JB Accepted Manuscript Posted Online 9 February 2015 J. Bacteriol. doi:10.1128/JB.02588-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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CodY regulates expression of the Bacillus subtilis extracellular proteases Vpr and Mpr

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Giulia Barbieria, Birgit Voigtb, Dirk Albrechtb, Michael Heckerb, Alessandra M. Albertinia,

9

Abraham L. Sonensheinc, Eugenio Ferraria*, and Boris R. Belitskyc#

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Dipartimento di Biologia e Biotecnologie “Lazzaro Spallanzani”, Università di Pavia, Pavia,

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Italya; Institute for Microbiology, Ernst-Moritz-Arndt University, Greifswald, Germanyb;

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Department of Molecular Biology and Microbiology, Tufts University School of Medicine,

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Boston, Massachusetts, USAc

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Running title: CodY-mediated regulation of B. subtilis exoproteases

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# Address correspondence to Boris Belitsky, [email protected]

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*Present address: Pronutria, Cambridge, Massachusetts, USA.

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1

Abstract

23 24

CodY is a GTP- and branched-chain amino acid-responsive global transcriptional

25

regulator in low G+C Gram-positive bacteria. By interacting with its two cofactors, it is able to

26

sense the nutritional and energetic status of the cell and respond by regulating expression of

27

adaptive genetic programs. In B. subtilis, more than two hundred genes, including peptide

28

transporters, intracellular proteolytic enzymes and amino acid degradative pathways, are

29

controlled by CodY.

30

proteases, Vpr and Mpr, is negatively controlled by CodY. By gel mobility shift and DNase I

31

footprinting assays, we showed that CodY binds to the regulatory regions of both genes, in the

32

vicinity of their transcription start points. The mpr gene is also characterized by the presence of a

33

second, higher-affinity CodY-binding site located at the beginning of its coding sequence. Using

34

strains carrying vpr- or mpr-lacZ transcriptional fusions in which CodY-binding sites were

35

mutated, we demonstrated that repression of both protease genes is due to the direct effect by

36

CodY and that thе mpr internal site is required for regulation. The vpr promoter is a rare example

37

of a sigma H-dependent promoter that is regulated by CodY. In a codY null mutant, Vpr became

38

one of the more abundant proteins of the B. subtilis exoproteome.

In this work, we demonstrated that expression of two extracellular

39 40

Importance

41

CodY is a global transcriptional regulator of metabolism and virulence in low G+C gram-

42

positive bacteria. In B. subtilis, more than two hundred genes, including peptide transporters,

43

intracellular proteolytic enzymes and amino acid degradative pathways, are controlled by CodY.

44

However, no role for B. subtilis CodY in regulating expression of extracellular proteases has

45

been established to date. In this work we demonstrate that, by binding to the regulatory regions

2

46

of the corresponding genes, B. subtilis CodY negatively controls expression of Vpr and Mpr, two

47

extracellular proteases. Thus, in B. subtilis, CodY can now be seen to regulate the entire protein

48

utilization pathway.

3

Introduction

49 50

First identified in Bacillus subtilis (1), CodY is a global transcriptional regulator whose

51

homologues are found almost ubiquitously in low G+C Gram-positive bacteria (2). DNA-

52

microarray and ChIP-to-chip experiments and, most recently, in vitro DNA-binding assays

53

coupled with massively parallel sequencing (the IDAP-Seq method) and genome-wide profiling

54

of transcription by RNA-Seq (3-5) have shown that in B. subtilis CodY regulates over 200 genes,

55

many of which encode components of metabolic pathways, are repressed during growth in the

56

presence of excess nutrients and are involved in the adaptation to poor growth conditions (6).

57

Although CodY acts mainly as a repressor, some B. subtilis genes are under positive CodY

58

regulation (3, 5, 7).

59

In all other CodY-expressing species examined to date, CodY also controls multiple

60

metabolic pathways (2, 8-12). In pathogenic species, key virulence genes are also under CodY

61

control (10-23). CodY can control transcription by binding in the vicinity of the promoter region

62

of the target genes, by competing with a positive regulator for binding, or by serving as a

63

roadblock to RNA polymerase (24).

64

Binding of CodY to DNA requires in most cases at least a moderately conserved version

65

of a 15-nt consensus motif (AATTTTCWGAAAATT) (4, 9, 25, 26) and is enhanced by its

66

interaction with two classes of effector molecules that act as signals of the nutritional status of

67

the cell: the branched-chain amino acids isoleucine, leucine, and valine (ILV) (27, 28) and GTP

68

(29-31). Varying the concentration of activated CodY results in a hierarchical, programmed

69

regulation of gene expression that presumably allows the cell to adapt in different ways to

70

varying levels of nutritional availability (5).

4

71

Extracellular proteases are thought to be involved in nutrient acquisition. B. subtilis

72

produces at least eight characterized extracellular or cell wall-associated proteases. The alkaline

73

serine protease subtilisin (AprE) and the neutral metalloprotease NprE, commonly referred to as

74

the major extracellular proteases, account for more than 95% of the total extracellular protease

75

activity of B. subtilis (32). The remaining protease activity is due to minor extracellular

76

proteases, which include the serine proteases Epr (33), bacillopeptidase F (Bpr) (34, 35) and Vpr

77

(36), the neutral protease B (NprB) (37), the cell wall-associated protease (WprA) (38), and the

78

metalloprotease (Mpr) (39, 40). None of them is essential for growth or sporulation of B. subtilis,

79

but transcription of most of the protease genes is tightly controlled by different regulators, such

80

as AbrB, DegU, ScoC, SinR, and SpoIIID, that allow their expression to be induced during

81

nutrient exhaustion in stationary phase (37, 41-46).

