Bacterial growth conditions.

All E. coli and B. subtilis strains were grown in LB broth or on LB agar. Antibiotics were used at the following concentrations for B. subtilis: chloramphenicol, 5 μg/ml; erythromycin plus lincomycin (MLS), 1 μg/ml and 25 μg/ml, respectively; kanamycin, 5 μg/ml; spectinomycin, 100 μg/ml; and tetracycline, 10 μg/ml. The β-galactosidase chromogenic indicator 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal; RPI) was used at a concentration of 100 μg/ml. Isopropyl β-d-1-thiogalactopyranoside (IPTG; RPI) was used at a final concentration of 1 mM unless otherwise noted. Antibiotics were used at the following concentrations for E. coli: ampicillin, 100 μg/ml; chloramphenicol, 10 μg/ml; and kanamycin, 50 μg/ml.

β-Galactosidase activity assays.

Cultures were grown overnight in LB broth at 30°C, and 20-μl amounts were spotted in triplicate onto LB agar. The plates were incubated at 37°C for 6 h. Samples were harvested from the plates and resuspended in 500 μl of Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 50 mM β-mercaptoethanol, pH 7.0). Resuspended cells (250 μl) were transferred to a 96-well plate, and measurements of the optical density at 600 nm (OD₆₀₀) were taken using a Tecan F50 (Tecan). The remaining cells were permeabilized by mixing with chloroform and 2% Sarkosyl (28, 42). The permeabilized cells (100 μl) were added to 96-well plates with 10 mg/ml ortho-nitrophenyl-β-galactoside (ONPG, 50 μl; RPI), and the OD₄₀₅ was measured every 1 min for 45 min. β-Galactosidase activity units were determined using the following calculation (μmol of ONP formed min⁻¹) × 10³/(OD₆₀₀ × ml of cell suspension) as previously described (43). Experiments were performed in triplicate. Means and standard deviations are shown.

Determination of MIC.

The MIC was determined by diluting overnight cultures 1:100 and growing cells to an OD₆₀₀ of 1.5 in LB containing 1 mM IPTG. The cultures were corrected to an OD₆₀₀ of 0.8 and diluted 1:50 in LB containing 1 mM IPTG. The cell mixtures (50 μl) were inoculated into 200 μl of LB containing 1 mM IPTG with hen egg white lysozyme ranging from 100 μg/ml to 0.10 μg/ml in a round-bottom 96-well plate. The cells were grown for 16 h at 37°C. Growth was defined as an OD₆₀₀ greater than 0.05. All assays were performed in triplicate on the same day, and each MIC determination was performed on three different days.

Expression of 6×His-tagged RsiV.

Overnight cultures of E. coli BL21λDE3 containing pKBW202 (pDEST17-6×His-rsiV^59-285) were grown at 30°C in LB with 100 μg/ml ampicillin. The following day, cell cultures were diluted 1:100 into 500 ml of fresh LB with ampicillin in 2-liter baffled flasks and incubated at 30°C until reaching an OD₆₀₀ of 0.5 to 0.6. IPTG was added to a final concentration of 1 mM to induce protein production, and the cultures incubated for an additional 4 h. At that time, the cells were chilled on ice and collected by centrifugation at 5,000 × g. The cell pellets were stored at −80°C until time for purification.

Purification of 6×His-tagged RsiV.

Cell pellets were thawed on ice and resuspended in 5 ml lysis buffer (50 mM sodium phosphate, 250 mM NaCl, 10 mM imidazole, pH 8.0) per 500 ml of initial culture volume. The cells were lysed by passaging through a Microfluidics LV1 high-shear microfluidizer (Newton, MA) twice. Lysate was centrifuged at 15,000 × g for 30 min at 4°C to pellet cellular debris. The cleared lysate was applied to a nickel affinity column to bind 6×His-tagged protein (Qiagen). The column was washed with 10 column volumes of wash buffer (50 mM sodium phosphate, 250 mM NaCl, 20 mM imidazole, pH 8.0). Protein was eluted with elution buffer (50 mM sodium phosphate, 250 mM NaCl, 250 mM imidazole, pH 8.0) and collected in 0.5-ml fractions. Samples from each fraction were analyzed by SDS-PAGE, and elution fractions containing the desired protein were combined. The combined fractions were then dialyzed into lysis buffer to remove the excess imidazole. The protein was further purified with a GE Healthcare ÄKTA fast protein liquid chromatography (FPLC) system (GE Healthcare Sciences, Pittsburg, PA) using a HisTrap HP nickel affinity column. The fractions containing 6×His-RsiV^59-285 were again combined, dialyzed into a storage buffer (25 mM Tris, 200 mM NaCl, 5% glycerol, pH 8.0), and frozen at −80°C until use.

