as previously described (15
). Because whole bacterial cells would have interfered with the LC-MS/MS-MRM procedure, we used a cell wall-enriched fraction of B. subtilis
cells, which we have previously shown to be the cellular fraction that contains the majority of pro-CSF processing activity (15
). Under the prescribed chromatography conditions, the retention time of synthetic CSF was 14.5 min. Neither the pro-CSF peptides nor the cell wall fraction of B. subtilis
separately resulted in a significant LC-MS/MS-MRM response for CSF (data not shown). In contrast, when the cell wall fraction was incubated with pro-CSF-WT, a significant MRM response was recorded at the appropriate retention time (Fig. ), indicating that pro-CSF-WT was cleaved into the CSF pentapeptide. When the cell wall fraction was incubated with the mutant pro-CSF peptides, the level of the MRM response was not more than 10% of the level observed with pro-CSF-WT (Fig. ). The greater defect in cleavage of pro-CSF to CSF for the mutant peptides determined by this assay than by the biological assays could have been due to the change in the profile or levels of pro-CSF processing proteases that occurred during preparation of the cell wall-enriched fraction of cells. The data obtained demonstrate that the amino acid substitutions at the −2, −3, and −4 positions significantly decreased proteolytic cleavage of ERGMT from a precursor peptide.
CSF processing proteases, subtilisin and Vpr, cleave mutant pro-CSF peptides less efficiently.
We previously showed that cells lacking the secreted serine proteases subtilisin, Vpr, and Epr had a defect in production of CSF and that purified subtilisin and Vpr were able to cleave synthetic pro-CSF to CSF (15
). To further support the hypothesis that subtilisin and Vpr have a direct role in processing pro-CSF to CSF in vivo, we examined whether the amino acid substitutions in PhrC that decreased production of CSF in vivo similarly affected the cleavage of pro-CSF by subtilisin or Vpr. Purified subtilisin and Vpr were incubated separately with pro-CSF substrates having substitutions at positions −2 to −4 relative to the cleavage site, and the levels of CSF produced were determined using the biological assay for CSF (Fig. ).
FIG. 4. Cleavage of mutant pro-CSF substrates by subtilisin and Vpr. Purified subtilisin or Vpr was incubated with the pro-CSF substrates indicated. The level of CSF produced was normalized to the level of CSF produced after incubation with the wild-type pro-CSF (more ...)
The Phe-to-Lys substitution at the −4 position resulted in severely reduced cleavage of pro-CSF (98% reduction for subtilisin [P = <0.0001, n = 3] and 90% reduction for Vpr [P = 0.001, n = 3]). The His-to-Ala substitution at the −3 position had a more modest effect on cleavage (47% [P = 0.03, n = 3] and 79% [P = 0.003, n = 3] reductions in pro-CSF cleavage by subtilisin and Vpr, respectively). The Val-to-Glu substitution at the −3 position had the most disparate effects on pro-CSF cleavage (97% [P = <0.0001, n = 3] reduction in cleavage to CSF by subtilisin and 42% [P = 0.02, n = 4] reduction in cleavage by Vpr). Collectively, these data indicate that substitutions at positions −2 to −4 decreased the efficiency of cleavage of pro-CSF to CSF by subtilisin and Vpr, and they support the hypothesis that subtilisin and Vpr have a direct role in processing CSF in vivo.
Defining the amino acids that are tolerated at the −4 position of pro-CSF and allow cleavage.
To begin to determine the rules that govern what amino acid sequences can be recognized for cleavage that releases mature Phr pentapeptides, we changed the amino acid at the −4 position of pro-CSF to the other 19 canonical amino acids in order to determine which amino acids support cleavage that releases CSF. The −4 position was chosen for this analysis as substitutions at this position resulted in the greatest defects in CSF production. CSF production by strains individually expressing 1 of the 19 mutant PhrC-F32 proteins was assessed using the biological assay (Fig. ).
FIG. 5. Amino acid substitutions at the −4 position relative to the cleavage site of PhrC. The Phe at position 32 of PhrC was replaced by the other 19 canonical amino acids. The levels of endogenous CSF production by cells expressing the mutant PhrC proteins (more ...)