82

The ability of CodY to regulate proteolytic activity was well documented in Lactococcus

83

lactis (28), while the effect of CodY on expression of B. subtilis extracellular proteases has been

84

uncertain. Although the aprE and nprE genes were identified as CodY-binding targets by ChIP-

85

to-chip and IDAP-Seq experiments, no effect of CodY was detected on expression of these genes

86

in microarray and RNA-Seq experiments (3-5). In contrast, though global analysis of gene

87

expression and IDAP-Seq experiments indicated that vpr and mpr are negatively regulated by

88

CodY, these genes were not identified as CodY targets by ChiP-to-chip experiments (3-5).

89

In this work we sought to test whether vpr and mpr are direct targets of B. subtilis CodY

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in vivo and in vitro. The results show that these two protease genes are indeed regulated by CodY

91

and that, when regulation by CodY is eliminated, Vpr becomes a major component of the

92

exoproteome.

5

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Materials and Methods

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Bacterial strains and culture media. The B. subtilis strains constructed and used in this study

95

were all derivatives of strain SMY (47) and are reported in Table 1. They were grown at 37°C in

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DS nutrient broth medium or in TSS minimal medium with 0.5% glucose as carbon source and

97

0.2% NH4Cl as nitrogen source (48). The same media with the addition of agar were used for

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growth of bacteria on plates. The TSS medium was supplemented as indicated with a mixture of

99

16 amino acids (aa) (49), which contained all amino acids commonly found in proteins except

100

for glutamine, asparagine, histidine, and tyrosine; the branched-chain amino acids ILV were

101

added at a final concentration of 200 μg/ml each. In TSS + 13 aa, ILV were omitted. For

102

proteomics experiments, TSS was supplemented with 2 mM CaCl2  2H2O and 10 µM MnSO4 

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4H2O. Escherichia coli strains JM107 (50) or DH5α (51) were used for isolation of plasmids and

104

were grown in LB medium (52). The following antibiotics were used when appropriate:

105

tetracycline (15 μg/ml) or spectinomycin (50 μg/ml) or chloramphenicol (5 µg/ml) or the

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combination of erythromycin (0.5 μg/ml) and lincomycin (12.5 μg/ml) for B. subtilis strains, and

107

ampicillin (50 μg/ml) for E. coli strains.

108

DNA manipulations. Methods for common DNA manipulations and bacterial transformation

109

were previously described (26, 53). Chromosomal DNA of B. subtilis strain SMY or plasmids

110

constructed in this work were used as templates for PCR. Plasmids isolated from E. coli strain

111

DH5 α were subjected to rolling circle amplification using the “Illustra TempliPhi 100

112

Amplification Kit” (GE Healthcare) before being used to transform B. subtilis competent cells.

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The oligonucleotides used in this work are described in Table 2. All cloned PCR-generated

114

fragments were verified by sequencing.

115

Gel mobility shift experiments using ≤1 fmole of end-labeled DNA fragments and DNase

6

116

I footprinting experiments using 20-40 fmoles of labeled DNA were performed following the

117

procedures described in detail previously (26, 54).

118

Construction of transcriptional lacZ fusions. To construct plasmid pGB3, containing a vpr-

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lacZ transcriptional fusion, a 680-bp vpr product, corresponding to positions -656 to +24 with

120

respect to the vpr start codon, was synthesized by PCR using oligonucleotides oGB9 and oGB10

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and cloned between the XbaI and HindIII sites of an integrative plasmid pHK23 (erm) (26).

122

Plasmid pGB4 (mpr-lacZ) was created as described above for pGB3, by cloning the 302-bp mpr

123

PCR product, obtained with oGB13 and oGB14 and corresponding to positions -260 to +42 with

124

respect to the start codon.

125

B. subtilis strains carrying the vpr-lacZ or mpr-lacZ fusion at the amyE locus (Table 1)

126

were isolated after transforming strain BB2511 (amyE::spc lacA) with the appropriate plasmids,

127

selecting for resistance to erythromycin conferred by the plasmids, and screening for loss of the

128

spectinomycin

129

recombination event. Strain BB2511 and all of its derivatives have very low endogenous β-

130

galactosidase activity due to a null mutation in the lacA gene (55).

131

Mutations in the CodY-binding sites. Mutations in the vpr regulatory region were introduced

132

by two-step overlapping PCR. In the first step, 0.6-kb products containing the 5’ part of the vpr

133

regulatory region were synthesized by using oligonucleotide oGB9 as the forward primer and

134

mutagenic oligonucleotides oGB11 (vprp1), oGB17 (vprp2), or oGB19 (vprp3) as the reverse

135

primer. In a similar manner, 0.1-kb products containing the 3’ part of the regulatory region and

136

the first 24 bp of the vpr coding sequence were synthesized by using mutagenic oligonucleotides

137

oGB12 (vprp1), oGB16 (vprp2), or oGB18 (vprp3) as the forward primer and oligonucleotide

138

oGB10 as the reverse primer. The appropriate pairs of PCR products were used in a second step

resistance

marker,

which

indicated

a

double-crossover,

homologous

7

139

of PCR as overlapping templates to generate modified fragments containing the entire vpr

140

regulatory region; oligonucleotides oGB9 and oGB10 served as forward and reverse PCR

141

primers, respectively. The final PCR products were digested with XbaI and HindIII and cloned in

142

pHK23 as described above to create pGB7 (vprp1-lacZ), pGB9 (vprp2-lacZ), and pGB10 (vprp3-

143

lacZ).

