4 salika m. shakir - journal of bacteriology · 2009. 11. 13. · shakir et al. a...

Regulatory Interactions of a Virulence-Associated Serine/Threonine 1

Phosphatase-Kinase Pair in Bacillus anthracis 2

3

Salika M. Shakir1, Katie M. Bryant

1, Jason L. Larabee

1, Elaine E. Hamm

1, Julie Lovchik

2, C. 4

Rick Lyons2

and Jimmy D. Ballard1*

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1Department of Microbiology and Immunology, The University of Oklahoma Health Sciences 8

Center, Oklahoma City, Oklahoma 73104, 2Department of Internal Medicine, University of New 9

Mexico Health Sciences Center, Albuquerque, NM, 87131 10

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Running title: A virulence-associated B. anthracis ser/thr phosphatase-kinase pair 12

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*Address Correspondences to: Jimmy D. Ballard, University of Oklahoma Health Sciences 16

Center, BRC-362A. 975 NE 10th

Street, Oklahoma City, OK 73104, Tel: 405-271-3855, 17

Fax: 405-271-3874, email: [email protected] 18

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01221-09 JB Accepts, published online ahead of print on 13 November 2009

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A virulence-associated B. anthracis ser/thr phoshatase-kinase pair

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ABSTRACT 19

In the current study we examined the regulatory interactions of a serine-threonine phosphatase 20

(BA-Stp1), serine-threonine kinase (BA-Stk1) pair in Bacillus anthracis. B. anthracis STPK101, 21

a null mutant lacking BA-Stp1 and BA-Stk1, was impaired in its ability to survive within 22

macrophages, and this correlated with an observed reduction in virulence in a mouse model of 23

pulmonary anthrax. Biochemical analyses confirmed that BA-Stp1 is a PP2C phosphatase and 24

dephosphorylates phosphoserine and phosphothreonine residues. Treatment of BA-Stk1 with 25

BA-Stp1 altered BA-Stk1 kinase activity, indicating that the enzymatic function of BA-Stk1 can 26

be influenced by BA-Stp1 dephosphorylation. Using a combination of mass-spectrometry and 27

mutagenesis approaches, three phosphorylated residues T165, S173, and S214 in BA-Stk1 were 28

identified as putative regulatory targets of BA-Stp1. Further analysis found that T165 and S173 29

were necessary for optimal substrate phosphorylation, while S214 was necessary for complete 30

ATP-hydrolysis, autophosphorylation, and substrate phosphorylation. These findings provide 31

insight into a previously undescribed Stp/Stk pair in B. anthracis. 32

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INTRODUCTION 42

A profile of the intracellular signaling proteins that regulate transition of Bacillus anthracis from 43

dormancy to expression of virulence factors is emerging. Like many prokaryotes, B. anthracis 44

utilizes two-component histidine kinase systems to regulate physiological changes and the 45

expression of virulence factors. These systems include the Spo0 histidine kinase based 46

phosphorelay pathway (32, 37) and the Bacillus respiratory response A and B involved in 47

regulating toxin expression (36). Unlike histidine kinase systems, little is known about reversible 48

serine/threonine phosphorylation events in B. anthracis. These systems are common to 49

eukaryotic cells (3, 14, 25, 40), but were only recently found in prokaryotes to modulate a variety 50

of metabolic and physiological processes (1, 2, 7, 11, 12, 15, 17, 24, 28, 35, 38). Whether 51

reversible serine/threonine phosphorylation contributes to similar events in B. anthracis is not 52

known. 53

54

The current paradigm for prokaryotic serine/threonine kinases (STK) is based in part on the 55

structure of PknB, a serine-threonine kinase from Mycobacterium tuberculosis that is structurally 56

related to eukaryotic Hanks-type kinases (39). PknB autophosphorylates and is dephosphorylated 57

by a M. tuberculosis phosphatase, PstP, in order to alter kinase activity (4). Similar to the 58

findings on PnkB, Madec et al. identified critical autophosphorylated residues and 59

autophosphorylated domains of PrkC, an STK from Bacillus subtilis (22), which suggested the 60

phosphorylation state of these residues impacts the activation of PrkC (22). These studies 61

suggested prokaryotic STKs exhibited similar activities to their eukaryotic homologs and were 62

regulated by cognate phosphatases. Hence, studies on serine-threonine phosphatase (STP)/STK 63

pairs may help define a core regulatory module in bacterial physiology and virulence, wherein 64


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the kinase autophosphorylates following interaction with stimuli and is subsequently down 65

regulated by a cognate phosphatase when stimuli levels decline. 66

67

Analysis of the B. anthracis genome indicates this organism has a single phosphatase/kinase pair 68

encoded within a putative operon. This operon, between nucleotides 3588319 and 3678099 in the 69

genome of B. anthracis Sterne, contains eight candidate open reading frames (ORF). Six of the 70

potential ORFs encode proteins involved in translation and DNA metabolism, while the 71

phosphatase-encoding ORF (stp1) and the kinase-encoding ORF (stk1) are paired at the 3’ end of 72

this operon. Examination of the genome sequences of several other Gram-positive bacteria 73

indicates this putative operon and the general orientation of stp1 and stk1 are conserved among 74

members of the Firmicutes group of bacteria. BA-Stp1 and BA-Stk1 homologs influence a 75

variety of bacterial processes. For example, homologs of BA-Stp1 and BA-Stk1 regulate growth 76

in Bacillus subtilis (12), cell viability and segregation in Streptococcus agalactiae (28), 77

competence in Streptococcus pneumoniae (26), and virulence in both Streptococcus pyogenes 78

(17) and Staphylococcus aureus (9). Although kinases homologous to BA-Stk1 influence several 79

bacterial processes in different species, the tandem association of this kinase with a phosphatase 80

does not vary. This observation led us to hypothesize that the phosphatase (BA-Stp1) influences 81

Ba-Stk1 activity by dephosphorylation. 82

83

In the current study we analyzed the importance of BA-Stp1 and BA-Stk1 in the virulence of B. 84

anthracis and assessed the biochemical interactions between these two proteins. Results from 85

these studies indicate this phosphatase/kinase pair contributes to the virulence of B. anthracis, as 86

mutants lacking BA-Stp1 and BA-Stk1 exhibit decreased lethality in a mouse model of 87


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inhalational anthrax. Furthermore, a series of biochemical analyses reveal an interaction between 88

BA-Stk1 and BA-Stp1 where BA-Stk1 autophosphorylates in order to enhance kinase activity 89

and is dephosphorylated by BA-Stp1 as a putative step in down-regulating kinase activity as 90

levels of stimuli subsides. Moreover, we have identified candidate serine and threonine residues 91

that appear to modulate kinase activity. These findings provide insight into a previously 92

undescribed serine-threonine phosphatase/kinase system in B. anthracis. 93

94

MATERIALS AND METHODS 95

Bacterial strains, cell lines, and growth conditions. B. anthracis Sterne strain 7702, (as 96

described by Cataldi et al.(6)) and Escherichia coli were grown in Brain Heart Infusion Broth 97

(BHI) and Luria-Bertani (LB) medium respectively at 37°C. 98

99

Abelson murine leukemia virus-transformed murine macrophages derived from ascites of 100

BALB/c mice (termed RAW 264.7) were obtained from the American Type Culture Collection 101

(ATCC, Manassas, VI). The RAW 264.7 cell line was maintained in tissue culture-grade T-75 102

flasks grown in the presence of Dulbecco’s Modified Essential Medium (DMEM) (ATCC) 103

supplemented with 10% Fetal Bovine Serum (FBS) (ATCC) at 37°C with 5% CO2 in a 104

humidified incubator. Cells were used between passages 10 and 20 for the infection experiments. 105

106

Genetic construction of a ∆ba-stp1/∆ba-stk1 double mutant in B. anthracis Sterne. Allelic 107

replacement was used to construct a double mutant lacking expression of BA-Stp1 and BA-Stk1. 108

Briefly, a 3.75 kb fragment comprising of 1,500 bp flanking regions sharing sequence similarity 109

to the upstream and downstream region of the ba-stp1 gene was PCR amplified from B. 110


