1 2 3 ACCEPTED - Journal of Bacteriology › content › jb › early › 2008 › 04 › 04 › JB.00265...2008/04/04 · 11 Marburg, D35043, Germany 12 2Department of Molecular

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EspA, an orphan hybrid histidine protein kinase, regulates the timing of 4

expression of key developmental proteins of Myxococcus xanthus 5

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Penelope I. Higgs1*, Sakthimala Jagadeesan

1, Petra Mann

1, and David R. Zusman

2 8

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1Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, 10

Marburg, D35043, Germany 11

2Department of Molecular and Cell Biology, University of California, Berkeley, CA 12

94720-3204, USA 13

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Running title: EspA control of development 15

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* Corresponding author. 17

Mailing address: 18

Max Planck Institute for Terrestrial Microbiology 19

Karl-von-Frisch Strasse 1 20

35043 Marburg, Germany 21

Tel. (+49) (0)6421 178301 22

Fax (+49) (0)6421 178309 23

Email: [email protected] 24

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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00265-08 JB Accepts, published online ahead of print on 4 April 2008

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ABSTRACT 25

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Myxococcus xanthus undergoes a complex starvation-induced developmental 27

program that results in cells forming multicellular fruiting bodies by aggregating into 28

mounds and then differentiating into spores. This developmental program requires at 29

least seventy-two hours and is mediated by a temporal cascade of gene regulators in 30

response to intra- and extra-cellular signals. Mutants in espA, encoding an orphan 31

hybrid histidine kinase, alter the timing of this developmental program, greatly 32

accelerating developmental progression. Here, we characterized EspA, and 33

demonstrated that it autophosphorylates in vitro on the conserved histidine residue 34

and then transfers the phosphoryl group to the conserved aspartate residue in the 35

associated receiver domain. The conserved histidine and aspartate residues were both 36

required for EspA function in vivo. Analysis of developmental gene expression and 37

protein accumulation in espA mutants indicated that the expression of the A-signal 38

dependent spi gene was not affected but that the MrpC transcriptional regulator 39

accumulated earlier resulting in earlier expression of its target, the FruA 40

transcriptional regulator. Early expression of FruA correlated with acceleration of 41

both the aggregation and sporulation branches of the developmental program as 42

monitored by early methylation of the FrzCD chemosensory receptor and early 43

expression of the sporulation-specific dev and Mxan_3227 (Ω7536) genes. These 44

results show that EspA plays a key role in the timing of expression of genes necessary 45

for progression of cells through the developmental program. 46

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INTRODUCTION 47

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Myxococcus xanthus is a Gram-negative rod shaped bacterium that is a model 49

system for social behavior in prokaryotes. M. xanthus resides in the soil or on 50

herbivore dung and obtains nutrients by secreting antibiotics and enzymes (such as 51

proteases and lysozyme) that digest macromolecules from prey microorganisms or 52

decaying organic matter (43). Communities of M. xanthus exhibit cooperative growth 53

which is dependent on the amount of secreted enzymes that break down complex 54

macromolecules for subsequent uptake by the individual cells (44). Under starvation 55

conditions, M. xanthus enters an elaborate developmental program in which cells first 56

aggregate into mounds of approximately 100000 cells and then within these mounds 57

(fruiting bodies), cells differentiate into metabolically quiescent, environmentally 58

resistant spores [reviewed in (50)]. Upon sensing nutrient rich conditions, spores 59

germinate and reenter the vegetative cycle. It is presumed that formation of spores 60

within fruiting bodies allows M. xanthus to germinate in groups providing an 61

advantage for cooperative feeding behaviors. 62

The M. xanthus developmental program is temporally regulated in association 63

with a series of intra- and inter-cellular signaling events [reviewed in (13)]. A core 64

pathway of molecular events have been identified which are necessary for formation 65

of mature fruiting bodies (summarized in Fig. 7). First, starvation is sensed via the 66

stringent response resulting in increased guanosine tetra- or penta- phosphate 67

[(p)ppGpp] (10, 51). Rising (p)ppGpp levels trigger A-signaling (51), proposed to be 68

a population (quorum) sensing mechanism (14, 23) in which individual cells are 69

induced to release amino acids and peptides generated by extracellular proteolysis in a 70

manner dependent on genes of the asg class (20-22, 38). When sufficient A-signal is 71

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produced, several genes necessary for the developmental program are induced (14, 72

23). 73

One of the genes whose transcriptional upregulation depends upon A-74

signaling is mrpC which encodes a transcriptional regulator of the cAMP receptor 75

(CRP) family (56, 57). The MrpC protein is subject to complex post-translational 76

regulation which appears to control its affinity for identified target promoters: 77

phosphorylated MrpC (MrpC-P) affinity for target sequences is reduced, whereas the 78

proteolytically processed MrpC2 protein shows increased affinity (34, 35). In addition, 79

MrpC-P does not appear to be processed into MrpC2. MrpC2 binds to the promoter 80

and is thought to induce transcriptional upregulation of the key developmental 81

transcriptional regulator gene, fruA (60). FruA is an orphan response regulator of the 82

two-component signal transduction family containing a DNA-binding output domain 83

(5, 36). Genetic evidence suggests that FruA is activated by phosphorylation of a 84

conserved aspartic acid in the receiver domain (5). 85

FruA activation is proposed to occur in response to the C-signal pathway (5). 86

C-signal is encoded by the csgA gene and csgA mutants are unable to form proper 87

fruiting bodies or to sporulate (8, 9, 16). C-signal arises from proteolytic processing of 88

the cell-surface associated CsgA protein (28) and has been proposed to be sensed by 89

an unidentified receptor protein on a neighboring cell. As a result of cell-cell contact, 90

CsgA expression is upregulated (15, 17) and C-signaling is amplified (7). 91

In a current model [reviewed in (13, 52)], FruA activated in response to C-92

signaling stimulates development through a branched pathway with one branch 93

leading to aggregation and a second branch leading to sporulation. FruA is proposed 94

to induce the aggregation branch by, in an unknown mechanism, stimulating 95

methylation of the FrzCD methyl-accepting chemotaxis protein (MCP) which directs 96

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cells to aggregate into mounds (2, 30, 31, 48, 53). Increased cell-cell contact in 97

mounds is proposed to lead to increased C-signaling and increased activation of FruA. 98

It is then proposed that the higher levels of activated FruA induce the sporulation 99

branch of the developmental pathway. It has been demonstrated that FruA activates 100

transcription of the dev locus (63) which is in turn necessary for expression of Tn5 101

lacZ Ω7536 (27), which resides in gene Mxan_3227 (J. Jakobsen, personal 102

communication). Both the dev locus and Mxan_3227 are required for sporulation (27, 103

58). In this manner, C-signaling and the FruA protein are proposed to coordinate the 104

formation of spores with the completion of fruiting bodies. 105

Generation of mature fruiting bodies is a relatively slow process; under 106

laboratory conditions, spore-filled fruiting body formation takes at least 72 hours. 107

Several mutants in two-component signal transduction genes have been described 108

[espA (4), todK (41), espC (25), and redCDEF (11)] that progress through the 109

developmental program more quickly forming more disorganized fruiting bodies but 110

otherwise displaying no defect in the ability to form spores. These observations 111

suggest that in wildtype cells, these respective gene products act to repress 112

progression through the developmental program until a specific condition or set of 113

conditions is met. However, it is unclear how these proteins mediate this repression. 114

