acetylation of the response regulator rcsb controls ... · 59 encoding rna rpra , which activates...

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Acetylation of the Response Regulator RcsB 1

Controls Transcription from a Small RNA Promoter 2

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Linda I. Hu1, Bui Khanh Chi2, Misty L. Kuhn3, Ekaterina V. Filippova3, Arti J. Walker-4

Peddakotla1, Katrin Bäsell2, Dörte Becher2, Wayne F. Anderson3, Haike Antelmann2, and 5

Alan J. Wolfe1# 6

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1Department of Microbiology and Immunology 8

Loyola University Chicago 9

Stritch School of Medicine 10

2160 S. First Ave. Bldg. 105 11

Maywood, IL 60153, USA 12

2Institute for Microbiology, 13

Ernst-Moritz-Arndt-University of Greifswald, 14

F.-L.-Jahn-Str. 15, D-17487 Greifswald, Germany 15

3Center for Structural Genomics of Infectious Diseases, Northwestern University, Feinberg School of 16

Medicine, Department of Molecular Pharmacology and Biological Chemistry, Chicago, IL 60611, USA 17

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*Running title: Acetylation regulates transcription 19

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To whom correspondence should be addressed: Alan J. Wolfe, Department of Microbiology and 21

Immunology, Loyola University Chicago, Stritch School of Medicine, 2160 S. First Ave. Bldg. 105, 22

Maywood, IL 60153, Tel: (708) 216-5814; Fax: (708) 216-9574; E-mail: [email protected] 23

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00383-13 JB Accepts, published online ahead of print on 12 July 2013

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Abstract 24

Nε-lysine acetylation was recently discovered on many bacterial proteins that function in diverse 25

cellular processes. Thus, many questions remain unanswered. For example, what mechanisms 26

regulate lysine acetylation? Does acetylation affect physiology? To help answer these questions, 27

we studied the Escherichia coli response regulator and transcription factor RcsB, which is 28

reported to be acetylated in vitro. To characterize RcsB acetylation, we monitored transcription 29

from the rprA promoter, which requires RcsB. The conventional view is that RcsB is activated by 30

phosphorylation through either the Rcs phosphorelay or acetyl phosphate. We affirmed that rprA 31

transcription requires phosphorylated RcsB and showed that AcP is a phosphoryl group donor to 32

RcsB. However, a mutant that accumulates AcP (ackA) exhibited reduced rprA transcription 33

instead of the predicted increase. rprA transcription also diminished in the cobB mutant, which 34

lacks the only known E. coli protein deacetylase. This suggests the existence of an inhibitory 35

mechanism that involves lysine acetylation, a supposition supported by the observation that RcsB 36

isolated from the ackA or cobB mutants was hyperacetylated. Finally, we used a genetic approach 37

to identify an AckA- and CobB-sensitive lysine (Lys-154) that controls RcsB activity. We 38

propose that acetylation inhibits RcsB activity and that some of this inhibition acts through the 39

acetylation of Lys-154. 40

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Introduction 42

More than 300 post-translational modifications (PTMs) have been identified (1,2). By 43

decorating proteins in various combinations, PTMs can alter charge, size, hydrophobicity or 44

conformation, and thus a protein’s activity, stability, cellular location, and/or affinity for its 45

binding partners (3). Phosphorylation is one of the most common PTMs. In bacteria and lower 46

eukaryotes, phosphorylation of histidinyl and aspartyl residues allows two-component signal 47

transduction systems to sense environmental change and, in response, to appropriately alter 48

cellular behavior. The Rcs phosphorelay is a complex two-component signal transduction 49

pathway composed of three proteins: the hybrid sensor RcsC, the histidine phosphotransferase 50

RcsD and the response regulator (RR) RcsB (Figure 1A). In response to certain extracellular 51

stimuli, the hybrid sensor RcsC autophosphorylates its conserved histidinyl residue and donates 52

that phosphoryl group to a conserved aspartyl residue within its receiver (REC) domain. Next, the 53

phosphoryl group transfers to a conserved histidinyl residue on RcsD, which passes the 54

phosphoryl group to aspartyl residue 56 (Asp-56) within the REC domain of the RR RcsB. Once 55

phosphorylated, RcsB is reported to either form an RcsB homodimer or form heterodimers with 56

its co-activator RcsA (4) or the LuxR-type transcription factors BglJ (5) and GadE (6). The result 57

is regulated transcription of more than 150 genes in Escherichia coli (7), including the small non-58

encoding RNA rprA, which activates translation of the stationary phase sigma factor RpoS (8). 59