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anthracis Sterne 7702 genomic DNA using forward primer, 5’-CC 111

GGAATTCGGCATTAGATGCTAGAAAAGTGC-3’ and reverse primer, 5’-112

CCGGAATTCGTCTAATTTGATAAGGGCCTTTAC-3’ incorporating EcoRI restriction sites. 113

The PCR product was subsequently ligated to pGEM-T-easy vector (Promega) using TA 114

overhangs to generate the plasmid construct pGEM-PK101 that was transformed into E. coli 115

JM109. Transformed clones were screened for ampicillin resistance and DNA sequencing of the 116

isolated plasmid confirmed clones positive for insert. Inverse PCR was then performed using 117

pGEM-PK101 as a template with right primer, 118

5’-CGCGGATCCAGTGCAACGTGCTGATTGGAAAAC-3’ and left primer, 5’-119

CGCGGATCCCTCGTCACCTCGTCTCTTCTTTGC-3’ that amplify a gene sequence inverse 120

to ba-stp1 incorporating BamHI restriction sites. The inverse PCR product comprising the 121

homologous flanking regions of ba-stp1 (inserted in pGEM-T-easy) was then ligated to BamHI 122

digested apha-3 gene (Ω-km-2 cassette) isolated from pUTE618 (31) conferring kanamycin 123

resistance. The resulting clone was then digested with EcoRI to release ∆ba-stp1-kanr which 124

was then sub-cloned into pUTE568 (31), a generous gift from Dr. Theresa Koehler, producing 125

pPK101. E. coli strain JM110 (∆dam ∆dcm) was then transformed with pPK101 to obtain 126

unmethylated DNA that was subsequently electroporated into B. anthracis Sterne 7702. 127

Transformants were selected on solid LB-agar plates containing kanamycin (100 µg/ml) and 128

erythromycin (5 µg/ml). To select for the double-recombination event, isolated clones were 129

serially passaged in antibiotic-free medium and clones exhibiting a Kanr

Erms phenotype were 130

identified. The mutant STKP101 was confirmed by PCR and maintained in medium containing 131

kanamycin (100 µg/ml). 132

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Spore preparation and infection experiments. Vegetative bacilli of B. anthracis Sterne 7702 134

parent strain and B. anthracis STPK101 mutant strain (grown in the presence of kanamycin (100 135

µg/ml) were induced to sporulate by growth in sporulation medium (PA broth) containing: 0.6 136

mM CaCl2, 2H2O; 0.8 mM MgSO4, 7H2O; 0.3 mM MnSO4, H2O; 85.5 mM NaCl and 8 g/L 137

Nutrient Broth (pH 6.0) as described by McKevitt et al. (23). Spore suspensions were diluted to 138

working concentrations necessary for the corresponding experiment, and heated to 70°C for 30 139

min to kill any residual vegetative cells as well as to heat-activate the spores. RAW 264.7 140

macrophages were seeded into 24-well tissue culture-grade plates at a concentration of 7.5 x 105 141

cells/well in DMEM-10% FBS and incubated overnight at 37ºC in 5% CO2. Macrophages were 142

then infected with spores of B. anthracis Sterne 7702 parent or B. anthracis STPK101 mutant 143

strain at a multiplicity of infection (MOI) of 10. Subsequently, the plates were centrifuged at 250 144

x g for 10 min. The plates were then incubated at 37ºC, 5.0% CO2, for 30 min after which 145

gentamicin (10 µg/ml) was added to kill extracellular vegetative bacilli for an additional 30 min. 146

Next, the infected macrophages were washed ten times with DMEM-10% FBS and then 147

incubated for up to 10 hours. At two-hour intervals, the culture media was removed and the 148

macrophages were scraped from the wells with PBS, briefly vortexed and subsequently serially 149

diluted and plated on solid media. The total number of colony forming units (CFU) were 150

enumerated following an overnight incubation. The average and standard deviation were 151

calculated from two independent experiments performed in triplicate. 152

153

In vivo analysis of B. anthracis STPK101 was performed using a mouse model of pulmonary 154

anthrax similar to that previously described (20, 23). Briefly, female DBA/2 mice (housed in 155

accordance with approved IACUC guidelines, University of New Mexico Health Sciences 156


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Center) were anesthetized and the trachea was exposed by a small incision under the skin. 157

Spores were injected in a 50 ml volume using a bent 30-guage needle. To determine the number 158

of spores deposited per group, 2 mice were sacrificed and the total CFU was determined from 159

homogenized lung tissue. The animals were monitored twice daily for survival and clinical 160

signs. 161

162

Monitoring spore germination and sporulation efficiency. Spore germination was determined 163

using the protocol described by McKevitt et al. (23). Briefly, spore preparations (3x107) of B. 164

anthracis Sterne 7702 parent and B. anthracis STPK101 mutant strains were heat activated (20 165

min at 65°C) and resuspended in 200 µl of medium (PBS, L-alanine, inosine, or 25% BHI) in a 166

well of a 100 well Honeycomb 2 (Thermo Electron Corp) microplate. Germination was 167

monitored at 37°C by loss of optical density at 600 nm caused by changes in the refractivity of 168

the spore. Germination was analyzed as percent decrease in optical density measured every 3 169

minutes for 30-60 min under shaking conditions. Experiments were carried out in triplicate and 170

mean absorbance was determined. 171

172

To analyze sporulation, B. anthracis Sterne parent and B. anthracis STPK101 mutant vegetative 173

cells were cultured in sporulation medium containing: 0.6mM CaCl2⋅2H2O, 0.8mM 174

MgSO4⋅7H2O, 0.3mM MnSO4⋅H2O, 85.5mM NaCl and 8 g/L Nutrient Broth (pH 6.0) for 3 days 175

at 30°C. The samples were heated at 70°C for 15 min to kill vegetative bacilli and he number of 176

heat resistant CFU were determined by serial dilution and plating. The percentage of heat 177

resistant CFU from the total CFU was calculated to determine the sporulation efficiency. 178

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Construction of His-tagged BA-Stp1 and His-tagged BA-Stk1cat. Genomic DNA was isolated 180

from an overnight culture of B. anthracis Sterne 7702 in BHI. To construct His-BA-Stp1, a 753-181

bp fragment was PCR amplified with forward primer 5'-182

GGAATTCCATATGGAAGGAGCGCAAAGAAGAGAC-3' and reverse primer 5'-183

GGCGCGGATCCGCTTATAACGGTCATTTAAGA-3' from isolated genomic DNA using 184

High Fidelity Polymerase (Roche). Similarly, the putative catalytic domain of BA-Stk1cat was 185

PCR amplified from genomic DNA using the following oligonucleotides; 5’-186

GGGATTCATATGGTGCTGATTGGAAAACGCTTAAATG-3’ 187

and 5’-CGGATCCTTGTCGCTTCCATATCTTCCGG-3’. The primers were designed to 188

incorporate NdeI and BamHI restriction enzyme sites respectively. The PCR product was cleaned 189

with QIAquick PCR Purification Kit (Qiagen) and subsequently digested with NdeI and BamHI 190

(New England Biolabs) overnight at 37°C. The digested PCR products were then ligated using 191

T4 DNA ligase (Roche) into the expression vector, pET-15b, and transformed into E. coli XL1-192

Blue to obtain the recombinant plasmids, pET-15b-BA-Stp1 and pET-15b-BA-Stk1cat. DNA 193

sequence analysis of the BA-Stp1 insert at the Laboratory for Genomics & Bioinformatics 194

Sequencing Core (OUHSC), demonstrated that it was identical to the locus BAS3714 of B. 195

anthracis Sterne 7702. 196

197

Expression and purification of His-tagged BA-Stp1 and His-tagged BA-Stk1cat. The 198

recombinant plasmids pET-15b-BA-Stp1 and pET-15b-BA-Stk1cat were transformed into E. coli 199

BL21 (DE3) cells (Novagen) and transformed clones were selected on LB agar plates containing 200

ampicillin (50 µg/ml) (American Bioanalytical). Single colonies were grown in cultures to an 201