The identified consensus sequence of the residues that precede the cleavage site for Phr peptides indicated that a hydrophobic residue is important at the −4 position. Consistent with this, the hydrophobic residues, such as Val, Ile, and Leu, supported levels of CSF production similar to the levels exhibited by the wild-type strain with a Phe residue at the −4 position. Furthermore, substitution of a hydrophobic Met residue at this position resulted in levels of CSF production that were 1.8-fold-higher than the levels observed with the Phe residue (P = 0.03, n = 4). The only exceptions to this were the hydrophobic Ala and Trp substitutions, which resulted in 20% (P = 0.03, n = 3) and 50% (P = 0.03, n = 3) decreases in CSF production, respectively. Trp is the largest amino acid, and the defect in CSF production may have been due to the large side chain sterically hindering cleavage. At the other extreme, Ala is the smallest hydrophobic amino acid, and the defect in CSF production may have been due to the small size of the side chain, which was not able to stabilize the interaction with pro-CSF processing proteases.
As further support for the hypothesis that a hydrophobic residue at the −4 position is important, some polar residues at this position decreased production of CSF. As noted above, a Lys substitution severely decreased CSF production; in addition, Arg, Cys, and Asp substitutions decreased CSF production 72% (P = 0.05, n = 3), 68% (P = 0.008, n = 3), and 44% (P = 0.03, n = 5), respectively. However, some polar amino acids could be tolerated at this position; a Glu, Asn, or Gln substitution did not result in a statistically significant difference in CSF production. The latter finding indicates that the prediction based on the consensus sequence that there is a hydrophobic residue at this position is not strictly correct. While there is a preference for hydrophobic residues at the −4 position, some polar residues can be tolerated.
Conclusions and implications.
In this study, we identified residues that are important for release of the CSF signaling peptide of B. subtilis
from its precursor protein, PhrC. We previously identified a loose consensus sequence for five residues preceding the cleavage sites that produce the mature Phr pentapeptides of B. subtilis
) (Table ). Analysis of substitution of these five amino acid residues in PhrC, the precursor protein for the Phr peptide CSF, revealed the importance of these residues in directing cleavage of the precursor protein to release CSF. However, the amino acid substitution data also revealed that a relatively wide variety of sequences can be tolerated at these positions and that this toleration may be due to the fact that multiple proteases are able to cleave PhrC.
PhrC appears to tolerate a relatively wide variety of sequences in the residues preceding the cleavage site, resulting in CSF production that is relatively robust to mutational perturbation. For example, it was observed that only 4 of 19 amino acid substitutions at the −4 position resulted in a >2-fold defect in CSF production. This robustness to mutational perturbation appears to be due to the presence of multiple, redundant proteases that process CSF. If subtilisin were the only protease that processes PhrC to CSF, we would have observed that a Val-to-Glu substitution at the −2 position severely decreased CSF production. Instead, this substitution had a moderate effect on CSF production because Vpr is able to process PhrC with this substitution, when subtilisin cannot process it. It is interesting that a Glu residue occurs at the −2 position of PhrE and PhrH (with a one-residue gap allowed for the PhrH alignment, although it is difficult at this time to accurately predict the sequence of the mature PhrH peptide). Given the ability of Vpr to process a PhrC substrate with a Glu residue at the −2 position, we predict that Vpr and not subtilisin plays a significant role in processing of the PhrE and PhrH peptides. Further work could determine whether production of these Phr peptides exhibits robustness to mutational perturbation similar to that of CSF.
These studies contributed to our goal of identifying the proteases that process Phr peptides of B. subtilis
and other bacteria. Even though CSF production was tolerant to many amino acid substitutions, identifying substitutions such as the Phe-to-Lys substitution at the −4 position relative to the cleavage site for CSF should allow us to test whether production of other Phr peptides is similarly disrupted by such a substitution. Of course, one method to test whether subtilisin, Vpr, or Epr has a role in cleaving other Phr peptides is to test these proteases in vitro to determine whether they have this processing activity, as we have done with PhrA (15
). Showing that analogous amino acid substitutions that affect processing of CSF by subtilisin and Vpr in vitro also affect processing of a Phr peptide, such as PhrA, would provide in vivo support for the hypothesis that these Phr peptides are processed by the same proteases. One question that is important to answer before substitutional analyses of other Phr precursor proteins are performed is whether there is flexibility in the position of the residues that direct cleavage of Phr peptides. As shown in Table , in order to obtain maximal alignment of the Phr precursor proteins, it was necessary to introduce a one-residue gap between the cleavage site and the consensus sequence for a few of the Phr peptides. Future studies need to address how this additional amino acid affects the residues required for cleavage of the Phr precursor proteins. In the long term, these studies have laid the foundation for determining the mechanism of production of Phr peptides in B. subtilis
and other bacteria.