Production of anti-RsiV polyclonal antibodies.

Purified 6×His-RsiV^59-285 protein was sent for production of polyclonal antibodies in New Zealand White rabbits (ProSci Inc., San Diego, CA). Serum from the final bleed showed high specificity in the detection of RsiV at dilutions of 1:10,000.

Immunoblot analysis.

For immunoblot analysis, cultures were grown overnight at 30°C and subcultured at a dilution of 1:50 into 5 ml LB broth supplemented with 1 mM IPTG or 0.5 mM IPTG (GFP constructs). Cells were grown at 37°C with rotation to mid-log phase (OD₆₀₀ of 0.8 to 1.0) and were left untreated as a control or were treated for 10 min with hen egg white lysozyme (2 μg/ml unless otherwise noted). The cells (1.5 ml) were then pelleted by centrifugation, resuspended in 100 μl of 2× sample buffer (65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% [wt/vol] glycerol, 0.01% bromophenol blue, and 5% β-mercaptoethanol), and lysed by sonication. Samples were electrophoresed on a 15% SDS–polyacrylamide gel (Bio-Rad). The proteins were then blotted onto nitrocellulose and incubated with one of the following primary antibodies: anti-RsiV (1:10,000), anti-GFP (1:10,000), or anti-σ^A (1:15,000) antibodies. The primary antibody was detected by incubation with goat anti-rabbit IRDye 800CW antibody (1:10,000, LiCor), and the immunoblots were imaged on an Odyssey CLx (LiCor).

The site 2 protease RasP is required for σ^V activation.

In B. subtilis, the only anti-σ factor known to be degraded by a proteolytic cleavage cascade is RsiW, which controls the activation of σ^W. After the degradation of RsiW is initiated by PrsW, it is trimmed by an unknown protease and then cleaved by the site 2 protease RasP (17–19). We sought to determine if the site 1 protease PrsW and site 2 protease RasP were required for σ^V activity. We examined the effects of prsW and rasP null mutations on σ^V activity in response to lysozyme by monitoring the β-galactosidase activity from a P_sigV-lacZ reporter. In wild-type cells, P_sigV-lacZ expression is induced ~65-fold in the presence of lysozyme (Fig. 2). As expected, in the absence of σ^V, P_sigV is not induced in response to lysozyme (Fig. 2). In the rasP mutant background, P_sigV induction is limited to low basal levels even in the presence of lysozyme (Fig. 2). The defect in P_sigV-lacZ expression in the rasP mutant could be complemented by expressing rasP⁺ from an ectopic locus (Fig. 2), suggesting that RasP is required for σ^V activation. In contrast, the expression of P_sigV-lacZ was induced ~68-fold in response to lysozyme in the prsW mutant (Fig. 2), which suggests that PrsW is not required for σ^V activation. It appears that the expression of P_sigV-lacZ is induced to a much greater level in the prsW mutant than in the wild type when lysozyme is present (Fig. 2). However, the basal level of P_sigV-lacZ is also significantly higher in the prsW mutant, and thus, the fold induction of P_sigV-lacZ in both the wild type and the prsW mutants is ~65-fold (Fig. 2). We found that a sigW mutation does not result in an increase similar to that in the prsW mutant (Fig. 2). This suggests that the effect of the prsW mutation is not due to an inability to activate σ^W. It was recently demonstrated that PrsW altered the levels of several membrane proteins in a σ^W-independent manner, suggesting that PrsW has additional roles in the cell (46). Our results do demonstrate that RasP is required for the lysozyme induction of σ^V activity and that PrsW is dispensable.

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Fig 2

Activation of σ^V is dependent upon the site 2 protease RasP but not the site 1 protease PrsW. B. subtilis P_sigV-lacZ (CDE1546), ΔsigV::kan P_sigV-lacZ (CDE1547), ΔprsW::erm P_sigV-lacZ (CDE1549), ΔrasP::tet P_sigV-lacZ (JLH653), and ΔrasP::tet P_hs-rasP⁺ P_sigV-lacZ (JLH640) strains were grown overnight at 30°C and spotted (20 μl) in triplicate on plates containing LB supplemented with IPTG (1 mM) and LB supplemented with IPTG (1 mM) plus lysozyme (10 μg/ml). Plates were incubated at 37°C for 6 h. Cells were collected and resuspended in Z buffer. β-Galactosidase activity was calculated as described in Materials and Methods. Wild type (WT) induction in the presence of lysozyme was set as 100.

RasP is required for site 2 cleavage of RsiV.