144

Plasmids pGB8 (mprp1-lacZ), pGB11 (mprp3-lacZ) and pGB12 (mprp2-lacZ) were

145

constructed as described above for pGB4, by using oGB13 as the forward primer and mutagenic

146

oligonucleotides oGB15, oGB25 or oGB24 as the reverse primer for PCR, respectively.

147

CodY overexpression and purification. Wild-type CodY with a C-terminal five-histidine tag

148

was overexpressed and purified to near homogeneity as described previously (26).

149

Labeling of DNA fragments. A 769- or 812-bp PCR product containing the regulatory region of

150

the vpr gene was synthesized by using pGB3 as the template and either oGB9 and oBB253 or

151

oBB67 and oBB102 as the forward and reverse primers, respectively. A 391- or 434-bp PCR

152

product containing the regulatory region of the mpr gene was synthesized by using plasmid

153

pGB4 as the template and either oGB13 and oBB253 or oBB67 and oBB102 as the forward and

154

reverse primers, respectively. The vector-specific primer oBB67 starts 96 bp upstream of the

155

XbaI site, used for cloning; oBB102 and oBB253 start 36 and 89 bp downstream of the HindIII

156

site that serves as a junction between the promoters and the lacZ part of the fusions, respectively.

157

The reverse primer oBB102 or oBB253 was labeled with T4 polynucleotide kinase and [γ-

158

32P]ATP (6,000 Ci/mmol, Perkin Elmer).

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Enzyme assays. β-galactosidase specific activity was determined as described previously (56).

160

Preparation of protein extracts and 2D-PAGE. Cells of B. subtilis strains SMY (wild-type) or

161

BB1043 (codY) were grown in the TSS + 16 aa medium and harvested by centrifugation (10,000

8

162

rpm, 4°C, 10 min) during exponential growth (OD600 =1.3), in the transition phase (OD600 =4.0)

163

or in stationary phase (OD600 =5.6-6.1). Extracellular proteins were prepared by precipitation

164

from the supernatant with TCA (57). The concentration of the re-dissolved extracellular proteins

165

was determined with RotiNanoquant (Roth). Isoelectric focusing in the pH range of 3-10 was

166

done with 100-µg protein samples using commercially available IPG strips (Serva). Isoelectric

167

focusing and separation in the second dimension was performed according to Büttner et al. (58).

168

Gels were stained with Flamingo Fluorescent Gel Stain (Bio-Rad Laboratories) according to the

169

manufacturer’s instructions.

170

Protein spot quantification and protein identification. Gels were scanned and the images

171

were analyzed with the Delta2D software version 4.3 (Decodon). Protein spot quantification was

172

conducted according to Wolf et al. (59). Briefly, gel images for proteins from wild-type and

173

mutant strains were overlaid and a fusion gel image, which was generated in silico using the

174

image fusion function of the Delta2D software, was used for spot detection. After spot editing,

175

protein spots were transferred to the single-gel images. Spot quantities were calculated with the

176

Delta2D software as a fraction (%) of the total intensity of all protein spots present on the gel

177

attributable to the intensity of an individual protein spot.

178

For identification, spots were excised from gels with the Ettan Spot Picker (GE

179

Healthcare) using pick lists generated with the Delta2D software. Digestion with trypsin and

180

spotting of the resulting peptide solutions onto MALDI targets were performed in the Ettan Spot

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Handling Workstation (GE Healthcare). MS analysis was done by MALDI-TOF-MS/MS using

182

the Proteome Analyzer 4800 (Applied Biosystems) (59). Peak lists were searched against a B.

183

subtilis database with the MASCOT search engine version 2.1.0.4 (Matrix Science) using search

184

parameters as in Wolf et al.(59).

9

185 186

Results CodY-mediated regulation of the vpr gene.

187

To assess the ability of CodY to regulate vpr expression, a vpr-lacZ transcriptional fusion

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containing a 680-bp fragment including the entire intergenic region upstream of vpr and the first

189

24 nucleotides of the coding sequence was constructed (Fig. 1A). Under conditions of maximal

190

CodY activity, in a glucose-ammonium minimal medium containing ILV and a mixture of 13

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other amino acids (hereafter referred to as the TSS + 16 aa medium), expression of the vpr fusion

192

under steady-state growth conditions was about 10-fold higher in the codY null mutant strain

193

GB1011 than in the wild-type strain GB1003 (Table 3). In the wild-type strain, the activity of the

194

fusion increased 3-fold when ILV was omitted (TSS + 13 aa) and was completely derepressed

195

when TSS was unsupplemented with amino acids (Table 3). Though this pattern of amino acid-

196

dependent expression is common to CodY-regulated genes, the inability of partly active CodY,

197

present in TSS-growing cells, to repress vpr at all is rather unusual (26).

198

Binding of CodY to the vpr regulatory region.

199

The ability of CodY to bind to the vpr promoter was established by gel mobility shift

200

assay (Fig. 1B) and DNase I footprinting (Fig. 1C). In a gel mobility shift assay, CodY bound to

201

the vpr regulatory region with an apparent KD (equilibrium dissociation constant, defined as the

202

protein concentration needed to shift 50% of the DNA fragments under conditions of CodY

203

excess over DNA) of ∼10 nM (Fig. 1B). A DNase I footprinting experiment revealed that CodY

204

protects a 30-nt site, corresponding to positions -20 to +10 with respect to the putative vpr

205

transcription start point determined in a tiling array transcriptome analysis of B. subtilis (60)

206

(Fig. 1A and 1C). This binding site overlaps the transcription start point and fully encompasses a

10

207

core CodY-binding site, at positions -11 to +5, previously identified by IDAP-Seq as a sequence

208

in which each base-pair is essential for CodY binding (4).

