Induction and regulation of a secreted peptidoglycan hydrolase by a membrane Ser/Thr kinase that detects muropeptides

Authors


*E-mail jonathan.dworkin@columbia.edu; Tel. (+1) 212 342 3731; Fax (+1) 212 305 1468.

Summary

Here, we report that the model Gram-positive organism, Bacillus subtilis, expresses and secretes a muralytic enzyme, YocH, in response to cell wall-derived muropeptides derived from growing cells but not lysed cells. This induction is dependent on PrkC, a membrane Ser/Thr kinase that binds to peptidoglycan and that belongs to a broadly conserved family including the essential PknB kinase of M. tuberculosis. YocH stimulates its own expression in a PrkC-dependent manner demonstrating the presence of an autoregulatory loop during growth. Cells lacking YocH display a survival defect in stationary phase but enzymes secreted by other cells in the culture rescue this defect. The essential translation factor EF-G is an in vivo substrate of PrkC and this phosphorylation occurs in response to muropeptides. Therefore, we hypothesize that YocH is used by the bacterium to digest peptidoglycan released by other bacteria in the milieu and that the presence of these fragments is detected by a membrane kinase that modifies a key regulator of translation as well as to stimulate its own expression.

Introduction

The bacterial cell wall is composed largely of peptidoglycan, a polymer that provides the characteristic cell shape and the ability to withstand osmotic pressure. Peptidoglycan consists of repeated glycan strands composed of β(1,4) linkages between N-acetylglucosamine and N-acetylmuramic acid (NAM) sugars that have covalent cross-links between NAM-associated peptides. During growth, bacteria turn over their cell wall material due to the actions of peptidoglycan hydrolases and/or amidases that process mature peptidoglycan to allow the insertion of new material (Doyle et al., 1988). These fragments serve as signals in a range of host–microbe interactions including Bordetella pertussis infection and Vibrio fischeri-squid symbiosis (Cloud-hansen et al., 2006) and stimulate the innate immune response (Hasegawa et al., 2006) by binding to host proteins like Nod1/2 (Girardin et al., 2003) and Toll-like receptor 2 (Asong et al., 2009). In addition to this central role in host–bacterial interactions, peptidoglycan fragments act as an inter-bacterial signal that simulates growth of dormant Bacillus subtilis spores (Shah et al., 2008).

Dormant cells of Micrococcus luteus are stimulated to divide (resuscitate) by exposure to non-dormant M. luteus cells (Votyakova et al., 1994; Mukamolova et al., 1998). This stimulation requires resuscitation-promoting factor (Rpf), a secreted 17 kDa protein (Mukamolova et al., 2002). A homologue of M. luteus Rpf from M. tuberculosis is structurally similar to lysozyme (Cohen-gonsaud et al., 2005; Ruggiero et al., 2009), a protein known to digest peptidoglycan by breaking the β(1,4) linkage between the N-acetyl glucosamine and the N-acetyl muramic acid and Rpf proteins from both M. luteus and M. tuberculosis are capable of digesting peptidoglycan in vitro (Mukamolova et al., 2006). M. tuberculosis contains five Rpf proteins, and a mutant strain lacking all five still grows in vitro, but is defective in restoration of growth from stationary phase (Kana et al., 2008). While strains lacking multiple Rpf proteins are defective in animal models of tuberculosis (Tufariello et al., 2004; Downing et al., 2005; Russell-goldman et al., 2008), the delayed reactivation observed for the single rpfB mutant (Tufariello et al., 2006) indicates that it may be sufficient for exit from dormancy. RpfB interacts with a peptidoglycan endopeptidase, RipA, at cell division sites (Hett et al., 2007; Hett et al., 2008) suggesting that RpfB plays a role in peptidoglycan metabolism. However, despite the clear importance of these proteins in the life cycle of this pathogen, the molecular mechanism underlying their regulation remains mysterious tBLAST analysis of the B. subtilis genome with the RpfB sequence reveals a distant homologue, YabE (Ravagnani et al., 2005). While YabE is predicted to be an integral membrane protein and therefore, unlike RpfB, is probably not secreted, the YabE paralogue YocH is secreted (Tjalsma et al., 2004). YocH contains two LysM domains that mediate peptidoglycan binding (Buist et al., 2008) and three aspartate residues in its C-terminal ‘Stationary phase survival’ (Sps) domain (Ravagnani et al., 2005) that are involved in catalysis by the Escherichia coli lytic transglycosylase MltA (Blackburn and Clarke, 2001; Van straaten et al., 2007). Thus, YocH could function analogously to Rpf proteins by digesting peptidoglycan present in the extracellular milieu.

The essential two-component YycFG system in B. subtilis senses aspects of cell wall metabolism (Dubrac et al., 2008; Winkler and Hoch, 2008). The membrane-bound sensor kinase YycG responds to an as yet unidentified signal at the division septum and phosphorylates the response regulator YycF to coordinate growth and division with cell wall restructuring (Bisicchia et al., 2007; Fukushima et al., 2008). YycF regulates a number of genes that encode proteins with putative cell wall degrading and/or modifying activities including yocH (Howell et al., 2003). YycG interacts with the extracellular domains of the membrane-bound proteins YycH and YycI (Szurmant et al., 2005) and a deletion of YycI results in increased levels of expression from PyocH (Szurmant et al., 2007). In addition to these direct effects, the YycFG system downregulates IseA (YoeB), an inhibitor of autolysins (Salzberg and Helmann, 2007; Yamamoto et al., 2008). While the molecule that activates this system is unknown, the involvement of the YycFG in cell wall metabolism suggests that it could be a cell wall component such as peptidoglycan.