OD600=0.7 before inducing for 4 hours at 37°C with 1mM Isopropyl β-D-1-202


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thiogalactopyranoside (Denville Scientific). Cells were then harvested by centrifugation at 9000x 203

g for 20 min and the pellets were resuspended in 20 mM Tris-HCl, 500 mM NaCl, and 10 mM 204

imidazole pH 7.9 and lysed using a cell disruptor. Recombinant BA-Stp1 and BA-Stk1cat were 205

purified using the His•Bind kit (Novagen) according to the manufacturer’s instructions and 206

desalted in 20 mM Tris-HCl using PD-10 columns (GE Healthcare). The purified proteins were 207

visualized on a 12.5% SDS-PAGE stained with Coomassie Brilliant Blue. When necessary, the 208

His-tag of purified BA-Stp1 was cleaved using thrombin (Novagen) following manufacturer’s 209

instructions. 210

211

In vitro phosphatase assays using p-NPP and phosphopeptides. All reactions were performed 212

in 50 µl reaction buffer containing 50 mM Tris-HCl, pH 8.0, 5 mM MnCl2, 5 mM MgCl2, 0.5 213

mM DTT (Invitrogen). Single tablets of p-NPP (Sigma) were dissolved in 5 ml of reaction 214

buffer immediately before use to give a working stock of 10 mM. Fifty microliters of this 215

working stock was added to each well in triplicate, and the reaction was initiated by adding 3 µg 216

of purified BA-Stp1 to the appropriate wells. Absorbance at 405 nm was measured every 5 min 217

for 90 min in a 96-well plate reader at 37°C. 218

219

Dephosphorylation of phosphopeptides was examined using a malachite green/molybdate 220

phosphatase detection system (10). Briefly, 5 mM phosphopeptides, RRApTVA (Promega), 221

RRApSVA and RRLIEDAEpYAARG (Upstate) were added to the reaction buffer 222

containing 50 mM Tris-HCl pH 8.0, 5mM MnCl2, and 0.02% (v/v) β-mercaptoethanol. To 223

initiate the reaction, BA-Stp1 was added to the reaction mixture across a range of concentrations 224

from 0-600 mM, in a total final volume of 50 µl. The reactions were incubated at 37°C for 15 225


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min. Subsequently, 50 µl of the molybdate dye/Additive mixture (Promega) was added to stop 226

the reaction and the plate was incubated for 15 min at room temperature to allow for color 227

development. Absorbance of the malachite green-molybdate complex was measured at 600 nm 228

and the amount of free phosphate released was determined using a standard curve. For enzymatic 229

analysis, 1.3 µM BA-Stp1 and phosphopeptides concentrations ranging from 5-400 µM were 230

used to determine the initial reaction rates. The Km and Vmax values were calculated by using non-231

linear regression analysis (Prism software). 232

233

Oligonucleotide site-directed mutagenesis. Mutagenesis of aspartic acid residues was 234

performed using QuikChange site-directed mutagenesis kit (Stratagene) according to 235

manufacturer’s instructions. Briefly, 50 ng of pET-15b-BA-Stp1 was used as a template. Point 236

mutations were inserted using synthetic oligonucleotide pairs as follows, D18A forward primer 237

5'-AAAAGTTCGTCAACATAATGAAGCTAGTGCAGGAGTTTTTCACAAT-3', reverse 238

primer 5'-AATTGTGAAAAACTCCTGCACTAGCTTCATTATGTTGACGAACTTTT-3', 239

D36A forward primer 5'-TTTAGCGGTAGTAGCAGCTGGAATGGGAGGTCATC- 3', reverse 240

primer 5'-GATGACCTCCCATTCCAGCTGCTACTACCGCTAAA-3' D194A forward primer 241

5'-GATCAGCTACTTCTTTGCTCTGCTGGATTATCTAACAAAGTTTCT-3', reverse primer 242

5'-AGAAACTTTGTTAGATAATCCAGCAGAGCAAAGAAGTAGCTGATC-3', D233A 243

forward primer 5'-TGATCGTGGCGGGGAAGCTAATATCACCCTTGTTC-3', reverse primer 244

5'-GAACAAGGGTGATATTGACTTCCCCGCCACGATCA-3'. Mutations were confirmed by 245

DNA sequencing. 246

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Dephosphorylation of BA-Stk1cat. Phosphate release was measured by malachite 248

green/molybdate reactivity after dephosphorylation of recombinant BA-Stk1cat by BA-Stp1 wild 249

type or aspartic acid mutants. Enzymatic analysis of BA-Stp1 with BA-Stk1cat as substrate was 250

performed. Briefly, BA-Stk1cat concentrations ranging from 2-50 µM were used to determine the 251

initial reaction rates. The Km and Vmax values were calculated by using non-linear regression 252

analysis (Prism software). kcat (s-1

) was calculated using the formula Vmax/Et. The catalytic 253

efficiency of the reaction (s-1

mM-1

) was determined by the formula kcat / Km . 254

255

Western blot analysis of dephosphorylation of BA-Stk1cat. Dephosphorylation reactions of 2 256

µM BA-Stk1cat were initiated by the addition of equimolar amounts (5 µM) of either purified 257

BA-Stp1, D18A mutant, or a control of shrimp alkaline phosphatase (Roche) diluted in reaction 258

buffer. Reactions were incubated at 37°C and stopped at 5, 15, and 30 min with the addition of 259

4X SDS-Laemmli buffer, and boiled for 5 min. Subsequently, 10 µl of the reaction products 260

were separated on a 10% SDS-PAGE and transferred to a PVDF membrane, which was probed 261

with 1:1000 concentration of Phospho-(Ser) 14-3-3 Binding Motif Antibody (Cell Signaling). 262

The blot was then washed with TBS, 0.1% Tween-20 five times for 5 min each and incubated 263

with goat anti-rabbit IgG (H+L)-horse radish peroxidase (HRP) conjugate antibody (Bio-Rad) 264

for 1 h on a shaker at room temperature. The bands were visualized using enhanced 265

chemiluminescence (Amersham) according to manufacturer’s instructions followed by exposure 266

to film. 267

268

Dephosphorylation of autophosphorylated BA-Stk1cat. Autophosphorylation was performed 269

by addition of 1 µCi of [γ-32P] adenosine triphosphate (ATP) (Perkin Elmer) to 10 µg of purified 270


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BA-Stk1cat in 100 µl kinase reaction buffer (4 mM MOPS, pH 7.2, 5 mM MnCl2, 0.05 mM DTT 271

and 10 µM ATP). The reaction was incubated at 37°C and unincorporated [γ-32P] ATP was 272

removed using a G-50 sephadex spin column (Amersham). One microgram of phosphorylated 273

BA-Stk1cat was incubated with 3 µg of purified BA-Stp1 in phosphatase reaction buffer 274

containing 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 0.5 mM DTT. Reactions were stopped at 0, 275

30, 60, and 90 min using 4X SDS-Laemmli buffer and boiled for 5 min. Samples were resolved 276

by SDS-PAGE, and the dried gel was exposed and visualized by autoradiography. To test BA-277

Stk1 kinase activity after dephosphorylation with BA-Stp1, ATP and [γ-32P] ATP were replaced 278

by thio-γATP and [35

S]ATP-γS. 279

280

Measurement of BA-Stk1cat kinase activity in vitro. Kinase activity of 1.25 µM BA-Stk1cat 281

wild type, alanine mutants (T165A, S173A, S214A, T290A), or aspartic acid mutants (T165D, 282

S173D, S214D, T290D) was assayed in reaction buffer (4 mM MOPS, pH 7.2, 5 mM MnCl2, 283

0.05 mM DTT) with 10 µM ATP (Cell Signaling Technology). After incubating for 90 min at 284

37°C, kinase activity was assayed using the Kinase-Glo Luminescent Kinase Assay Platform 285

(Promega) and the luminescent signal was measured in a Victor 3 (Perkin Elmer) plate reader. 286

The luminescent signal inversely correlates to the amount of kinase activity. The percent relative 287

activity of BA-Stk1cat site-directed mutants was calculated as an inverse of units of 288

luminescence, compared to the wild type BA-Stk1cat. All reactions were performed in triplicate. 289

