Correspondence: Bernard Weisblum, Department of Medicine, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, WI 53706, USA. Tel.: 608-262-0972; e-mail: Weisblum@wisc.edu
Streptococcus pneumoniae contains a single Ser/Thr kinase-phosphatase pair known as StkP-PhpP. Here, we report the interaction of StkP-PhpP with S. pneumoniae UDP-N-acetylmuramoyl:L-alanine ligase, MurC, an enzyme that synthesizes an essential intermediate of the cell wall peptidoglycan pathway. Combinatorial phage display using StkP as target selected the peptide sequence YEVCGSDTVGC as an interacting partner and subsequently confirmed by ELISA. The phage peptide sequence YEVCGSDTVGC aligns closely with the MurC motif spanning S. pneumoniae amino acid coordinates 31–37. We show that MurC is phosphorylated by StkP and that phosphoMurC is dephosphorylated by PhpP. These data suggest a link between StkP-PhpP with the coordinated regulation of cell wall biosynthesis via MurC.
The Ser/Thr kinases together with their cognate phosphatases play a major role in cell signaling. For reviews of the protein kinases and protein phosphatases in prokaryotes (Kennelly, 2002; and Deutscher & Saier, 2005), StkP, a eukaryotic-type serine-threonine kinase belongs to the signaling network of Streptococcus pneumoniae where it was shown (Echenique et al., 2004) to regulate the expression of competence and virulence. Nováková et al. (2005) reported that autophosphorylated StpK is dephosphorylated by PhpP, a mammalian-type PP2C-type phosphatase encoded by an adjacent open reading frame in the genome, while Osaki et al. (2009) showed that StkP and PhpP act as a functional pair in vivo.
The enzymes or processes found to be regulated by StkP and/or PhpP include: phosphoglucosamine mutase, GlmM, responsible for synthesis of the early intermediate of cell wall biosynthesis, N-acetylglucosamine, (Nováková et al., 2005), ribosomal protein S1 and the alpha subunit of RNA polymerase (Nováková et al., 2010); DivIVA cell division protein (Nováková et al., 2010); FtsZ, cell division protein (Giefing et al., 2010); enzymes of pyrimidine biosynthesis cell division protein, DNA repair, iron uptake, the oxidative stress response, and competence (Nováková et al., 2010); choline-binding protein A (CbpA), (Standish et al., 2007); and, RR06/HK06, one of twelve two-component signal transduction systems (Agarwal et al., 2012).
Having shown regulation of heme transport, nucleotide synthesis, and DNA repair to be mediated by the orphan response regulator RitR, we further showed that RitR expression is regulated by StkP. In the present studies, we have attempted to learn how StkP, and by inference together with PhpP, interact with as yet undiscovered pathways.
StkP and its orthologous Ser/Thr kinases contain an intracellular kinase domain and four environmentally responsive extracellular PASTA domains, (Gordon et al., 2000), in crystallographic studies, to bind to the ß-lactam cefuroxime. It has been shown that the extracellular PASTA-containing domain of StkP binds to both chemically synthesized fragments of cell wall peptidoglycan and to ß-lactam antibiotics that mimic the terminal portions of the peptidoglycan pentapeptide moiety (Maestro et al., 2011). Thus, the StkP extracellular PASTA domains likely bind and monitor various bacterial peptidoglycan precursors (Gordon et al., 2000; Yeats et al., 2002; Rajagopal et al., 2003; Jones & Dyson, 2006) using the level detected to make adaptive changes in genomic expression.
Bacterial cell wall biosynthesis requires the essential Mur enzymes (MurA-MurF), which catalyze successive early steps in the formation of the peptidoglycan precusor monomeric unit, (van Heijenoort, 2001; El Zoeiby et al., 2003; Smith, 2006). The studies undertaken suggest multiple levels of control of cell wall synthesis that phosphorylation of MurC, in part, mediates this control.
Materials and methods
Purification of recombinant S. pneumoniae StkP, PhpP, MurC, and MurD
GST-StkP was prepared as described previously (Ulijasz et al., 2009). The open reading frame (ORF) associated with the Ser/Thr kinase, StkP, from S. pneumoniae strain TIGR4, was cloned into plasmid pGEX-6P-1 digested with EcoRI and BamHI. Protein was expressed as a GST fusion in Escherichia coli BL21 and purified using a gluthione-sepharose 4B affinity column. PhpP was purified as described previously (Ulijasz et al., 2009). The ORF associated with MurC was amplified from S. pneumoniae TIGR4 genomic DNA, and cloned into plasmid pET28b (EMD Biosciences) digested with EcoRI and BamHI, which added an N-terminal His tag. The recombinant gene was expressed in E. coli BL21 and purified by Ni++ affinity chromatography. Likewise, the gene for MurD (UDP-N-acetylmuramoyl L-alanine D-glutamine ligase) was cloned in to pET28b (EMD Biosciences) and purified by Ni++ affinity chromatography as described above.