Digested peptidoglycan and purified muropeptides germinate dormant B. subtilis spores via PrkC, the eukaryotic-like membrane Ser/Thr kinase which contains PASTA (‘Penicillin and Ser/Thr kinase Associated’) repeats in its extracellular domain that bind peptidoglycan (Shah et al., 2008). During spore germination and logarithmic growth, PrkC phosphorylates EF-G (Gaidenko et al., 2002; Shah et al., 2008), the essential ribosomal GTPase involved in mRNA and tRNA translocation as well as ribosome recycling (Savelsbergh et al., 2009). Recently, the ribosome-associated GTPases CpgA and EF-Tu have also been reported to be in vitro PrkC substrates (Absalon et al., 2009), further supporting a role for PrkC in regulating translation. PrkC may also play a role in controlling cell division because the PrkC homologues M. tuberculosis PknB (Dasgupta et al., 2006; Parikh et al., 2009), Streptococcus agalactiae Stk1 (Silvestroni et al., 2009) and Corynebacterium glutamicum PknB (Fiuza et al., 2008; Schultz et al., 2009) phosphorylate proteins involved in peptidoglycan synthesis and/or cell division.

The germination of spores in response to peptidoglycan fragments and the presence of a pathway responsive to cell wall metabolism suggested that exposure of growing B. subtilis cells to peptidoglycan fragments could induce specific genes. Here we show that incubation of growing cells with small soluble peptidoglycan fragments results in the induction of yocH, which we further demonstrate encodes a secreted peptidoglycan hydrolase. This induction of yocH is dependent on PrkC and YocH autoregulates its own expression via the PrkC pathway.

Results

Peptidoglycan-derived muropeptides induce yocH

Bacteria release significant amounts of peptidoglycan into the extracellular milieu during growth (Doyle et al., 1988). To identify B. subtilis genes induced by this exogenous peptidoglycan, we incubated B. subtilis cells with cell-free supernatants derived from growing B. subtilis cultures that contain large quantities of peptidoglycan fragments (Mauck et al., 1971). In an initial microarray-based screen, we identified several genes induced by this treatment and chose to focus on yocH. This gene encodes a protein with similarity in its C-terminus to the catalytic domain of the E. coli lytic transglycosylase MltA (Ravagnani et al., 2005), and it is regulated by the cell wall-sensing YycFG system (Howell et al., 2003). The induction of yocH by cell-free supernatants was dependent on the growth phase of the culture they were derived from because yocH levels were higher following incubation with supernatants isolated from growing cells as compared with those isolated from non-growing cells (Fig. 1A).

Figure 1.

m-Dpm type peptidoglycan induces yocH expression.
A. Cell-free supernatants from B. subtilis isolated at different OD600 values (◆) were added to log phase cultures of B. subtilis at an OD600∼0.3 for 30 min. yocH induction ratios (o) were measured for each sample. Dashed line indicates no induction.
B. Mutanolysin-digested B. subtilis peptidoglycan (m-Dpm) and lysostaphin-digested S. aureus peptidoglycan (l-lys) were added to log phase cultures of B. subtilis at an OD600∼0.3 for 30 min.
C. 25 µM m-Dpm-containing disaccharide tripeptide (P3; ‘purified m-Dpm’), 20 µM m-Dpm-containing synthetic muropeptide (DHl-100; ‘synthetic m-Dpm’), and 20 µM l-lys-containing synthetic muropeptide (DHl-138, ‘synthetic l-lys’) were added to log phase cultures of B. subtilis at an OD600∼0.3 for 30 min. RT-PCR using yocH specific primers was performed with DNase I-treated RNA isolated from both cultures and untreated controls. yocH band intensities were measured using ImageJ (NIH) and induction ratios determined. All experiments were performed in triplicate. The induction rations are listed in Table S1.

Consistent with the known presence of peptidoglycan fragments in culture supernatants, yocH was induced ∼5-fold when growing B. subtilis cells were incubated with mutanolysin-digested B. subtilis peptidoglycan (Fig. 1B). Incubation of lysostaphin-digested Staphylococcus aureus peptidoglycan that contains an l-lys with log-phase B. subtilis cells did not induce yocH, indicating that an m-Dpm residue in the peptidoglycan stem peptide was likely necessary (Fig. 1B). The specific inducing molecule was identified using synthetic muropeptides. As with mutanolysin-digested B. subtilis peptidoglycan, m-Dpm containing muropeptides activated yocH (Fig. 1C), indicating that a muropeptide was sufficient for this induction. The inability of synthetic l-lys-containing muropeptides to induce yocH was consistent with the specificity of induction observed with peptidoglycan fragments (Fig. 1C).