290

Measurement of myelin basic protein (MBP) phosphorylation by BA-Stk1cat in vitro. To 291

analyze the effect of alanine or aspartic acid substitutions on the kinase activity of BA-Stk1cat, 2 292

µM of purified wild type BA-Stk1cat or site-directed mutant protein was incubated with 20 µM 293


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myelin Basic protein (Upstate) in kinase buffer containing 1 µCi of [γ-32P] ATP. The reactions 294

were incubated for 30 min at 37°C and stopped using 4X SDS-Laemmli buffer and boiled for 5 295

min. Samples were resolved by SDS-PAGE, the dried gel was exposed and visualized by 296

autoradiography. The level of 32

P incorporation was determined by phosphorimage analysis. 297

298

Mass spectrometric analysis of BA-Stk1cat. Mass spectrometry analysis was performed in the 299

Molecular Biology Proteomics Facility (University of Oklahoma Health Sciences Center). 300

Following SDS-PAGE and staining of autophosphorylated and dephosphorylated BA-Stk1cat, 301

protein bands were excised and then digested with trypsin within the gel utilizing the Pierce in-302

gel tryptic digestion kit and then extracted from the 1.0 % trifluoroacetic acid/water and 303

combined with the digestion solution. The samples were subjected to LC/MS and LC/MS/MS on 304

a Dionex Ultimate 3000 nanoscale HPLC connected to an Applied Biosystems QSTAR Elite 305

mass spectrometer operated in the positive ion mode at a voltage of 2.3 KV. The peptide 306

separation was performed on a PepMap C18 column (75 micron ID x 150 mm length, packed 307

with 3 micron particles) at a flow rate of 250 nanoliters/min. For LC/MS/MS, data was collected 308

in the Information Dependent Acquisition mode over a mass range of 300-2000 m/z. Peptides 309

with a +2 to +5 charge status were selected for MS/MS fragmentation. A survey scan in the MS 310

mode was performed, followed by MS/MS fragmentations of the three most abundant ions in the 311

mass range of 300-1400 m/z for five seconds for each ion selected. The MS/MS data was 312

collected over a mass range of 50 to 2400 m/z. The MS data file was used to search the NCBI 313

non-redundant protein database (release 02/16/2007) utilizing the MASCOT search engine 314

(software version 2.2), both of which are installed on an on-site MASCOT server. The search 315

parameters consisted of the database search of all Eubacteria, Firmicutes with trypsin selected as 316


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the enzyme, allowing one missed cleavage. Fixed modifications were set to 317

carboxamidomethylation of cysteine residues, while oxidation of methionine, deamidation of 318

Asn/Gln and phosphorylation of serine and threonine were chosen for variable modifications. 319

Protein identifications were made based upon MASCOT Mowse scores greater than the 320

significance threshold value at p < 0.05. The elution point and relative ionization intensity of 321

phosphorylated peptides and their non-phosphorylated versions were determined by LC/MS 322

analysis over a mass range of 300- 2000 m/z. The differentially phosphorylated versions were 323

observed by plotting the extracted-ion current for each peptide utilizing a 1.0 amu window 324

spanning the three most abundant isotopic forms of +3 charged peptide. 325

326

RESULTS 327

Identification and characterization of a serine/threonine phosphatase-kinase pair in B. 328

anthracis. A search of the B. anthracis Sterne genome revealed putative orthologs of eukaryotic 329

STKs and phosphatases. In all, four putative STKs and six putative serine/threonine 330

phosphatases (STPs) were identified. Of particular interest to our studies was a pair of ORFs 331

juxtaposed at the 3’ end of a putative operon, as this was the only closely related STP/STK pair 332

identified in B. anthracis. The STP (termed BA-Stp1) and STK (termed BA-Stk1) are encoded 333

by BAS3714 and BAS3713 (GenBank accession numbers AAT56015 and AAT56016) 334

respectively. No other closely associated STP/STK pair was identified within the genome of B. 335

anthracis. 336

337

To characterize BA-Stp1/BA-Stk1 in B. anthracis Sterne, a double mutant (∆stp1/∆stk1) was 338

constructed by allelic replacement using a Ω kanr cassette consisting of the apha3 gene 339


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conferring kanamycin resistance and the mutant was termed B. anthracis STPK101. In vitro 340

analysis of B. anthracis STPK101 found that the mutant grew in liquid medium, sporulated, and 341

germinated with efficiency similar to the parent strain (data not shown). Thus, we next 342

determined whether the virulence of B. anthracis STPK101 was similar to parent strain. 343

344

Macrophages are considered the primary site of B. anthracis spore germination following 345

infection (13), hence B. anthracis STPK101 was assessed for growth and survival in these cells. 346

When RAW 264.7 macrophage-like cells were infected with spores of the parent B. anthracis 347

Sterne strain or B. anthracis STPK101, the total number of colony forming units of B. anthracis 348

STPK101 recovered at 10 h post-infection were significantly reduced compared to the parent 349

strain (Fig. 1A). A comparison between STPK101 and the parent strain found no differences in 350

the rate of growth in tissue culture medium alone (data not shown), suggesting that the observed 351

defect was due to attenuated germination, growth, or survival of the mutant within the 352

macrophage. 353

354

Next, to assess the importance of the phosphatase-kinase pair in virulence of B. anthracis, the 355

50% lethal dose (LD50) of the parent B. anthracis Sterne strain and B. anthracis STPK101 was 356

compared using a mouse model of pulmonary anthrax. As shown in Fig. 1B and Fig. 1C, the 357

LD50 of B. anthracis STPK101 (4.5 x 105

spores) was 10-fold higher than the LD50 of the B. 358

anthracis parent strain (4.5 x 104 spores). Collectively, these data indicate that the phosphatase-359

kinase pair contributes to the survival of B. anthracis during infection of cultured macrophages 360

and following infection in a mouse model of pulmonary anthrax. 361

362


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Characterization of BA-Stp1. Because BA-Stp1 and BA-Stk1 appeared to be important in B. 363

anthracis virulence, we began to analyze these proteins in more detail. We predicted that BA-364

Stp1 could dephosphorylate BA-Stk1, which made it critical to first establish that BA-Stp1 is 365

indeed a phosphatase. Experiments using p-nitrophenyl phosphate (p-NPP) as synthetic 366

substrate found that BA-Stp1 exhibits phosphatase activity and requires divalent metal ions, 367

Mn2+

or Mg2+

, to hydrolyze p-NPP (data not shown). Such a dependence on divalent cations 368

suggested that BA-Stp1 could be a PP2C family phosphatase (1, 3, 5). This was confirmed by 369

further biochemical analysis which revealed that BA-Stp1 is inactivated by non-specific 370

inhibitors such as sodium pyrophosphate, but is unaffected by specific PPP and PP2B 371

phosphatase inhibitors such as okadaic acid and calyculin A (data not shown). 372

373

Next, we performed experiments designed to identify the types of residues dephosphorylated by 374

BA-Stp1. Synthetic phosphopeptides with phosphate groups at threonine (RRApTVA), serine 375

(RRApSVA), or tyrosine (RRLIEDAEpYAARG) residues were used as substrates in a 376

malachite-green phosphate detection assay. Phosphate release increased when phosphothreonine 377

or phosphoserine peptides were used as substrates, but not with the phosphotyrosine peptide 378

(Fig. 2A). Using these synthetic substrates, we also determined the kinetic parameters of the 379

dephosphorylation reaction. The Vmax and Km values for the phosphothreonine peptide were 380

calculated to be 115.3 pmole min-1

µg-1

and 121.5 µM respectively. Similarly, the Vmax and Km 381

values for the phosphoserine peptide were found to be 172.7 pmole min-1

µg-1

and 248.6 µM 382

respectively. The Km value of BA-Stp1 for the phosphotyrosine peptide was above the range for 383

accurate determination (data not shown), which is consistent with low levels of Pi detected in the 384

phosphate release assay. 385


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386

Examination of BA-Stp1 identified residues (D18, D36, D194, and D233) which corresponded to 387

metal-complexed sites in human PP2Cα, leading us to predict that BA-Stp1 might share a 388

common enzymatic mechanism with human PP2Cα (8, 16). To address this possibility, single 389

aspartic acid mutants D18A, D36A, D194A, and D233A were generated in BA-Stp1 and each 390

mutant was assayed for phosphatase activity using RRApSVA or RRApTVA as substrates. 391