209

CodY binding has been associated with the presence of a 15-bp consensus sequence

210

(AATTTTCWGAAAATT) (4, 9, 25, 26) hereafter referred as the CodY-binding motif. A

211

bioinformatics search revealed that the vpr CodY-binding site includes two partially overlapping

212

15-nt CodY-binding motifs, I and II, with four and three mismatches, respectively, with respect

213

to the consensus sequence (positions -15 to -1 and -6 to +9 with respect to the vpr transcription

214

start point) (Fig. 1A).

215

Mutagenesis of the vpr CodY-binding site.

216

Three different double-nucleotide substitution mutations were introduced upstream of the

217

transcription start point and within the two overlapping CodY-binding motifs of the vpr gene

218

(Table 3). All mutations were aimed at decreasing the similarity of one or both of the motifs to

219

the CodY-binding consensus sequence. As reported in Table 3, all three mutations significantly

220

reduced the ability of CodY to repress expression from the vpr promoter. Moreover, the vprp2

221

mutation strongly decreased the affinity of CodY for the vpr regulatory region (Fig. 1D; the p1

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and p3 mutations have not been tested).

223

The decreased activities of the vprp1- and vprp3-lacZ fusions in the absence of CodY

224

suggest that the p1 and p3 mutations, being located in the proximity of the -10 region of the

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promoter, affect its intrinsic activity (Table 3, compare strains GB1011, GB1015, and GB1030).

226

CodY-mediated regulation of the mpr gene.

227

The ability of CodY to regulate mpr expression was tested using an mpr-lacZ

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transcriptional fusion comprising the entire 260-bp intergenic region upstream of the gene and

229

the first 14 codons of the coding sequence (Fig. 2A). Expression of the fusion in TSS + 16 aa

11

230

medium was almost 13-fold higher in the codY null mutant strain GB1012 than in the wild-type

231

strain GB1004. In the wild-type strain, the activity of the fusion was derepressed 4-fold when

232

ILV were omitted, and was further increased 2.5-fold in TSS medium without added amino acids

233

(Table 4).

234

The expression of mpr-lacZ (and vpr-lacZ) in the codY null mutant was unaffected by the

235

amino acid composition of the medium, indicating that CodY is the major relevant regulator of

236

the mpr and vpr genes under the conditions tested.

237

Binding of CodY to the mpr regulatory region.

238

CodY binding to the mpr region was demonstrated by gel mobility shift (Fig. 2B) and

239

DNase I footprinting experiments (Fig. 2C). Purified CodY bound a labeled mpr fragment with

240

an apparent KD of ∼30 nM (Fig. 2B). As revealed by a DNase I footprinting experiment, CodY

241

binding occurred at two sites. The upstream, lower-affinity site I mapped to positions -24 to +23

242

with respect to the putative transcription start point of the gene (60); the best possible CodY-

243

binding motif in this region (positions -9 to +6) contains 5 mismatches to the consensus

244

sequence. The higher-affinity site II extended from positions +63 to +99 with respect to the

245

transcription start point of mpr (positions -7 to +30 with respect to the translation start site of the

246

gene) and included a 15-bp CodY motif with four mismatches to the consensus (positions +15 to

247

+29 with respect to the translation start site) (Fig. 2A and 2C). The locations and relative

248

strengths of the two CodY-binding sites were correctly predicted by IDAP-Seq: the two core

249

CodY-binding sites were located at positions -10 to +9 and +75 to +94 with respect to the

250

putative transcription start point of the gene, respectively (4) (Fig. 2A).

251

Mutagenesis of the internal mpr CodY-binding site.

12

252

Three different double-nucleotide substitution mutations aimed at decreasing the

253

similarity of the CodY-binding motif associated with the higher-affinity mpr CodY-binding site

254

II to the consensus motif were introduced separately 84 to 93 bp downstream of the transcription

255

start point and corresponding to positions 23 and 24, 20 and 21, or 15 and 16 with respect to the

256

mpr translation start site (Table 4). While the introduction of the p1 mutation reduced by 6-fold

257

the ability of CodY to repress mpr-lacZ transcription, both p2 and p3 abolished CodY-dependent

258

regulation of mpr (Table 4). Even the least effective mutation, p1, strongly decreased the affinity

259

of CodY for the mpr regulatory region (Figure 2D; the p2 and p3 mutations have not been

260

tested). CodY-mediated repression of mpr expression appears therefore to be mediated mainly by

261

binding at the high-affinity site II.

262

Somewhat unexpectedly, two of the mutations affected expression of the mpr-lacZ fusion

263

in a negative (p1) or positive (p3) way, even in a codY null strain, indicating that they altered

264

either the intrinsic activity of the promoter or stability of mpr-lacZ mRNA or efficiency of lacZ

265

translation (Table 4).

266

Role of the sigma H factor in vpr expression.

267

Based on DNA-microarray experiments, the vpr gene was reported to be expressed from

268

a σH-dependent promoter (61). σH, a product of the sigH (spo0H) gene, is an alternative sigma

269

factor of RNA polymerase, which controls induction of many genes during the transition from

270

exponential to stationary phase and is required for sporulation (62). In TSS + 16 aa medium,

271

expression of the vpr-lacZ fusion was almost completely abolished in a sigH null mutant,

272

indicating that the vpr promoter is indeed dependent on σH even during steady-state growth

273

(Table 3). In contrast, expression of the mpr-lacZ fusion, which is not known to require σH, was

274

not affected by a sigH mutation (Table 4). Thus, the vpr promoter is the second example of a σH-

13

275

dependent promoter that is repressed by CodY; previously, the ureAp2 promoter was found to be

276

under CodY control (63).