YocH is a secreted protein with peptidoglycan binding and muralytic properties

YocH is a member of the extracellular proteome of B. subtilis (Tjalsma et al., 2004) and consistent with this observation, YocH-FLAG is found in the secreted fraction of log-phase B. subtilis cells (Fig. 2A). YocH contains two LysM peptidoglycan-binding domains (Steen et al., 2003) and His6-YocH purified from E. coli remained bound (∼40%) to the insoluble peptidoglycan pellet (Fig. S1). This binding is similar to that observed with the extracellular domain of PrkC (Shah et al., 2008). To test its putative muralytic activity, purified YocH-His6 was subjected to zymogram analysis and a clearance band was seen at the appropriate molecular weight (Fig. 2B). As the catalytic activity of MltA absolutely requires residue D308 (Van straaten et al., 2005), a mutant form of the His-tagged protein (His6-YocHD264A) that has an alanine in the equivalent position was purified. The absence of a clearance band associated with this protein in a zymogram (Fig. 2B) indicates that YocH likely uses a similar catalytic mechanism to digest peptidoglycan.

Figure 2.

YocH is a secreted peptidoglycan binding protein with muralytic activity.
A. Cellular lysates (L) and concentrated supernatants (S) from growing wild-type (PY79), WT; ΔyocH (JDB1881), ΔyocH; ΔyocH PyocH-gfp Pspac-yocH-FLAG (JDB2090), WT YocH-FLAG; and ΔyocH PyocH-gfp Pspac-yocHD264A-FLAG (JDB2091), D264A YocH-FLAG cultures were subjected to 8% SDS-PAGE and immunoblotted with α-FLAG antibodies. The upper band (top arrow) presumably is the unprocessed form whereas the lower band (bottom arrow) is the processed form.
B. YocH-his6 and YocHD264A-his6 purified from supernatants generated by growing ΔyocH PyocH-gfp Pspac-yocH-his6 (JDB2016) and ΔyocH PyocH-gfp Pspac-yocHD264A-his6 (JDB2092) cultures were subjected to zymogram analysis.

PrkC is required for muropeptide-mediated yocH induction

The B. subtilis membrane Ser/Thr kinase PrkC is required for germination of spores in response to peptidoglycan and to muropeptides (Shah et al., 2008). To examine whether PrkC was also required for peptidoglycan-mediated yocH induction, RNA was isolated from wild-type and ΔprkC cultures that were either untreated or treated with mutanolysin-digested peptidoglycan. As before, yocH was induced ∼5-fold in wild-type cultures, but treatment of ΔprkC cultures with peptidoglycan fragments did not lead to yocH induction (Fig. 3A). This requirement for PrkC was not observed for other genes induced by peptidoglycan such as wprA that encodes an extracellular protease (Fig. S2). The kinase activity of PrkC was necessary because peptidoglycan fragments did not induce yocH (Fig. 3A) in a strain expressing a PrkC mutant allele (PrkCK40A) that eliminates PrkC kinase activity in vitro (Madec et al., 2002). Additionally, overexpression of PrpC, the phosphatase belonging to the phylogenetically diverse PPM family that is co-expressed with PrkC and dephosphorylates PrkC (Obuchowski et al., 2000), blocked induction of yocH by peptidoglycan (Fig. 3B). By contrast, overexpression of a mutant PrpC (PrpCD36N) that carries an inactivating mutation in a conserved active site residue (D36) (Obuchowski et al., 2000) did not inhibit yocH induction.

Figure 3.

Induction of YocH is dependent on PrkC kinase.
A. RNA was isolated from wild-type (PB2), ΔprkC (PB705) and ΔprkC Pspac-prkCK40A-FLAG (JDB2227) cultures following incubation with or without mutanolysin-digested B. subtilis peptidoglycan.
B. RNA was isolated from wild-type (PB2), ΔprpC Pspac-prpC (JDB2094) and ΔprpC Pspac-prpCD36N (JDB2097).
C. RNA was isolated from log-phase cultures of wild-type B. subtilis (PY79) that were subjected to either no treatment or treatment with either 10 µM bryostatin (bryostatin) or with mutanolysin-digested peptidoglycan (muropeptides) or with mutanolysin-digested peptidoglycan along with 100 pM staurosporine (muropeptides + staurosporine) for 30 min. RT-PCR was conducted with yocH-specific primers. The ratios of treated cells compared with the non-treated cells for three separate samples were calculated. The induction rations are listed in Table S1.

Kinase stimulatory or inhibitory molecules affect yocH induction

The similarity of the kinase domain of the PrkC homologue M. tuberculosis PknB with eukaryotic Ser/Thr kinases (Ortiz-lombardia et al., 2003; Young et al., 2003) suggested that small molecules which either inhibit or activate these kinases would similarly affect PrkC. Bryostatin, a natural product synthesized by a marine bacterium, potently activates eukaryotic Ser/Thr kinases through direct binding to the phorbol ester binding site (Hale et al., 2002) and stimulates germination of B. subtilis spores in a PrkC-dependent fashion (Shah et al., 2008). Exposure of growing cultures to bryostatin caused ∼3-fold induction of yocH (Fig. 3C) and this induction was dependent on the presence of PrkC because it was not observed in a ΔprkC strain (data not shown). Staurosporine, a small molecule ATP mimic produced by some strains of Streptomyces, inhibits eukaryotic Ser/Thr kinases by blocking ATP binding (Ruegg and Burgess, 1989). Growing cultures of B. subtilis incubated with peptidoglycan fragments in the presence of staurosporine reduce yocH induction as compared with in its absence (Fig. 3C), consistent with the loss-of-function phenotype of the PrkC K40A active site mutant (Fig. 3A).