As shown in Fig. 2B, substituting alanine for these critical aspartic acid residues eliminated 392

phosphatase activity of BA-Stp1, supporting our prediction that BA-Stp1 is similar 393

mechanistically to the human PP2Cα. 394

395

BA-Stk1 is a substrate of BA-Stp1. Having confirmed that BA-Stp1 is a phosphatase we next 396

sought to determine the interaction of this phosphatase with BA-Stk1. To test whether BA-Stp1 397

dephosphorylates phospho-residues on BA-Stk1, the catalytic domain of BA-Stk1, termed BA-398

Stk1cat was cloned, expressed, and purified from E. coli. Western blot analysis with antibodies to 399

phosphoserine and phosphothreonine residues demonstrated that BA-Stk1cat was 400

autophosphorylated (data not shown), which is similar to previous findings on B. subtilis PrkC 401

(21, 22). Phosphorylated BA-Stk1cat was then used as a substrate for BA-Stp1. As shown in Fig. 402

3A, when BA-Stk1cat was added to the reaction there was an increase in detectable levels of 403

phosphate relative to the negative control, suggesting that BA-Stp1 hydrolyzes the phosphate 404

groups on BA-Stk1cat. Kinetic analysis of dephosphorylation of BA-Stk1cat suggested that BA-405

Stp1 dephosphorylates phospho-residues on BA-Stk1cat with a high affinity (Km 9.65 µM) and a 406

catalytic efficiency, kcat/Km of 113.9 s-1

mM-1

. Moreover, phosphate release was significantly 407


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reduced when each of the aspartic acid mutants, D18A, D36A, D194A, or D233A was 408

substituted for wild type BA-Stp1 in the reaction (Fig. 3B). 409

410

BA-Stp1 inactivates BA-Stk1. Because BA-Stk1cat was isolated from E. coli in a recombinant 411

form, it was necessary to show that the residues dephosphorylated by BA-Stp1 arose from 412

autophosphorylation and not through cross-phosphorylation by E. coli kinases during isolation of 413

the protein. To accomplish this we incubated BA-Stk1cat with [γ-32P] ATP thereby allowing the 414

predicted autophosphorylation to occur. The 32

P labeled BA-Stk1cat was then incubated with BA-415

Stp1, or buffer control, and the level of incorporated 32

P was determined by autoradiography. As 416

shown in Fig. 3C, when labeled BA-Stk1cat was incubated with BA-Stp1 there was a precipitous 417

decrease in the level of detectable phosphorylated kinase. Within 30 min following the 418

incubation start there was a greater than 90% reduction in the level of detectable phosphorylated 419

kinase indicating that BA-Stp1 dephosphorylates autophosphorylated BA-Stk1. 420

421

To confirm our prediction that BA-Stk1cat serine residues were dephosphorylated by BA-Stp1, 422

we examined the dephosphorylated kinase using anti-phosphoserine antibodies (Fig. 4A). As 423

shown in the immunoblot, upon incubation with shrimp alkaline phosphatase, there was no 424

detectable decrease in the phosphoserine signal. However, when BA-Stp1 was added to BA-425

Stk1cat, a precipitous reduction in levels of phosphoserine was observed. Prolonged incubation 426

(30 min) resulted in hydrolysis of the phosphate group from serine residues on BA-Stk1cat to 427

levels that were no longer detectable by the anti-phosphoserine antibodies. Finally, when a 428

similar analysis was performed using an inactive phosphatase mutant, BA-Stp1D18A there was no 429


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detectable change in the level of phosphorylated BA-Stk1cat; further suggesting this reaction 430

requires catalytically active phosphatase. 431

432

Previous work by Madec et al. demonstrated that autophosphorylation is critical to the kinase 433

activity of B. subtilis PrkC (22), suggesting that BA-Stp1-mediated dephosphorylation could 434

alter the kinase activity of BA-Stk1. Hence, we next analyzed the impact of BA-Stp1 435

dephosphorylation on the kinase activity of BA-Stk1. To accomplish this, his-tagged BA-Stk1cat 436

was incubated with recombinant BA-Stp1, or BA-Stp1D36A, both of which had the his-tags 437

removed. This approach was taken in order to separate BA-Stk1cat and BA-Stp1 prior to 438

performing the kinase assay. After separating the two proteins using nickel-affinity 439

chromatography, BA-Stk1cat was incubated with [γ-32P] ATP and levels of autophosphorylated 440

kinase were determined across a time-course. As shown in Fig. 4B, pretreatment of the kinase 441

with BA-Stp1 resulted in a reduction in the amount of 32

P incorporated during 442

autophosphorylation. In contrast, pretreatment of BA-Stk1cat with the inactive phosphatase 443

mutant, BA-Stp1D36A, did not alter levels of kinase activity. 444

Because it was difficult to absolutely exclude the effects of trace amounts of phosphatase in 445

these experiments a second experimental approach was utilized to confirm that 446

dephosphorylation reduced the kinase activity of BA-Stk1. After pretreatment of BA-Stk1 with 447

BA-Stp1, BA-Stk1 autophosphorylation and kinase activity was measured using thio-γATP 448

instead of ATP. The rational for this approach was that thiophosphorylated substrates are 449

resistant to dephosphorylation by protein phosphatases (4, 19). BA-Stk1 was pre-incubated with 450

BA-Stp1 for 30 min and the kinase activity was then measured in a time course using [35

S] ATP-451

γS and myelin basic protein as substrates. As shown in Fig. 4C, pretreatment of BA-Stk1 with 452


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BA-Stp1 reduced its autophosphorylation and kinase activity towards myelin basic protein. 453

Collectively, these data suggest that phosphorylation of BA-Stk1cat renders it active and that BA-454

Stp1 modulates this activity by dephosphorylation. 455

456

Identification of autophosphorylated residues on BA-Stk1. Phosphorylation and 457

dephosphorylation appeared to be important for regulating the kinase activity of BA-Stk1cat, and 458

this heightened our interest in identifying residues phosphorylated in BA-Stk1cat. Indeed, we 459

predicted that such residues are involved in important aspects of BA-Stk1’s enzymatic activity 460

and might be subjected to regulation via BA-Stp1 dephosphorylation. Thus, in the next series of 461

experiments we mapped phosphorylated regions in BA-Stk1. Autophosphorylated BA-Stk1cat 462

was excised from an SDS-PAGE gel, trypsin digested, and the phosphopeptides were isolated 463

using immobilized gallium. The enriched phosphopeptide pool was then subjected to 464

LC/MS/MS analysis. The MS/MS spectra identified four phosphorylated peptides of BA-Stk1cat 465

based on sequences from the NCBI non-redundant protein database bank (Table 1). Of these 466

peptides BA-Stk1cat (150-183) with the sequence, 467

VTDFGIATATSATTITHTNSVLGSVHYLSPEQAR was predicted to have a cluster of seven 468

phospho-residues. When BA-Stk1cat was subjected to dephosphorylation by BA-Stp1, we 469

observed a loss in the level of phosphorylated amino acids within this peptide fragment, 470

indicating that BA-Stp1 hydrolyzes phosphate groups of this phosphopeptide (data not shown). 471

A similar observation was made on the peptide BA-Stk1cat (208-221) with the sequence, 472

QPFSGESAVAIALK, indicating that serine residues on this peptide are dephosphorylated by 473

BA-Stp1. Similar peptides were found by Madec et al. to be phosphorylated in PrkC (22), 474

suggesting PrkC and BA-Stk1 share autophosphorylation domains. 475


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476

Kinase activity of serine/threonine mutants of BA-Stk1cat. Madec et al. performed an 477

extensive mutational analysis of candidate phosphorylated residues in the activation loop of 478

PrkC, by substituting alanine for threonines and serines in this domain of the kinase. PrkC 479

mutants that could no longer phosphorylate substrates were then identified (22). Yet, whether 480

these mutants were defective in ATP hydrolysis and autophosphorylation, or whether sustained 481

phosphorylation of these residues influenced activity was not determined. Moreover, whether 482

