The Penicillin-Binding Protein PbpP Is a Sensor of β-Lactams and Is Required for Activation of the Extracytoplasmic Function σ Factor σ^P in Bacillus thuringiensis

Bacillus thuringiensis, B. cereus, and B. anthracis contain two open reading frames in the sigP region that encode predicted penicillin-binding proteins (PBPs). In Bacillus thuringiensis subsp. kurstaki HD73, these PBPs are called pbpP (HD73_3488) and bt3491 (HD73_3491). We also identified a third open reading frame that appears to be found only in Bacillus thuringiensis subsp. kurstaki HD73, called bt3487 (HD73_3487) (Fig. 1A). Although they are not located in the same operon as sigP and rsiP, we hypothesized that they may play a role in the response of σ^P to β-lactams because PBPs have been well characterized as targets of β-lactam antibiotics (9, 35). Additionally, genes involved in the same signaling system are often located in the neighboring regions. To determine if BT3487, PbpP, and BT3491 were required for the response of σ^P to β-lactams, we generated strains with in-frame deletions of each of the genes and measured the effect on ampicillin resistance. We found that the deletion of pbpP led to a dramatic decrease in the ampicillin MIC similar to that of a ΔsigP mutant (Table 1) (33, 34). In contrast, strains with deletions in bt3487 and bt3491 had no effect on ampicillin resistance (not shown). We also determined that a ΔpbpP mutant is more sensitive to cefoxitin and cefmetazole than the wild type (WT) (Table 1).

We noted that a ΔsigP-rsiP mutant is more sensitive to β-lactams than a ΔpbpP mutant. We hypothesized that a ΔpbpP mutant may block σ^P activation in response to β-lactams but retains a basal level of σ^P activation that allows a low level of resistance to β-lactams. To monitor σ^P activity, we took advantage of the fact that σ^P is required for the transcription of its promoter (P_sigP); thus, we inserted a P_sigP-lacZ promoter fusion into the thrC locus (33, 34). To determine if PbpP played a role in σ^P activation, we tested the effect of a pbpP deletion on σ^P activity by monitoring P_sigP-lacZ expression. Interestingly, we did not observe activation of σ^P in the ΔpbpP mutant in the presence of cefoxitin (see Fig. S1A in the supplemental material). We complemented the ΔpbpP mutant with pbpP⁺ on a plasmid under the control of its native promoter. We found that σ^P was activated in the presence of cefoxitin to an extent similar to that observed for the WT (Fig. S1A). To reinforce our finding that ΔpbpP results in the loss of σ^P activation, we conducted β-galactosidase assays to quantify the effect on σ^P activation. As previously reported, P_sigP-lacZ expression is induced in a dose-dependent manner in response to increased cefoxitin concentrations in the WT (Fig. 1C) (34). Consistent with previous observations, we did not observe induction of P_sigP-lacZ in the ΔsigP-rsiP mutant because σ^P is required for transcription from P_sigP (34). We found that the deletion of pbpP resulted in the loss of P_sigP-lacZ expression at every concentration tested (Fig. 1C). Taken together, our data suggest that PbpP is required for the activation of σ^P, thereby altering the transcription of the σ^P regulon and β-lactam resistance.

Because our data suggest that PbpP is required for σ^P activation, we hypothesized that PbpP is required for RsiP degradation. To test this, we compared the effects of cefoxitin on the degradation of green fluorescent protein (GFP)-RsiP in WT, ΔpbpP, and ΔrasP mutant strains. We previously showed that GFP-RsiP is functional and localized to the membrane (34). We found that the levels of full-length GFP-RsiP decreased in the WT in the presence of cefoxitin (Fig. 1D) (34). When a ΔrasP mutant, which lacks the site 2 protease, was incubated with cefoxitin, we observed a decrease in full-length GFP-RsiP and the buildup of an intermediate GFP-RsiP fragment, indicating the loss of site 2 cleavage (Fig. 1D) (34). This GFP-RsiP fragment is approximately the predicted size for a site 1 protease cleavage product. In contrast, we found that full-length GFP-RsiP levels did not decrease in the ΔpbpP mutant when grown in the presence of cefoxitin (Fig. 1D). This suggests that PbpP is required for site 1 cleavage of RsiP and, thus, σ^P activation.

