A Eukaryotic Type Serine/Threonine Kinase and Phosphatase in Streptococcus agalactiae Reversibly Phosphorylate an Inorganic Pyrophosphatase and Affect Growth, Cell Segregation, and Virulence*

Lakshmi Rajagopal, Anne Clancy, and Craig E. RubensDagger

From the Division of Infectious Disease, Childrens Hospital and Regional Medical Center, Seattle, Washington 98105

Received for publication, December 13, 2002, and in revised form, January 17, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein phosphorylation is essential for the regulation of cell growth, division, and differentiation in both prokaryotes and eukaryotes. Signal transduction in prokaryotes was previously thought to occur primarily by histidine kinases, involved in two-component signaling pathways. Lately, bacterial homologues of eukaryotic-type serine/threonine kinases and phosphatases have been found to be necessary for cellular functions such as growth, differentiation, pathogenicity, and secondary metabolism. The Gram-positive bacteria Streptococcus agalactiae (group B streptococci, GBS) is an important human pathogen. We have identified and characterized a eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Biochemical assays revealed that Stk1 has kinase activity and localizes to the membrane and that Stp1 is a soluble protein with manganese-dependent phosphatase activity on Stk1. Mutations in these genes exhibited pleiotropic effects on growth, virulence, and cell segregation of GBS. Complementation of these mutations restored the wild type phenotype linking these genes to the regulation of various cellular processes in GBS. In vitro phosphorylation of cell extracts from wild type and mutant strains revealed that Stk1 is essential for phosphorylation of six GBS proteins. We have identified the predominant endogenous substrate of both Stk1 and Stp1 as a manganese-dependent inorganic pyrophosphatase (PpaC) by liquid chromatography/tandem mass spectrometry. These results suggest that these eukaryotic-type enzymes regulate pyrophosphatase activity and other cellular functions of S. agalactiae.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein phosphorylation is a principal mechanism of signal transduction governing various cellular processes such as physiology, growth, and development. Regulation of protein function or enzyme activity by covalent and reversible phosphorylation is crucial for the regulation of cellular responses of both prokaryotes and eukaryotes to dynamic internal and external environmental conditions. Signal transduction in prokaryotes was thought to occur primarily by histidine kinases that activate transcription by phosphorylation of cognate response regulators at aspartate residues (1). However, phosphorylation by serine kinases have also been described in prokaryotes. Examples of signal transduction by serine kinases, specifically in Gram-positives, include the well characterized and novel HPr system with bifunctional kinase/phosphatase activity necessary for carbon catabolite repression (for review, see Ref. 2) and the isocitrate dehydrogenase kinase/phosphatase system (3, 4). Other examples of serine phosphorylation involves cognate pairs of kinases and phosphatases that regulate stress responses in Bacillus subtilis (5). Interestingly, although these serine kinases are homologous to the two-component histidine kinases (6), SpoIIE, which regulates sporulation in B. subtilis, is homologous to eukaryotic-type protein phosphatases (7).

In eukaryotes, reversible protein phosphorylation via serine, threonine, and tyrosine protein kinases and phosphatases is essential for cell cycle control, cell type determination, and differentiation (8). In contrast to the prokaryotic two-component systems, these eukaryotic kinases and phosphatases often comprise a network of protein phosphorylation cascades that coordinate responses to a variety of signals. The eukaryotic serine/threonine kinases (STK)1 show a high degree of conservation in their amino acid sequence at their N-terminal catalytic domains and are termed the Hanks' I-XI domains (8).

The recent discovery of eukaryotic-type or Hanks' STK in a number of prokaryotes such as Myxococcus xanthus (9-11), Streptomyces sp. (12, 13), Anabaena sp. (14, 15), Cyanobacteria (for review, see Ref. 16), B. subtilis (17), and a few archeabacterial sp. (for review, see Ref. 18) has sparked interest in the function and evolution of these eukaroytic-type signal transduction systems in prokaryotes. It has been described that mutations in STK homologues in Myxococcus sp., Streptomyces sp., and other spore-forming bacteria cause premature sporulation or abnormal development (for reviews, see Refs. 19 and 20). Eukaryotic-type STK essential for virulence was reported in Yersinia psuedotuberculosis (21) and Pseudomonas aeruginosa (22). The Yersinia STK (YpkA) is distinct from other prokaryotic homologues because kinase activation occurs only upon binding to host cytoplasmic factors such as actin (23, 24). A number of STK have also been described in Mycobacterium tuberculosis (25-27). However, the phenotypes of mutations in these genes and their biological role in pathogenesis have yet to be reported.

Eukaryotic serine/threonine phosphatases (STP) have been classified into two distinct structural families: PP1/2A/2B and PP2C. These phosphatases are defined by their amino acid sequences and tertiary structures (for review, see Refs. 28 and 29). The action of cognate pairs of eukaryotic kinases and phosphatases tightly regulate functions of target proteins (30). The existence of STP was also presumed to be exclusive to eukaryotes; however, bacterial homologues of eukaryotic-type STP have been described (31). SppA in Streptomyces coelicolor (32) and Pph1 in M. xanthus (33) are PP2C-type STP that are important for vegetative growth and development. A recent study on a eukaryotic-type STK and STP in B. subtilis indicated the requirement of these enzymes for normal biofilm and spore formation (17). Although STK and STP have been identified and biochemically characterized in a number of prokaryotes, their functions are not well understood because of the lack of information on their endogenous phosphorylation targets and activating signals.

Streptococcus agalactiae (group B streptococci, GBS) are Gram-positive cocci that are the causal agents of human neonatal pneumonia, sepsis, and meningitis. GBS is also an emerging pathogen in immunocompromised adults (34). Eukaryotic-type STK and STP have not been reported in the Gram-positive pathogenic cocci such as Streptococcus sp., Staphylococcus sp., and Enterococcus sp., although recent advances in genome analysis have revealed the presence of their putative homologues. The role of signal transduction pathways essential for regulation of GBS growth in response to environmental conditions, e.g. during infection, have not been thoroughly elucidated. A few virulence determinants of GBS like capsular polysaccharide beta -hemolysin and CAMP factor have been described (for review, see Ref. 35). Recent reports on GBS demonstrated that tyrosine phosphorylation by CpsD regulates capsular polysaccharide production (36), suggesting regulation of virulence factors by signal transduction mechanisms.

In this study, we describe the existence of a eukaryotic-type serine/threonine kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Mutants defective for Stk1 or both Stp1 and Stk1 expression exhibit pleiotropic effects on growth, cell segregation, and virulence, suggesting an important role for these enzymes in the regulation of various cellular processes. The kinase and its cognate phosphatase are also essential for reversible phosphorylation of a manganese-dependent inorganic pyrophosphatase (PpaC). These studies suggest that one of the functions of these eukaryotic-type enzymes is to regulate pyrophosphatase (PpaC) activity in GBS.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Growth Conditions and DNA Manipulations-- GBS strains were grown either in Todd Hewitt Broth (Difco Laboratories) or chemically defined medium (37) in 5% CO2 at 37 °C. A909 (WT) is a type Ia capsular polysaccharide clinical isolate of GBS (38). Recombinant DNA and other techniques were performed as described (39). Open reading frame (ORF) and BLAST homology searches were performed using the NCBI Internet server (www.ncbi.nlm.nih.gov). PSORT (psort.nibb.ac.jp) and SOSUI (sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html) were used to identify secretion signals and transmembrane domains. Multiple sequence alignments of gene products were created with Clustal X (40).

Construction of Alkaline Phosphatase Fusions-- To identify ORFs encoding secreted or membrane-associated proteins in GBS, we utilized the alkaline phosphatase gene fusion technology, as described previously (41). Briefly, random Sau3AI fragments of GBS chromosomal DNA were ligated into the BamHI site of the expression plasmid pAN200 and electroporated into Escherichia coli MC1061 as described (41). E. coli colonies containing the recombinant plasmids were screened for blue color on Luria Agar plates containing 5-bromo-4-chloro-3-indolyl phosphate (XP, 40 µg/ml). Plasmids were isolated from the blue colonies, and the region cloned within the BamHI site of pAN200 was sequenced using the primer oml110 (5'-GACGCTGAATCGGTG-3'). Sequence analysis was also used to confirm that the translational fusions were "in frame" to the coding sequence of phoZ.