PrkC phosphorylates EF-G in response to peptidoglycan fragments

The ribosomal GTPase EF-G is an in vivo and in vitro substrate of PrkC (Gaidenko et al., 2002). Because EF-G is phosphorylated by PrkC during spore germination in response to muropeptides (Shah et al., 2008), we examined whether PrkC also phosphorylated EF-G in log-phase cells in response to peptidoglycan fragments. Immunoprecipitated EF-G from log-phase cells that had been incubated with mutanolysin-digested peptidoglycan was probed with an anti-phosphothreonine antibody (Fig. 4). Although equivalent amounts of EF-G were immunoprecipitated from wild-type and ΔprkC cells treated with muropeptides, the greater phosphorylation seen in wild-type cells indicated that PrkC phosphorylates EF-G in the presence of muropeptides (Fig. 4, Fig. S3). EF-Tu was reported to be a substrate of PrkC in vitro (Absalon et al., 2009); however, probing immunoprecipitated EF-Tu with phosphothreonine antibodies did not reveal a signal corresponding to EF-Tu (Fig. S4). Threonine phosphorylation of proteins that migrated at ∼100–130 kDa was observed, but the identity of those proteins remains unknown.

Figure 4.

PrkC phosphorylates EF-G in response to peptidoglycan fragments. Protein lysates were generated from wild-type B. subtilis (PB2) or from a ΔprkC (PB705) strains incubated with peptidoglycan isolated from log-phase B. subtilis cells for 60 min. Lysates were immunoprecipitated with α-EF-G and subjected to Western blotting with α-EF-G and α-phosphothreonine antibodies.

YocH regulates its own expression

As expression of yocH is induced by mutanolysin-digested peptidoglycan (Fig. 1B) and YocH is itself a peptidoglycan hydrolase (Fig. 2B), YocH could regulate its own expression. That is, would the digestion of peptidoglycan by YocH generate fragments that induce PyocH? To examine this possibility, yocH was placed under inducible control in a strain lacking endogenous yocH and expression of a PyocH reporter was measured so if yocH were subject to autoregulation, expression of YocH would activate PyocH. In support of this model, addition of inducer (IPTG) to a strain where yocH was under the control of Pspac increased the activity of a PyocH reporter ∼20-fold as compared with the same strain grown in the absence of inducer (Fig. 5). As expression of YocHD264A, a mutant form of YocH that is unable to digest peptidoglycan (Fig. 2B) had no effect on PyocH reporter RNA levels (Fig. 5), this autoregulation depends on the muralytic activity of YocH. The requirement of PrkC for induction of yocH in response to peptidoglycan (Fig. 3A) suggested that PrkC mediates the observed yocH autoregulation. We examined this possibility by introducing a ΔprkC mutation into the strain carrying Pspac-yocH and the PyocH reporter. This strain no longer increased PyocH reporter expression in response to yocH induction (Fig. 5). Thus, YocH appears to autoregulate its own expression by generating muropeptides that are in turn sensed by the membrane kinase PrkC.

Figure 5.

YocH autoregulation requires functional YocH and PrkC. Early exponential phase cultures (OD600∼0.2) of strains carrying inducible ΔyocH Pspac-yocH-FLAG PyocH-gfp (‘YocHwt PrkCwt’; JDB2090), ΔyocH Pspac-yocHD264A-FLAG PyocH-gfp (‘YocHD264A PrkCwt’; JDB2091) and ΔprkCΔyocH Pspac-yocH-FLAG PyocH-gfp strain (‘YocHWTΔprkC’; JDB2296) were treated with IPTG (1 mM) for 2 h at 37°C. RNA was isolated from treated and untreated cultures and RT-PCR was performed with gfp-specific primers. Following agarose electrophoresis, the ratios of the signals from treated and untreated sample were obtained. They are listed in Table S1.

Deletion of yocH results in a post-exponential phase survival defect

YocH contains an Sps domain in its C-terminus. Proteins containing this domain have been proposed to facilitate survival in stationary phase by analogy with the Rpf proteins of M. tuberculosis and M. luteus (Ravagnani et al., 2005). Consistent with this possibility, a ΔyocH strain (Fig. 6A, dashed line) exhibits a post-exponential phase survival defect as compared with the wild-type parent (Fig. 6A, solid line) using buffered Luria–Bertani (LB) medium (Gaidenko et al., 2002). This defect is rescued by the expression of YocH (Fig. 6B, solid line). The muralytic activity of YocH was necessary for this complementation because expression of the YocHD264A mutant that lacks muralytic activity (Fig. 2A) did not restore wild-type levels of post-exponential survival (Fig. 6B, dashed line).

Figure 6.

Absence of YocH decreases post-exponential phase survival.
A. Exponential and post-exponential phase survival of wild-type (◆, solid line; JDB3) and ΔyocH (inline image, dashed line; JDB1881) strains.
B. Exponential and post-exponential phase survival of ΔyocH Pspac-yocH (◆, solid line; JDB2090) and ΔyocH Pspac-yocHD264A (inline image, dashed line; JDB2091) strains.
C. Exponential and post-exponential phase survival of wild-type (◆; PB2), ΔyocH (inline image; JDB2338), ΔprkC (Δ; PB705) and ΔyocHΔprkC (□; JDB2340) strains. Cultures were grown in buffered LB at 37°C and OD600 was measured at designated intervals.

A strain carrying a ΔprkC mutation also exhibits a post-exponential phase survival defect (Gaidenko et al., 2002). As PrkC is necessary for induction of yocH in response to peptidoglycan fragments (Fig. 3A), we examined whether the ΔyocH phenotype involved the PrkC pathway by comparing the post-exponential phase survival defects of strains carrying single ΔyocH and ΔprkC mutations and a strain carrying both mutations. As the defect of strains carrying either single mutation was the same as that of a strain carrying both mutations (Fig. 6C), YocH and PrkC likely function in the same pathway.