277

Overproduction of the Vpr protein in codY mutant cells.

278

The ability of CodY to affect expression of the vpr gene at the protein level in TSS + 16 aa

279

medium was determined after separating B. subtilis extracellular proteins by 2D-PAGE and

280

identifying individual protein spots using MALDI-TOF-MS/MS analysis. In three independent

281

experiments, the average abundance of all identified Vpr spots in a codY null mutant strain

282

increased 15- to 25-fold compared to that in a wild-type strain; the abundance of the principal

283

Vpr spot group, labeled as “Vpr” and comprising at least 88% of total Vpr in a codY null strain,

284

increased 32- to 50-fold (Fig. 3, Table 5). As expected, the effect of the codY mutation was

285

similar during exponential and stationary phases of growth due to the presence of excess amino

286

acids in the TSS medium. In the absence of CodY, Vpr was one of the more abundant proteins of

287

the extracellular proteome, comprising about 1% of total extracellular protein (Table 5).

288 289

Unfortunately, the Mpr protein co-migrated in our experiments with pectate lyase, the product of the pel gene, which made quantitation of Mpr expression impossible.

14

290

Discussion

291

In response to the intracellular levels of ILV, L. lactis CodY is able to regulate the

292

expression of genes encoding products involved in protein degradation and peptide assimilation

293

for nitrogen supply (9). In nitrogen-rich media, L. lactis CodY represses expression of the PrtP

294

proteinase, Opp transporter and PepN, PepC, and PepO1 peptidases (25). In B. subtilis, the

295

genes, encoding peptide transporters Dpp and App, an intracellular peptidase DppA and

296

intracellular protease IspA, are known to be negatively regulated by CodY (1, 3-5, 64).

297

In this work, we demonstrated that the genes, coding for two B. subtilis minor

298

extracellular proteases Vpr and Mpr (36, 39, 40, 65, 66), are also repressed by CodY. CodY

299

repression at the vpr locus is exerted by binding at the single CodY-binding site located in the

300

vicinity of the transcription start point of the gene, implying that the mechanism of repression is

301

by competition with RNA polymerase. The repression of mpr expression appears to be mediated

302

mainly by binding of the repressor at the high-affinity site II, internal to the coding sequence of

303

the gene. CodY binding at sites located within the coding regions of target genes was previously

304

reported to cause efficient repression of gene expression by transcriptional roadblocking (4, 54,

305

67, 68). It seems likely that CodY binding to the mpr site II also creates a transcriptional

306

roadblock for elongating RNA polymerase. In this case, however, the proximity of mpr site II to

307

the translation initiation codon may also cause the stalled RNA polymerase to interfere with

308

translation initiation. The location and relative strengths of the vpr and mpr CodY-binding sites,

309

determined in this work by DNase I footprinting and gel mobility shift experiments, were found

310

to correlate very well with the results of IDAP-Seq (4).

311

Mpr was shown to be a broad-range (glutamate-specific) endopeptidase (69). This is in

312

accord with the assumption that the principal function of extracellular proteases is to supply

15

313

amino acids for growth via degradation of extracellular proteins, although B. subtilis

314

exoproteases have also been ascribed additional physiological roles. Extracellular proteases,

315

including Vpr, may affect multicellular behavior, such as swarming motility and biofilm

316

formation (70, 71). Interestingly, Vpr was identified as one of the most abundant proteins in

317

biofilms produced by some B. subtilis cells (71). Moreover, this minor serine protease was

318

demonstrated to play a role in the processing of the peptide antibiotic subtilin (72) and

319

production of two quorum sensing signaling peptides, PhrA and CSF, by cleavage of their

320

precursors (73).

321

Interestingly, CodY negatively regulates the rapA-phrA operon (3-5, 74). RapA is a

322

Spo0F phosphatase involved in the regulation of sporulation; PhrA is processed extracellularly to

323

produce a pentapeptide that inhibits RapA activity (75). Therefore, CodY regulates the activity

324

of RapA both at the level of rapA-phrA expression and at the level of Vpr-dependent proteolytic

325

processing of the full-length PhrA protein.

326

In ongoing work, we have found that two other B. subtilis genes coding for extracellular

327

proteases, aprE and nprE, for which no CodY-mediated regulation was previously detected (3, 5)

328

are, in fact, direct targets of CodY, but also of a second regulator whose expression is repressed

329

by CodY (Barbieri et al., manuscript in preparation). Thus, CodY is a direct repressor of at least

330

four B. subtilis extracellular proteases.

16

331

Acknowledgments

332

This work was supported by a research grant from the U. S. National Institute of General

333

Medical Sciences (GM042219) to A. L. Sonenshein. The content is solely the responsibility of

334

the authors and does not necessarily represent the official views of the National Institutes of

335

Health or of the NIGMS. G. Barbieri was supported by University of Pavia PostDoc Fund and

336

AA-FAR 2012-13.

17

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repression of the Bacillus subtilis dipeptide permease operon. Mol Microbiol 15:689-702.