Data Deposition-- The sequence of the GBS serine/threonine kinase and phosphatase operon was submitted to GenBankTM with accession number AF459092.

Reverse Transcriptase PCR-- Total RNA was isolated from early and mid-log phase cultures of GBS using the Gene Choice RNA Spin mini kit from PGC Scientifics (Gaithersburg, MD). The RNA was subsequently treated with RNase-free DNase (Promega) as described by the manufacturer and was purified using the RNeasy Mini Kit (Qiagen, San Diego, CA). Reverse transcriptase PCR was performed using the GeneChoice Thermo-RTII kit as per the manufacturer's instructions using stk1 gene-specific primer 3 (5'-GTTGAACTTACTTGTAGAAGTGC-3').

Internal primers used in PCR amplification for co-transcription analysis of stp1 and stk1 were primer 1 (5'-GGGCAACGTCGTTCCAATAATC-3') and primer 2 (5'-GCCATTGCTCGTGCTTCTCT-3'). PCR amplification was carried out for 25 cycles as per the manufacturer's instructions, and the products were analyzed by agarose gel electrophoresis. Positive control with genomic DNA as template and negative controls with reverse transcriptase omitted in the cDNA synthesis reaction were also performed and analyzed.

Construction and Expression of GST Fusion Proteins-- The stp1 and stk1 genes were amplified by PCR and individually cloned into the GST fusion vector pGEX4T3 (Amersham Biosciences). The PCR products were amplified from A909 chromosomal DNA using Expand High Fidelity PCR systems (Roche Molecular Biochemicals). The primers used to amplify stk1 were pkn1b (5'-CGGGTCGACTCGATGATTCAGATTGGCAAATTA-3') and lrpk2 (5'-ATAGTTTAGCGGCCGCTGTACTATCACTTGGT-3'), and the primers used to amplify stp1 were lrpp1 (5'-CGCGGATCCAGAAAAAAATATATGGAA-3') and lrpp2 (5'-GCCGCTCGAGATCATTTAAACCGCCTC-3'). The recombinant plasmids pLR1 (GST-Stk1 or rStk1) and pLR2 (GST-Stp1 or rStp1) were constructed by ligating BamHI-XhoI and NotI-SalI digested PCR products into pGEX4T3, respectively. The plasmids were electroporated into E. coli BL21DE3. Fusion protein expression and purification was performed as per the manufacturer's instructions. Because Stk1 is a membrane protein, a modified protocol that included solubilization in the presence of sodium laurylsarcosine (42) was used to purify the Stk1 fusion protein (rStk1). The recombinant proteins, rStp1 and rStk1, reacted with an anti-GST antibody in Western blots (data not shown).

Protein Phosphorylation and Phosphoamino Acid Analysis-- Autophosphorylation was performed by the addition of 10 µCi of [gamma -32P]ATP to 2 µg of rStk1 in kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 1 µM ATP) as described previously (43). The reaction was stopped by the addition of SDS-PAGE sample buffer, and the products were analyzed on a 10% SDS-PAGE followed by autoradiography. Phosphorylation of casein or myelin basic protein (Sigma) was performed by the addition of 5 µg of each to the kinase (rStk1) autophosphorylation reactions described above. The phosphorylated products were transferred to polyvinylidene difluoride membranes, and the proteins were hydrolyzed as described (44). The samples were spotted on Silica Gel G glass plates (Analtech Inc., Newark, DE) along with control phosphoserine, phosphothreonine, and phosphotyrosine (Sigma). The first and second dimension ascending chromatography was performed as described (45). The amino acid standards were visualized by ninhydrin staining followed by autoradiography to visualize radiolabeled amino acids.

Phosphatase Assays-- Hydrolysis of p-nitrophenyl phosphate (pNPP) was carried out as described (46). Briefly, each reaction contained 20 mM pNPP, phosphatase buffer (50 mM Tris-HCl, pH 8.0, and 2 mM MnCl2, MgCl2, or CaCl2) and 2 µg of rStp1. 2 µg of pure GST was also included in similar reaction conditions as a control. Hydrolysis of pNPP was monitored by increase in absorbance of p-nitrophenol (pNP) at 405 nm. A standard curve that determines the relationship between absorbance (A405) of pNP to its molar concentration under the above experimental conditions was performed, as previously described (47). Phosphatase activity of rStp1 on pNPP is expressed as the number of nmol of liberated pNP/µg of protein. The optimal concentration of MnCl2 was also determined by varying MnCl2 in the reaction conditions mentioned above.

To determine the velocity of the reaction, the amounts of rStp1 were varied in 10-fold dilutions (from 30 µg/ml to 0.3 µg/ml) while keeping the concentration of pNPP (20 mM) constant to determine the optimum range of enzyme where product (pNP) formation was linear in the 60-min assay period, as described (47). When the optimum enzyme concentration was determined (3 µg/ml), the concentration of pNPP was varied (1, 2.5, 5, 10, or 20 mM), and hydrolysis was monitored in the linear range of the reaction (i.e. every 10 min for a period of 60 min). The initial velocity of the reaction was determined for each substrate concentration, and a Lineweaver-Burk plot was derived to calculate the Km and Vmax, respectively.

For time course dephosphorylation, rStk1 was autophosphorylated, and unincorporated [gamma -32P] ATP was removed using a G50 Sephadex spin column. Phosphorylated rStk1 was incubated with 2 µg of rStp1 in phosphatase buffer containing 3 mM MnCl2. Aliquots were removed at various time points and analyzed on 10% SDS-PAGE followed by autoradiography.

Construction of Insertion and Allelic Exchange Mutations-- The entire stp1 gene, with a flanking sequence of 0.6 kb on either end, was PCR-amplified from WT chromosomal DNA and cloned in pBS KS+ (Stratagene, La Jolla, CA) resulting in pLR3. The primers used to amplify stp1 and flanking sequences from chromosomal DNA are lrfpp3 (5'-CTAGTCTAGACTATCCCAAGATGAATCTAG-3') and lrrpp4 (5'-CCCAAGCTTCTGTAGGGCAATAGTAACTGC-3'). A BamHI digest of plasmid pCIV2 yields a 2.2-kb Omega km-2 fragment that confers kanamycin resistance (48). The Omega km-2 fragment from pCIV2 was ligated into the BamHI site located in coding region of stk1 in pLR3. The 4.0-kb insert of the resulting plasmid pLR4 was excised and ligated into the SalI-NotI-linearized GBS temperature-sensitive vector pHY304 (39) to give pLR5. The plasmid pLR5 was used to obtain chromosomal insertion mutations in stk1 (see below).

To obtain allelic replacement of stp1 with chloramphenicol acetyltransferase on GBS chromosomal DNA, inverse PCR was performed on pLR3 using primers iopks (5'-ACATGCATGCAATACAGGAACAGCTAACAATCC-3') and ioppr (5'-TTTTCTGCAGATGTCAGTTTCCTCCTAGAC-3'). The product lacks the stp1 gene. The cat gene was amplified from the plasmid pDC125 (49) using primers icatppf (5'-AAACTGCAGAAAAAAATATATGAACTTTAATAAAATT-3') and pcatrs (5'-CATGCATGCTAAAAGCCAGTCAATAGG-3'). Both PCR products were digested with PstI-SphI and ligated to give pLR6. The 1.7-kb insert from the MCS of pLR6 was digested and ligated to SalI-NotI-linearized pHY304 to give pLR7. Plasmid pLR7 was used to obtain allelic replacement of stp1 with cat in GBS. To construct a double mutation in both stp1 and stk1, the 2.2-kb BamHI from pCIV2 was ligated to BamHI-linearized pLR7 (containing the chloramphenicol acetyltransferase replacement of stp1), resulting in pLR8. Plasmid pLR8 was used to construct chromosomal mutations in both stp1 and stk1.