In the absence of an extracytoplasmic stimulus, the Rcs phosphorelay works in reverse 60

with RcsD and RcsC functioning as a net phosphatase, dephosphorylating phosphorylated RcsB 61

(Figure 1B). Under such conditions, it is proposed that the phosphoryl group donor for RcsB is 62

acetyl-phosphate (AcP), the intermediate of the Pta-AckA pathway (9). Disruptions of this central 63

metabolic pathway alter intracellular AcP concentrations (10) and result in correlative changes to 64


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in vivo RcsB activities (11). While AcP has been shown to function as the phosphoryl donor to 65

various response regulators in vitro (12,13), its ability to act in this capacity for RcsB has not been 66

demonstrated. 67

Another PTM is Nε-lysine acetylation, which is thought to use acetyl-CoA (AcCoA) as 68

the acetyl donor (3). It was recently reported that RcsB is one of several E. coli transcription 69

factors acetylated in vitro by Pat, a Salmonella enterica Gcn5-like acetyltransferase (GNAT) (14). 70

Both Pat and its E. coli homolog YfiQ (15,16) (also called Pka (17) or PatZ (18)) acetylated RcsB 71

lysine residue 180 (Lys-180) (14). Deacetylation of Lys-180Ac was catalyzed by CobB (14), a 72

member of the sirtuin family of NAD+-dependent deacetylases (19). Since Lys-180 is located in 73

the DNA binding helix-turn-helix motif of RcsB, Lys-180 acetylation reduced RcsB’s affinity for 74

its DNA site at the flhDC promoter (14). Whether RcsB is acetylated in vivo and whether 75

acetylated RcsB has physiological relevance remained unknown (14,20). 76

Here, we provide evidence that the RcsB-dependent rprA promoter is regulated by both 77

phosphorylation and acetylation of RcsB. First, we identify a novel RcsB-dependent behavior and 78

demonstrate that it depends on RcsB phosphorylation. We then show that this behavior is 79

inhibited by loss of CobB’s deacetylase activity or by deletion of ackA, which causes AcP to 80

accumulate. The relevant target of CobB and AckA appears to be RcsB, as mass spectrometry 81

detected several acetylated RcsB lysines, some of which were sensitive to both CobB and AckA. 82

One of those acetylated lysines (Lys-154) appears to play an essential role in RcsB’s ability to 83

activate rprA transcription, but not in its ability to inhibit migration through semi-solid agar. To 84

the best of our knowledge, this is the first report that acetylation of a bacterial transcription factor 85

could have an effect in vivo. 86

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Materials and Methods 88

Bacterial strains, plasmids, and bacteriophage. All bacterial strains used in this study are listed in 89

Table 1. Derivatives were constructed by generalized transduction with P1kc, as described 90

previously (21). The transcriptional fusion φ(PrprA-lacZ), carried by λPrprA as described 91

previously (8), was a generous gift from Susan Gottesman (National Institutes of Health, 92

Bethesda, MD). Construction of monolysogens was performed and verified as described 93

previously (22,23). 94

95

Culture conditions. For strain construction, cells were grown in LB containing 1% (w/v) tryptone, 96

0.5% (w/v) yeast extract, and 0.5% (w/v) sodium chloride; LB plates also contained 1.5% agar. 97

For promoter activity assays, cells were grown in TB7, which contains 1% (w/v) tryptone 98

buffered at pH 7.0 with potassium phosphate (100 mM). Transformations were performed by 99

electroporation, or through the use of either transformation buffers 1 and 2 (24) or transformation 100

and storage solution (25). Cell growth was monitored spectrophotometrically (DU640; Beckman 101

Instruments, Fullerton, CA) by determining the absorbance at 600 nm (A600). Spectinomycin (100 102