In this paper, we focus on EspA, a histidine protein kinase homolog, 115

previously proposed to be necessary for regulating only the timing of sporulation (4). 116

Two particular features of EspA make it difficult to predict how EspA may function 117

to alter the developmental program: First; it is an orphan [i.e. not genetically 118

organized together with a cognate response regulator gene (49)] so that it is not clear 119

as to the nature of the output mechanism. Second, EspA is a hybrid kinase containing 120

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an associated receiver domain located at the carboxy terminus of the protein and the 121

role of the receiver in EspA activity is unclear. 122

Here we show that the histidine kinase activity is required early during the 123

developmental program to inhibit developmental progression, both aggregation and 124

sporulation. Our data suggests that in espA mutants, while A-signaling is unaffected, 125

MrpC accumulates earlier compared to wildtype resulting in earlier expression of 126

FruA and premature induction of the aggregation and sporulation branches of the 127

developmental program. Analysis of double mutants between espA and key 128

developmental regulators confirms that A-signaling and FruA are required for EspA-129

mediated modulation of developmental progression, but shows that C-signaling can 130

be partially bypassed. 131

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MATERIALS AND METHODS 133

134

Strains and growth conditions. Bacterial strains and plasmids used are listed 135

in Table 1. M. xanthus strains were grown vegetatively at 32°C on CYE agar plates 136

[1% Casitone, 0.5% Yeast extract, 10 mM 3-(N-morpholino)propanesulfonic acid 137

(MOPS) pH 7.6, 4 mM MgSO4, 1.5% agar] or in CYE broth (CYE lacking agar). 138

Plates were supplemented with 100 µg ml-1

kanamycin or 10 µg ml-1

oxytetracycline 139

where necessary. E. coli cells were grown under standard laboratory conditions in 140

Luria-Bertani (LB) broth supplemented with 100 µg ml-1

ampicillin or 50 µg ml-1

141

kanamycin where necessary (29). 142

Analysis of M. xanthus developmental phenotypes. Development was 143

assayed under submerged culture conditions modified from (19). Briefly, cells were 144

grown in vegetative conditions overnight in CYE broth and then diluted to an optical 145

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density at 550 nm (OD550) of 0.035 in fresh CYE media. 16 ml or 0.5 ml of diluted 146

cells was added to 9 cm Petri plates or per well to 24 well tissue culture plates, 147

respectively, and incubated at 32°C for 24 hours. To initiate the developmental 148

program, CYE media was replaced by an equivalent volume of MMC starvation 149

media (10 mM MOPS pH 7.6, 2 mM CaCl2, 4 mM MgSO4) and plates were incubated 150

at 32°C for the respective times indicated. For analysis of development on CF agar 151

plates, cells were grown to mid-log in CYE broth, washed and resuspended to 0.35 152

OD550 in MMC starvation media and 10 µl cells were spotted on CF plates (0.15% 153

Casitone, 0.2% sodium citrate, 0.1% sodium pyruvate, 0.02% (NH4)2SO4, 10mM 154

MOPS pH 7.6, 8mM MgSO4, 1mM KH2PO4, 1.5% agar) and incubated at 32°C. 155

Developmental phenotypes were recorded at the times indicated with a Leica MZ8 156

stereomicroscope and attached Leica DFC320 camera. 157

Construction of mutants. In-frame deletion of the receiver domain in espA 158

was generated by homologous recombination modified from a previously reported 159

method (61). Briefly, approximately 500 bp PCR fragments corresponding to regions 160

upstream and downstream of the deletion were separately amplified and then fused 161

together by overlap extension PCR. The resulting approximately 1000 bp PCR 162

fragments were then cloned into the EcoRI and BamHI sites of pBJ114. Clones were 163

sequenced to confirm sequences were error-free at the codon level. The plasmids were 164

integrated into the genome by homologous recombination and selected for by 165

resistance to kanamycin. Loss of the integrated plasmid via a second homologous 166

recombination event was then screened for by galK-mediated counter-selection on 167

CYE plates containing 2.5% galactose. Resulting kanamycin-sensitive (kanS), 168

galactose-resistant (galR) colonies were then screened by PCR for those that generated 169

a deletion as opposed to the original wildtype. Deletions were confirmed by Southern 170

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Blot analysis. The espAH407A or espAD696A substitution mutations were generated using 171

a similar procedure except the overlap PCR generated a GCG (Ala) codon in 172

replacement of the CAC (His407) or GAT (Asp696) codons, respectively. 173

Furthermore, kanS gal

R colonies were screened by PCR using primers containing 174

either the wildtype codon or the mutant GCG codon at the extreme 3’ end. Desired 175

codon substitutions were then confirmed by PCR amplifying the relevant areas of the 176

espA gene using the mutant strain genomic DNA as template, cloning the PCR 177

products into pGEX-4T-1 and sequencing the cloned product. 178

Insertion mutants in asgA were generated by cloning internal fragments 179

containing bp 257-797 of the asgA sequence into the EcoRI/BamHI sites of pBJ114, 180

generating pPH127. pPH127 was introduced by electroporation into strains DZ2 181

(wildtype) and DZ4227 (DZ2 ∆espA) and plasmid integration was selection for by 182

kanamycin resistance, resulting in strains PH1010 and PH1011, respectively. Insertion 183

mutants in fruA were similarly generated except bp 30-451 of fruA were cloned 184

generating plasmid pPH128 and strains PH1013 and PH1012, respectively. 185

To generate csgA mutants in the DZ2 background, genomic DNA isolated 186

from strain DK9035 was electroporated into either DZ2 or DZ4227 and resulting 187

double homologous recombination events were selected by oxy-tetracycline resistance 188

generating strains PH1014 and PH1015, respectively. Strain PH1016 (DZ4227 189

frzCD::Tn5) was similarly generated by electroporating genomic DNA from strain 190

DZ4169 into DZ4227 with kanamycin selection. 191

Cloning, overexpression, and purification of GST-EspA kinase proteins. 192

Over-expression plasmids pPH141 and pPH156 encode the glutathione S-transferase 193

(GST) affinity tag fused at the amino-terminus to the EspA kinase region (EspAkin) 194

which constitutes the HisKA and HATPase_c domains; Fig. 1A) or to the kinase 195

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mutant (EspAkinH407A), respectively (Fig. 1B). pPH141 and pPH156 were constructed 196

by PCR-amplifying the kinase region of espA (codons 390-646) using genomic DNA 197

isolated from strains DZ2 (wild type) or PH1008 (DZ2 espAH407A), respectively, as a 198

template. The resulting PCR products were cloned into the EcoRI site of pGEX4T-1. 199

Plasmids pPH143 and pPH157 encoding GST fused to the kinase and receiver 200

domains of EspA (EspAkin-rec) (Fig. 1A), or to the kinase and point-mutated receiver 201

(EspAkin-recD696A), respectively, were similarly constructed by amplifying the region of 202

espA encoding the HisKA to protein end (codons 390-768) from DZ2 or PH1009 203

(DZ2 espAD696A) genomic DNA, respectively (Fig. 1B). All constructs were 204

sequenced to confirm absence of PCR generated errors. E. coli BL21λDE3 was 205

transformed with the relevant over-expression plasmids and expression of the GST-206

tagged proteins was induced at approximately 0.7 OD550 by the addition of 0.1 mM 207