Plasmids pLR5, pLR7, and pLR8 were individually electroporated into WT GBS strain, A909 to derive mutations in stk1, stp1, and a double mutant in stp1 and stk1, respectively. Selection for double recombination events using kanamycin or chloramphenicol selection and screening for loss of plasmid replication (erythromycin sensitivity) was carried out as described previously (39). At least four independent colonies were obtained from A909/pLR5 that were stk1 mutants (Stk1-). A representative clone was designated LR113. Independent kanamycin-resistant, erythromycin-sensitive colonies were also obtained from A909/pLR8 that had mutations in both stp1 and stk1 (Stp1- Stk1-, representative clone designated LR114). Southern hybridization of genomic DNA isolated from LR113 (Stk1-) and LR114 (Stp1- Stk1-) was performed using a probe internal to stk1 and confirmed the insertion of Omega km-2 in stk1 (data not shown). Reverse transcriptase PCR confirmed that the lack of stp1 gene expression in LR114 (Stp1- Stk1-; data not shown). However, these colonies were chloramphenicol-sensitive, indicating that the endogenous promoter transcribing the cat gene was weak for chloramphenicol selection. We were therefore unable to isolate allelic exchange mutations in stp1 (A909/pLR7) using chloramphenicol selection by this method.

Construction of Complementation Constructs-- DNA fragments containing stk1 and both stp1 and stk1 were amplified from wild type A909 chromosomal DNA using high fidelity PCR. The primer pairs fpk2 (5'-CGGGGTACCAGATTTAATCACTCTAGC-3') and rppk2 (5'-CTAGTCTAGATCCTGTATTAGTTGTACT-3), and fppk2 (5'-CGGGGTACCTGTCTAGGAGGAAACTGAC-3') and rppk2 were used for amplification of stk1 and both stp1 and stk1, respectively. The PCR products was digested with XbaI-KpnI and ligated downstream to the tetracycline promoter, Ptet in the GBS complementation vector pDC123 (49). Plasmid pLR9 was derived and encodes wild type stk1 allele, and pLR10 was generated and encodes wild type stp1 and stk1alleles. The vector pDC123 was also introduced into each of the mutant strains as negative controls.

Virulence Analysis-- Time-mated, barrier-sustained, Caesarean-delivered female Sprague-Dawley rats were obtained from Charles River Laboratories. Virulence analysis was performed using a neonatal rat sepsis model of infection as described (50). GBS strains (A909, LR113, and LR114) were grown to an OD600 nm of 0.3, washed, resuspended in phosphate-buffered saline, and used as the infecting inoculum. Groups of five pups were given 10-fold dilutions of each strain by intraperitoneal injection, and the pups were checked for signs of morbidity and mortality every 8 h for 72 h. The LD50 values were determined using the methods of Reed and Meunch (51), and three independent experiments were performed.

Electron Microscopy-- Bacterial cells from log and stationary phase cultures were fixed, adhered, dried, and sputter-coated with gold on glass coverslips as described (52). They were then examined under a JSM 6300F (JEOL, Tokyo, Japan) scanning electron microscope, using an accelerating voltage of 15 kV, and photographed at 1k-5kx.

In Vitro Phosphorylation-- Preparation of cell extracts and in vitro phosphorylation were performed as described (53). The extracts were either used directly or soluble and membrane proteins were separated by ultracentrifugation at 100,000 × g for 30 min at 4 °C. In all cases, the protein concentrations were estimated using the Bradford method, and equal amounts of protein were used for each reaction. 10 µCi of [gamma -32P]ATP was added to 100 µg of cell extract, in the presence or absence of 2 µg of rStk1. The reaction was incubated at 37 °C for 15 min, and then the products were analyzed on a 10% SDS-PAGE followed by autoradiography. For complementation experiments, in vitro phosphorylation reactions were carried out as described in the absence of rStk1.

Purification and Identification of P35-- Two-dimensional gel electrophoresis on total soluble proteins from LR114 indicated that phosphorylated P35 (P35p) had a pI of 4.25 (data not shown). We therefore purified P35p from soluble fractions of LR114 using an anion exchange column, subsequent to in vitro phosphorylation in the presence of rStk1. The sample was exchanged into 30 mM Tris-HCl, pH 7.5, using a Centriprep YM-10 (Millipore Corp., Bedford, MA), and ~5 mg of total protein was injected into a Bio-logic Duoflow HPLC equipped with a UNO Q-1 anion exchange HPLC column (7 × 35 mm; Bio-Rad). The proteins were eluted in a 50-ml linear gradient of 0-0.5 M NaCl in 30 mM Tris-HCl, pH 7.5. Aliquots of all fractions were analyzed on a 10% SDS-PAGE gel, and the proteins were stained with Coomassie followed by autoradiography. Proteins from the fraction that showed an intense signal at ~35 kDa in the autoradiograph were desalted, concentrated, analyzed on an SDS-PAGE gel, and stained with Coomassie Blue. The band corresponding to P35 was precisely excised from the gel, and in-gel tryptic digestion was carried out as described (54). The tryptic peptides were analyzed by microcapillary liquid chromatography and tandem mass spectroscopy (MS), with automated switching to tandem mass spectrum (MS/MS) mode for peptide fragmentation and sequence analysis (using a Finnigan LCQDECA XP mass spectrometer with electrospray ionization and surveyor HPLC, 55). The collision-induced dissociation spectra were compared with streptococcal genome databases against all six ORFs using the SEQUEST program that matches theoretical and acquired tandem mass spectra (56).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Stk1 and Stp1

In a study to identify secreted and membrane-associated proteins of GBS, we cloned random fragments of GBS chromosomal DNA, downstream to the promoter of the Enterococcus faecalis alkaline phosphatase gene phoZ in the plasmid pAN200, as described previously (Ref. 41; also see "Experimental Procedures"). The phoZ gene in pAN200 lacks its endogenous signal sequence (41). E. coli colonies containing recombinant pAN200 plasmids with ORFs that either encode a signal sequence or hydrophobic domain enable secretion of the alkaline phosphatase and are blue on XP agar (41). Recombinant plasmids from approximately 60 independent blue colonies were sequenced, and a number of ORFs of GBS that either encoded secretion signals or transmembrane domains were identified (data not shown). Interestingly, one of these clones showed homology to prokaryotic homologues of eukaryotic-type STK. The portion of the STK gene cloned in pAN200 was ~0.53 kb in length and comprises a transmembrane domain of 23 amino acids (indicated by solid lines in Fig. 1B). A combination of arbitrary primed PCR amplification (57) and genomic DNA sequencing was used to further analyze the ORF encoding the STK and to identify other ORFs in this region (Fig. 1A). Sequence analysis revealed that the gene encoding STK (stk1) spans a region of 1.81 kb with a predicted gene product of 69.0 kDa. Stk1 does not posses a signal sequence, indicating that the protein is not secreted. The N-terminal region contains the consensus Hanks' XI domains (8), identified as the catalytic domain of eukaryotic STK. A homology sequence alignment of Stk1 with other prokaryotic and eukaryotic STK is shown in Fig. 1B. The predicted transmembrane domain of 23 amino acids (amino acids 345-368, identified in the pAN200 clone) is located at the C terminus of Stk1, which presumably localizes the Stk1 protein to the cell membrane of GBS (Fig. 1, A and B). Topology analysis predicted that Stk1, similar to other membrane-associated STK, localizes to the cytoplasmic membrane. The analysis also predicted that the N-terminal region of Stk1 is located within the cytoplasm and that the C-terminal domain is extracellular.