Since RasP is required for σ^V activity and is a known site 2 protease for other ECF anti-σ factors, we tested the effects of a rasP mutant on RsiV degradation. Cells expressing rsiV from an IPTG-inducible promoter were grown to mid-log phase and were left untreated or treated with lysozyme (2 μg/ml) for 10 min. The state of RsiV was probed using anti-RsiV antibodies which detect only the C-terminal 230 amino acids of RsiV. As expected, RsiV was cleaved to near completion in both the wild-type and prsW mutant strain (Fig. 3A). Similarly, we found that a rasP mutation did not inhibit cleavage of RsiV in the presence of lysozyme (Fig. 3A). This is not surprising, since the C-terminal product would be the result of successful site 1 cleavage and our antibodies were raised against the C-terminal portion of RsiV.

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Fig 3

RsiV degradation is dependent upon the site 2 protease RasP. B. subtilis containing an IPTG-inducible copy of rsiV⁺ (JLH402) or gfp-rsiV⁺ (JLH453) was subcultured 1:100 into LB supplemented with IPTG (1 mM or 0.5 mM, respectively). Strains with a site 1 prsW mutation (JLH415 and JLH465) were grown in the same manner. Strains with a site 2 rasP mutation (JLH416 and JLH466) were subcultured 1:50 but otherwise grown similarly. At mid-log phase, cultures were incubated for 10 min without (−) or with (+) lysozyme (2 μg/ml) at 37°C. The immunoblot was probed with anti-RsiV antibodies (A and B) or anti-GFP antibodies (C).

RasP is known to perform site 2 cleavage of RsiW (47). To determine if RasP is required for site 2 cleavage of RsiV, we constructed a strain producing an N-terminal GFP-RsiV⁺ fusion protein which localizes to the cell membrane (see Fig. S2 in the supplemental material). Cells producing GFP-RsiV⁺ were treated as described above and then probed with either anti-RsiV or anti-GFP antibodies. When using anti-RsiV antibodies to follow GFP-RsiV⁺ degradation, we found that GFP-RsiV⁺ was degraded in response to lysozyme similarly to wild-type RsiV (Fig. 3B). Consistent with previous results, neither the absence of PrsW nor of RasP altered the degradation of RsiV (Fig. 3B). However, when the same samples were probed using anti-GFP antibodies to detect the N-terminal portion of the GFP-RsiV fusion protein, we were able to detect further cleavage events (Fig. 3C). In the presence of lysozyme, a faint band corresponding to a truncated form of GFP-RsiV⁺ could be observed in both wild-type and prsW mutant cells (Fig. 3C). In the absence of RasP, we observed an increase in this truncated product of GFP-RsiV only when the cells had been treated with lysozyme (Fig. 3C). This suggests that RasP is required for site 2 cleavage of RsiV.

PT inhibits degradation of GFP-RsiV at site 2.

Although B. subtilis RasP is not essential for growth, a rasP mutation has pleiotropic effects. Mutants of rasP grow more slowly in liquid, are not competent, cannot activate σ^W, have cell division defects, and have decreased long-term survival (26, 47, 48). Since the deletion of rasP affects several other cellular processes, we sought to temporarily inhibit RasP function by using an inhibitor to avoid the secondary effects due to long-term growth without RasP. The site 2 intramembrane metalloproteases are dependent upon the coordination of zinc for enzymatic activity (49). The chelator 1,10-phenanthroline (PT) inhibits site 2 protease function by chelating the zinc molecule (49). Using the GFP-RsiV⁺ construct to follow RsiV degradation, cells were grown to mid-log phase and then treated for 30 min with increasing concentrations of PT. The cells were then incubated with lysozyme (2 μg/ml) for 10 min. At the highest concentration of PT, a truncated product of GFP-RsiV accumulates that is the same size as the product that accumulates in the rasP mutant (Fig. 4). As a control, we also compared the effects of PT on the degradation of GFP-RsiV in a rasP mutant. We did not detect any further accumulation of the site 1 product in the rasP mutant even with the highest levels of PT. This suggests that the effect of PT is likely via inhibition of the site 2 protease RasP. This provides further evidence that RasP is required for site 2 cleavage of RsiV and indicates that, like other site 2 proteases, zinc is likely necessary for RasP function.

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Fig 4

1,10-phenanthroline inhibits site 1 protease cleavage. B. subtilis cells of P_hs-gfp-rsiV (JLH453) and P_hs-gfp-rsiV ΔrasP::cat (JLH466) strains were grown overnight and then subcultured 1:100 (JLH453) or 1:50 (JLH466) into 5 ml LB supplemented with IPTG (0.5 mM). When cultures reached mid-log phase, samples were treated with increasing concentrations of 1,10-phenanthroline (0, 0.5, 1, and 5 mM) for 30 min and then treated (+) with lysozyme (2 μg/ml) for 10 min at 37°C. The immunoblot was probed with anti-GFP antibodies or anti-σ^A antibody, which was used as a loading control.