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552

Table 1. B. subtilis strains used in this work Strain SMY BH1 PS251 BB1043 BB2511 GB1004 GB1007 GB1008 GB1011 GB1012 GB1015 GB1016 GB1025 GB1026 GB1027 GB1028 GB1029 GB1030 GB1031 GB1032 BB3949 BB3950

Genotype wild-type sigH::cat trpC2 pheA1 codY::(erm::spc) trpC2 codY::(erm::spc) amyE::spc lacA ΔamyE::Φ(mpr-lacZ erm) lacA::tet ΔamyE::Φ(vprp1-lacZ erm) lacA::tet ΔamyE::Φ(mprp1-lacZ erm) lacA::tet ΔamyE::Φ(vpr-lacZ erm) lacA::tet codY::(erm::spc) ΔamyE::Φ(mpr-lacZ erm) lacA::tet codY::(erm::spc) ΔamyE::Φ(vprp1-lacZ erm) lacA::tet codY::(erm::spc) ΔamyE::Φ(mprp1-lacZ erm) lacA::tet codY::(erm::spc) ΔamyE::Φ(vprp2-lacZ erm) lacA::tet ΔamyE::Φ(vprp3-lacZ erm) lacA::tet ΔamyE::Φ(mprp2-lacZ erm) lacA::tet ΔamyE::Φ(mprp3-lacZ erm) lacA::tet ΔamyE::Φ(vprp2-lacZ erm) lacA::tet codY::(erm::spc) ΔamyE::Φ(vprp3-lacZ erm) lacA::tet codY::(erm::spc) ΔamyE::Φ(mprp2-lacZ erm) lacA::tet codY::(erm::spc) ΔamyE::Φ(mprp3-lacZ erm) lacA::tet codY::(erm::spc) ΔamyE::Φ(vpr-lacZ erm) lacA::tet codY::(erm::spc) sigH::cat ΔamyE::Φ(mpr-lacZ erm) lacA::tet codY::(erm::spc) sigH::cat

Source or referencea (47) (76) P. Serror SMY × PS251 DNA (26) BB2511 × pGB4 BB2511 × pGB7 BB2511 × pGB8 GB1003 × BB1043 DNA GB1004 × BB1043 DNA GB1007 × BB1043 DNA GB1008 × BB1043 DNA BB2511 × pGB9 BB2511 × pGB10 BB2511 × pGB11 BB2511 × pGB12 GB1025 × BB1043 DNA GB1026 × BB1043 DNA GB1027 × BB1043 DNA GB1028 × BB1043 DNA GB1011 × BH1 DNA GB1012 × BH1 DNA

553 554

a

The symbol × indicates transformation by plasmid or chromosomal DNA

555

28

556

Table 2. Oligonucleotides used in this work Oligonucleotide type and name

Sequence (5'-3')a

Flanking primers Forward CGGACTCTAGACAGCCGCCTTTCTTTGGTATG oGB9 oGB13 oBB67 Reverse oGB10 oGB14 oGB15 oGB24 oGB25 oBB102 oBB253

Internal mutagenic primers Forward oGB12

CAGACTCTAGAGGAAAGCCGATAACAAAAGCC GCTTCTAAGTCTTATTTCC

Specificity

vpr mpr ermb

CGCAAAAGCTTAAAGCGAATGATCCCCTTTTTC

vpr mpr CGCACAAGCTTGTAAGCGAACCATTGTTTTCTGAATC mprp1 CGAACAAGCTTGTAAGCGAACCATTGTTTggTGAATC mprp2 CGAACAAGCTTGTAAGCGAACCATTGTTTTCTccATCTTGGAAC mprp3 CAACAAGCTTGTAAGCGAACCATTGTTTTCTGAATCccGGAACTAATTTC spoVG-lacZ b CACCTTTTCCCTATATAAAAGC GGTTTTCCCGGTCGAC lacZ b

GACACAAGGATTTTTTTccATTTTCAAGAAATATATAC

oGB16 oGB18

GACACAAGGATTTTTTTGAATTggCAAGAAATATATAC GACACAAGGATTTggTTGAATTTTCAAG

Reverse oGB11

GTATATATTTCTTGAAAATggAAAAAAATCCTTGTGTC

oGB17 oGB19

GTATATATTTCTTGccAATTCAAAAAAATCCTTGTGTC CTTGAAAATTCAAccAAATCCTTGTGTC

vprp1 vprp2 vprp3

vprp1 vprp2 vprp3

557 558

a

559

underlined.

560

b

The altered nucleotides in the CodY-binding motives are in lowercase. Restriction sites are

These targets are located on plasmid pHK23.

561

29

562

Table 3. Expression of vpr-lacZ fusionsa

Strain

Relevant genotypeb

Fusion version

vpr CodY-binding motifsc

GB1003

wild-type

vpr-lacZ

ATTTTTTTGAATTTTCAAGAAATA

GB1011

Additions to the medium

β -Galactosidase activityd Miller Units

%

None 13 aa 16 aa

7.9 ± 1.3 2.3 ± 0.5 0.7 ± 0.1

103 30 9

codY

None 13 aa 16 aa

8.0 ± 1.8 7.0 ± 1.2 7.6 ± 1.5

105 91 100

BB3949

codY sigH

16 aa

0.7 ± 0.03

10

GB1007

wild-type

16 aa

0.3 ± 0.1

38

GB1015

codY

16 aa

0.8 ± 0.1

100

GB1025

wild-type

16 aa

5.7 ± 1.1

83

GB1029

codY

16 aa

6.8 ± 1.0

100

GB1026

wild-type

16 aa

1.2 ± 0.03

58

GB1030

codY

16 aa

2.1 ± 0.4

100

vprp1-lacZ

vprp2-lacZ

vprp3-lacZ

ATTTTTTTccATTTTCAAGAAATA

ATTTTTTTGAATTggCAAGAAATA

ATTTggTTGAATTTTCAAGAAATA

563 564

a Cells were grown in TSS glucose-ammonium medium, unsupplemented or containing a mixture of 13 aa or the same mixture with

565

ILV added (16 aa). β-Galactosidase activity was assayed and expressed in Miller units ± standard deviation. All values are averages

30

566

of at least two experiments.