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Fig. 1.   A, physical map of the serine/threonine kinase and phosphatase operon in GBS. The thick arrows denote ORFs. Promoter and terminator regions are represented as P and T, respectively. The genes sunL, stp1, and stk1 encode RNA methyltransferase, PP2C phosphatase, and serine/threonine kinase, respectively. The gene hk1 encodes a histidine kinase, and the question mark indicates an ORF with no homology to the data base. TM denotes transmembrane domain, and I-XI denote Hanks' conserved domains. Primer 1 was designed toward coding regions in stp1, and primers 2 and 3 corresponds to coding regions in stk1. The lower panel shows co-transcription of stp1 and stk1 in GBS. Primer 3 was used to amplify stk1-specific cDNA from total RNA. DNA-PCR products were subsequently amplified using primer pairs 1 and 2. Lanes 1 and 3 represent co-transcription analysis of stp1 and stk1 on RNA isolated from early and mid-log phase cultures of wild type GBS, respectively. Lanes 2 and 4 represent negative controls (without reverse transcriptase) for cDNA synthesis by using the RNA samples used in lanes 1 and 3, respectively. Lane 5 represents PCR amplification using genomic DNA as the template and is the positive control. M represents the DNA size marker (1 kb). B, amino acid sequence alignment of STK using Clustal X and BoxShade. Identical residues are shaded in black, and similar residues are shaded in gray. The consensus amino acid sequence is also shown; uppercase letters represent conserved residues, and lowercase letters represent frequent residues at that position. Sag, S. agalactiae (GBS) STK; Spy, S. pyogenes STK; lmo, Listeria monocytogenes STK; Sav, Staphylococcus aureus STK; HumCDK7, Homo sapiens CDK-activating kinase; HumMARK4, H. sapiens microtubule affinity-regulating STK. Hanks' conserved domains are depicted in the N-terminal region by the domain numbers (I-XI). The transmembrane domain of the GBS Stk1 is denoted by thick lines. The region of stk1 cloned in the alkaline phosphatase fusion vector pAN200 that spans the amino acid region from 280-458 is marked by a bracket. C, amino acid sequence alignment of PP2C STP using Clustal X and BoxShade. Identical residues are shaded in black, and similar residues are shaded in gray. The consensus amino acid sequence is also shown; uppercase letters represent conserved residues, and lowercase letters represent frequent residues at that position. Sag, S. agalactiae (GBS) STP; Spy, S. pyogenes STP; lmo, L. monocytogenes STP; Sav, S. aureus STP; HumIA6Q, H. sapiens PP2C phosphatase. Conserved domains I-XI of PP2C phosphatases are depicted throughout the coding sequence.

Immediately upstream of stk1 is an ORF (stp1) that shows homology to PP2C-type STP. Sequence alignment revealed XI conserved domains in Stp1, observed in all PP2C-type protein phosphatases (58). For homology sequence alignment see Fig. 1C. The gene stp1 encodes a putative cytoplasmic protein of 26.5 kDa. There is a one-base overlap in the ORFs of stp1 and stk1, suggesting that the genes are co-transcribed. The putative promoter sequence for these genes, -10 (TATAAT) and -35 (TTGACA) were identified within the 3' end of the gene for RNA methyl transferase (sunL), upstream to stp1. Putative terminator sequences were identified downstream of stk1 (Fig. 1A).

Co-transcription of stp1 and stk1

Sequence analysis revealed a one-nucleotide overlap between the ORFs of Stp1 and Stk1; it therefore seemed likely that the genes stp1 and stk1 might be co-transcribed. To test this hypothesis, we performed reverse transcriptase PCR analysis as detailed under "Experimental Procedures." As shown in Fig. 1A, primer 3 was used to amplify stk1 specific cDNA from RNA isolated at early and mid-log phase cultures, respectively. The internal primers 1 and 2 (Fig. 1A) were subsequently used to amplify the PCR product from the template stk1 cDNA. Lanes 1 and 3 in Fig. 1A represent RNA isolated from early and mid-log phase cultures of WT GBS. A PCR product of 915 bp is observed in these lanes that is also seen in the DNA control (i.e. lane 5). Corresponding negative control reactions were also performed on RNA without reverse transcriptase (lanes 2 and 4). This confirms that stp1 and stk1 are co-transcribed and suggests that these genes might be regulated in a similar manner.

Stk1 Is a Functional Serine/Threonine Kinase

To characterize the biochemical function of Stk1, we constructed an N-terminal GST fusion to stk1. The fusion protein (rStk1) was purified using GST-Sepharose and had the expected molecular mass of 96 kDa. To determine whether Stk1, like most STK, would autophosphorylate in vitro, autophosphorylation reactions were performed in the presence of 10 µCi of [gamma -32P]ATP, and the products were separated on SDS-PAGE followed by autoradiography. A 96-kDa phosphorylated protein was observed (Fig. 2A, lane 1), indicating rStk1 was capable of autophosphorylation, in contrast to the control GST protein (Fig. 2A, lane 2).


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Fig. 2.   Stk1 has serine/threonine kinase activity. Autophosphorylation of Stk1. Autophosphorylation reactions were performed by the addition of 2 µg of rStk1 or control GST protein in the presence of [gamma -32P]ATP, respectively. The reaction was stopped after 15 min, and the products were analyzed on 10% SDS-PAGE followed by autoradiography. A, lane 1 shows autophosphorylation of rStk1, and lane 2 represents control GST protein. Positions and molecular masses (kDa) of protein standards are indicated on the left. B, serine residues are phosphorylated in Stk1. C and D, Stk1 phosphorylates casein and myelin basic protein at threonine residues. Phosphorylation of rStk1, casein, and myelin basic protein was performed as described under "Experimental Procedures." The phosphorylated proteins were hydrolyzed in 6 N HCl and subjected to two-dimensional, ascending thin layer chromatography. The open circles denote the positions of the nonradioactive phosphoamino acid standards identified by ninhydrin staining. pS, phosphoserine; pT, phosphothreonine; pY, phosphotyrosine. The plates were subsequently exposed to autoradiography. The numbers and the arrows indicate the first and second dimensions used for separation of the phosphoamino acids, respectively.

To identify the phosphorylated amino acid residues in rStk1, the radiolabeled phosphorylated protein was hydrolyzed, and amino acids were separated by two-dimensional thin layer chromatography. Fig. 2B shows that rStk1 was predominantly phosphorylated at serine residues, demonstrating that it is a serine kinase.

STK have been previously shown to phosphorylate casein or myelin basic protein at threonine residues (59). We observed that rStk1 phosphorylated both of these proteins at threonine residues (Fig. 2, C and D). Neither casein nor myelin basic protein underwent autophosphorylation in control experiments (data not shown). These results confirm that Stk1 can phosphorylate both serine and threonine residues.

Stp1 Has Phosphatase Activity and Dephosphorylates Stk1

To study whether Stp1 was a functional phosphatase enzyme, we constructed an N-terminal GST fusion to stp1. Previously characterized protein phosphatases hydrolyze pNPP in the presence of specific divalent cations (46). Hydrolysis of pNPP was measured by an increase in absorbance at 405 nm over time, and phosphatase activity of Stp1 is expressed as nmol of liberated pNP/µg protein. We observed that purified recombinant Stp1 (rStp1, 52 kDa) hydrolyzed pNPP only in the presence of Mn2+ (Fig. 3A). Hydrolysis was not observed with GST alone, confirming that Stp1 is a functional phosphatase. The concentration of Mn2+ also seemed to influence the reaction, and hydrolysis was most effective at 2 mM Mn2+ (data not shown).


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Fig. 3.   Stp1 has phosphatase activity. A, phosphatase activity of rStp1 was monitored by the hydrolysis of pNPP phosphate into pNP over time, in the presence of various divalent cations. Purified GST protein was included as a control, in similar reaction conditions. The nmol of pNP liberated/µg protein was calculated as described under "Experimental Procedures." Note that hydrolysis of pNPP was observed only in the presence of Mn2+. B, the kinetic parameters of pNPP hydrolysis by rStp1 was measured at various substrate concentrations (1, 2.5, 5, 10, and 20 mM) over time (60 min). The reaction velocity was calculated in the linear range of the reaction for each substrate concentration, and a Lineweaver-Burk plot was derived. The Km and Vmax was determined as 3.677 ± 0.014 mM and 150 ± 6.4 pmol/min/µg, respectively. C, Stp1 dephosphorylates Stk1. Phosphorylated rStk1 was incubated with rStp1 in phosphatase buffer containing Mn2+ as described under "Experimental Procedures." Aliquots of the reaction were removed at various time intervals (0-40 min), and the reaction products were analyzed on 10% SDS-PAGE followed by autoradiography.