YocH acts in a cell-autonomous fashion

YocH is secreted and found in the extracellular milieu (Fig. 2A; Tjalsma et al., 2004), suggesting that it could function cell autonomously. That is, in a culture containing both wild-type and ΔyocH cells, does YocH secreted from wild-type cells increase the survival of cells unable to produce YocH? The survival of a ΔyocH strain was increased in the presence of a wild-type strain, suggesting that YocH was able to rescue the mutation ( Table 1). The muralytic activity of YocH was necessary for this rescue because a strain expressing a mutant YocH that does not hydrolyse peptidoglycan (YocHD264A) failed to rescue the survival defect of the ΔyocH strain (Table 1).

Table 1.  Cell autonomous activity of YocH.
Strainscfu ml–1 (T0)cfu ml–1 (T24)% T0
  1. Single cultures of a wild-type strain (JDB1760; cmR), a ΔyocH strain (JDB1881; ermR), a ΔyocH Pspac-yocH (JDB2090; specR) and ΔyocH Pspac-yocHD264A(JDB2091; specR) were grown in buffered LB medium up to OD600∼2.0. Then single or mixed cultures containing approximately equal cfus of each strain (T0) were assayed for cfus following growth in buffered LB medium for 24 h at 37°C (T24). To determine cfus, aliquots were plated on appropriate antibiotic plates The cfus obtained for each strain at T0 were normalized to 100% to determine the fraction of survivors at T24.

Wild type5.6 × 1083.08 × 10855 ± 5
ΔyocH5.5 × 1081.37 × 10825 ± 4
Wild type2.4 × 1081.15 × 10848 ± 2
ΔyocH2.2 × 1081.2 × 10855 ± 8
ΔyocH Pspac-yocH5.16 × 1083.1 × 10860 ± 5
ΔyocH Pspac-yocH2.2 × 1081.2 × 10854 ± 4
ΔyocH2.45 × 1081.1 × 10845 ± 3
ΔyocH Pspac-yocHD264A5.5 × 1081.08 × 10820 ± 5
ΔyocH Pspac-yocHD264A2.39 × 1080.55 × 10823 ± 6
ΔyocH2.18 × 1080.48 × 10822 ± 8

Discussion

Growing bacteria turn over their cell wall and consequently release peptidoglycan fragments into the environment. These molecules therefore would serve as an inter-bacterial signal for the presence of growth-permissive conditions and their detection would allow bacteria to monitor the hospitability of the environment for continued growth. Muropeptides are recognized by dormant bacterial spores of a number of species and induce germination of these spores (Shah et al., 2008). Here we have demonstrated that these molecules are also recognized by growing B. subtilis and induce the expression of a secreted peptidoglycan hydrolase. The enzymatic activity of this secreted protein is necessary for this induction and indicates the presence of an auto-regulatory loop.

Induction of yocH

Supernatants derived from cultures of growing cells triggered yocH activation (Fig. 1A). Consistent with the presence of peptidoglycan fragments in these supernatants, m-Dpm-containing peptidoglycan isolated from growing B. subtilis cells and treated with mutanolysin was also effective at inducing yocH (Fig. 1B). As m-Dpm-containing muropeptides or a synthetic m-Dpm disaccharide tripeptide induced yocH (Fig. 1C), muropeptides were likely the inducing molecule. The inability of a synthetic l-lys-containing muropeptide (Fig. 1B) or digested S. aureus peptidoglycan to induce yocH (Fig. 1C) indicates the presence of an l-lys residue in the stem peptide was sufficient to prevent induction. This specificity was similar to that observed during B. subtilis spore germination where the presence of a m-Dpm residue was necessary to promote induction (Shah et al., 2008) and, interestingly, is similar to that observed with eukaryotic proteins that interact with muropeptides (Girardin et al., 2003; Boneca, 2005; Guan and Mariuzza, 2007).

Supernatants derived from cultures of growing cells triggered yocH activation better than those derived from cells in stationary phase (Fig. 1A). This result indicates that the muropeptides contained in these supernatants report not only cell density, but also the growth phase of the culture by detecting peptidoglycan fragments released as the products of growth but not those released during cell lysis. The ability of a cell to differentiate between these molecules would allow it to respond only to conditions conducive to growth. While the molecular basis of these differences is unknown, one possibility is that hydrolysis of the glycosidic linkage between MurNAc and the GlcNAc residues by a lytic transglycosylase results in the formation of a 1,6-anydroMurNAc containing disaccharide peptide whereas digestion by a lysozyme results in a terminal reducing MurNAc (Vollmer et al., 2008). Thus, if lysozymes and lytic transglycosylases were differentially active during exponential and post-exponential phases, then the presence of the 1,6-anhydro ring containing molecules would indicate the growth phase of the culture. As lytic transglycosylases and penicillin biding proteins (Vollmer et al., 1999) form complexes involved in cell elongation, the presence of anhydro-containing muropeptides would signal that conditions were permissive for growth.