BA-Stk1 utilized similar residues for autophosphorylation was not known. Thus, we examined 483

representative serine/threonine residues for their contribution to regulation of BA-Stk1cat activity. 484

The first two residues T165 and S173 were selected as representatives of targets phosphorylated 485

within the activation loop (T165) or outside the activation loop (S173) of the enzymatic domain 486

of BA-Stk1cat. S214 was selected from the identified phosphopeptide BA-Stk1cat (208-221). 487

Finally, T290 was selected as a representative of targets phosphorylated in the juxtamembrane 488

region of BA-Stk1 (spanning 55 residues) that is implicated in the regulation of eukaryotic 489

kinases (34). Alanine substitutions of these residues in BA-Stk1cat were made and the impact of 490

these mutations on kinase activity and autophosphorylation was determined. Kinase activity of 491

the mutants was measured in terms of ATP hydrolysis by analyzing the consumption of ATP in a 492

luminescence-based assay. As shown in Fig. 5A, kinase activity of mutants S173A and S214A is 493

reduced compared to wild type BA-Stk1cat, suggesting that phosphorylation at residues, S173 and 494

S214 is important for BA-Stk1cat kinase activity. Mutants T165A and T290A, however, did not 495

show reduced kinase activity as levels of ATP hydrolysis were comparable to wild type BA-496

Stk1cat. 497

498


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To further test the importance of phosphorylation on these residues, aspartic acid substitutions 499

were used to mimic a phosphorylated state of BA-Stk1 and the mutated proteins were tested for 500

kinase activity by measuring levels of ATP hydrolysis. As shown in Fig. 5B, the kinase activity 501

of S173D and S214D was similar to wild type BA-Stk1cat suggesting that the phosphorylated 502

state of residues S173 and S214 is critical for kinase activity. 503

504

To confirm the role of these residues in the BA-Stk1 kinase activity, both alanine and aspartic 505

acid mutants of T165, S173, S214, and T290 were analyzed in a myelin basic protein (MBP) 506

phosphorylation assay using radiolabeled ATP. Analysis of autophosphorylation activity of the 507

BA-Stk1 mutants demonstrated that the mutants undergo autophosphorylation to levels slightly 508

reduced or similar to wild type BA-Stk1cat (Fig. 5C). However, comparison of MBP 509

phosphorylation by the mutants and wild type BA-Stk1cat shows that kinase activity of both 510

alanine and aspartic acid mutants of T165 and S173 is significantly reduced (Fig. 5C). 511

Calculation of the percent phosphorylation of MBP shows that mutants of T165 and S173 exhibit 512

only 20% and <2% phosphorylation, respectively, compared to wild type BA-Stk1cat (Fig. 5D). 513

These results suggest that the presence of the phosphate moiety on these residues is required for 514

binding and/or phosphorylation of substrates. Though T165A and T165D had no detectable 515

defect in ATP hydrolysis as demonstrated in Fig. 5A and Fig. 5B, both mutants were attenuated 516

in substrate phosphorylation (Fig. 5C and Fig. 5D). More importantly, the native phosphoserine 517

at position 173 is essential for both ATP hydrolysis (Fig. 5A) and for substrate phosphorylation 518

(Fig. 5C and Fig. 5D). 519

520


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Analysis of MBP phosphorylation by mutant S214A also shows reduced activity; however, this 521

defect is rescued by the aspartic acid substitution on the same site (Fig. 5D) suggesting that the 522

phosphorylation state of residue S214 is important not only for hydrolysis of ATP (Fig. 5A) but 523

for phosphorylation of substrates as well (Fig. 5D). Though residue T290 was predicted as being 524

phosphorylated in kinases homologous to BA-Stk1, alanine or aspartic acid substitutions at this 525

site had no apparent effect on autophosphorylation or substrate phosphorylation (Fig. 5D). 526

527

DISCUSSION 528

The current findings provide insight into a paired serine/threonine phosphatase and kinase in B. 529

anthracis. Several recent studies have shown that Stp/Stk pairs contribute to bacterial virulence 530

and physiology but the underlying molecular mechanisms and how these proteins are regulated 531

remains poorly understood. To this end, the data provided herein addresses important gaps in our 532

understanding of how the activity of a bacterial Stk is regulated by a partner phosphatase through 533

dephosphorylation. The results of this study indicate that BA-Stp1 regulation of BA-Stk1 is a 534

complex system of multiple residues targeted for dephosphorylation, with some phospho-535

residues important only for autophosphorylation, while other residues are necessary for both 536

ATP hydrolysis and substrate phosphorylation. 537

538

Analysis of the enzyme kinetic parameters (Km and Vmax) indicated that BA-Stp1 539

dephosphorylates phosphothreonine and phosphoserine residues with Km values similar to those 540

reported for phosphatases, such as PphA from Synechocystis PPC 6803(30), and PrpZ from S. 541

enterica (18). Thus, these biochemical and enzymatic characterizations of BA-Stp1 all 542

correspond to the expected activities of a PP2C phosphatase. The data shown in Fig. 3 indicate 543


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that BA-Stp1 utilizes this PP2C phosphatase activity to modulate BA-Stk1, wherein 544

dephosphorylation leads to a decline in kinase activity. Importantly, this reaction appears to be 545

more specific as BA-Stp1 exhibited a higher affinity, Km 9.65 µM vs 248.6 µM, towards BA-546

Stk1 compared to generic substrates. 547

548

A graphical summary of our data is presented in Fig. 6. Unlike histidine kinases whose 549

phosphorylation status can either turn on/off a single phosphotransferase mechanism, BA-Stk1 550

exhibits a complex assembly of critical autophosphorylated residues, some of which contribute 551

to ATP hydrolysis and autophosphorylation, and others that contribute only to substrate 552

phosphorylation. For example, in the absence of phosphorylation of S173 and S214, substrate 553

phosphorylation is attenuated but autophosphorylation increases. The phosphorylation of S173 554

and S214 may, therefore, shift the kinase from an autophosphorylating state to substrate 555

phosphorylation. While it is difficult to absolutely exclude the influence of subtle conformational 556

changes occurring from the alanine substitution, the aspartic acid substitutions further support 557

the idea that the balance between these two biochemical activities is influenced by the 558

phosphorylation of these residues. Indeed, when phosphorylation is simulated at S214 by 559

aspartic acid substitution, autophosphorylation is reduced, and substrate phosphorylation is 560

enhanced. In a corresponding manner, when the residue can not be phosphorylated due to an 561

alanine substitution, Stk1 increases autophosphorylation and decreases substrate 562

phosphorylation. Conversely, mimicking phosphorylation of S173 by aspartic acid substitution 563

reduced autophosphorylation and substrate phosphorylation, indicating the addition of a 564

phosphate group to this residue has an overall negative effect on kinase activity. Finally, aspartic 565

acid substitution at T165 slightly increased autophosphorylation, suggesting that phosphorylation 566


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of this residue enhances kinase activity. We predict that T165 is one of the key residues 567

dephosphorylated by BA-Stp1 leading to down regulation of kinase activity and that the presence 568

of the threonine at position 165 is necessary to allow for binding and/or phosphorylation of target 569

substrates. Collectively, we observed that phosphorylation of some residues in BA-Stk1 promote 570

kinase activity, while phosphorylation of other residues represses kinase activity. These results 571

reveal a sophisticated mechanism of control for BA-Stk1 kinase activity, wherein a network of 572

phosphorylated residues are altered to either activate or inactivate the enzymatic activity of this 573

signaling protein in B. anthracis. 574

575

A comparison of our findings on Ba-Stp1 relative to B. subtilis PrpC is instructive. Like Ba-576

Stp1, PrpC is believed to be functionally coupled with its partner kinase, PrkC. Moreover, PrpC 577

was found to dephosphorylate PrkC in earlier work by Obuchowski and colleagues (27) and this 578

is similar to our current results on Ba-Stp1 dephosphorylation of Ba-Stk1. However, the impact 579

of dephosphorylation on kinase activity has not been determined for the PrpC/PrkC pair. 580