To determine the velocity of the reaction, the substrate concentration (pNPP) was varied between 1 mM and 20 mM while keeping the enzyme concentration constant (3 µg/ml), as described under "Experimental Procedures." Phosphatase activity (nmol of pNP/min/µg) of Stp1 at each substrate concentration was determined over time, and the initial reaction velocity was calculated. A Lineweaver-Burk plot of the analysis is shown in Fig. 3B, and the Km and Vmax values were determined as 3.677 ±.0.014 mM and 150 ± 6.4 pmol/min/µg, respectively.

Because the genes encoding Stp1 and Stk1 are genetically linked, we hypothesized that Stp1 and Stk1 might function as cognate phosphatase and kinase enzymes, respectively. To test this hypothesis, we examined dephosphorylation of phosphorylated rStk1 by rStp1. Recombinant Stp1 was incubated with autophosphorylated rStk1, the aliquots were removed at various time points, and the products were analyzed on SDS-PAGE followed by autoradiography. Fig. 3B shows that rStp1 dephosphorylated rStk1 and dephosphorylation increased with time. Also, we did not detect a phosphorylation product corresponding to rStp1 (52 kDa; data not shown), indicating that Stk1 did not phosphorylate Stp1. These results demonstrate that Stk1 is a substrate for Stp1.

Mutagenesis of stk1 and stp1

To evaluate the role of Stk1 and Stp1 in GBS, we constructed isogenic mutant strains defective for expression of Stk1 (Stk1-, strain LR113) and a double mutant defective in Stp1 and Stk1 (Stp1- Stk1-, strain LR114). Recombinant plasmids complementing these mutations were also constructed and transformed into their respective mutants. The plasmid pLR9 has the wild type stk1 allele and plasmid pLR10 has both stp1 and stk1 alleles in the complementation vector pDC123 (49). Introduction of pLR9 (pStk1+) and pLR10 (pStp1+ Stk1+) into LR113 and LR114 resulted in strains LR117 (Stk1-/pStk1+) and LR120 (Stp1- Stk1-/pStp1+ Stk1+), respectively. A strain deficient in Stp1 expression was obtained by the introduction of pLR9 into LR114 resulting in LR119 (Stp1- Stk1-/pStk1+). The vector pDC123 was also electroporated into WT, LR113, and LR114 as a control resulting in strains LR115, LR116, and LR118, respectively.

Evaluation of Phenotypes of LR113 and LR114

Effects on Growth-- Growth characteristics in rich (Todd Hewitt Broth) and chemically defined media indicated that the mutants LR113 and LR114 had an extended lag phase of 60-90 min compared with WT (Fig. 4A). However, the growth and doubling time of these mutants was comparable with WT during exponential phase. The extended lag phase was not due to the presence of the Omega km-2 insertion, because a control strain (DS101) with an Omega km-2 insertion in an unknown region on the chromosome showed similar growth as WT. There were also no changes in pH or the presence of growth inhibitors in the spent media to explain the growth lag. Further, the addition of glucose to the growth medium had no effect on the growth lag, indicating that the phenotypes of the mutations are not alleviated by carbon sources.


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Fig. 4.   A, LR113 and LR114 show an extended lag phase during growth. Growth curves of WT A909, Stk1- mutant LR113, Stp1- Stk1- mutant LR114, and a control strain DS101 are shown. The cells were incubated in Todd Hewitt Broth at 37 °C in the presence of 5% CO2. Cell growth was monitored by measuring the optical density at 600 nm over a 5-h period. B, Stk1 and Stp1 are required for cell segregation. Scanning electron microscopy was performed on GBS strains grown to log and stationary phase as described under "Experimental Procedures." Microphotographs at 1000-fold magnification of derivatives of WT (LR115), Stk1- mutant (LR116), and Stp1- Stk1- mutant (LR118) show extensive chain formation. The complemented mutant strains LR117 (Stk1-/pStk1+) and LR120 (Stp1- Stk1-/pStp1+Stk1+) are restored for normal chain length. Note that LR119 (Stp1- Stk1-/pStk1+) defective in Stp1 biosynthesis, shows abnormal chaining.

Complementation of mutant strains (LR116 and LR118) restored growth similar to WT (data not shown). LR119 demonstrated an extended lag phase that was intermediate between WT and LR116 or LR118, indicating that Stp1 expression is necessary for complete restoration of normal GBS growth. These results demonstrate that Stk1 and Stp1 influence growth of GBS.

Effects on Cell Morphology-- A number of eukaryotic-type STK and a few STP mutants were shown to cause developmental abnormalities (19, 31). We performed scanning electron microscopy to assess changes in cell morphology. WT cells (A909 and LR115 in Fig. 4B) grew primarily as diplococci occasionally forming short chains of 5-8 cells as previously described (60). In contrast, both Stk1- (LR113 and the control LR116) and Stp1- Stk1- mutants (LR114 and the control LR118) formed extensive chains (50-100 cells/chain; Fig. 4B).

Mutant strains containing the complementing clones (i.e. LR117 and LR120) demonstrated normal chain length. The strain LR119 that remains Stp1- did not display normal cell segregation. These data suggest that both Stk1 and Stp1 are essential for normal cell segregation and chain length in GBS.

Effects on Virulence-- We performed standard LD50 virulence analysis of these mutants using a neonatal rat sepsis model, as described (50). Bacteria grown to log phase are used as inoculum in this model of infection, which pre-empts the bacteria from entering lag phase in vivo. Therefore, this model was valuable to assess virulence effects of the mutations, independent from their effects on lag phase growth. The LD50 of the mutant strains LR113 and LR114 compared with the isogenic WT strain A909 from three independent experiments is shown in Table I. The LD50 values of LR113 and LR114 were significantly higher ranging from 25- to 100-fold. The double mutant LR114 was slightly more attenuated than LR113. The variability observed between animal experiments is expected because of variations in both the age of the neonatal rats and the susceptibility between animal lots. We also observed that the decrease in virulence was not due to changes in known GBS virulence traits such as capsular polysaccharide, CAMP factor, and beta -hemolysin production. Collectively, these results indicate that Stk1 contributes to normal virulence of GBS. The effects of a mutation in stp1 alone on the virulence of GBS remains to be characterized. We speculate that the attenuation in virulence of these strains is perhaps a consequence of misregulation of factors crucial for GBS virulence or survival in vivo.


                              
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Table I
Stk1- strains of S. agalactiae (GBS) are attenuated for virulence
Virulence analysis was performed using a neonatal rat sepsis model of infection as described under "Experimental Procedures." GBS strain A909 represents WT, LR113 is an Stk1- strain, and LR114 is a Stp1- Stk1- strain.

Stk1 and Stp1 Are Essential for Reversible Phosphorylation of a 35-kDa Protein (P35)

Based on our observations that mutations in Stk1 and Stp1 affected normal functions of GBS and the prediction that Stk1 localizes to the cytoplasmic membrane with its N-terminal catalytic domain directed toward the cytoplasm, we hypothesized that this signal transduction pathway may regulate post-translational modification of endogenous GBS proteins. Therefore, to gain insight into the possible phosphorylation target(s) of Stk1, we performed in vitro phosphorylation reactions on cell extracts from WT, LR113 and LR114 in the presence of [gamma -32P]ATP. We hypothesized that phosphorylation substrates of Stk1 would not be phosphorylated in cell extracts of Stk1- and Stp1- Stk1- strains. Further, the addition of rStk1 protein to these cell extracts should restore phosphorylation of the target proteins. The results are shown in Fig. 5. The band at ~65 kDa corresponding to autophosphorylated Stk1 was observed in WT (Fig. 5A, lane 1) but was absent in both LR113 and LR114 (Fig. 5A, lanes 4 and 7), confirming that these mutants were defective in Stk1 biosynthesis.