IPTG followed by growth for 3 hours at 25°C. Cells were then harvested, resuspended 208

in 0.05 volumes PBS lysis buffer (135 mM NaCl, 3.6 mM KCl, 8 mM Na2HPO4, 2 209

mM KH2PO4) containing 1mg ml-1

lysozyme and 1:200 dilution mammalian protease 210

inhibitor cocktail (Sigma) and incubated on ice for 30 minutes. Cells were lysed by 211

French press three times at approximately 18 000 psi and then incubated with rotation 212

in the presence of 1% Triton X-100 for 40 minutes at 4°C. Insoluble protein was 213

removed by centrifugation at 600 x g for 30 minutes and the supernatant was 214

incubated for 3 hours at 4°C with 1 ml of GST bind resin (Novagen) pre-equilibrated 215

with PBS. The resin was washed with 30 ml of PBS prior to elution of protein with 3 216

ml of elution buffer [50 mM Tris pH 8.0, 10 mM reduced glutathione]. The eluted 217

fractions were analyzed by sodium dodecyl sulphate polyacrylamide gel 218

electrophoresis (SDS-PAGE), peak elution fractions were pooled, and the protein was 219

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dialyzed overnight against TGMNKD buffer [50 mM Tris-HCl pH 8.0, 10% (v/v) 220

glycerol, 5 mM MgCl2, 150 mM NaCl, 50 mM KCl, 1 mM DTT] for further assays. 221

Radiolabeled in vitro phosphorylation and ATPase assays. in vitro 222

phosphorylation of proteins was carried out in TGMNKD buffer (above) containing 223

0.5 mM [γ-32

P]ATP (14.8 GBq mmol-1

; Amersham) and 5 µM of EspA in 25 µl total 224

volume for 120 minutes at room temperature (RT). Aliquots of 10 µl were quenched 225

with 5 µl of 3x SDS/EDTA loading dye [7.5% (w/v) SDS, 90 mM EDTA, 37.5 mM 226

Tris-HCl pH 6.8, 37.5% glycerol, 0.3 M DTT], loaded without prior heating on a 11% 227

polyacrylamide gel and separated by SDS-PAGE. Gels were exposed to 228

phosphoimager screen over night and images were detected on a Typhoon Trio 229

phosphorimager (Amersham Biosciences) and analyzed using the ImageQuant version 230

5.0 software (Molecular Dynamics). Gels were subsequently stained by Coomassie 231

dye to detect protein. 232

ATPase activity was measured under the conditions of the in vitro 233

phosphorylation assay except that 0.5 mM [α-32

P]ATP (110 TBq mmol−1

; Amersham) 234

was used as a substrate. The products of [α-32

P]ATP hydrolysis were analyzed by thin 235

layer chromatography (TLC) as per (42). Briefly, the reaction products were separated 236

from EspA proteins using Microcon-Y10 ultrafiltration columns (Millipore). Adenine 237

nucleotides were separated by spotting 0.5 µl of the elute to a poly (ethyleneimine)-238

cellulose F TLC plate (Merck) with 2.4 M formic acid as the solvent system for 20 239

minutes at RT. The labeled nucleotides on the TLC plates were visualized on a 240

phosphoimager and analyzed using ImageQuant software as described above. 241

Real-time polymerase chain reaction analyses. RNA for real-time 242

polymerase chain reaction (RT-PCR) analyses was harvested from cells developing 243

under submerged culture. RNA was isolated by the hot-phenol method as per (29, 37). 244

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Briefly, cells were harvested at the indicated time points, pelleted, and immediately 245

stored at -80°C. Pellets were resuspended in 1 ml ice cold Solution I (0.3 M sucrose, 246

0.01 M sodium acetate pH 4.5) and added to 1 ml Solution II (2% SDS, 0.01M 247

sodium acetate pH4.5) at 65°C. 2 ml 65°C phenol was added followed by incubation 248

at 65°C for 5 min. Reactions were flash cooled and the aqueous layer was again 249

extracted with 2 ml 65°C phenol followed by 2 ml phenol/chloroform/isoamyl alcohol 250

as above. RNA was precipitated from the final aqueous layer with 0.1 volume sodium 251

acetate pH 4.5 and 2 volumes ethanol followed by centrifugation. Pellets were washed 252

with 75% ethanol and resuspended in RNase-free water. 10 ug RNA was treated with 253

DNase I (Fermentas) at 37°C for 60 min and then purified with RNeasy RNA 254

purification columns (Qiagen) according to manufacture’s instructions. 1ug DNA-free 255

RNA was reverse transcribed into cDNA using random hexamer primers (Amersham) 256

and Superscript III reverse transcriptase (Invitrogen) in a 20ul reaction according to 257

manufacture’s instructions. Real-time PCR was performed on 2 µl of a 1:50 dilution 258

of the cDNA reaction using SYBR green PCR master mix (Applied Biosystems) and 259

primers specific to the indicated gene in a 7300 Real Time PCR System (Applied 260

Biosystems). For each sample, a control reaction was performed on RNA to be certain 261

no contaminating DNA was present. All samples were normalized to the wildtype 262

strain at zero hours of development. Representative RT-PCR patterns are shown but 263

similar patterns were obtained from at least two biological replicate experiments. 264

Immunoblot analysis. Protein lysates for Western Blot analyses were 265

obtained from cells developing under submerged culture for the indicated time points. 266

Lysates were prepared from cells pelleted and frozen at -20°C. Pellets were 267

resuspended in 0.4 ml MMC starvation buffer containing 1:200 dilution mammalian 268

protease inhibitor cocktail (Sigma) and disrupted by sonication with cooling using a 269

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Branson 250 sonifier equipped with a microtip. Cell lysates were quantitated using a 270

Bradford assay (Bio-Rad Laboratories), resuspended in 2x or 3x Laemmli sample 271

buffer (LSB) (24) to 0.5 µg µl-1

, heated at 99°C for 5-7 minutes and stored at -20°C. 272

10 µg protein lysates were resolved by denaturing SDS-PAGE using the following 273

acrylamide concentrations: 8% for EspA, 12% for MrpC and FrzCD, and 13% for 274

FruA. CsgA was resolved on Tris-Tricine gels as per (45). Proteins were transferred 275

to polyvinyldenedifluoride (PVDF) membrane using a semi-dry transfer apparatus 276

(Hoeffer). Western Blot analyses were performed using the following antibody 277

dilutions: α-EspA polyclonal antibodies (pAb) at 1:1500; α-FruA pAb, α-MrpC pAb 278

(35), and α-FrzCD (32) at 1:5000; α-CsgA C-terminal pAb (28) at 1:2500; and α-PilC 279

pAb (V. Jakovljevic and L. Sogaard-Andersen, unpublished) at 1:10 000. Secondary 280

α-rabbit IgG-horseradish peroxidase (HRP) antibody (Pierce) was used at 1:20 000 281

and signals were detected with enhanced chemiluminescence substrate (Pierce) 282

followed by exposure to autoradiography film. Representative immunoblot patterns 283

are shown but similar patterns were obtained from at least two biological replicates. 284

285

RESULTS 286

287

Analysis of EspA kinase activity. EspA (768 amino acids) is a two-288

component signal transduction hybrid histidine protein kinase homologue (4) (Fig. 289