567

b All strains contained a lacA null mutation.

568

c Sequences of positions -15 to +9 with respect to the putative transcription start point (double underlined) of the vpr-lacZ fusions.

569

CodY-binding motifs I and II are italicized and in boldface, respectively. The core CodY-binding site is underlined. The mutated

570

nucleotides are in lowercase.

571

d β-Galactosidase activity of each fusion in TSS+16 aa medium in a strain containing a codY null mutation was normalized to 100%.

572

31

573

Table 4. Expression of mpr-lacZ fusionsa

Strain

Relevant genotypeb

Fusion version

mpr CodY-binding motifc

GB1004

wild-type

mpr-lacZ

ATGAAATTAGTTCCAAGATTCAGAAAACA

GB1012

Additions to the medium

β -Galactosidase activityd Miller Units

%

None 13 aa 16 aa

11.2 ± 1.5 4.5 ± 0.8 1.2 ± 0.2

71 29 8

codY

None 13 aa 16 aa

14.3 ± 1.4 14.8 ± 1.8 15.7 ± 1.2

91 94 100

BB3950

codY sigH

16 aa

19.0 ± 0.9

121

GB1008

wild-type

16 aa

2.9 ± 0.5

52

GB1016

codY

16 aa

5.5 ± 1.0

100

GB1027

wild-type

16 aa

19.1 ± 0.3

93

GB1031

codY

16 aa

20.6 ± 1.5

100

GB1028

wild-type

16 aa

29.5 ± 2.0

89

GB1032

codY

16 aa

33.2 ± 3.2

100

mprp1-lacZ

mprp2-lacZ

mprp3-lacZ

ATGAAATTAGTTCCAAGATTCAccAAACA

ATGAAATTAGTTCCAAGATggAGAAAACA

ATGAAATTAGTTCCggGATTCAGAAAACA

574 575

a Cells were grown and β-galactosidase activity (expressed in Miller units ± standard deviation) was assayed as described for Table

576

3.

32

577

b All strains contained a lacA null mutation.

578

c Sequences of positions +70 to +98 with respect to the transcription start point of the mpr-lacZ fusions. The leftmost three nucleotides

579

correspond to mpr initiation codon. The CodY-binding motif is in boldface and the core CodY-binding site is underlined. The mutated

580

nucleotides are in lowercase.

581

d β-Galactosidase activity of each fusion in TSS+16 aa medium in a strain containing a codY null mutation was normalized to 100%.

582 583

33

584 585 586 587 588 589 590

Table 5. Abundance of Vpr in the B. subtilis exoproteome

591

growth

592 593

Protein spots

wild-type

codY

codY/wild-type ratio

Main Vpr spot group

0.019±0.008

0.611±0.591

31.9

All Vpr spots

0.051±0.018

0.748±0.615

14.6

Exponential

594 595

Transition phase

596

Main Vpr spot group

0.020±0.012

0.823±0.620

42.1

597

All Vpr spots

0.040±0.024

0.929±0.705

23.0

598 599

Stationary phase

600

Main Vpr spot group

0.018±0.005

0.889±0.321

50.1

601

All Vpr spots

0.045±0.018

1.121±0.327

24.8

602 603 604

Cells were grown in TSS + 16 aa medium, and the extracellular proteins were isolated at

605

different stages of growth and analyzed as described in Materials and Methods. All values are

606

averages of three independent experiments plus/minus standard deviation. Several protein spots

607

containing Vpr, but of different intensities, were identified in the exoproteome. Protein spot

608

abundance for the main spot group, labeled as Vpr (Fig. 3), and for all Vpr spots was calculated

609

as a fraction of total protein in all spots on the gel.

34

610

Figure legends

611

Figure 1. Binding of CodY to the vpr regulatory region. A. The sequence of the vpr insert used

612

to construct the vpr-lacZ fusions. Coordinates are reported with respect to the putative

613

transcription start point (60), indicated by the bent arrow. The core CodY-binding site identified

614

by IDAP-Seq (4) is in boldface. Two overlapping CodY-binding motifs with 4 and 3 mismatches

615

to the consensus are underlined; two more motifs with 5 mismatches to the consensus can be

616

found in the same region and are not shown. The CodY-protected region, detected in DNase I

617

footprinting experiments, is underlined with the dashed line. The directions of translation of vpr

618

and ywcI are indicated by the long arrows. The initiation codons of the two genes are in boldface.

619

B. Gel mobility shift assay of CodY binding to a radioactively labeled vpr PCR fragment,

620

obtained with oligos oBB67 and oBB102, in the presence of 10 mM ILV. CodY concentrations

621

(nM of monomer) are reported below each lane. C. DNase I footprinting analysis of CodY

622

binding to the vpr fragment. The labeled vpr PCR fragment, obtained with oligos oGB9 and

623

oBB253, was incubated with increasing amounts of purified CodY in the presence of 10 mM

624

ILV and 2 mM GTP and then with DNase I. Concentrations of CodY (nM of monomer) are

625

reported above each well. The corresponding A + G sequencing ladder of the bottom DNA

626

strand is on the left. The protected area is shown by the vertical line and the corresponding

627

sequence is reported; the bent arrow indicates the transcription start point and the direction of

628

transcription. The core CodY-binding site identified by IDAP-Seq is in boldface. D. Gel mobility

629

shift assay of CodY binding to a radioactively labeled vprp2 fragment, obtained with oligos

630

oBB67 and oBB102, in the presence of 10 mM ILV. CodY concentrations (nM of monomer) are

631

reported below each lane.