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Fig. 5.   Reversible phosphorylation of a 35-kDa protein by Stk1 and Stp1. A, in vitro phosphorylation reactions were performed by the addition of 100 µg of total cell extracts from either WT, LR113 (Stk1-), or LR114 (Stp1- Stk1-) to 10 µCi of [gamma -32P]ATP in the presence and absence of rStk1 for 15 min, respectively. Recombinant Stp1 was also added subsequent to the in vitro phosphorylation reactions in the presence of rStk1, as described under "Experimental Procedures." The products of the reactions were analyzed on 10% SDS-PAGE followed by autoradiography. Lanes 2, 5, and 7 represent addition of 2 µg of rStk1, and lanes 3, 6, and 9 represent addition of 2 µg of rStp1, subsequent to addition of rStk1. Positions and molecular masses (kDa) of protein standards are indicated on the right. The position of phosphorylated rStk1, Stk1, and P35 are also indicated. B, Stp1 influences phosphorylation of P35. In vitro phosphorylation reactions were carried out by the addition of 10 µCi of [gamma -32P]ATP to 100 µg of soluble or membrane proteins isolated from WT, LR113, and LR114 in the presence and absence of rStk1 respectively. The reaction was stopped after 15 min, and the products of the reactions were analyzed on 10% SDS-PAGE followed by autoradiography. M denotes membrane, and S denotes soluble fractions. Lanes 1-4 represent soluble and membrane fractions isolated from WT, and lanes 5-8 represent soluble and membrane fractions isolated from LR113 (Stk1-). Lanes 9-12 also represent soluble and membrane proteins extracted from WT, and lanes 13-16 represent soluble and membrane proteins isolated from LR114 (Stp1- Stk1-). The reactions containing rStk1 are indicated. The positions and molecular masses (kDa) of protein standards are indicated on the left. The positions of phosphorylated rStk1, Stk1, and P35 are also indicated. Note increase in phosphorylation of P35 in the presence of rStk1 in the soluble fraction of LR114 (Stp1- Stk1-).

Upon addition of rStk1, a band at ~96 kDa corresponding to the autophosphorylation product of rStk1 was observed (Fig. 5A, lanes 2, 5, and 8), demonstrating functional activity of rStk1 in the cell extracts. A phosphorylated product of ~35 kDa was observed in the presence of rStk1 (Fig. 5A, lanes 2, 5, and 8). These results suggested that the 35-kDa protein (designated P35) was a phosphorylation substrate for Stk1.

To test whether Stp1 would dephosphorylate P35p, rStp1 was added to the extracts subsequent to the phosphorylation reactions by rStk1. We observed that rStp1 dephosphorylates Stk1, rStk1, and P35 in WT, LR113, and LR114 (Fig. 5A, compare lane 2 with lane 3, lane 5 with lane 6, and lane 8 with lane 9). Collectively, these data show that P35 is an endogenous substrate of both Stp1 and Stk1.

Localization of Phosphorylated P35 Protein

Because phosphorylation of P35 was observed only in the presence of rStk1 in the WT, we hypothesized that the presence of Stp1 in the cell extracts might function to either dephosphorylate and/or bind to P35, preventing its phosphorylation. Sequence analysis predicted Stp1 and Stk1 to be soluble and membrane-associated proteins, respectively. We therefore performed in vitro phosphorylation reactions on soluble and membrane fractions of cell extracts to distinguish between the activities of the two enzymes.

Phosphorylation profiles of WT, LR113, and LR114 are shown in Fig. 5B. The endogenous kinase Stk1 was seen primarily in the membrane fraction of the WT cells (Fig. 5B, lane 3), confirming that Stk1 is a membrane-associated kinase. The P35p protein also primarily localized to the membrane fraction in the WT (Fig. 5B, lane 3). P35p was observed in the WT even in the absence of exogenous rStk1 (Fig. 5B, lane 3), consistent with the hypothesis that P35 is an endogenous substrate of Stk1. P35p was not observed in LR113 or LR114 in the absence of rStk1 (Fig. 5B, lanes 7 and 15), confirming that Stk1 is essential for phosphorylation of P35.

The addition of rStk1 induced an increase in P35 phosphorylation (Fig. 5B, compare lanes 3 and 4). The levels of P35p and its localization in LR113 were similar to WT (Fig. 5B, lanes 1-8), indicating that phosphorylation is not required for membrane localization. In contrast to WT and LR113, P35p protein was observed in substantial amounts in the soluble fraction of LR114 (Fig. 5B, lane 14). Quantitative analysis using a PhosphorImager (Strom 840; Molecular Dynamics Inc.) revealed a ~20-fold increase in levels of P35p in the soluble fraction of LR114 relative to WT (Fig. 5B, compare lanes 14 and 10). Because Stp1 is a soluble protein phosphatase, an increase in P35p in the soluble fraction of LR114 indicates that Stp1 negatively regulates phosphorylation of P35. These data also indicate that P35 protein is present in both soluble and membrane fractions of GBS. Although we have utilized in vitro phosphorylation to identify target proteins, the altered phosphorylation profile of the double mutant (LR114) compared with LR113 and WT indicates the role of a genetic mutation in stp1 on P35 phosphorylation. These data indicate that the in vitro phosphorylation experiments provide significant evidence into phosphorylation mechanisms that occur in vivo.

Complementation Restores Phosphorylation of P35

We next examined whether complementation of the mutant strains restored Stk1-dependent P35 phosphorylation. We performed in vitro phosphorylation on the complemented mutant strains as described under "Experimental Procedures." As observed previously, the mutant strains with vector control LR116 and LR118 were deficient for Stk1 and P35 phosphorylation (Fig. 6, lanes 5-8). The complemented strains, LR120 and LR117, demonstrated phosphorylation and membrane localization of Stk1 and P35 (Fig. 6, lanes 3 and 9) similar to that of the WT (lane 1). These results suggest a link between reversible phosphorylation of P35 and phenotypic changes observed in the Stk1- and Stp1- Stk1- mutants. Although P35 phosphorylation was observed in LR119 (Fig. 6, lane 11), the strain had defects in growth and cell segregation, suggesting that regulated phosphorylation of P35 and possibly other downstream signaling events may be crucial for normal cellular functions of GBS.


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Fig. 6.   Complementation restores phosphorylation and membrane localization of Stk1 and P35. In vitro phosphorylation reactions were carried out by the addition of 10 µCi of [gamma -32P]ATP to 100 µg of soluble or membrane proteins isolated from derivatives of WT, mutant, and complemented strains, respectively. LR115 (WT/pDC123) represents WT; mutant strains are LR116 (Stk1-/pDC123) and LR118 (Stp1- Stk1-/pDC123); and complemented strains are LR117 (Stk1-/pStk1+) and LR120 (Stp1- Stk1-/pStp1+Stk1+). LR119 represents an Stp1-deficient strain (Stp1- Stk1-/pStk1+). The reaction was stopped after 15 min, and the products were analyzed on 10% SDS-PAGE followed by autoradiogaphy. M denotes membrane, and S denotes soluble fractions. The positions and molecular masses (kDa) of protein standards are indicated on the left. The positions of phosphorylated Stk1 and P35 are indicated. SDT1-STD5 indicate additional proteins that are phosphorylated in an Stk1-dependent manner.

Interestingly, we observed that five additional proteins (Fig. 6, lanes 1, 3, and 9; labeled SDT1-SDT5 for serine/threonine kinase downstream targets 1-5) are also phosphorylated in the presence of Stk1. Stk1-dependent phosphorylation of these target proteins was consistently observed in the in vitro phosphorylation experiments. It is possible that these proteins are a part of a signal transduction cascade required for Stk1-dependent P35 phosphorylation.