Regulation of YocH

Expression of yocH has been previously shown to be regulated by the essential YycFG two-component system in B. subtilis (Howell et al., 2003). Here we demonstrate that the kinase activity of PrkC is necessary for yocH induction in response to muropeptides because strains lacking PrkC or expressing a mutant PrkC carrying a point mutation in its active site do not respond (Fig. 3A). The ability of the extracellular domain of PrkC to bind insoluble peptidoglycan (Shah et al., 2008) suggests that PrkC directly binds these muropeptides. However, given that PrkC is a Ser/Thr kinase, it is unlikely that it directly activates PyocH. In two-component systems such as YycFG, the membrane kinase phosphorylates a DNA binding protein on an aspartic acid residue, thereby activating transcription of a particular set of genes. Thus, PrkC could phosphorylate YycF on a Ser or Thr residue, thereby changing the ability of YycF to activate transcription. Alternatively, PrkC could phosphorylate proteins involved in cell division as is observed with its homologues M. tuberculosis PknB (Kang et al., 2005; Parikh et al., 2009) and S. agalactiae Stk1 (Silvestroni et al., 2009). These modifications would result in the activation of PyocH through the YycFG system that detects changes in cell wall metabolism (Dubrac et al., 2008). A third possibility is that PrkC phosphorylates an unknown protein that directly regulates PyocH expression. The gene encoding this protein could be detected in a genetic screen using a PyocH-lacZ reporter if constitutively active PrkC mutants were known but as yet these have not been identified.

When yocH is placed under IPTG-inducible control, treatment with IPTG increases the expression of a PyocH reporter. Importantly, only YocH capable of digesting peptidoglycan can regulate its own expression because the enzymatically inactive YocHD264A does not similarly affect expression of the reporter (Fig. 5). This autoregulatory loop (Fig. 7) also depends on PrkC (Fig. 5) as was observed with yocH induction in response to exogenous muropeptides. This is consistent with the demonstrated muralytic activity of YocH as well as its presence in the supernatant (Fig. 2A and B). We do not know if YycFG is necessary for this loop because these proteins are essential, although their role in sensing cell wall perturbations suggests that they are involved.

Figure 7.

Model of YocH function. YocH (blue circles) is secreted into the extracellular milieu where it digests peptidoglycan that derives from either the same cell or other cells in the culture. The digested muropeptides bind to the PrkC kinase (orange) in the membrane. This binding induces yocH through an unknown pathway (?) that possibly acts in parallel with the two-component YycFG system (red) and results in the production of additional YocH molecules.

Physiological role of YocH

Absence of YocH reduces post-exponential phase survival in liquid culture (Fig. 6A). This phenotype can be attributed to YocH function because inducible expression of wild-type YocH but not a catalytically inactive mutant (YocHD264A) complements this survival defect in liquid cultures (Fig. 6B). As YocH can function in a cell-autonomous manner (Table 1), it is not clear from these complementation experiments whether the source of this peptidoglycan is from the same cell or another cell in the culture. Thus, the inability to digest extracellular peptidoglycan into fragments is detrimental to the cell's survival in post-exponential phase. While peptidoglycan could serve as a nutritional source given that it is composed of disaccharide peptides, the inability of l-lys-containing peptidoglycan fragments to induce yocH (Fig. 1B) suggests that this is not likely.

Alternatively, the decrease in post-exponential phase survival of the ΔyocH strain could be due to a lack of stimulation of PrkC. Absence of PrkC results in a similar reduction in survival and because a ΔprkCΔyocH strain is as defective as a ΔprkC strain (Fig. 6C), the genes probably act in the same pathway. In contrast to PrkC, absence of PrpC, the partner phosphatase to PrkC, increases post-exponential phase survival as compared with wild type (Gaidenko et al., 2002). These opposite phenotypes suggest that increased phosphorylation of a common substrate of PrkC and PrpC is responsible for this increased survival. As the one identified common substrate of PrkC and PrpC is EF-G, its phosphorylation state (see below) may play a role in post-exponential phase survival. In addition, PrkC homologues play central roles in the physiology of a number of species is situations where post-exponential phase is important including Streptococcus pneumoniae StpK in competence (Echenique et al., 2004) and Enterococcus faecalis PrkC in persistence (Kristich et al., 2007).

The preferential induction of yocH by supernatants from growing cells as compared with cells in stationary phase (Fig. 1A) may indicate that this induction signals the presence of growing cells. Like YocH, S. aureus IsaA is a putative soluble lytic transglycosylase (Sakata et al., 2005; Stapleton et al., 2007). Production of IsaA is regulated by the S. aureus YycG and YycF homologues (Dubrac and Msadek, 2004) and expression of isaA was stimulated during exponential growth and repressed during post-exponential phase (Sakata and Mukai, 2007). As B. subtilis yocH is activated in a similar growth phase-dependent fashion, this phenomenon may be common to wide range of bacteria.

Role of EF-G phosphorylation

EF-G is an abundant cellular protein with several activities in regulating translation (Savelsbergh et al., 2009). First, EF-G plays an essential role in the translocation of mRNAs and tRNAs in the ribosome during translation (Shoji et al., 2009). Second, EF-G participates along with ribosome recycling factor in the process of ribosome recycling where the 70S ribosomes are split into subunits and the mRNA and tRNA are released (Hirokawa et al., 2006). The activity of eEF-2, its eukaryotic homologue, is regulated by phosphorylation (Ryazanov et al., 1988) and amino acid withdrawal leads to increases in EF-2 phosphorylation in yeast (Wang et al., 1998). While it is not known whether EF-G is similarly regulated, EF-G is phosphorylated in growing cells in response to muropeptides (Fig. 4). Thus, binding of muropeptides to PrkC would induce EF-G phosphorylation and thereby change the translational capacity of the cell as has been suggested previously for proteins containing an Sps domain (Ravagnani et al., 2005).