RasP constitutively degrades an RsiV intermediate.

In other systems, site 2 cleavage is preceded by and dependent upon site 1 cleavage to remove the extracellular domain of the target anti-σ factor (17, 20, 48, 50). To mimic site 1 cleavage of RsiV, we constructed a strain producing GFP-RsiV^1-60, which lacks the extracellular domain. Wild-type cells expressing full-length GFP-RsiV⁺ showed lysozyme-dependent degradation of RsiV (Fig. 5). In the absence of RasP, full-length GFP-RsiV⁺ develops a truncated form of GFP-RsiV only in the presence of lysozyme (Fig. 5). When we examined wild-type cells producing GFP-RsiV^1-60, lacking the extracellular domain, the fusion protein was undetectable in the presence and absence of lysozyme (Fig. 5). Note that free GFP accumulates in all strains producing the RsiV^1-60 truncation, suggesting that the truncated fusion protein is inherently unstable and may be degraded by other proteases as well (Fig. 5). However, in the rasP mutant, we observed accumulation of GFP-RsiV^1-60 regardless of whether cells had been exposed to lysozyme or not (Fig. 5). This suggests that, similar to other ECF σ factors, which are regulated by intramembrane proteases, RasP cleavage of RsiV occurs after site 1 cleavage and is constitutive once site 1 processing has occurred.

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Fig 5

Truncation of RsiV results in constitutive degradation in a RasP-dependent manner. Immunoblot comparing full-length RsiV from B. subtilis P_hs-gfp-rsiV (JLH453) and P_hs-gfp-rsiV ΔrasP::cat (JLH466) strains and RsiV without the extracellular domain from P_hs-gfp-rsiV^1-60 (JLH516) and P_hs-gfp-rsiV^1-60 ΔrasP::cat (JLH536) strains. WT strains (JLH453 and JLH516) were subcultured 1:100 into 5 ml LB supplemented with IPTG (0.5 mM). Strains containing rasP mutations (JLH466 and JLH536) were subcultured 1:50 into 5 ml LB supplemented with IPTG (0.5 mM). Cultures were grown to mid-log phase and then left untreated (−) or treated (+) with lysozyme (2 μg/ml) for 10 min at 37°C. The immunoblot was probed with anti-GFP antibodies or anti-σ^A antibody, which was used as a loading control. Cytoplasmic GFP was used as a size control (TPM1263).

The site 2 protease RasP is required for σ^V activation.

Our data indicate that RasP, the site 2 protease for RsiW, is also required for site 2 cleavage of RsiV. In the absence of site 2 cleavage, either through a rasP mutant or by inhibition of RasP activity using a zinc chelator, a truncated form of RsiV accumulates that cannot be degraded. Our data suggest RasP is the site 2 protease, although we cannot rule out the possibility that additional factors may be involved in site 2 processing. In contrast to the site 1 proteases, which appear highly specific for each substrate, it is becoming increasingly clear that the site 2 proteases can recognize a wide range of substrates. In addition to cleaving anti-σ factors RsiV and RsiW, RasP also cleaves the cell division protein FtsL (26). Furthermore, it is thought that RasP aids in clearing signal peptides from the cell membrane (27). RsiV and RsiW do not share any homology at the amino acid level; however, both are single-pass transmembrane anti-σ factors. Although it is still unclear how site 2 proteases recognize their substrate, one hypothesis is that site 2 proteases sense membrane proteins with helix-destabilizing amino acids rather than recognizing a specific amino acid sequence, which is consistent with the ability of RasP to act on multiple membrane proteins (26, 47).

Our work also suggests that RasP activity is constitutive. We constructed a putative site 2 substrate which lacked the extracellular domain of RsiV. This substrate was degraded by RasP even in the absence of lysozyme. Thus, while lysozyme is required to initiate the proteolytic cascade leading to RsiV destruction, lysozyme is not required to induce RasP activity.

Site 1 cleavage is the regulated step in σ^V activation.