1A). The sensor module in EspA consists of a Fork-head associated (FHA) domain 290

and two adjacent PAS/PAC domains. In this paper, we focused on the signal 291

transmitter/output domains: (i) residues 395-468 encode a HisKA dimerization 292

domain containing a conserved histidine at position 407 (H407) predicted to be the 293

site of autophosphorylation; (ii) residues 518-624 encode a HATPase_c domain 294

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predicted to be necessary for ATP binding and catalysis; and (iii) residues 650-760 295

encode a receiver domain containing a conserved aspartic acid at position 696 (D696) 296

predicted to be a phospho-acceptor residue. 297

To determine whether EspA has histidine kinase activity, we purified a GST-298

tagged recombinant protein containing EspA’s kinase region (HisKA and HATPase_c 299

domains) as well as a version bearing a H407 to alanine substitution (GST-EspAkin 300

and GST-EspAkinH407A, respectively) (Fig. 1A and B). These two proteins were 301

assayed for auto-phosphorylation in the presence of [γ-32

P]ATP. While a radioactive 302

band corresponding to GST-EspAkin could be readily detected, the corresponding 303

band for GST-EspAkinH407A was not detected, indicating that the kinase domain of 304

EspA is capable of autophosphorylation on His 407 (Fig. 2A and B, lanes 1 and 2). To 305

determine what effect the receiver domain has on this reaction, we purified and 306

analyzed a protein containing both the kinase and receiver domains (GST-EspAkin-rec) 307

and the same protein bearing a substitution of the conserved aspartic acid in the 308

receiver domain (GST-EspAkin-recD696A) (Fig. 1B). When these proteins were analyzed 309

under the same conditions as those employed for the kinase, no radio-labeled GST-310

EspAkin-rec product was detected, whereas the GST-EspAkin-recD696A band was readily 311

detected (Fig. 2A and B, lanes 3 and 4). The GST-EspAkin-rec protein was active 312

because we could detect turnover of ATP to ADP when the proteins were incubated in 313

the presence of [α-32

P]ATP and the reaction supernatant was subsequently analyzed 314

by thin layer chromatography for [α-32

P]ADP accumulation (Fig. 2C and D). These 315

results suggest that EspA becomes autophosphorylated on His407 followed by two 316

possibilities: 1) the phosphoryl group is transferred to D696 of the receiver domain 317

but is subsequently released as inorganic phosphate such that the kinase signal is 318

depleted either due to intrinsic instability or because EspA’s kinase domain also 319

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displays phosphatase activity, or 2) the intact receiver domain prevents 320

autophosphorylation but not ATP hydrolysis. 321

To determine whether kinase activity is necessary for EspA activity in vivo, 322

we generated a strain (PH1008) bearing a substitution of the H407 to alanine 323

(espAH407A) at the native espA locus (Fig. 1C). Analysis of the developmental 324

phenotypes of the espAH407A mutant in parallel with wildtype (strain DZ2) and the 325

espA-null mutant (∆espA; strain DZ4227) under submerged culture conditions 326

demonstrated that the espAH407A mutant behaved like the ∆espA mutant: the cells 327

began to aggregate between 12-18 hours, approximately 12 hours earlier than the 328

wildtype (Fig. 3A). Like the ∆espA mutant, the espAH407A mutant also produced 329

spores earlier than wildtype and produced spores outside of fruiting bodies (data not 330

shown). The espAH407A and ∆espA mutants also exhibited similar phenotypes when 331

development was induced on clone fruiting (CF) agar plates (data not shown). In 332

addition, immunoblot analysis indicated that EspAH407A is produced as a stable protein 333

(Fig. 3B, data described below). Together, these data indicate that the conserved H407 334

residue is absolutely required for EspA function and imply that kinase activity is 335

necessary for EspA-mediated control over developmental progression. 336

To analyze the role of the associated receiver domain in EspA, we similarly 337

generated mutants bearing either an alanine substitution of the conserved D696 in the 338

receiver domain (espAD696A; strain PH1009) or a deletion of the entire receiver domain 339

(espA∆REC; strain PH1007) (Fig. 1C). When subject to the same analyses described 340

above, these mutants both phenocopied the espAH407A and ∆espA mutants (Fig. 3A), 341

indicating that the receiver domain and in particular the D696 residue are absolutely 342

required for EspA function. 343

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Expression of EspA during development. Analysis of espA-lacZ fusions 344

demonstrated that espA is transcribed at the onset of starvation and peaks at 345

approximately 24 hours of development in the DZ2 strain (4). To determine the 346

protein expression pattern of EspA, we analyzed cell lysates harvested from a 347

developmental time course by immunoblot analysis using affinity purified antibodies 348

generated against the full-length EspA protein. An EspA-specific band was detected 349

which migrated at 83 kDa, near the predicted molecular mass for EspA (Fig. 3B). In a 350

developmental time-course, EspA was barely detectable under vegetative conditions, 351

but increased after the onset of starvation, with peak expression at 18-24 hours, after 352

which expression decreased (Fig. 3B). When we examined EspA expression in the 353

espAH407A, espAD696A and espA∆REC strains, we detected EspA proteins at the predicted 354

molecular masses of 83, 83, and 71kDa, respectively (Fig. 3B). Interestingly however, 355

we observed that mutations rendering EspA inactive changed the expression pattern 356

of the mutant EspA. While both EspA and mutant EspA showed similar levels from 357

0-6 hours, during the 6-12 hour period mutant EspA showed maximal expression 358

between 12-18 hours rather than at 18-24 hours observed in the wildtype. Furthermore, 359

the mutant EspA proteins were not detected after 18 hours while expression continued 360

in the wildtype, albeit at decreased levels after 24 hours. It should be noted that the 361

relative amount of EspA protein detected was the same between EspAH407A, 362

EspAD696A, and wildtype EspA. A less intense signal for EspA∆REC likely arose from 363

fewer epitopes in this deletion mutant. The early decrease in EspA signal in the espA 364

mutants is likely a secondary effect resulting from early sporulation in these mutants; 365

either EspA is not released from spores under the lysis conditions employed or is not 366

present in spores. However, our data suggest that wildtype EspA activity may be 367

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necessary for delaying espA transcription and/or protein accumulation from 6 to 12 368

hours and that EspA is involved in regulating its own expression. 369

We were therefore interested in determining whether the transcription of the 370

espA gene was upregulated earlier in an EspA-inactive strain compared to a strain in 371

which EspA was active. Quantitative real-time PCR analysis of espA expression from 372

wildtype versus the espAH407A mutant suggested that espA was indeed upregulated in 373

the espAH407A mutant between 6-12 hours compared to wildtype (Fig. 3C). These 374

observations suggest that EspA likely is involved in a phosphorylation-dependent 375

signaling pathway that ultimately downregulates its own transcription between 6-12 376

hours of development. 377

Developmental marker gene expression analysis in espA mutants. ∆espA 378

mutants aggregate and sporulate earlier than wildtype suggesting that in wildtype cells 379

EspA transiently represses development progression. However, it is unclear how and 380

when in the developmental pathway EspA mediates this developmental repression. 381

The M. xanthus developmental program is characterized by temporal cascades of gene 382

expression. To gain insight into the nature of EspA control, we examined the 383

expression of several characterized developmental genes (see Fig. 7) in wildtype vs. 384

∆espA strains in submerged culture conditions to determine how defects in EspA 385

affect other genes involved in the developmental pathway. 386

To address whether ∆espA mutants were altered in production or reception of 387

the A-signal, we analyzed the expression of the A-signal responsive gene, spi (21) 388