A defining feature of PBPs is the ability to covalently bind β-lactams (9, 36). We sought to determine if PbpP has the capacity to bind β-lactams. We tested if PbpP could bind Bocillin-FL (Boc-FL), a fluorescent β-lactam consisting of penicillin V and BODIPY FL dye (37). We found that Bocillin-FL was degraded when σ^P was activated (Fig. S3B). In a ΔsigP-rsiP mutant, we found that Bocillin-FL was not degraded, suggesting that σ^P-regulated β-lactamases are likely responsible for Bocillin-FL degradation (Fig. S3B). To perform Bocillin-FL labeling experiments, we expressed pbpP from an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter in a ΔsigP-rsiP mutant. We labeled cells with Bocillin-FL and blotted them with anti-PbpP antisera (37). We observed a fluorescent band at approximately 66 kDa with both Bocillin-FL and anti-PbpP antisera. This band was the predicted size of PbpP; it increased in intensity with increasing IPTG concentrations and was not observed in the empty vector (EV) control (Fig. 2A and Fig. S3A). This demonstrates that PbpP binds β-lactams. We also noted that the lack of a fluorescent band corresponding to PbpP in the EV suggests that the levels of PbpP in wild-type cells are not high enough to be detected by Bocillin-FL labeling.

All PBPs have an active-site serine that is acylated by β-lactams (36). We identified serine 301 (S301) as the likely active-site residue required for transpeptidation based on homology to other PBPs. To determine if S301 is the active-site serine, we mutated it to an alanine by site-directed mutagenesis and expressed pbpP^S301A under the control of an IPTG-inducible promoter. In the strain producing PbpP^S301A, the 66-kDa band was lost when imaging for Bocillin-FL (Fig. 2A and Fig. S3A). However, immunoblotting using anti-PbpP antisera detected a 66-kDa band corresponding to PbpP^S301A, which is produced at levels similar to those of the WT protein (Fig. 2A; Fig. S3A). Thus, PbpP^S301A cannot covalently bind Bocillin-FL. This suggests that PbpP is a penicillin-binding protein, S301 is required for binding β-lactams, and S301 is likely the active-site serine.

We sought to determine if β-lactam binding to PbpP was required for σ^P activation using a PbpP^S301A active-site mutant. We complemented the ΔpbpP mutant with pbpP⁺ and pbp^S301A under the control of their native promoter in a single copy by integrating constructs at the B. subtilis integrative conjugative element (ICEBs1) site in the B. thuringiensis chromosome (38). We found that PbpP⁺ restored P_sigP-lacZ expression in the presence of cefoxitin (Fig. 2B). In contrast, when we complemented the strain with pbpP^S301A, we observed no increase in P_sigP-lacZ expression in the presence of cefoxitin (Fig. 2B). These data suggest that binding of PbpP to β-lactams is required for β-lactams to activate σ^P.

We noted that the basal level of P_sigP-lacZ expression was higher in the strains complemented with pbpP⁺ and pbp^S301A integrated at ICEBs1 than in WT B. thuringiensis (Fig. 2B). We reasoned that this might be due to higher basal levels of expression of pbpP and pbpP^S301A at the ICEBs1 site. Thus, we sought to determine the effect of the overexpression of pbpP⁺ and pbpP^S301A on σ^P activation. We expressed pbpP⁺ or pbpP^S301A from a tetracycline-inducible promoter on a multicopy plasmid (34, 39). We observed that increased expression of pbpP⁺ or pbpP^S301A leads to a dose-dependent increase in the expression of P_sigP-lacZ, in the absence of β-lactams (Fig. 3A). We also found that the addition of cefoxitin led to a further increase in P_sigP-lacZ expression when pbpP⁺ was overexpressed (Fig. S4B). We noted increased basal levels of P_sigP-lacZ expression in the absence of anhydrotetracycline (ATc) and concluded that this is likely due to leaky expression of P_tet-pbpP and P_tet-pbpP^S301A (Fig. 3A and Fig. S4B). These data suggest that the overexpression of both the WT and the active-site mutant (S301A) can activate σ^P even in the absence of β-lactams. We interpret this to mean that the requirement for β-lactam binding to PbpP can be compensated for by increased levels of PbpP; however, β-lactam binding to PbpP further enhances σ^P activation (Fig. S4B). The activation of σ^P in WT cells is likely not due to β-lactam-induced pbpP transcription as the expression of pbpP is not induced by β-lactams (Fig. S1B and C). The pbpP^S301A mutant also fails to induce σ^P activation when expressed under the control of its native promoter, further suggesting that pbpP is not induced by β-lactams (Fig. 2B).