Phage display on StkP
Wells were coated with 1 μg purified GST-StkP for 1 h and blocked with 10 mg mL−1 ovalbumin for 1 h. Ovalbumin alone was used as binding control. All washes were performed with PBS + 0.1% TX-100. 108 pfu purified phage were added to each well, incubated for 30 min, washed, followed by incubation for 30 min with anti-M13 phage HRP antibody at 1/20K dilution, washed, and tetramethylbenzidine (TMB) substrate added. Binding was determined by TMB substrate conversion measured at 650 nm. Phage display was performed as described by Sparks et al., 1996;. 96-well protein-binding microtiter plates (Nunc) were coated with purified GST-StkP protein and used to select M13 phage from a combinatorial phage peptide display library ‘X10C’ - ten random amino acids with a C-terminal CySH. The library was a gift from BK Kay (Gee et al., 1998). The M13 phage clones from the third round of selection were plaque-purified, and 10 individual clones were selected and sequenced. The phage sequence was associated with Desulfovibrio vulgaris MurC on the basis of a BLAST search (Altschul et al., 1990) and aligned with a set of selected MurC sequences using Multalin (Corpet, 1988).
Interaction between StkP and phage was performed following established phage ELISA protocols (Sparks et al., 1996). All washes were performed with PBS + 0.1% Tween-20. 1 μg of purified GST-StkP per well was added to protein-binding plates (Nunc). For phage ELISA, 1 × 108 pfu of purified phage was added/well to those coated with GST-StkP protein or to control wells coated with ovalbumin. Detection of purified M13 phage binding was performed with anti-M13 phage horseradish peroxidase (HRP) conjugate monoclonal antibody (GE Healthcare) at 1/20 000 dilution. The amount of phage bound was determined spectrophotometrically by measuring the conversion of TMB substrate at 650 nm.
In vitro enzymatic phosphorylation-dephosphorylation
GST-StkP was autophosphorylated in a 20 μL reaction volume containing: 50 mM Tris–HCl (pH 8.0), 1 mM DTT, 5 mM MgCl2, 100 μM ATP, 1 μCi of [γ-32P]-ATP (Specific activity, > 6000 Ci mmole−1) and 100 ng GST–StkP, followed by incubation at 30 °C for 20 min. To phosphorylate MurC, 1 μg purified MurC was added to the above reaction and incubated at 30 °C for an additional 20 min. The reaction was stopped by addition of sodium dodecyl sulfate loading buffer, and the samples were fractionated by SDS-PAGE, mounted in cellophane, dried, and analyzed by autoradiography. Dephosphorylation of phosphorylated MurC (MurC-P) was performed by subsequent addition of 5 mM MnCl2 and 1 μg of purified PhpP to the completed MurC phosphorylation reaction above, followed by incubation for 40 min at 30 °C and analysis as described. Phosphorylation reactions for the purified MurD was also performed as described above. Autophosphorylation controls of MurC and MurD alone were performed by adding 1 μCi of [γ-32P]-ATP directly to MurC and MurD and incubating as described above.
Phage binding to StkP/peptide and sequence analysis
Phage display against purified StkP as target yielded after three rounds of selection plus enrichment a single peptide sequence, YEVCGSDTVGC, present in all 10 plaque-purified phage clones that were selected. A blast search (Altschul et al., 1990) of the peptide yielded several partial sequence matches with S. pneumoniae MurC. The most extensive match was with D. vulgaris, where 7 of 10 consecutive identical amino acids were aligned. Moreover, the variant forms of the displayed peptide in the sample organisms cited were located in MurC, all between 25 and 30 amino acid resides from the N-terminus, as shown in Fig. 1.
Phage-based ELISA with StkP-coated wells and YEVCGSDTVGC-bearing phage confirmed the specificity of binding of peptide-bearing phage to StkP. Results are shown in Fig. 2. As negative control, Phage bearing the E9 sequence (Ulijasz et al., 2009), specific for PhpP, VADGMGGR, had no detectible affinity toward StkP.