µg/ml), ampicillin (100 µg/ml), and chloramphenicol (25 µg/ml for experiments using pCA24n-103

yfiQ or 17.5 µg/ml for experiments with pCC1-derived plasmids) were added to growth media 104

when needed. To induce expression of genes carried from various plasmids, β-D-1 105

thiogalactopyranoside (IPTG) was added at the indicated concentrations. 106

107

Promoter activity assays. To monitor promoter activity from φ(PrprA-lacZ), cells were grown 108

aerobically at 37°C in TB7 overnight. The overnight cultures were diluted into fresh TB7 and 109

grown aerobically at 37°C until early stationary phase. At regular intervals, 50 µl aliquots were 110


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harvested and added to 50 µl of All-in-One β-galactosidase reagent (Pierce Biochemical). β-111

galactosidase activity was determined quantitatively using a microtiter format, as described 112

previously (26). As a blank, 50 µl of sterile TB7 was used. Promoter activity was always 113

determined across the entire growth curve and was plotted versus either time or A600; for some 114

experiments, however, only peak activity during exponential phase is shown. Each experiment 115

included three biological replicates unless otherwise mentioned. The values represent the means 116

with standard deviations. All experiments were performed at least twice. 117

118

RcsB cloning into a single copy vector. To construct a single copy plasmid that permits 119

expression of moderate amounts of WT RcsB, we cloned the WT rcsB allele into a derivative of 120

CopyControlTM pCC1TM (EpiCentreR). We began with pVEC, a derivative of pCC1TM, in which 121

the plasmid-encoded lacZα is disrupted by a kanamycin resistance cassette (Table 1, (27)). To 122

replace the kanamycin resistance cassette with the WT rcsB allele, we first constructed a 123

derivative of pVEC that lacked the kanamycin cassette. pVEC was BamHI-digested, ligated, 124

transformed into TransforMaxTM EPI300TM-T1R Chemically Competent E. coli (EpiCentreR), and 125

plated onto an LB plate containing chloramphenicol (17 µg/ml) and 5-bromo-4-chloro-indolyl-β-126

D-galactopyranoside (X-gal). A blue transformant was isolated and grown overnight at 37°C in 127

LB supplemented with chloramphenicol (17 µg/ml) to maintain the plasmid and with 128

CopyControlTM BAC Autoinduction Solution (EpiCentreR) to amplify the plasmid. The plasmid 129

DNA was purified and this pCC1 derivative was designated pLIH001 (Table 1). We then 130

amplified the WT rcsB allele from the chromosome of strain AJW3759 (Table 1), using primers 131

rcsBFBamHI and rcsBRBamHI (Table 1). The resulting amplicon was purified using the 132

CloneJETTM PCR Purification Kit (Thermo Scientific) and cloned into pJET1.2 using the 133


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CloneJETTM PCR Cloning Kit (Thermo Scientific). The ligated product was transformed into 134

competent DH5α and plated onto LB ampicillin plates. The WT rcsB allele was excised from 135

pJET1.2 with BamHI, gel-extracted, subcloned into BamHI-digested pLIH001, transformed into 136

EPI300 cells, and plated onto LB plates containing chloramphenicol and X-gal. White colonies 137

were used to inoculate LB supplemented with chloramphenicol and CopyControlTM BAC 138

Autoinduction Solution (EpiCentreR) and shaken overnight at 37°C. Plasmid DNA was purified 139

and sequenced using primer pCC1RBZ (Table 1). The resulting plasmid was named pLIH002 140

(Table 1). For experiments that involved pLIH002 or derivatives, pVEC functioned as the 141

negative control. 142

143

Site-directed mutagenesis. Site-directed mutagenesis of RcsB in pLIH002 was conducted using 144

QuikChangeR Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies) in 145

accordance with the manufacturer’s instructions using the mutagenic primers listed in Table 1. 146

147

Cloning, Recombinant Expression, and Purification. To purify proteins used for in vitro studies, 148

we transformed the chloramphenicol resistant ASKA plasmids that encode YfiQ or RcsB with a 149

noncleavable N-terminal polyhistidine (His6) tag (28) into kanamycin resistant BL21 Magic cells 150