PrpC/PrkC also differs from BA-Stp1/BA-Stk1 in that the phosphatase/kinase pair appears to 581

regulate late-stationary growth phase events (e.g. sporulation and biofilm formation) in B. 582

subtilis, an effect we did not observe in B. anthracis. Despite several attempts, we could not 583

detect or demonstrate any difference in sporulation efficiency between the parent B. anthracis 584

Sterne strain and B. anthracis STPK101 (data not shown). In fact extensive analysis of B. 585

anthracis STPK101 did not find any notable growth defect under in vitro conditions, and a 586

prominent phenotype only became apparent when infection models were used. These data 587

suggest BA-Stp1/BA-Stk1 may have been refined by B. anthracis for growth and virulence in 588

vivo, while PrpC/PrkC have evolved to regulate events more important to survival of B. subtilis 589


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in the environment. Very recently, Shah et al. (33) reported a role for B. subtilis PrkC in sensing 590

muropeptides released during germination, and B. anthracis was among the list of other bacteria 591

for which this group defined this system. The study by Shah et al. found that null mutants 592

lacking the gene for PrkC and BA-Stk1 were unable to sense muropeptide and peptidoglycan as 593

a germinant. Thus, although we did not observe a defect in germination of the mutant in vitro, it 594

is reasonable to suspect that the attenuation of virulence could be due to defects in the ability of 595

B. anthracis to regulate peptidoglycan-related germination during the establishment of disease. 596

597

Our findings report the importance of a critical serine/threonine phosphatase-kinase pair in the 598

virulence of B. anthracis and highlight a mechanism by which a bacterial phosphatase modulates 599

the activity of its partner kinase at the molecular level. We hypothesize that BA-Stk1 acts as a 600

sensor for regulating virulence in response to stimuli and does so by autophosphorylation, and as 601

the stimuli subsides BA-Stp1 serves to inactive the kinase by dephosphorylation. Further studies 602

on the phosphatase-kinase association between BA-Stp1 and BA-Stk1 will provide new insights 603

into identification of novel target substrates and the importance of reversible phosphorylation as 604

a regulatory mechanism in B. anthracis. 605

606

Acknowledgements 607

We thank Dr. Ken Jackson and Stephen Snow at the Molecular Biology-Proteomics Facility 608

(OUHSC) for their help with the mass spectrometric analysis of BA-Stk1. This work was 609

supported by Public Health Service grant U54A1057156, Western Region Center for Biodefense 610

and Emerging Infectious Diseases (J.D.B. and C.R.L.) 611

612


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25. Mumby, M. C., and G. Walter. 1993. Protein serine/threonine phosphatases: structure, 687

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27. Obuchowski, M., E. Madec, D. Delattre, G. Boel, A. Iwanicki, D. Foulger, and S. J. 693

Seror. 2000. Characterization of PrpC from Bacillus subtilis, a member of the PPM 694

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28. Rajagopal, L., A. Clancy, and C. E. Rubens. 2003. A Eukaryotic Type 696

Serine/Threonine Kinase and Phosphatase in Streptococcus agalactiae Reversibly 697

Phosphorylate an Inorganic Pyrophosphatase and Affect Growth, Cell Segregation, and 698

Virulence. J. Biol. Chem. 278:14429-14441. 699

29. Reed, L. J., and H. Muench. 1938. A Simple Method Of Estimating Fifty Per Cent 700

Endpoints. Am. J. Epidemiol. 27:493-497. 701

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phosphatase PphA from Synechocystis PCC 6803 controls dephosphorylation of the 703

signalling protein PII. Mol Microbiol 44:855-64. 704


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31. Saile, E., and T. M. Koehler. 2006. Bacillus anthracis Multiplication, Persistence, and 705

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3174. 707

32. Scaramozzino, F., A. White, M. Perego, and J. A. Hoch. 2009. A Unique GTP-708

Dependent Sporulation Sensor Histidine Kinase in Bacillus anthracis. J. Bacteriol 709

191:687-692. 710

33. Shah, I. M., M.-H. Laaberki, D. L. Popham, and J. Dworkin. 2008. A Eukaryotic-like 711

Ser/Thr Kinase Signals Bacteria to Exit Dormancy in Response to Peptidoglycan 712

Fragments. Cell 135:486-496. 713

34. Thiel, K. W., and G. Carpenter. 2007. Epidermal growth factor receptor 714

juxtamembrane region regulates allosteric tyrosine kinase activation. Proceedings of the 715

National Academy of Sciences 104:19238-19243. 716

35. Treuner-Lange, A., M. J. Ward, and D. R. Zusman. 2001. Pph1 from Myxococcus 717

xanthus is a protein phosphatase involved in vegetative growth and development. Mol 718

Microbiol 40:126-40. 719

36. Vetter, S. M., and P. M. Schlievert. 2007. The Two-Component System Bacillus 720

Respiratory Response A and B (BrrA-BrrB) Is a Virulence Factor Regulator in Bacillus 721

anthracis. Biochemistry 46:7343-7352. 722

37. White, A. K., J. A. Hoch, M. Grynberg, A. Godzik, and M. Perego. 2006. Sensor 723

Domains Encoded in Bacillus anthracis Virulence Plasmids Prevent Sporulation by 724

Hijacking a Sporulation Sensor Histidine Kinase. J. Bacteriol. 188:6354-6360. 725

38. Wiley, D. J., R. Nordfeldth, J. Rosenzweig, C. J. DaFonseca, R. Gustin, H. Wolf-726

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A (YpkA) is necessary for full virulence in the mouse, mollifying phagocytes, and 728

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39. Young, T. A., B. Delagoutte, J. A. Endrizzi, A. M. Falick, and T. Alber. 2003. 730

Structure of Mycobacterium tuberculosis PknB supports a universal activation 731

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40. Zhou, H., J. D. Watts, and R. Aebersold. 2001. A systematic approach to the analysis 733

of protein phosphorylation. Nat Biotech 19:375-378. 734

735

736

737

738

739

740

741

742

743

744

745

746

747

748

749

750

751

752

753

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755


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Table 1. Phosphopeptides of BA-Stk1cat identified by Mass spectrometry 756

Peptide name Peptide Sequence

Stk1:45-55 DY[pS]NNEEFIKR

Stk1:150-183 VTDFGIA[pT][pT][pS]A[pT][pT]I[pT]H[pT]NSVLGSVHYLSPEQA

R

Stk1:208-221 QPFSGE[pS]AVAIALK

Stk1:264-273 DIE[pT]ALYPER

757

Phosphopeptides identified in autophosphorylated Ba-Stk1 by LC-MS/MS analysis of tryptic 758

digests. Residues in parentheses were identified as predicted phosphorylation sites based on a 759

search of the NCBI non-redundant protein database search of all Eubacteria, Firmicutes group. 760

761

762

763

764

765

766

767

768

769

770

771

772


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773

Figure Legends: 774

775

Figure 1. Analysis of survival and virulence of B. anthracis Sterne STPK101. Panel A: 776

Survival of B. anthracis Sterne STPK101 in cultured macrophage-like cells. RAW 264.7 777

macrophages were infected with spores at a MOI of 10 as described in materials and methods. At 778

0, 2, 6, 8, and 10 h post infection, spores and vegetative bacilli were collected and the total 779

number of colony forming units were enumerated by plate count method for parent strain (black 780

bars) and B. anthracis Sterne STPK101 (white bars). Statistical analysis was performed using 781

unpaired two-tailed Student’s T test (P value 0.0173). Panel B and C: Virulence of B. anthracis 782

Sterne STPK101 in a mouse model of pulmonary anthrax. Kaplan-Meier survival curves of 783

DBA/2 mice treated with the parent strain B. anthracis Sterne (panel B) and B. anthracis Sterne 784

