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
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ABSTRACT |
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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.
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 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.
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 [ 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 [ 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
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 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 [ 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).
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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES
-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.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
-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.
km-2 fragment that
confers kanamycin resistance (48). The
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).
). 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
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.
-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.
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ABSTRACT
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RESULTS
DISCUSSION
REFERENCES
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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
[-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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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).
|
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 km-2 insertion, because a control strain
(DS101) with an
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.
|
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 -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.
|
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
[-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.
|
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.
|
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 [-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).
|
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.
|
|
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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DISCUSSION |
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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
-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.
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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.
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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.
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
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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.
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