The P35 Protein Is a Homologue of a Manganese-dependent Inorganic Pyrophosphatase

To identify P35, the protein was purified from soluble fractions of LR114. Total soluble proteins from LR114 were phosphorylated in the presence of rStk1 and [gamma -32P]ATP. The proteins were purified using a HPLC anion exchange column as described under "Experimental Procedures." Fig. 7A (lanes 1 and 2) shows Coomassie-stained total soluble and membrane proteins from LR114, after phosphorylation by rStk1. Lane 3 shows Coomassie-stained proteins from the HPLC-purified fraction that predominantly contained P35p. The gel was subsequently subjected to autoradiography (Fig. 7A, lanes 4-6). An obvious protein band was observed in lane 3 that was phosphorylated (lane 6), and the position of the band corresponded to the size of P35p. Phosphoamino acid analysis on P35p from this fraction revealed that phosphorylation occurred at serine residues on this protein (Fig. 7B).


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Fig. 7.   A, purification of P35p by HPLC. HPLC purification of phosphorylated [gamma -32P]P35 was performed on soluble fractions of LR114 as described under "Experimental Procedures." An aliquot of the HPLC-purified fraction containing the highest concentration of phosphorylated P35 was analyzed on an SDS-PAGE along with (rStk1 phosphorylated) soluble and membrane fractions of LR114, prior to HPLC purification. The gel was stained with Coomassie followed by autoradiography. The left panel shows Coomassie-stained proteins in the molecular mass range of 55-25 kDa, and the corresponding autoradiograph is shown in the right panel. Lanes 1 and 2 represent soluble and membrane proteins of LR114. The fraction (fraction 28) containing the highest concentration of P35p protein is seen in lane 3. Lanes 4-6 represent the corresponding autoradiograph. Note that P35p is observed in lanes 4-6, with the highest intensity in lane 6. B, P35 is phosphorylated at serine residues. Phosphoamino acid analysis was performed on purified P35p. The phosphorylated protein was hydrolyzed in 6 N HCl and subjected to two-dimensional, ascending thin layer chromatography. The open circles denote the positions of the nonradioactive phosphoamino acid standards identified by ninhydrin staining. pS, phosphoserine; pT, phosphothreonine; pY, phosphotyrosine. The plate was subsequently exposed to autoradiography. The numbers and arrows indicate the first and second dimensions used for separation of the phosphoamino acids.

The band corresponding to P35p was precisely excised from an SDS-PAGE gel and digested with trypsin. The resulting peptides were analyzed using liquid chromatography and tandem MS, as described (55). The collision-induced dissociation MS/MS for each of the eight peptides (Table II, for a sample collision-induced dissociation spectral, see Fig. 8) was compared with predicted spectra from the annotated genome sequence databases of S. agalactiae (recently released at www.tigr.org) (61). The peptide sequences from MS analysis showed homology to a manganese-dependent, inorganic pyrophosphatase (designated PpaC) in S. agalactiae and also to PpaC homologues in Streptococcus pyogenes and Streptococcus gordonii (62). Phosphorylated serine residues were not present on the tryptic peptides of P35 identified by the MS/MS analysis, indicating that these modified residues are located else where on P35/PpaC.


                              
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Table II
Peptide sequences identified from the mass spectrometric analysis of purified P35
Peptide sequences, cross-correlation score (Xc), experimental and predicted ions (Ions) and MW (M+H)+ from the mass spectrometric analysis of tryptic digests of P35p are indicated. The peptide sequences identified by MS analysis of P35p are underlined in the protein sequence of PpaC. Contiguous peptides were identified at two different regions.

The amino acid sequence of PpaC (P35) is MSKILVFGHQNPDSDAIGSSVAFAYLAKEAWGLDTEAVALGTPNEETAYVLDYFGVQAPRVVESAKAEGVETVILTDHNEFQQSISDIKDVTVYGVVDHHRVANFETANPLYMRLEPVGSASSIVYRMFKENGVSVPKELAGLLLSGLISDTLLLKSPTTHASDIPVAKELAELAGVNLEEYGLEMLKAGTNLSSKTAAELIDIDAKTFELNGEAVRVAQVNTVDINDILARQEEIEVAIQEAIVTEGYSDFVLMITDIVNSNSEILALGSNMAKVEAAFEFTLENNHAFLAGAVSRKKQVVPQLTESYNA.



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Fig. 8.   Identification of P35. Tryptic digests of purified P35 protein and subsequent liquid chromatography/tandem mass spectrometric analysis were performed as described under "Experimental Procedures." A sample tandem mass spectrum derived by the collision-induced dissociation of a peptide precursor ion [(M+H)2+ = 821.54] and the peptide sequence predicted by SEQUEST are indicated. Adjacent b- and y-type ions are also indicated.

The peptide sequences and scores obtained from the MS/MS analysis are shown in Table II. The MS data covered 113 of the 311-amino acid PpaC protein. The predicted size and pI of PpaC compare well with the observed size and pI of P35p (Table III). These results indicated that P35 and PpaC are homologues of the same protein. A comparison of PpaC from S. agalactiae to homologues from other Gram-positive bacteria indicated conservation in the amino acid sequence, pI, and size of the protein (Table III). Collectively, these results suggest that Stk1 and Stp1 are required for reversible phosphorylation of a manganese-dependent inorganic pyrophosphatase (PpaC/P35).


                              
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Table III
Comparison of mass, pI, and amino acid homology of inorganic pyrophosphatases in gram-positive bacteria


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The importance of eukaryotic-type kinases and phosphatases in prokaryotic signal transduction has been steadily gaining interest since their first discovery in M. xanthus (9). Although a large number of STK and a few STP have been identified in both pathogenic and nonpathogenic bacteria (20), a significant lack of information exists on their physiological substrates and signal transduction cascades, which has limited our understanding of these systems. The results presented in this paper report two significant findings. First, we have identified and characterized a eukaryotic-type STK (Stk1) and its cognate phosphatase (Stp1) that affect normal growth, virulence, and cell segregation of the Gram-positive pathogen, S. agalactiae. Second, using in vitro phosphorylation and mass spectrometric analysis, we identified that a manganese-dependent inorganic pyrophosphatase is reversibly phosphorylated by Stk1 and Stp1.

Mutations in stk1 and stp1 caused an extended lag phase during growth in vitro. In E. coli and Saccharomyces cerevisiae, an extended lag phase was associated with the accumulation of cAMP (63, 64). However, cAMP levels were not altered in the GBS mutants (data not shown). Stk1 and Stp1 are also required for normal cell segregation and chain length of GBS. Mutations in these genes caused extensive chain formation. The chain formation was not likely due to defects in cell division because the number of colony-forming units for the mutant strains are similar to WT, for any given point in growth phase.

The cellular events involved in the regulation of chain length in chain forming cocci have yet to be elucidated. Spontaneous "opacity variants" of GBS demonstrated changes in chain length from 5 to 25 cells/chain (65). These variants were opaque in appearance, grew only on GC agar media and showed varied susceptibility to polymorphonuclear neutrophils. In contrast, Stk1- and Stp1- Stk1- mutants were not opaque, grew in rich and defined growth media, had longer chain lengths (50-100 cells/chain), and were comparable with WT in susceptibility to polymorphonuclear neutrophils (data not shown). These results suggest that the genetic basis of the opacity variants does not lie in the stp1 or stk1 genes. Mutations in a bacterial tyrosine kinase in Caulobacter crescentus and a PP2C-type phosphatase in S. cerevisiae were observed to affect cell division and cell separation (43, 66). Collectively, these observations indicate that signal transduction pathways via serine, threonine, tyrosine kinases, and phosphatases influence growth, cell division, and segregation in many organisms.