In addition to the phosphorylation of EF-G, PrkC is necessary for the signal transduction cascade that results in the induction of yocH. A possible connection between these two effects arises in the recent characterization of genes whose expression changed following treatment of S. aureus cells with fusidic acid, a molecule that interferes with release of EF-G following translocation. Six out of nine peptidoglycan hydrolase genes under control of YycFG including the secreted peptidoglycan hydrolase IsaA were upregulated by fusidic acid (Delgado et al., 2008). The interaction between the PrkC homologue S. agalactiae Stk1 and the two-component regulator CovR in cytotoxin expression (Rajagopal et al., 2006) suggests that a fruitful direction for future research will be examining the functional overlap between the PrkC and YycFG pathways.

Role of muralytic enzymes in exit from dormancy

The human pathogen M. tuberculosis can persist in the host for decades following infection before initiating disease. While the transition between latency and reactivation is thought to be essential for the bacterium to cause disease, the mechanism underlying this transition is unclear. Rpf proteins secreted by M. tuberculosis are important for this transition because an rpfB mutant of M. tuberculosis Erdman displayed delayed kinetics of reactivation in a mouse model of dormancy (Tufariello et al., 2006) and mutants of M. tuberculosis H37Rv lacking various combinations of rpf-like genes were attenuated for growth in mice (Downing et al., 2005). The muralytic activity of M. tuberculosis Rpf proteins and the homologous Rpf from M. luteus suggests that they facilitate reactivation though cell wall remodelling (Mukamolova et al., 2006; Telkov et al., 2006).

Although B. subtilis YocH is not by sequence similarity an Rpf homologue, the work described here demonstrates that it is functionally analogous given that it is secreted (Fig. 2A), muralytic (Fig. 2B) and that its absence results in a post-exponential phase survival defect (Table 1). Our data also demonstrate that yocH induction in response to muropeptides is dependent on the kinase activity of PrkC (Fig. 3). M. tuberculosis PknB is a homologue of PrkC that is essential and is therefore a high-priority drug target (Fernandez et al., 2006). Our work suggests that, by analogy, PknB may respond to muropeptides that are generated as a result of the muralytic activity of the Rpf proteins. A plausible connection between PknB activation and exit from dormancy is the ability of PrkC to phosphorylate EF-G (Gaidenko et al., 2002; Shah et al., 2008), a protein involved in ribosome recycling. As this process is necessary for the transition from stationary phase to growth in E. coli (Janosi et al., 1998), phosphorylation of EF-G could be a trigger for M. tuberculosis reactivation.

Experimental procedures

Bacillus subtilis strains used in this study are listed in Table 2 and details of their construction are described in Supporting information. B. subtilis strains were grown in LB except for experiments involving survival in post-exponential phase where the cultures were grown in buffered LB (Gaidenko et al., 2002). For experiments where growing cells were treated with cell free supernatants, 3 ml of cultures was grown in TSS medium [per litre: 10 ml of 50% glucose, 10 ml of 1.2% of MgSO4, 10 ml of 0.4% FeCl3-citrate (1:1), and 100 ml of 10× TSS salts (20 g of NH4Cl, 3.6 g of K2HPO4, 60 g of Tris base) and the pH was adjusted to 7.5 with HCI]. The cells were grown to an OD600 = 0.3, harvested and resuspended in 3 ml of cell-free supernatants isolated from cells grown to OD600 of 0.3, 0.6, 0.9, 1.0, 1.3, 1.6 and 1.9, shaken for 30 min and harvested for RNA isolation. For experiments where growing cells were treated with PG, 3 ml of cultures was grown to an OD600 = 0.3 and treated with either of the following agents: 5 mg of digested or undigested m-Dpm or lys-type peptidoglycan, 25 µM purified disaccharide-tripeptide P3 (m-Dpm), 20 µM of synthetic disaccharide-dimer DHl-100 (m-Dpm) or 20 µM of synthetic disaccharide dimer DHl-138 (l-lys) for 30 min at 37°C prior to harvesting and RNA isolation. P3 was a gift from Dr David Popham (Virginia Tech) and DHl-100 and DHl-138 were gifts from Dr Shahriar Mobashery (Notre Dame) and their synthesis is described in Lee et al. (unpublished data). Lysostaphin and mutanolysin were obtained from Sigma.

Table 2. B. subtilis strains.
StrainGenotypeSource
PY79Wild typeLab collection
PB2trpC2Gaidenko et al. (2002)
PB705trpC2 prkCΔ1Gaidenko et al. (2002)
JDB1881ΔyocH::ermThis study
JDB2014ΔyocH::erm sacA: PyocH-gfp cmThis study
JDB2016ΔyocH::erm sacA: PyocH-gfp cm amyE::Pspac-yocH-his6 specThis study
JDB2090ΔyocH::erm sacA: PyocH-gfp cm amyE::Pspac- yocH-FLAG specThis study
JDB2091ΔyocH::erm sacA::PyocH-gfp cm amyE::Pspac-yocHD264A-FLAG specThis study
JDB2092ΔyocH sacA: PyocH-gfp cm amyE::Pspac- -yocHD264A-his6This study
JDB2094trpC2ΔprpCΔ1 amyE::Pspac-prpC specThis study
JDB2097trpC2ΔprpCΔ1 amyE::Pspac-prpCD36N specThis study
JDB2227trpC2ΔprkCΔ1 amyE::Pspac-FLAG-prkCK40A specShah et al. (2008)
JDB2296trpC2ΔprkCΔ1ΔyocH::erm sacA::PyocH-gfp cm amyE::Pspac- -yocH-FLAG specThis study
JDB2338trpC2ΔyocH::ermThis study
JDB2340trpC2ΔprkCΔ1ΔyocH::ermThis study