(originally identified as Tn5 lac Ω4521) (18), by real-time PCR as the cells 389

progressed through the developmental program. Our analyses indicated that in 390

wildtype DZ2 cells, spi was upregulated by 30 minutes after induction of starvation 391

with peak expression by 2-4 hours (Fig. 4A). We observed no significant difference in 392

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transcription of spi between wildtype and ∆espA mutant, suggesting that espA mutants 393

are not impaired in production or reception of the A-signal. 394

We next analyzed initial mrpC transcription which is also indirectly 395

responsive to A-signal (56). In DZ2, mrpC expression was upregulated after onset of 396

starvation and continued to rise over the course of the 12 hours of development for 397

which it was analyzed (Fig. 4B). In the espA strain, mrpC expression was similar to 398

that of wildtype (Fig. 4B). 399

Analysis of fruA gene transcription in DZ2 showed that fruA was upregulated 400

after onset of starvation in DZ2 with peak expression between 12-24 hours (Fig. 4C). 401

In the espA mutant, fruA expression was essentially similar to wildtype from 0-6 402

hours. However, by 12 hours, fruA transcript was 5.6 fold higher in the ∆espA cells, 403

after which it appeared to decrease (Fig. 4C). This early upregulation of fruA was 404

consistently observed in several biological replicates (data not shown). 405

Expression of the dev locus depends at least in part on regulation by FruA (62) 406

and expression of Mxan_3227 (Tn5 lacZ Ω7536) depends upon the dev locus (27). In 407

DZ2, devR was upregulated between 6-12 hours of development and began to 408

decrease between 24-30 hours (Fig. 4D). In the espA strain, consistent with the 409

premature expression of fruA, devR was expressed approximately 16 times higher 410

from 6-12 hours in the espA mutant and began to decrease between 12-18 hours (Fig. 411

4D). In a similar manner, expression of Mxan_3227 was markedly upregulated in the 412

espA mutant between 24-30 hours compared to wildtype, which peaks between 30-36 413

hours (Fig. 4E). Together, these data suggest that in espA mutants the developmental 414

program begins to be perturbed after A-signaling but before transcription of fruA. 415

416

Developmental marker protein expression analysis in espA mutants. 417

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418

To further examine how the early expression of fruA might be controlled in the ∆espA 419

mutant, we examined the protein expression patterns of several key developmental 420

regulators (see Fig. 7). First, we determined that the expression of FruA was 421

upregulated earlier in the ∆espA mutant (between 0-6 hours) compared to wildtype 422

(12-18 hours) (Fig. 5A), consistent with the early expression of fruA mRNA as was 423

detected by real-time PCR (Fig. 4C). 424

MrpC protein is required for induction of fruA transcription (34, 35, 60); we 425

therefore examined the accumulation of MrpC. It had previously been determined that 426

in wildtype M. xanthus strain DZF1 (FB), MrpC can be detected in two forms: full-427

length MrpC present in vegetative and developing cells and proteolytically processed 428

MrpC2 present in only developing cells (35). Immunoblot analysis of MrpC 429

expression in wildtype DZ2 cells revealed four MrpC bands (Fig. 5B). We attribute 430

the full-length form (Fig. 5B solid arrow) to the species detected in vegetative cells. 431

The smaller MrpC2 species (Fig. 5B open arrow) was detected between 24-30 hours 432

in DZ2. We attribute the additional more slowly migrating bands (Fig. 5B dashed 433

arrows) to phosphorylated forms of MrpC which were inferred, but not detected, in 434

the earlier study (35). It is not clear whether the detection of these putative 435

phosphorylated forms is due to differences in immunoblot techniques or due to the 436

difference of development in strains DZ2 versus DZF1. The DZ2 strain develops 437

significantly more slowly than the DZF1 strain, perhaps allowing detection of more 438

transient isoforms of MrpC. In the ∆espA mutant, all four forms of MrpC were 439

detected except that the respective MrpC isoforms began to accumulate 6 to 12 hours 440

earlier than in the wildtype (Fig. 5B). in vitro data suggests that fruA is regulated by 441

MrpC2 (35, 60). However, in both the wildtype and espA mutant, the upregulation of 442

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fruA expression correlated with accumulation of the full-length MrpC isoforms (at 18 443

and 6 hours development, respectively), although low levels of MrpC2 can be 444

detected at earlier time points on extended exposures (data not shown). The 445

observation that MrpC protein accumulation is higher in the espA mutants, whereas 446

the mrpC transcription is the same as wildtype during the first 12 hours of 447

development (Fig. 4B) suggests that EspA may play a role in transiently repressing 448

MrpC accumulation early during the developmental program. 449

FruA activity is proposed to be modulated by the C-signal pathway (5). We 450

next examined whether we could detect differential expression of CsgA. A sharp 451

increase of CsgA was observed between 6-12 hours in the espA mutant compared to a 452

more gradual accumulation between 6-18 hours in the wildtype strain (Fig. 5C). These 453

results suggest that the C-signal amplification pathway is also functioning earlier in 454

the espA mutant. 455

It has been suggested that activated FruA directly or indirectly influences the 456

activity of the Frz chemotaxis pathway (53). Development-related signaling of the Frz 457

chemosensory pathway can be followed by the increased methylation of FrzCD 458

receptor, which is a methyl-accepting chemotaxis protein (MCP) that becomes highly 459

methylated during development (31). We therefore next examined the developmental 460

expression pattern of FrzCD in the ∆espA and wildtype strains. FrzCD with different 461

methylation, demethylation or deamidation levels can be detected as multiple bands 462

on immunoblots using anti-FrzCD serum (30). Under the culture conditions used here, 463

FrzCD harvested from cells incubated in submerged culture with vegetative media for 464

24 hours was primarily present as unmethylated species (Fig. 5D closed arrow). After 465

onset of development (vegetative media replaced with starvation media) between 0-12 466

hours, FrzCD was detected primarily as methylated species (Fig. 5D open arrow). 467

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Between 12-24 hours, FrzCD became partially less methylated and then finally after 468

24 hours, commensurate with formation of mounds (Fig. 3), FrzCD was detected as 469

fully methylated species. This pattern of FrzCD methylation over the course of the 470

developmental program in which FrzCD undergoes partial transient demethylation 471

just before mound formation differs from previously published methylation patterns 472

using wildtype strains DZF1 (FB) (31) or DK1622 (53). As discussed above for MrpC, 473

the more slowly developing wildtype DZ2 strain may allow for detection of more 474

transient FrzCD methylation states. In the espA strain, the same overall pattern of 475

FrzCD methylation was detected except that the pattern was shifted six hours earlier 476

(between 6-12 hours development) than in the wildtype. In the ∆espA strain, mound 477

formation was observed between 12-18 hours indicating that the FrzCD pool 478

remained as partially demethylated after mounds began to form (Fig. 3). These results 479

suggest that in the ∆espA mutant, the FrzCD-methylation status is also modulated 480

earlier than in the wildtype, consistent with the early aggregation phenotype displayed 481

by the ∆espA mutant. 482

As a control for loading, we also examined the expression of PilC, a pilus 483

associated protein (64, 65) which is not predicted to be developmentally regulated. 484