632

Figure 2. Binding of CodY to the mpr regulatory region. A. Sequence of the mpr insert used to

35

633

construct the mpr-lacZ fusions. Coordinates are reported with respect to the putative

634

transcription start point (60), indicated by the bent arrow. The core CodY-binding sites identified

635

by IDAP-Seq (4) are in boldface. The CodY-binding motifs of sites I and II are underlined. The

636

CodY-protected regions, detected in DNase I footprinting experiments, are underlined with the

637

dashed lines. The directions of translation of purT and mpr are indicated by the long arrows. The

638

mpr initiation codon is in boldface. B. Gel mobility shift assay of CodY binding to a

639

radioactively labeled mpr PCR fragment, obtained with oligos oBB67 and oBB102, in the

640

presence of 10 mM ILV. CodY concentrations (nM of monomer) are reported below each well.

641

C. DNase I footprinting analysis of CodY binding to a radioactively labeled mpr PCR fragment

642

obtained with primers oGB13 and oBB253. Increasing amounts of purified CodY were incubated

643

with the mpr fragment in the presence of 10 mM ILV and 2 mM GTP before treatment with

644

DNase I. The corresponding A + G sequencing ladder of the bottom DNA strand is shown on the

645

left. The protected areas corresponding to sites I and II are shown by the continuous and dashed

646

vertical lines, respectively, and their sequences are reported in the corresponding boxes. The core

647

CodY-binding sites identified by IDAP-Seq are in boldface. Concentrations of CodY (nM of

648

monomer) are reported above each well. D. Gel mobility shift assay of CodY binding to a

649

radioactively labeled mprp1 fragment, obtained with oligos oBB67 and oBB102, in the presence

650

of 10 mM ILV. CodY concentrations (nM of monomer) are reported below each lane.

651

Figure 3. Expression of extracellular Vpr in B. subtilis during exponential (A), transition (B) and

652

stationary (C) phase of growth. A section of a 2D-PAGE containing protein spots corresponding

653

to Vpr is shown. The presence of multiple Vpr spots and spot groups may be caused by

654

carbamidomethylation or other unknown modifications of proteins. Additional minor Vpr spots

655

were detected on the gel but are not shown. The quantification of Vpr spots is reported in Table

36

656

5.

37

A -600 CAGCCGCCTT TCTTTGGTAT GTACGCTGAG CCGAATAGAC CGCGGCAGCC GCAGTTTTTC TGTCCGGCGT CACCCAGTTC ATCAAAAAGA -510 CCATCCACAC CCGTAAAGAT ACAAGCAAAC GTTTCATAGA AAAACATCCC TCCGCTTCTT TTTGGCAGGC AGCCTTTTTA GCAGCCCGTT ywcI -420 TTCTCAGCCG CAGCCCGCAA GAAAAGACGG -330 TGGAAAGAGA ATTCTTTGTC ACAATATGAG -240 TGGAGAACCG CTTTGAAAAC TTTATACACA -150 ATCTATATTT TGTATACGAA CGTATATTCC

CCGATTTCTT TTCTCGCAAA CTAGCGGCTC TAGCAAAACT CATGACTCTA TGATAGACCG TGAAAAAACC AACTAGTTTT TAGAAGTTTT GTTGAAAGCT GAAAGAATTG AAATGAAAAT AGTTATCCCA AAGATAAGAA CAACTTAATC ACAAGAGATA TCCACATGTC CACAAACTCT TAACTATATA TATACACAGG TTTATTCACT TATACACAGG GTTCTGTGTA TAACTCCTTC

- 60 GTTATACACA AACAAAATCC AATAAATGGT CCAAATGACA CAAGGATTTT TTTGAATTTT CAAGAAATAT ATACTAGATC TTTCACATTT motif I motif II DNase I footprint + 30 TTTCTAAATA CAAAGGGGGA AACACATTGA AAAAGGGGAT CATTCGCTTT A/G

vpr

B

1.6 0

6.3 3.1

25 12.5

100 50

400 nM 200

C

0 1.6 3.1 6.2 12.5 25 50 100 200 400 nM

D

0 3.1 6.2 12.5 25 50 100 200 400 800 nM

G T T C C A A A A A A A C T T A A A A G T T C T T T A T A

A

-191 GGAAAGCCGA TAACAAAAGC CGGACGTCGT ATGGCAGTTG CGCTTTCTGC TGCTGATTCA GTTGAAACGG CAAGAGAGAA purT -111 TGCAAAGAAA GCGTTGGACC AGCTAATTTT AAAATAGAGT TTGAACAGGT CTTGTCATGG GACAAGGCCT GTTTTTTTCT

- 31 TTCTCCGTAA AAGTTTTATC ATAAGAATCA GAAACCTGAT TATAATGTAA AAGTCTTCCA TCGATACGGG TGGTTGACAC motif I DNase I footprint (site I) + 50 TAAAGGAGGG AGATGACAAA ATGAAATTAG TTCCAAGATT CAGAAAACAA TGGTTCGCTT AC motif II DNase I footprint (site II) mpr A/G

3.1 0

B

6.3

12.5 50 200 800 nM 25 100 400

C

site I

0 3.1 6.2 12.5 25 50 100 200 400 800 nM site II

D

0 3.1 6.2 12.5 25 50 100 200 400 800 nM

A T T T T C A A A A T A G T A T T C T T A G T C T T T G G A C T A A T A T T A C A T T T T C A

A C T G T T T T A C T T T A A T C A A G G T T C T A A G T C T T T T G T T

wild-type

A

B

C

codY