Peptidoglycan isolation

Peptidoglycan was isolated from growing B. subtilis or S. aureus cells as described (Shah et al., 2008). Resuspended B. subtilis peptidoglycan (50 mg ml–1) was digested with mutanolysin (10 µg ml–1) overnight at 37°C prior to inactivation of mutanolysin at 80°C for 20 min. S. aureus peptidoglycan (50 mg ml–1) was digested with lysostaphin (0.5 mg ml–1) overnight at 37°C prior to inactivation of the enzyme at 80°C for 20 min.

RNA isolation and RT-PCR analysis

RNA was isolated from cell pellets using RNeasy kit (Qiagen) according to the manufacturer's instructions. Prior to treatment with lysozyme, RNA was stabilized with Bacterioprotect (Qiagen). Isolated RNA was treated with DNase I (37°C, 50 min) followed by heat inactivation (65°C, 10 min). RT-PCR reactions were carried out in 25 µl volumes with 100 ng of RNA for each sample using SSIII RT/Platinum Taq enzyme (Invitrogen) according to the manufacturer's instructions. Briefly, reverse transcription was carried out at 50°C for 30 min followed by PCR [Denaturation: 94°C (15 s), PCR: 30 cycles of 94°C (15 s), 55°C (30 s) and 68°C (1 min kb–1)]. Total sample volumes were loaded on 0.8% agarose gels followed by electrophoresis and staining with SYBR gold (Invitrogen). ImageJ (NIH) was used for gel quantification.

Analysis of secreted proteins

Cells were grown in 3 ml LB medium until OD600 = ∼0.1. For expression of proteins from Pspac, the cultures were induced with 1 mM IPTG for 2 h. The cultures were centrifuged (5000 g, 5 min) and the supernatant added to ice-cold ethanol (5× volume, 15 min). Following centrifugation (3000 g, 10 min), the precipitated pellets were air-dried and directly resuspended in 80 µl 2× SDS-loading dye. For cellular protein extracts, the pellets were resuspended in 500 µl 10 mM Tris, pH 8.0, 10% sucrose and treated with 1 mg ml–1 lysozyme for 15 min prior to resuspension in 80 µl 2× SDS-loading dye. Resuspended pellets were subjected to SDS-PAGE followed by immunoblotting with α-FLAG antibodies (Sigma) and by detection by ECL (Amersham).

Purification of secreted proteins

Bacillus subtilis strains were grown in 30 ml LB medium until OD600 = ∼0.1. For expression of his-tagged proteins from Pspac, 30 ml of cultures was induced with 1 mM IPTG for 2 h prior to removal of cells by centrifugation. The supernatants were concentrated (Vivaspin columns, MWCO = 10 kDa) to a final volume of 0.5 ml and purified using Ni2+ affinity chromatography as described (Shah et al., 2008).

Zymogram analysis

Zymogram analysis was carried out as described (Piuri and Hatfull, 2006). Briefly, gels were cast with 0.01% SDS and 30 µl (∼5 mg) B. subtilis peptidoglycan resuspension. Following electrophoresis, the gel was incubated at 37°C for 16 h in 1% Triton X-100, 25 mM Tris-HCl pH 8.5, washed once in water and stained for 3 h with 0.5% methylene blue in 0.01% KOH. Clearance bands were visualized following destaining (5× water wash).

Immunoprecipitation and Western analysis

For detection of YocH in secreted fractions, α-FLAG antibodies (Sigma) were used (1:3000). For immunoprecipitation of EF-G, 3 ml of cultures of B. subtilis was harvested with or without treatment with B. subtilis peptidoglycan (10 mg) for 60 min, incubated with lysozyme (1 mg ml–1) in Tris-EDTA buffer (pH 8.0) for 15 min in a total volume of 400 µl and sonicated (45 s, 2 pulses). The samples were centrifuged and the resulting supernatants were added to 10 µl EF-G-Protein A Dynabead (Invitrogen) complex as described (Shah et al., 2008). Following immunoprecipitation for 1 h at 4°C, the beads were washed 3× with PBS and directly resuspended in 50 µl of 2× SDS-loading dye and boiled for 30 min. Fifteen microlitres of samples was loaded onto duplicate 8% gels for probing with α-EF-G antibodies (1:3000 dilution) and α-phosphothreonine antibodies (1:250 dilution, Invitrogen).

Acknowledgements

We thank members of our laboratory for advice and Dr Howard Shuman for comments on the manuscript. B. subtilis muropeptide P3 was a gift from Dr David L. Popham (Virginia Tech) and synthetic muropeptides DHl-100 and DHl-138 were gifts of Dr Shahriar Mobashery (Notre Dame). Dr Pascale Cossart (Institut Pasteur) generously provided the EF-Tu antibody. This work was supported by the Irma T. Hirschl Trust and by NIH R0185468.

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