From 0-18 hours PilC protein was detected at the same levels in the ∆espA strain 485

compared to wildtype (Fig. 5E), indicating that the early expression of MrpC, FruA, 486

or CsgA in the espA mutants was not simply due to overloaded protein samples. 487

Consistent with the premature sporulation observed in the espA mutant, PilC was 488

detected at reduced levels during the later developmental time points. 489

Analysis of espA double mutants with key developmental genes. Analysis 490

of developmental gene and protein expression markers in the espA mutant indicated 491

that EspA acts on the developmental program after A-signaling but before the action 492

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of FruA. We therefore generated asgA, fruA, and csgA mutants in the DZ2 wildtype 493

strain and asgA, fruA, csgA and frzCD mutants in the ∆espA (DZ4227) strain and 494

analyzed the developmental phenotypes. Mutants in asgA, fruA, and csgA in the DZ2 495

background yielded the expected phenotypes (Fig. 6) that have been previously 496

observed in the DK1622 or FB (DZF1) backgrounds (5, 9, 36, 39). 497

The analysis of spi expression in the ∆espA mutant suggested that A-signaling 498

is not affected in the espA mutant. Mutants in asgA do not produce A-signal and do 499

not develop. ∆espA asgA double mutants phenocopied the asgA single mutant 500

indicating that completion of A-signaling is a prerequisite of EspA-mediated 501

developmental repression (Fig. 6). Our model suggests that the early aggregation and 502

sporulation observed in the espA mutant is due to premature expression of FruA. 503

∆espA fruA double mutants displayed a fruA phenotype (Fig. 6) indicating that 504

EspA’s effect on aggregation and sporulation requires FruA. Current models for the 505

molecular mechanisms controlling the developmental program in M. xanthus suggest 506

that FruA activity is modulated in response to the C-signal amplification pathway (5). 507

Interestingly, the double ∆espA csgA mutant was able to aggregate and form spores, 508

albeit at a delayed and reduced level compared to wildtype or the ∆espA single mutant 509

(Fig. 6 and data not shown). This phenotype suggests that in espA mutants, C-510

signaling can be partially bypassed. Finally, analysis of ∆espA frzCD double mutants 511

yielded a mixed phenotype: like the frzCD parent mutant, cells formed frizzy 512

filaments rather than aggregation centers but produced elevated levels of spores 513

consistent with the espA parent (Fig 6). 514

515

DISCUSSION 516

517

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EspA was previously characterized as a putative histidine kinase that is 518

necessary for appropriate timing of sporulation during development (4). To gain 519

further insight as to how EspA affects the developmental program, we first set out to 520

analyze whether EspA was indeed functioning as a histidine protein kinase. Our 521

biochemical analyses indicated that the kinase domain of EspA is capable of in vitro 522

auto-phosphorylation on the conserved histidine residue at amino acid position 407, 523

and analyses of an espAH407A point mutation in vivo indicate that kinase activity is 524

required for EspA-mediated control over developmental progression. The observation 525

that espAD696A and espA∆REC mutants phenocopy the kinase-inactive espAH407A mutant 526

indicates that phosphotransfer to the receiver domain is a required step in EspA-527

mediated control over the developmental program. These results clarify our in vitro 528

phosphorylation results and suggest that the presence of the receiver domain disrupts 529

the accumulation of signal on the kinase domain because the phosphoryl group is 530

transferred from the histidine residue to the conserved aspartate at position 696, but 531

the phosphoryl group is rapidly released as inorganic phosphate. A similar result was 532

observed with the hybrid histidine kinase LuxN which controls luminescence 533

production in Vibrio species (6, 59). 534

If transfer of the phosphoryl group to the receiver domain is a required step of 535

signal transduction in EspA function, what is the signal output from EspA? One 536

possibility is that EspA participates in a four-step phospho-relay first to an 537

unidentified histidine phosphotransferase (Hpt) protein and then to a response 538

regulator protein again in a manner analogous to that of the LuxN(Q)/LuxU/LuxO 539

system. Hpt proteins involved in four step phospho-relay signal transmission are 540

difficult to identify based on sequence alone (1), and current annotation of the M. 541

xanthus genome does not identify convincing candidates that could serve as a partner 542

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for EspA. In addition, the M. xanthus genome encodes 64 orphan two-component 543

response regulators (49) indicating that identification of a cognate output protein(s) is 544

challenging. Another intriguing possibility is that EspA may mediate its output 545

directly, perhaps through direct protein interaction with other developmental 546

regulators. 547

To attempt to identify how EspA might affect the developmental program and 548

thus to pinpoint possible output mechanisms, we next sought to analyze when the 549

developmental program began to differ from wildtype in the espA mutant. We used 550

quantitative real-time PCR to analyze the expression patterns of several established 551

developmental marker genes known to be expressed in a specific order during the 552

developmental pathway (outlined in Fig. 7). These analyses determined that in the 553

espA mutant, expression of the A-signal dependent spi gene and initial upregulation of 554

mrpC expression (which is also partially dependent on A-signaling) was not altered 555

compared to wildtype, whereas expression of fruA, dev and Mxan_4227 mRNAs are 556

upregulated earlier in the espA mutant compared to wildtype (Fig. 4). These data 557

suggest that EspA normally begins to delay the developmental program after the 558

completion of A-signaling but before the upregulation of the major developmental 559

regulator, fruA. 560

Examination of the protein expression profile of MrpC, a key developmental 561

transcriptional regulator which is necessary for transcription of fruA, clarified why 562

fruA is upregulated earlier in the espA mutants. A key observation here is that MrpC 563

protein accumulates between 0-6 hours of development in the espA mutant 564

(approximately 12 hours earlier than in the wildtype) (Fig. 5B), although mrpC 565

expression is not different from wildtype during this time period (Fig. 4B). Our data 566

suggest that EspA is involved in regulating MrpC protein accumulation, perhaps at 567

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the level of translation or degradation. It should be noted that while the early MrpC 568

accumulation in the espA mutant correlated with early fruA transcription, we did not 569

observe an effect of MprC on mrpC transcription over wildtype in the first 12 hours of 570

development (Fig. 4B). While a possible explanation is that the MrpC2 isoform 571

displays higher affinity for the fruA promoter than the mrpC promoter, based on in 572

vitro studies (35), it is also likely that in vivo the situation is more complicated 573

involving factors not yet identified. 574

MrpC appears to be a key point of developmental control. First, it has 575

previously been determined that early development via premature fruA expression due 576

to modulation of MrpC is observed pkn8 or pkn14 mutants; in these mutants MrpC 577

cannot be phosphorylated and MrpC2, which is normally only detected during 578

development, is detected in vegetative conditions (34). Second, it has recently been 579

demonstrated that unphosphorylated MrpC acts as an anti-toxin for MazF, an mRNA 580

interferase that is necessary development because it induces a percentage of cells to 581

undergo autolysis (33). MrpC was also shown to be a mazF transcriptional activator 582

and the binding of MrpC to the mazF promoter was prevented by the MrpC-MazF 583

complex. Interestingly however, MrpC expression is apparently not altered in a mazF 584

mutant suggesting the interplay of the various forms of MrpC (MrpC, MprC2, MrpC-585