Since the loss of PbpP results in little to no degradation of RsiP in the presence of β-lactams, we tested if the increased expression of pbpP leads to the degradation of RsiP in the absence of β-lactams. We introduced P_tet-pbpP⁺ or P_tet-pbpP^S301A into a strain containing IPTG-inducible gfp-rsiP. We found that the overexpression of PbpP and PbpP^S301A leads to decreases in full-length GFP-RsiP levels, suggesting that PbpP can induce RsiP degradation and, thus, σ^P activation (Fig. 3B). This suggests that PbpP controls σ^P activation by controlling RsiP degradation.

The site 1 protease required for initiating RsiP degradation has not yet been identified. Since PbpP is required for site 1 cleavage of RsiP, the possibility exists that PbpP is the site 1 protease. We sought to determine if basal-level site 1 cleavage occurred in the absence of pbpP, which would suggest that another protein can cleave RsiP. Since site 2 cleavage is rapid (34), we expressed gfp-rsiP in a ΔpbpP ΔrasP double mutant, which should allow the buildup of any GFP-RsiP site 1 cleavage product. We observed the accumulation of a band corresponding to a GFP-RsiP fragment in the ΔrasP mutant in the absence of cefoxitin, and the intensity of this band increased in the presence of cefoxitin (Fig. 4A). We observed the same band in the ΔpbpP ΔrasP mutant; however, the band did not increase in the presence of cefoxitin. We concluded that in a ΔpbpP ΔrasP mutant, there is a basal level of site 1 cleavage of RsiP occurring in the presence and absence of cefoxitin (Fig. 4A). This suggests that site 1 cleavage can occur in the absence of PbpP, but it is not β-lactam inducible. Presumably, in this strain, the unidentified site 1 protease still retains its basal level of activity but cannot be further activated in the presence of cefoxitin due to the absence of PbpP.

To test if PbpP is sufficient for site 1 cleavage of RsiP, we introduced IPTG-inducible gfp-rsiP into the Bacillus subtilis chromosome (which does not encode a homolog of sigP or rsiP) and expressed pbpP using a xylose-inducible promoter. We grew the cells in the presence of 0.01 mM IPTG and increasing concentrations of xylose. We asked if PbpP was expressed and presumably properly folded by labeling with the fluorescent β-lactam Bocillin-FL. We observed a fluorescent band corresponding to PbpP that increased in intensity with increasing concentrations of xylose (Fig. 4B). We also monitored GFP-RsiP levels by performing immunoblot analysis using anti-GFP antisera. We did not observe degradation or a decrease in RsiP levels even at the highest levels of PbpP, indicating that PbpP is not sufficient for RsiP degradation in B. subtilis (Fig. 4C). Taken together, these data lead us to conclude that PbpP is not the site 1 protease but is required for sensing of β-lactams in B. thuringiensis.

Since PbpP is likely not acting as the site 1 protease, we hypothesized that PbpP functions as a sensor that binds β-lactams and subsequentially activates σ^P. Therefore, we hypothesized that the reason why some β-lactams do not activate σ^P is that they have a lower affinity for PbpP. To test this hypothesis, we determined the affinity of PbpP for eight different β-lactams by modifying a Bocillin-FL inhibition experiment previously described by Kocaoglu and colleagues (40). We calculated the 50% inhibitory concentration (IC₅₀) (the concentration of β-lactam at which 50% of Bocillin-FL labeling of PbpP is inhibited) to determine the binding affinity of different β-lactams. We found that while the β-lactams had different IC₅₀s for PbpP, the differences did not correlate with the ability of the β-lactams to activate σ^P (Fig. 5A and B). For example, we found that some of the nonactivating β-lactams (cefoperazone and cefsulodin) had IC₅₀s similar to those of activating β-lactams (Fig. 5A and B). Thus, the disparity in the β-lactams’ ability to activate σ^P is not simply due to the inability of PbpP to bind different β-lactams. These data also suggest that simple binding of any β-lactam to PbpP is not sufficient for σ^P activation.

We found that β-lactam binding to PbpP is not sufficient for σ^P activation because nonactivating β-lactams covalently bind PbpP with affinities similar to those of the activating β-lactams (i.e., cefsulodin and ampicillin have nearly identical binding affinities for PbpP). We hypothesize that the β-lactams that activate σ^P induce a conformational change in PbpP that permits a protein-protein interaction. If this hypothesis were true, the β-lactams that do not activate σ^P would be able to inhibit the activation of σ^P by occupying the PbpP active site. To test this, we pretreated cells with cefsulodin (a nonactivator of σ^P) and then added cefoxitin (an activator of σ^P). We found that cefsulodin inhibited the activation of σ^P by cefoxitin in a dose-dependent manner (Fig. 6). We also show that pretreatment with cefmetazole (an activator of σ^P) does not inhibit activation (Fig. 6). Therefore, nonactivating β-lactams inhibit σ^P activation presumably by occupying the active site of PbpP and preventing activating β-lactams from binding PbpP and activating σ^P (Fig. 7).