STPK101 (panel C). DBA/2 mice, 10/group, were subjected to an intra-tracheal challenge of 785

either 1x104, 1x10

5, or 1x10

6 spores of either the parent strain B. anthracis Sterne or B. anthracis 786

Sterne STPK101. Mice were observed for 14 days following challenge and the LD50 was 787

calculated using the Reed and Muench method (29) (inset within the panel). 788

789

Figure 2. Biochemical analysis of BA-Stp1. The biochemical characteristics of BA-Stp1 were 790

determined by examining the dephosphorylation of synthetic peptides with phosphate groups on 791

serine, threonine, or tyrosine residues. These activities were then compared with BA-Stp1 792

mutants containing alanine substitutions at critical aspartic acid residues. Panel A: Analysis of 793

phospho-residue specificity of BA-Stp1. Dephosphorylation of synthetic phosphopeptides by 794

BA-Stp1 was measured using a malachite-green phosphate detection system. BA-Stp1 795


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(concentrations ranging from 0-600 nM) was incubated with 50 µM phosphothreonine peptide-796

RRApTVA (black bars), 50µM phosphoserine peptide-RRApSVA (grey bars), and 50µM 797

phosphotyrosine peptide-RRLIEDAEpYAARG (white bars). Phosphatase activity was 798

detected at 600 nm and measured in terms of pmole of phosphate (Pi) released. Error bars 799

represent the standard deviation. Panel B: Analysis of phosphatase activity of site-directed 800

aspartic acid mutants of BA-Stp1. Phosphatase activity of site-directed aspartic acid mutants of 801

BA-Stp1 was measured in comparison to wild type BA-Stp1. Using 50 µM RRApSVA (black 802

bars) and 50 µM RRApTVA (grey bars) as substrates, phosphatase activity of 2 µM site-803

directed mutants, D18A, D36A, D194A, and D233A and 2 µM wild type BA-Stp1 was measured 804

as pmole of phosphate released. Error bars represent the standard error of the mean. Statistical 805

analysis was performed with ANOVA (p value<0.0001) 806

807

Figure 3. Analysis of BA-Stk1 as a candidate substrate of BA-Stp1. Panel A: 808

Dephosphorylation of recombinant BA-Stk1cat by BA-Stp1. The dephosphorylation of BA-Stk1 809

was assessed using a malachite-green phosphate detection assay in which the dephosphorylation 810

of BA-Stk1cat (0-25 µM) by BA-Stp1 is reported as pmole of Pi released following 30 min 811

incubation. Error bars represent the standard deviation of the mean. Statistical analysis was 812

performed by ANOVA (p value <0.0001). Panel B: Analysis of BA-Stk1 dephosphorylation by 813

BA-Stp1 mutants. BA-Stp1 or individual corresponding mutants were incubated with 2 µM BA-814

Stk1cat was in reaction buffer for 30 min and the pmole of Pi released was determined. Error 815

bars represent the standard deviation of the mean. Statistical analysis was performed with 816

ANOVA (p value <0.001) Panel C: BA-Stp1-dephosphorylation of autophosphorylated BA-817

Stk1. BA-Stk1 was autophosphorylated in the presence of [γ-32P] ATP for 30 min and then 818


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incubated in phosphatase buffer with or without purified BA-Stp1. Aliquots were removed at 0, 819

30, 60, and 90 min and the reaction products were analyzed on SDS-PAGE followed by 820

autoradiography to visualize the degree of dephosphorylation. 821

822

Figure 4. BA-Stp1 dephosphorylation of BA-Stk1 and corresponding decrease in 823

autophosphorylation. Purified BA-Stk1 was subjected to a dephosphorylation reaction by BA-824

Stp1 and examined for changes in autophosphorylation activity. Panel A: Western Blot analysis 825

of BA-Stk1 serine phosphorylation following incubation with BA-Stp1. BA-Stk1 was incubated 826

under the indicated conditions and examined for dephosphorylation using polyclonal anti-827

phosphoserine antibodies at the indicated time-points. Lanes 1-3, BA-Stk1 incubated in reaction 828

buffer alone; Lanes 4-6, BA-Stk1 incubated with shrimp alkaline phosphatase (SAP); Lanes 7-9, 829

BA-Stk1 incubated with BA-Stp1; Lanes 10-12, BA-Stk1 incubated with enzymatically inactive 830

BA-Stp1D18A. Panel B: Autoradiograph of BA-Stk1 autophosphorylation following treatment 831

with BA-Stp1. Prior to the autophosphorylation reaction, BA-Stk1 was incubated with BA-Stp1 832

or the enzymatically inactive BA-Stp1D36A for 30 min. Untagged BA-Stp1 and BA-Stp1D36A 833

were removed from the mixture by affinity chromatography and the kinase activity of His-tagged 834

BA-Stk1 was examined by analyzing autophosphorylation using [γ-32P] ATP. Reactions were 835

stopped at 0, 15, 30, and 60 min and the products were analyzed on SDS-PAGE followed by 836

autoradiography. Lanes 1-4, Control treated BA-Stk1; Lanes 5-8 BA-Stp1 treated BA-Stk1; 837

Lanes 9-12 BA-Stp1D36A treated BA-Stk1. Panel C: Autoradiograph of BA-Stk1 838

autophophosphorylation and MBP phosphorylation following treatment with BA-Stp1. BA-Stk1 839

was preincubated with BA-Stp1 or buffer control for 30 min to allow for the dephosphorylation 840

reaction. Subsequently, autophosphorylation and kinase activity was analyzed using MBP and 841


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[35

S] ATP-γS. Reactions were stopped at 15, 30, and 60 min and the products were analyzed on 842

SDS-PAGE followed by autoradiography. 843

844

Figure 5. Kinase activity of BA-Stk1 mutants. Alanine or aspartic acid-substituted site-845

directed mutants of BA-Stk1 were examined for changes in kinase activity in order to identify 846

serine and threonine residues critical to these events. Panel A: ATP hydrolysis activity of four 847

alanine site-directed mutants of BA-Stk1cat. Purified candidate serine (S173A and S214A) and 848

threonine (T165A and T290A) site-directed mutants of BA-Stk1cat were tested for activity in a 849

luminescence assay measuring consumption of ATP. Panel B: ATP hydrolysis activity of 850

aspartic acid mutants of BA-Stk1cat. Purified serine (S173D and S214D) and threonine (T165D 851

and T290D) site-directed mutants of BA-Stk1cat were tested for activity in a luminescence assay 852

as described above. Results were calculated as percent kinase activity relative to wild type BA-853

Stk1cat. Statistical analysis was performed with unpaired two-tailed Student’s T-test and all 854

calculated values were p<0.05. Panel C: Phosphorylation activity of BA-Stk1 mutants towards 855

myelin basic protein (MBP). Alanine or aspartic acid-substituted site-directed mutants of BA-856

Stk1 were examined for kinase activity towards MBP in order to identify serine and threonine 857

residues critical for substrate phosphorylation. Purified wild type BA-Stk1cat or alanine or 858

aspartic acid-mutants of BA-Stk1cat were incubated with MBP in the presence of [γ-32P] ATP for 859

30 min in kinase buffer. The reactions were stopped with SDS loading buffer and the reaction 860

products were analyzed on SDS-PAGE followed by autoradiography. Panel D. Quantification of 861

MBP phosphorylation. Levels of 32

P incorporation were obtained by PhosphorImage analysis 862

and the relative values are depicted as percent phosphorylation of MBP by BA-Stk1 mutants 863

compared to wild type BA-Stk1cat. Error bars represent standard deviation of the mean. 864


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Statistical analysis was performed with unpaired two-tailed Student’s T-test and all calculated 865

values were p<0.05. 866

867

Figure 6. Graphic summary of BA-Stk1 phospho-residues targeted by BA-Stp1 and their 868

contribution to specific kinase activities. Four residues are represented in the figure and 869

include the two serines and two threonines predicted as sites of phosphorylation 870

dephosphorylated by BA-Stp1. This model is derived from a series of experiments using a 871

combination of mutagenesis approaches and kinase assays to determine the role of each residue 872

in the activity of BA-Stk1. The symbols ↑ and ↓ denote increase or decrease in activity. Overall 873

kinase activity (KA) is measured in terms of levels of autophosphorylation plus substrate 874

phosphorylation. 875


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4 salika m. shakir - journal of bacteriology · 2009. 11. 13. · shakir et al. a...

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