Phosphorylation of MurC by StkP
The ability of StkP to phosphorylate MurC was tested in vitro (Fig. 3). Phosphorylation of MurC by StkP was performed by first autophosphorylating StkP (Fig. 3, Lanes 3 and 4), followed by addition of MurC (Fig. 3, Lanes 1 and 6). No autophosphorylation of MurC was seen (Fig. 3, lane 2). Addition of PhpP to autophosphorylated StkP results in a marked reduction in StkP phosphorylation (Fig. 3, Lane 5). PhpP also exhibits dephosphorylation activity upon phosphoryl MurC (Fig. 3, Lane 7).
We also tested the ability of StkP to phosphorylate MurD, which is involved, along with MurC, in the stepwise synthesis of the pentapeptide unit to the UDP-N-acetylmuramic acid scaffold. Autophosphorylated StkP was used in reactions to phosphorylate both MurC (Fig. 4, Lane 3) and MurD (Fig. 4, Lane 5). We observed phosphorylation of MurC but not MurD. No autophosphorylation of MurC or MurD alone was observed (Fig. 4, Lanes 2 and 4) Coomassie staining of the phosphorylation reactions run by SDS-PAGE showed that equal amounts of MurC and MurD were used (Fig. 4b).
We show that StkP phosphorylates MurC L-alanine ligase, an essential enzyme in the cell wall biosynthesis pathway and that the cognate phosphatase, PhpP, dephosphorylates phosphoryl MurC. By the use of phage display, we identified MurC as a target for StkP (Fig. 2). While phage-based ELISA confirmed the specific binding of the peptide YEVCGSDTVGC with StkP (Fig. 3). This peptide sequence closely aligns with the N-terminus of MurC, 25–30 amino acid residues downstream of the N-terminus.
Interestingly, Fiuza et al. (2008) determined that the Ser/Thr kinase, PknA, in Corynebacterium glutamicum, phosphorylated multiple sites within the C. glutamicum MurC L-alanine ligase. Phosphorylation of all three MurC domains, namely, the UDP- MurNac-binding domain, ATP-binding domain, and L-alanine-binding domain was found. Their findings include that phosphorylation is correlated with a decrease in MurC enzymatic activity, suggesting the nature of the role for phosphorylation of MurC in regulating cell wall biosynthesis. The list of S. pneumoniae phosphoproteins reported in studies of Sun et al. (2010) did not include MurC or RitR (Ulijasz et al., 2009) among the 84 phosphoproteins that were identified.
Although we established that StkP can phosphorylate MurC (Figs 3 and 4), the location and extent of MurC phosphorylation in S. pneumoniae remain to be determined as well as the extent to which phosphorylation alters the affinity of MurC for its substrates. Studies of M. tuberculosis showed that PknA, a mycobacterial eukaryotic-type Ser/Thr kinase, was able to phosphorylate MurD (Thakur & Chakraborti, 2008); however, in our hands, attempts to phosphorylate recombinant MurD from S. pneumoniae were unsuccessful (Fig. 4). Of interest is that in both C. glutamicum and M. tuberculosis, phosphorylation of Mur enzymes by a Ser/Thr kinase is carried out by PknA rather than by a PASTA domain-bearing Ser/Thr kinase-like StkP. These observations and others like these may be due to the presence of multiple Ser/Thr kinases in these strains, as opposed to the sole Ser/Thr kinase present in S. pneumoniae.
StkP acts as a global gene regulator in S. pnuemoniae (Sasková et al., 2007), which would be consistent with multiple regulatory systems linking with StkP-PhpP. In addition to targets identified by other groups, our previous work on StkP links up with an orphan response regulator RitR, modulating iron uptake (Ulijasz et al., 2009). The Ser/Thr kinase in S. agalactiae was shown to phosphorylate and regulate cytotoxin expression through the response regulator CovR (Rajagopal et al., 2003, 2006), further suggesting multiple system regulation through Ser/Thr kinases in the Streptococci. Lastly, recent results reported by (Beilharz et al., 2012) have provided additional information on how StkP plays an important role in the regulation of cell wall synthesis as well as the synthesis of the septum and its closure. In addition to the mechanisms already described, it will be interesting to learn how many more ways bacteria have of regulating cell wall biosynthesis.
We thank Brian K. Kay for his generous gift of the phage display library on which these studies were based. This work was supported, in part, by grant 5R03-AI081005 from the U.S. National Institutes of Health.