The principal finding of this work is the demonstration that PbpP is required for the activation of σ^P in response to some β-lactams. Based on our findings, we propose the following working model for how PbpP functions as a sensor for β-lactams. In WT cells in the absence of stress, RsiP binds σ^P and inhibits σ^P activation (Fig. 7). When activating β-lactams are present, they bind the active-site serine of PbpP. The binding of the activating β-lactams results in a conformational change in PbpP that allows it to interact with a component of the σ^P system. This interaction initiates regulated intramembrane proteolysis of RsiP and, thus, σ^P activation (Fig. 7). This model is supported by ample evidence: (i) deletion of pbpP blocks RsiP degradation and σ^P activation, (ii) mutants of PbpP unable to bind β-lactams fail to activate σ^P in response to β-lactams, and (iii) overexpression of PbpP or PbpP^S301A leads to constitutive RsiP degradation and σ^P activation. Thus, PbpP plays an essential role in sensing the presence of inducing β-lactams and controlling σ^P activation.

It is possible that PbpP is a site 1 protease that initiates RsiP degradation; however, we think that it is unlikely. While PbpP is required for site 1 cleavage of RsiP in response to β-lactams, the totality of our data does not support PbpP as the site 1 protease. First, the overexpression of PbpP in B. subtilis does not induce the degradation of RsiP as it does in B. thuringiensis. Our data indicate that PbpP is functional, folded, and localized properly when expressed in B. subtilis since it can be labeled on whole cells by Bocillin-FL. This argues that PbpP is not sufficient for site 1 cleavage of RsiP and suggests that an unidentified B. thuringiensis protease is required. Second, in B. thuringiensis, we observed low-level site 1 cleavage of RsiP in the absence of PbpP. This argues that PbpP is not absolutely required for site 1 cleavage. If PbpP were a site 1 protease, there must be a second protease in B. thuringiensis that has low basal activity and cleaves RsiP at site 1 in the absence of PbpP. Finally, PbpP lacks any predicted protease domains. Future work will be required to identify the protease(s) required for site 1 cleavage of RsiP and, thus, σ^P activation.

We hypothesize that PbpP is the sensor of β-lactams for the σ^P system. In support of this, we found that σ^P is not activated in the ΔpbpP mutant or when pbpP^S301A is expressed from the native P_pbpP promoter. However, the overproduction of either PbpP or PbpP^S301A results in the activation of σ^P in the absence of β-lactams. This suggests that the overproduction of PbpP can compensate for β-lactam binding to PbpP to activate σ^P. Importantly, activation of σ^P is not due to inhibition of PbpP transpeptidase activity by β-lactams because PbpP^S301A is catalytically inactive yet does not result in σ^P activation. This loss of σ^P activity is not due to an instability of PbpP^S301A as it is produced at levels similar to those of WT PbpP. Activation of σ^P by β-lactams is not simply due to increased expression of pbpP since β-lactams do not induce pbpP expression. In addition, if increased expression of pbpP in response to β-lactams was responsible for σ^P activation, then we would have expected the pbpP^S301A allele to induce σ^P activation when expressed under the control of the native P_pbpP promoter. Taken together, these data suggest that PbpP interacts with some component of the signal transduction system.

In support of this hypothesis, we found that a subset of activating β-lactams bind PbpP with affinities similar to those of nonactivating β-lactams. We found that cefsulodin, a nonactivating β-lactam, can inhibit the activation of σ^P by an activating β-lactam, cefoxitin, presumably by competing for the active-site serine of PbpP. We hypothesize that nonactivating β-lactams do not induce the appropriate conformational change in PbpP to render it active and able to interact with its target. One obvious target for PbpP interaction is the anti-σ itself. However, we did not observe an interaction between the extracellular domains of RsiP^76–275 and PbpP^35–586 in vitro using a copurification assay (see Fig. S8 in the supplemental material). This raises the possibility that PbpP interacts with another protein like the as-yet-unidentified site 1 protease. Alternatively, it may interact indirectly with RsiP or the site 1 protease via an unknown protein. Future work will need to determine what PbpP interactions drive RsiP degradation and, thus, σ^P activation.