P, and MrpC-MazF) may effect MrpC’s activity on different target promoters (mrpC, 586

fruA, mazF) differently (33). It is worth noting that in the espA mutant, the relative 587

proportions of each MrpC isoform was not altered compared to wildtype, confirming 588

that the premature MrpC accumulation is not specifically due to differential 589

processing of MrpC and suggesting that MrpC’s interaction with MazF is not 590

perturbed. Differential protein accumulation of MrpC in the espA mutant may then 591

represent a third level of control of MrpC activity. 592

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We suggest that the premature accumulation of MrpC in the espA mutant is 593

the cause of early development in the mutant and can explain most of our results in 594

this paper (Fig. 7). First, A-signaling as monitored by spi gene expression was 595

unaffected in the espA mutant. It has previously been demonstrated that mrpC is 596

developmentally upregulated by the MrpAB two-component signal transduction 597

system (57) in a manner that is facilitated by A-signaling (56). The observation that 598

the initial upregulation of mrpC is not altered in the espA mutant is consistent with the 599

observation that A-signaling is not altered (Fig. 4A). In addition, espA asgA double 600

mutants display an asgA phenotype (Fig. 6) which would be expected since MrpC 601

expression should be reduced (56). Second, premature expression of MrpC induces 602

premature transcription of fruA and consequent production of FruA (Fig. 4C and 5A). 603

Premature FruA expression in turn leads to premature alteration of FrzCD 604

methylation patterns (Fig. 5D), which suggests that signaling is occurring through the 605

Frz chemosensory system promoting aggregation. FruA also acts as a transcription 606

factor for the dev locus (63), and devR is transcribed earlier in the espA strain (Fig. 607

4D). In addition, Mxan_3227 expression is dependent upon dev expression (27), and 608

the premature expression of Mxan_3227 in espA mutants (Fig. 4E) is likely a 609

consequence of premature expression of dev. dev and Mxan_3227 are both required 610

for sporulation inside of fruiting bodies (27, 58) and their premature expression is 611

consistent with the early sporulation phenotype in espA mutants [(4) and data not 612

shown]. Furthermore, espA fruA double mutants display a fruA phenotype (Fig. 6) 613

consistent with the fact that FruA is an essential component of the pathway affected in 614

espA mutants. However, espA frzCD double mutants display a mixed phenotype: the 615

observed frizzy aggregation is frzCD-like but the elevated sporulation is espA-like. 616

This result is to be expected if premature FruA expression requires FrzCD to promote 617

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normal aggregation, but can still promote sporulation through the dev and Mxan_3227 618

loci. 619

We observed that expression of CsgA is slightly early in espA mutants (Fig. 620

5C) which presumably arises because early FruA accumulation stimulates aggregation 621

through the FrzCD pathway. As the cells begin to stream into aggregates, end-to-end 622

contact stimulates C-signaling and CsgA expression is amplified. Interestingly 623

however, espA csgA mutants are able to aggregate and sporulate albeit at delayed and 624

reduced levels compared to wildtype (Fig. 6), suggesting that espA mutants can 625

partially bypass the requirement for C-signaling. This result suggests EspA may play 626

an additional unidentified role during development downstream of C-signal. It is 627

interesting that a similar partial bypass of C-signaling (but not of FruA) has been 628

previously observed with mutants in the unusual histidine protein kinase, rodK (40). 629

But what advantage does EspA’s repressive function provide for the 630

developmental program? We speculate that EspA aids in coordinating development 631

by delaying developmental progression until a threshold of an unidentified signal(s) 632

are sensed such that the multiple events (starvation sensing, population sensing etc) 633

that must be integrated to generate compact fruiting bodies are coordinated. In the 634

espA strains, premature aggregation and sporulation is associated with slightly 635

disorganized fruiting bodies and formation of spores amongst the peripheral rods. It is 636

likely that well organized compact fruiting bodies offer a selective advantage in 637

dissemination of groups of spores since we presume germination in groups allows for 638

more rapid growth based on the social aspect of M. xanthus feeding. Our results 639

suggest a model in which phosphorylated EspA acts after the completion of A-640

signaling to inhibit, via an unknown mechanism, accumulation of MrpC (Fig. 7). 641

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Upon sensing a change in an unidentified signal, EspA then relieves the inhibition, 642

MrpC protein is allowed to accumulate and the developmental program progresses. 643

The nature of the signal(s) sensed by EspA is currently unknown. Genetic 644

epistasis experiments suggest that EspA acts downstream from a signaling module 645

consisting of PktA5 and/or PktB8, two serine/threonine protein kinases, and EspB a 646

membrane protein (4, 55). Interestingly, EspA contains multiple sensing domains: a 647

Forkhead-associated domain which is likely necessary for interaction with PktA5 648

and/or PktB8 (55), and two adjacent PAS/PAC domains which are likely involved in 649

sensing the redox potential of the cell. We are currently investigating the exact role(s) 650

of the sensing domains in modulation of EspA activity. 651

The observation that mutants in several other histidine protein kinase 652

homologs [TodK (41), EspC (25), and RedCDEF (11)] display similar mutant 653

phenotypes to espA suggests that regulation of developmental progression is subject 654

to a sophisticated signal transduction network; we are currently exploring how these 655

other signaling systems control this complex developmental process. 656

657

ACKNOWLEDGMENTS 658

659

We gratefully acknowledge S. Inouye for anti-MrpC antibody, L. Søgaard-660

Andersen for anti-FruA and anti-PilC antibodies, and Namita Kothari for her 661

contributions to EspA phosphorylation analyses. We are also grateful to L. Søgaard-662

Andersen, K. Thorman, and S. Huntley for critical review of this manuscript. 663

This work was funded by the Max Planck Society (PIH) and the National 664

Institutes of Health (DZ) GM64463 and GM20509. 665

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Microbiol 23:109-21. 854

855

856

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859

Table 1. Bacterial strains and plasmids used in this study. 860

861

Strain or plasmid Relevant genotype Source or reference 862

863

M. xanthus strains 864

DZ2 Wild-type (3) 865

DZ4227 DZ2 ∆espA573 (4) 866

PH1008 DZ2 espAH407A This study 867

PH1009 DZ2 espAD696A This study 868

PH1007 DZ2 espA∆REC This study 869

PH1010 DZ2 asgA::pPH127 This study 870

PH1011 DZ4227 asgA::pPH127 This study 871

DK9035 DK1622 csgA::Tn5-132ΩLS205 872

∆frz(' CD-F):: Kan ' (54) 873

PH1014 DZ2 csgA:: Tn5-132ΩLS205 This study 874

PH1015 DZ4227 Tn5-132ΩLS205 This study 875

PH1013 DZ2 fruA::pPH128 This study 876

PH1012 DZ4227 fruA::pPH128 This study 877

DZ4169 DZ2 frzCD::Tn5 (47) 878

PH1016 DZ4227 frzCD::Tn5 This study 879

880

E. coli strains 881

TOP10 host for cloning Invitrogen 882

BL21λDE3 host for protein expression Novagen 883

884

Plasmids 885

pBJ114 backbone for deletions, galK, KmR (12) 886

pPH150 pBJ114 espAH407A This study 887

pPH148 pBJ114 espAD696A This study 888

pPH124 pBJ114 espA∆REC This study 889

pPH127 pBJ114 asgA This study 890

pPH128 pBJ114 fruA This study 891

pGEX-4T-1 GST expression plasmid, ApR Amersham 892

pPH141 pGEX-4T-1 espA kinase This study 893

pPH143 pGEX-4T-1 espA kinase-receiver This study 894

pPH156 pGEX-4T-1 espA kinaseH407A This study 895

pPH157 pGEX-4T-1 espA kinase-receiverD696A This study 896

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Figure Legends 898

899

Fig. 1. A. Domain organization of EspA protein depicted by SMART analysis (26, 900