Site 1 cleavage is the rate-limiting step in the activation of both σ^E in E. coli and σ^W in B. subtilis (18, 19, 50). Our data suggest that the activation of σ^V is also controlled at the site 1 cleavage step. First, we found that the accumulation of the truncated GFP-RsiV product was only detected in rasP mutants upon treatment with lysozyme (Fig. 3C), suggesting that the degradation of RsiV in response to lysozyme is tightly controlled. Second, when we mimicked the site 1-cleaved RsiV by constructing GFP-RsiV^1-60, which lacks the extracellular domain, GFP-RsiV^1-60 was unstable and was degraded even in the absence of lysozyme. However, in the absence of RasP, the truncated GFP-RsiV^1-60 accumulates. This mimics the putative site 2 substrate buildup observed when rasP mutant cells producing full-length GFP-RsiV⁺ are treated with lysozyme (Fig. 3C and ​and5).5). Taken together, these data suggest that site 2 cleavage of RsiV by RasP is not regulated by the presence of lysozyme but simply occurs as a consequence of the appearance of the appropriate substrate. This suggests that the regulation of σ^V activity, like several other ECF σ factors, is controlled at the level of the site 1 cleavage of the anti-σ factor RsiV.

The site 1 protease for RsiV is unknown.

We tested whether the known B. subtilis site 1 protease, PrsW, which is required for σ^W activation, was involved in RsiV degradation. Our data indicate that PrsW, the site 1 protease for RsiW, is not required for site 1 cleavage of RsiV. In the absence of PrsW, both P_sigV expression and RsiV degradation remain lysozyme inducible. It is interesting to note that the overall level of P_sigV-lacZ expression was greater in the absence of PrsW. The molecular mechanism for this is currently unclear. It was previously reported that prsW mutation leads to significant changes in the composition and levels of several membrane proteins (46). Some of these differences were due to an inability of the prsW mutant to activate σ^W (46). However, Zweers et al. also found that the absence of PrsW had effects on membrane proteins which could not be accounted for by an inability to activate σ^W, suggesting that PrsW may have substrates in the cell other than RsiW (46). One possible explanation for increased P_sigV induction in the absence of PrsW is that the levels of the site 1 protease responsible for cleaving RsiV may be increased. However, we were unable to detect an increase in the degradation of RsiV in the absence of lysozyme. In addition, we did not detect an increase of the RsiV site 2 substrate in a prsW rasP double mutant in the absence of lysozyme (data not shown). We also observed that the levels of RsiV appeared lower in the absence of RasP. In addition, it appears that the amount of RsiV site 1 cleavage is also lower in the absence of RasP. We hypothesize that both of these may be linked to the effect the absence of RasP can have on the accumulation of membrane proteins (46).

In an attempt to identify the site 1 protease, we performed multiple variations on a transposon screen to identify mutants which could not respond to lysozyme, and as yet, we have been unable to identify the site 1 protease. It is possible that multiple proteases are able to perform the initial site 1 cleavage of RsiV or that the site 1 protease is essential. However, if the site 1 protease is essential, it must have other roles in the cell, as σ^V itself is not essential. It is interesting that the site 1 proteases have been more difficult to identify in several cases, including ECF σ^L, σ^K, and σ^M in M. tuberculosis (23). In addition, while PrsW is absolutely required for site 1 cleavage of RsiW, there is another protease(s) that is required to trim RsiW before the site 2 protease RasP can cleave RsiW within the transmembrane domain (17). This protease(s) has not yet been identified. However, our data suggest that cleavage of RsiV at site 1 is the rate limiting step in σ^V activation, and thus, identifying the protease(s) required for this processing step is of interest.

Increased lysozyme resistance decreases σ^V activation.

Many stress response signal transduction systems detect a specific environmental stress and, in response, induce changes in gene expression to deal with this stress. By increasing the expression of repair mechanisms to correct damage associated with these stresses, cells downregulate the activation of the stress response system (55). The activation of σ^V causes an increase in lysozyme resistance through mechanisms that modify the structure of peptidoglycan (OatA) or lipoteichoic acid (Dlt), thus impairing the ability of lysozyme to cause damage and increasing bacterial survival (28, 31, 33). Our screen for mutants which block σ^V activation included an insertion that led to increased pdaC expression and increased lysozyme resistance (51). We found that increased lysozyme resistance when either pdaC (peptidoglycan deacetylase) or oatA (peptidoglycan O-acetyltransferase) was overexpressed caused a marked decrease in σ^V activation. This suggests that, like other cell stress response systems, σ^V is activated and induces genes which can decrease the damage generated by lysozyme, and in response, σ^V activity is then decreased.

This work was supported by National Institutes of Health grant R01AI087834 from the National Institute of Allergy and Infectious Diseases to C.D.E. and American Heart Association grant 13PRE14650053 to J.L.H.

We thank members of the Ellermeier laboratory for helpful comments and Theresa D. Ho (University of Iowa) for comments on the manuscript.