While the identification of a PBP required for the activation of an ECF σ factor is novel, there is precedence for a PBP transpeptidase-like domain functioning as a sensor of β-lactams. Found in diverse organisms, including Staphylococcus aureus and Bacillus licheniformis, BlaR1 (MecR1) contains an extracellular transpeptidase-like domain that senses β-lactams and a cytoplasmic protease domain. BlaR1 is a β-lactam sensor that directly binds β-lactams in its extracellular transpeptidase-like domain (41). The covalent bond formed with the β-lactam ring causes a conformational change in BlaR1 that activates the cytoplasmic protease domain (42). The protease domain cleaves the repressor of the β-lactamase operon, BlaI, thus activating the transcription of β-lactamase and increasing resistance to β-lactams (42). While the BlaIR system is clearly not synonymous with σ^P, it is worth noting that there is precedence for PBP domains that function as sensors of β-lactams.

All B. thuringiensis strains are isogenic derivatives of AW43, a derivative of B. thuringiensis subsp. kurstaki strain HD73 (43). All strains and genotypes can be found in Table 2. All B. thuringiensis strains were grown in or on LB media at 30°C unless otherwise specified. Liquid cultures of B. thuringiensis were grown with agitation in a roller drum. B. thuringiensis strains containing episomal plasmids were grown in LB medium containing chloramphenicol (Cam) (10 μg/ml; Ameresco) or erythromycin (Erm) plus lincomycin (Linc) (MLS) (1 μg/ml Erm [Ameresco] and 25 μg/ml Linc [Research Products International]). E. coli strains were grown at 37°C using LB-ampicillin (Amp) (100 μg/ml; Ameresco) or LB-Cam (10 μg/ml) medium. B. subtilis strains were grown on LB medium with antibiotics (Cam at 10 μg/ml, spectinomycin [Spec] at 100 μg/ml [Amresco], or Erm at 10 μg/ml). To screen for threonine auxotrophy, B. thuringiensis strains were patched onto minimal medium plates without or with threonine (50 μg/ml). The β-galactosidase chromogenic indicator 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal; Research Products International) was used at a concentration of 100 μg/ml. Anhydrotetracycline (ATc; Sigma) was used at a concentration of 100 ng/ml unless otherwise indicated. IPTG (Research Products International) and xylose (Acros) were used at the concentrations indicated in the figure legends. Additional β-lactams used in β-lactam-binding experiments were used at the concentrations indicated in the figure legends and were acquired from the following sources: cefsulodin, piperacillin, cefmetazole, and cefoxitin from Sigma-Aldrich; cephalothin from Chem-impex International Inc.; methicillin from Alfa Aesar; and cefoperazone from Toronto Research Chemical Inc.

All plasmids are listed in Table 3 and Table S1 in the supplemental material, which includes information relevant to plasmid assembly. Plasmids were constructed by isothermal assembly (44). Regions of plasmids constructed using PCR were verified by DNA sequencing. The oligonucleotide primers used in this work were synthesized by Integrated DNA Technologies (Coralville, IA) and are listed in Table S2. All plasmids were propagated using OmniMax 2-T1R as the cloning host and passaged through the nonmethylating E. coli strain INV110 before being transformed into a B. thuringiensis recipient strain.

To construct deletion mutants, we cloned 1 kb of DNA upstream and 1 kb downstream of the site of the desired deletion using primers listed in Table S2 into the temperature-sensitive pMAD plasmid (erythromycin resistant) between the BglII and EcoRI sites (45). Mutants were constructed by shifting temperatures as previously described (45).

B. subtilis ICEBs1 conjugation strains were constructed by transforming JAB932 as previously described (38). The resulting transformants or donor strains were grown in LB medium with d-alanine (100 μg/ml) for 2 h, at which point 1% xylose was added and cells were grown for 1 h. Recipient strains of B. thuringiensis were grown to an optical density at 600 nm (OD₆₀₀) of ∼0.8. The donor and recipient strains were mixed at equal concentrations, plated on LB medium containing d-alanine (100 μg/ml), and incubated for 6 h. Transconjugants were isolated by plating on LB plates containing chloramphenicol.