Analysis of the Stk1-deficient strains in a neonatal rat sepsis model demonstrated a significant attenuation in virulence. The LD50 values of the mutant strains were 25-100-fold higher, compared with wild type. Similar results have been reported for GBS for mutants in capsular polysaccharide expression (67) or in a screen for decreased virulence (50). Because log phase bacteria are used in this model of infection, the attenuation in virulence of these mutants is not due to their extended lag phase growth. It is possible that the attenuation in virulence may in part be due to defects in cell segregation. There were no changes in expression of known GBS virulence traits (i.e. capsular polysaccharide, CAMP factor, or beta -hemolysin) in LR113 and LR114, suggesting that Stk1 may be necessary for post-translational modification of novel factors that are crucial for GBS virulence. The double mutant LR114 (Stp1- Stk1-) showed a slight increase in attenuation of virulence compared with LR113 (Stk1-). However, because we were unable to isolate a mutation in stp1 alone, the role of Stp1 on virulence of GBS remains to be characterized. Complementation of the mutant strains was not assessed for virulence in vivo because the complementation vector is lost by at least 30% of the cells in the absence of antibiotic selection.

In vitro phosphorylation experiments revealed that Stk1 regulates phosphorylation of six GBS proteins. Using the gold standard technique of in vitro phosphorylation, we observed that P35 was the predominant substrate of Stk1 and Stp1. A significant increase in P35 phosphorylation was observed in the presence of rStk1. Likewise, a significant increase in P35 phosphorylation was also observed in the absence of Stp1 (LR114), confirming that Stk1 and Stp1 are essential for reversible phosphorylation of P35. Whether Stk1 and Stp1 directly mediate reversible phosphorylation of P35 or indirectly via the serine/threonine kinase downstream target (see "Results" and Fig. 6) proteins remains to be elucidated. Nevertheless, we observed that P35 undergoes reversible phosphorylation in an Stk1- and Stp1-dependent manner. The in vitro phosphorylation experiments demonstrate that Stp1 can dephosphorylate both Stk1 and P35, suggesting that Stp1 may be required for regulation of Stk1 and P35 in vivo. However, whether Stp1 acts upstream or downstream in the regulation of these enzymes in vivo is yet to be characterized.

Previous reports on identification of an endogenous phosphorylation target for a eukaryotic-type STK was reported for AfsK in Streptomyces (12). AfsK was shown to phosphorylate a transcriptional activator AfsR and regulate secondary metabolism in Streptomyces (68). However, AfsR phosphorylation was observed even in an AfsK mutant background, and the authors suggest that there may be redundancy in STK homologues in Streptomyces. In contrast, our results indicate that P35 phosphorylation occurs only in the presence of Stk1, and an increase in P35 protein phosphorylation is observed in the presence of an stp1 mutation, confirming that reversible phosphorylation of P35 is dependent on Stk1 and Stp1.

Mass spectrometric analysis indicated that P35 is a manganese-dependent inorganic pyrophosphatase (PpaC). PpaC belongs to a novel family of pyrophosphatases (Ppases), and homologues have been described in a number of Gram-positive bacteria (69). Ppases catalyze the hydrolysis of inorganic pyrophosphates (PPi) that are liberated during various cellular biosynthetic reactions such as carbohydrate metabolism, ATP hydrolysis, amino acid, and nucleotide biosynthesis to orthophosphate (Pi). A recent report indicated that dephosphorylation of the phosphocarrier protein Ser(P)-HPr in B. subtilis liberates pyrophosphate as the byproduct; these results suggest additional mechanisms of PPi synthesis within the cell (70). Because intracellular PPi concentrations are regulated by Ppase activity in the cell, changes in Ppase activity have been described to have global effects on cell metabolism, growth, and division of bacteria (71).

Evidence of transcriptional regulation of Ppase expression was observed in Legionella pneumophila, during intracellular infection of macrophages (72). The PpaC homologue of S. gordonii has been described to regulate expression of adhesins required for co-aggregation (62). These reports indicated the role of Ppases in virulence functions. Although transcriptional regulation of the GBS PpaC promoter has yet to be determined, our results suggest that regulation of PpaC in GBS occurs at the post-translational level. Mutations in bacterial Ppases have been previously described to be lethal (for review, see Ref. 71). Also, we did not identify other Ppase homologues from the recent genome sequence analysis of S. agalactiae (59, 73), suggesting that mutations in ppaC might be lethal to the organism. Therefore, regulation of essential gene products at the post-translational level by signal transduction systems may effectively regulate enzyme function.

P35/PpaC is the first example of a soluble bacterial pyrophosphatase that undergoes kinase-dependent phosphorylation and localizes to the membrane. Membrane localization of P35 is independent of its phosphorylation because P35 was observed in the membrane fractions of the mutant strains. Given that Stk1 is a membrane-associated kinase, we hypothesize that it may selectively modify membrane-bound P35, in vivo. Because Ppases and PPi levels regulate a wide variety of biosynthetic reactions, the distribution of Ppases to soluble or membrane fractions has been previously implied to be important in directing enzyme functions toward specific reaction pathways (71). In addition, incubation of soluble Ppases with phospholipids have been reported to convert the enzyme into the membrane-associated form that carries out the reverse reaction i.e. energy-dependent synthesis of PPi (74). It would be interesting to determine whether kinase-dependent phosphorylation of P35 catalyzes energy-dependent synthesis of PPi.

Our results indicated that Stk1 and Stp1 perform antagonistic functions on PpaC in vitro and are co-transcribed during various growth phases; therefore it might be expected that the Stk1- and Stp1-deficient strains will have opposing phenotypes. Surprisingly, we observed that these strains have similar phenotypes of altered cell growth and segregation. One possible explanation is that the phenotypes of these mutants are not solely due to misregulation of PpaC but may be either due to the additive effects of altered regulation of PpaC and other targets of these enzymes and/or misregulation of other signaling events that are yet to be characterized. In addition, the observations that PpaC localizes to both the soluble and membrane fractions of GBS and that Stk1 is a membrane-associated kinase and Stp1 is a soluble protein phosphatase increases the complexities of a simple co-relation between phenotypes of the mutants and enzymatic activity on a common target. Further characterization of PpaC and other targets of these enzymes and the role of reversible phosphorylation of these targets in vivo are under investigation and will provide more clues on the role of these eukaryotic-type enzymes in prokaryotic organisms.

In summary, our results suggest that these eukaryotic-type enzymes regulate fundamental metabolic processes that affect growth, cell segregation, and virulence of the Gram-positive pathogen, S. agalactiae. Interestingly, an increase in phosphorylation of a soluble inorganic pyrophosphatase and a mitogen-activated protein kinase was observed during the self-incompatibility response to pollen within the plant Papver rhoeas (75); these results suggest an existing link in signaling mechanisms between Ppases and kinases in eukaryotic systems as well. Also, the universal occurrence of Stk1, Stp1, and P35 homologues in pathogenic bacterial species such as Streptococci, Staphylococci, and Enterococci suggest that they may be well conserved, and further characterization of these enzymes and their targets could have widespread implications on their role in these pathogenic bacteria.

    ACKNOWLEDGEMENTS

We are grateful to Glen Tamura for suggestions throughout the course of this work and with the manuscript. We thank Donald Chaffin for assistance with the HPLC and Jonathan Buchholz for technical assistance. We are grateful to Daniel Shelver, Brandi Limbago, Christiane Beckmann, and Adam Griffith for critical review of the manuscript.

    FOOTNOTES

* This work was funded by Grant BWH 811501/NO1-AI-75326 from the Streptococcal Initiative of the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pediatrics, Div. of Infectious Disease, Mail Stop 8G-1, Children's Hospital and Regional Medical Center, 4800 Sandpoint Way NE, Seattle, WA 98105. Tel.: 206-987-2073; Fax: 206-987-3890; E-mail: craig.rubens@seattlechildrens.org.

Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M212747200

    ABBREVIATIONS

The abbreviations used are: STK, serine/threonine kinase(s); STP, serine/threonine phosphatase(s); GBS, group B streptococci; WT, wild type; ORF, open reading frame; GST, glutathione S-transferase; pNPP, p-nitrophenyl phosphate; pNP, p-nitrophenol; P35p, phosphorylated P35; HPLC, high pressure liquid chromatography; MS, mass spectroscopy; r, recombinant; Ppase, pyrophosphatase; HPr, histidine-containing protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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