46). FHA: Fork-head associated domain; PAS/PAC: energy sensing domains; HisKA 901

histidine kinase dimerization domain; HATPase_c: histidine kinase ATPase domain; 902

REC: receiver domain. B. Constructs for analysis of EspA kinase activity in vitro. C. 903

Mutations generated in the espA locus for in vivo analyses. 904

905

906

Fig. 2. Analysis of EspA kinase activity. A. Autoradiograph of GST-tagged -EspA 907

kinase (GST-EspAkin; lane 1), -EspA kinase H407A (GST-EspAkinH407A; lane 2), -908

EspA kinase-receiver (GST-EspAkin-rec; lane 3) and -EspA kinase-receiver D696A 909

(GST-EspAkin-recD696A; lane 4) incubated in the presence of [γ-32

P]ATP. B. Coomassie 910

stained gel corresponding to A. C. Thin layer chromatography analysis of [α-32

P]ADP 911

produced by [α-32

P]ATP (lane 1), GST-EspAkin (lane 2), GST-EspAkin-rec (lane 3), and 912

GST-EspAkin-recD696A (lane 4). D. Quantification of [α-32

P]ADP signal in C. as an 913

average from triplicate experiments. 914

915

Fig. 3. A. Developmental phenotype of espA mutants compared to wildtype. Cultures 916

of DZ2 (wildtype), DZ4227 (∆espA), PH1008 (espAH407A), PH1009 (espAD696A) and 917

PH1007 (espA∆REC) were induced to develop under submerged culture conditions at 918

32°C. Pictures were recorded at the indicated hours. Line: 1mm. B. Western Blot 919

analysis of EspA expression. 10 µg protein lysates prepared from cells in A. harvested 920

at the indicated hours development were subject to immunoblot with α-EspA 921

polyclonal antisera. Black arrows 83 kDa; white arrow 71 kDa. C. Quantitative real-922

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time PCR analysis of espA mRNA expression. Total RNA isolated from DZ2 923

wildtype (white diamonds) or PH1008 (espAH407A) (black squares) cells at the 924

indicated hours development was transcribed into cDNA. espA transcripts were 925

detected by real-time PCR. 926

927

Fig. 4. Quantitative real-time PCR analysis of developmental marker gene 928

transcription in wildtype (strain DZ2; white diamonds) and ∆espA (strain DZ4227; 929

black squares). Cells were developed in submerged culture and harvested at the 930

indicated time points. RNA was isolated and reverse transcribed into cDNA. Primers 931

specific for spi (A), mrpC (B), fruA (C), devR (D) or Mxan_3227 (E) were used for 932

real-time PCR analysis. 933

934

Fig. 5. Immunoblot analysis of developmental marker protein expression in wildtype 935

(strain DZ2) and ∆espA (strain DZ4227). Cells were grown in submerged culture and 936

harvested at the indicated time points. Samples containing 10ug protein were subject 937

to immunoblot analysis and probed with anti-FruA (A), anti-MrpC (B), anti-CsgA (C), 938

anti-FrzCD (D), or anti-PilC (E) polyclonal antisera. B: black arrows MrpC; white 939

arrows MrpC2; C: CsgA p25; D: black arrows FrzCD unmethylated species, white 940

arrows FrzCD methylated species. 941

942

Fig. 6. Developmental phenotype of key developmental genes in wildtype (DZ2; left 943

column) or ∆espA (DZ4227; right column) backgrounds. asgA (PH1010), espA asgA 944

(PH1011), fruA (PH1013) espA fruA (PH1012) csgA (PH1014), espA csgA (PH1015), 945

frzCD (DZ4169) and espA frzCD (PH1016) strains were induced to develop for 72 946

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hours on CF plates. Heat and sonication resistant spores were enumerated at 72 hours 947

development. Values are displayed as percent of wildtype spores. 948

949

Fig. 7. Molecular events during M. xanthus developmental program (top) in relation 950

to aggregation and sporulation (bottom). The roles of the developmental gene and 951

protein expression patterns that are assayed in the espA mutants are depicted; see text 952

for details. Pkn14 phosphorylates MrpC (not shown) which prevents MrpC2 953

accumulation (35). EspA may repress protein accumulation of MrpC. Solid lines 954

represent direct interactions; dashed lines indicate mechanisms of action are indirect 955

or unknown. Thick solid arrow represents time. Groups of M. xanthus cells (grey 956

rectangles) first responding to nutrient limitation and A-signal (i), begin to aggregate 957

(ii) into mounds (iii) and then form spores (grey circles) within the mounds (iv). 958

959

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Fig. 1

H407 D696

EspA

HEspAkin

AEspAkinH407A

H DEspAkin-rec

AHEspAkin-recD696A

A

B

CA D

EspAH407A

HEspA

∆REC

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Fig. 2

1 2 3 4

[α-32P]ADP

[α-32P]ATP

[α-3

2P

]AD

P

1 2 3 4

GST-EspAkin-rec

GST-EspAkin

GST-EspAkin-rec

GST-EspAkin

A

B

C

D

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

1 2 3 40.0

3.0

2.0

1.0

1 2 3 4

kDa

70

40

55

70

40

55

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A

B C

Fig. 3wt ∆espA espAH407A espAD696A

12

18

24

30

36

hours

dev

elopm

ent

espA∆REC

-0.5

0.5

1.5

2.5

3.5

4.5

0 6 12 18 24

hours development

0 12 18 24 306

wt

∆espA

espAH407A

espAD696A

espA∆REC

hours development

log2 i

ndu

ctio

n

wild type

∆espA

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spi

log2 i

ndu

ctio

n

hours development

A

mrpC

log2 i

ndu

ctio

n

hours development

B

devR

log2 i

ndu

ctio

n

hours development

D

Mxan_3227

log2 i

ndu

ctio

n

hours development

E

fruA

log2 i

ndu

ctio

n

hours development

C

Fig. 4

0

2

4

6

8

10

12

0 2 4 6 8 10 12

-1

0

1

2

3

0 2 4 6 8 10 12

0

2

4

6

8

10

0 6 12 18 24 30 36

0

2

4

6

0 6 12 18 24 30 36 42

-4

-2

0

2

4

6

8

0 6 12 18 24 30 36 42 48

wild type

∆espA

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wt

∆espA

MrpC

MrpC

wt

∆espA

FruA

FruA

Fig. 5

0 1812 24 306

A

D

C

B

E

hours development

FrzCD

FrzCD

wt

∆espA

CsgA

CsgA

wt

∆espA

wt

∆espA

PilC

PilC

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Fig. 6

asgA

csgA

frzCD

fruA

wt ∆espA

142%

40%

100%

19% 71%

<0.5%

<0.5%<0.5%

<0.5%

<0.5%

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Fig. 7

starvation

A-signal

spi mrpC

MrpC

MrpC2

fruA

FruA

FruA~P

dev

FrzCDCH3

aggregation

sporulation

Mxan_3227

C-signal

EspA

Pkn14

CsgA

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Documents

1 2 3 ACCEPTED - Journal of Bacteriology › content › jb › early › 2008 › 04 › 04 › JB.00265...2008/04/04 · 11 Marburg, D35043, Germany 12 2Department of Molecular