Plasmids were introduced into B. thuringiensis by electroporation (46, 47). Briefly, recipient cells were grown to late log phase at 37°C from a fresh plate. For each transformation, cells (1.5 ml) were pelleted by centrifugation (8,000 rpm) and washed twice in room-temperature (RT) sterile water. After careful removal of all residual water, 100 μl of filter-sterilized 40% polyethylene glycol 6000 (PEG 6000; Sigma) was used to gently resuspend cells. Approximately 2 to 10 μl of unmethylated DNA (>50 ng/μl) was added to cells and transferred to a 0.4-cm-gap electroporation cuvette (Bio-Rad). Cells were exposed to 2.5 kV for 4 to 6 ms. LB medium was immediately added, and cells were incubated at 30°C for 1 to 2 h prior to plating on selective media.

To quantify expression from the sigP promoter, we measured the β-galactosidase activity of cells containing a P_sigP-lacZ promoter fusion. Cultures grown overnight were diluted 1:50 in fresh LB medium and incubated to mid-log phase (OD of 0.8 to 1.5) at 30°C with or without ATc or IPTG. One milliliter of each subculture was pelleted (8,000 rpm), washed (in LB broth), and resuspended in 1 ml LB broth lacking or including specified antibiotics. After 1 h of incubation at 37°C, 1 ml of each sample was pelleted and resuspended in 1 ml of Z-buffer (0.06 M Na₂HPO₄, 0.04 M NaH₂PO₄*H₂O, 0.01 M KCl, 0.001 M MgSO₄). Cells were permeabilized by mixing with 16 μl of chloroform and 16 μl of 2% Sarkosyl (27, 48). Permeabilized cells (50 μl) were mixed with 100 μl of Z-buffer and 50 μl of 2 mg/ml chlorophenol red-β-d-galactopyranoside (CPRG; Research Products International) (50 μl), which is considerably more sensitive than X-Gal (49). The OD₅₇₈ was measured over time using an Infinite M200 Pro plate reader (Tecan). β-Galactosidase activity units [(micromoles of chlorophenol red formed per minute) × 10³/(OD₆₀₀ × milliliters of cell suspension)] were calculated as previously described (50). Experiments were performed in technical and biological triplicate, with the means and standard deviations shown.

To determine the MICs for various antibiotics, we diluted cultures of bacteria grown overnight (washed in LB medium) 1:1,000 in medium containing 2-fold dilutions of each antibiotic. All MIC experiments were performed in round-bottom 96-well plates. Each experiment was performed in triplicate, and the cultures were allowed to incubate for 24 h at 37°C before observing growth or no growth by centrifuging the plates at 1,000 rpm for 5 minutes and observing the presence or absence of pellets.

Samples were electrophoresed on a 15% SDS-polyacrylamide gel, and proteins were then blotted onto a nitrocellulose membrane (GE Healthcare, Amersham). Nitrocellulose was blocked with 5% bovine serum albumin (BSA), and proteins were detected with a 1:10,000 dilution anti-GFP antisera. Streptavidin IR680LT (1:10,000) was used to detect two biotin-containing proteins, PycA (HD73_4231) and AccB (HD73_4487), which served as loading controls (51, 52). To detect primary antibodies, the blots were incubated with a 1:10,000 dilution of goat anti-rabbit IR800CW (Li-Cor) and imaged on an Odyssey CLx scanner (Li-Cor) or Azure Sapphire (Azure Biosystems). All immunoblots were performed at room temperature a minimum of three times, with a representative example shown.

Cultures grown overnight at 30°C were diluted 1:50 and grown to an OD of ∼1.0. The cultures were aliquoted in 1-ml aliquots and pelleted at 8,000 rpm. The cells were washed twice in 500 μl of 1× phosphate-buffered saline (PBS) and resuspended in either 50 μl of 50 μg/ml Bocillin-FL (Thermo Fisher) or 50 μl of 10-fold dilutions of β-lactams (0.0005 to 5,000 μg/ml). The samples resuspended in β-lactams were incubated for 30 min at room temperature and then pelleted and resuspended in 50 μl or 50 μg/ml Boc-FL for 15 min. After incubation in Boc-FL, all the samples were pelleted and resuspended in 200 μl sample buffer with 5% β-mercaptoethanol (βME). The samples were sonicated, heated, and electrophoresed on a 12% polyacrylamide gel. The gels were imaged on an Azure Sapphire system (AzureBiosystems) by excitation at 488 nm and detection at 518 nm. The Bocillin-FL labeling experiment was performed in biological triplicate for each antibiotic, and the Bocillin-FL intensity for the PbpP band was quantified on each gel. The average intensity was used to calculate the IC₅₀ using GraphPad Prism, with means and standard errors or deviations shown.