(29). The ASKA-encoded, His6-tagged protein was expressed in the presence of 34 μg/ml 151

chloramphenicol and 35 μg/ml kanamycin, and purified with IMAC followed by size exclusion 152

chromatography as described (Kuhn et al., submitted, (30)). 153

154

Dynamic light scattering. For characterization of molecular size in solution, dynamic light 155

scattering (DLS) provides a measure of the translational diffusion coefficient of protein in 156


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solution. All DLS measurements were performed on a Zetasizer Nano-S Zen 1600 DLS 157

instrument with a 633 nm wavelength laser. The fluctuations in light intensity, due to the 158

Brownian motion of the molecules, were measured by a photodiode at a 90° angle. Photons were 159

counted and the time dependence of the light intensity fluctuations was analyzed by 160

autocorrelation. Assumptions include a solution viscosity equal to 1.019 and that the proteins are 161

spherical in nature. The proteins were centrifuged for 5 min with 14,000 x g prior to collecting 162

scattering data. Experiments were run at 25°C and at least 6 measurements were taken at each 163

condition. Regularization histogram analyses of samples were carried out using the Zetasizer 164

Nano software. 165

166

LTQ-Orbitrap Velos-LC-MS/MS analysis. For identification of in vivo acetylation sites, cells 167

transformed with pHis6rcsB (which expresses His6-tagged RcsB, (31)) were grown aerobically at 168

37°C at 250 rpm in TB7 supplemented with 50 µM IPTG and spectinomycin until entry into 169

stationary phase and cell lysates were prepared. For the identification of in vivo acetylation of 170

cells inhibited for sirtuin activity, 20 mM nicotinamide was added to the growth medium. For 171

identification of in vitro acetylation sites, His6-tagged RcsB and His6-tagged YfiQ proteins were 172

purified from E. coli cells (see below). The proteins in the in vivo and in vitro samples were 173

separated by SDS-polyacrylamide gel electrophoresis. To normalize the number of cells to lyse 174

per milliliter, the inverse of the culture A600 was used (32). To avoid cross-contamination, we 175

electrophoresed the samples from each strain and/or condition on separate SDS-polyacrylamide 176

gels with bioreplicates separated by an empty lane(s). Electrophoresis was terminated before 177

proteins could run off the bottom of the gel. The gel was cut down the middle of the empty lane to 178

separate the bioreplicates. Each half of the gel was stained with SimplyBlueTM SafeStain 179


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(Invitrogen) and destained with water. The bands were then excised and subjected to tryptic 180

digestion, as described previously (33). Peptides eluted from tryptic digests of gel pieces were 181

subjected to reversed phase column chromatography (self-packed C18 column, 100 μm diameter 182

x 200 mm length) operated on an Easy-nLC II (Thermo Fisher Scientific, Waltham, MA). Elution 183

was performed using a binary gradient of buffer A (0.1% (v/v) acetic acid) and buffer B (99.9 % 184

(v/v) acetonitrile; 0.1% (v/v) acetic acid) over a period of 100 min with a flow rate of 300 nl/min. 185

MS and MS/MS data were acquired with the LTQ-Orbitrap-Velos mass spectrometer (Thermo 186

Fisher Scientific) equipped with a nanoelectrospray ion source. The Orbitrap Velos was operated 187

in data-dependent MS/MS mode using the lock-mass option for real time recalibration. After a 188

survey scan in the Orbitrap (r = 30,000), MS/MS data were recorded for the 20 most intensive 189

precursor ions in the linear ion trap. For MS/MS analysis, singly charged ions were not taken into 190

account. 191

192

Identification of lysine acetylation sites. Post-translational lysine acetylation sites on proteins 193

were identified by searching all MS/MS spectra in .dta format against an E. coli database 194

(extracted from the Uniprot-KB database: 195

http://www.uniprot.org/uniprot/?query=Escherichia+coli+K12&sort=score), using Sorcerer™-196

SEQUEST® (Sequest v. 2.7 rev. 11, Thermo Electron including Scaffold_3_00_05, Proteome 197

Software Inc., Portland, OR). The Sequest search was carried out considering the following 198

parameters: a parent ion mass tolerance - 10 ppm, fragment ion mass tolerance of 1.00 Da. Up to 199

two tryptic mis-cleavages were allowed. Methionine oxidation (+15.99492 Da), cysteine 200

carbamidomethylation (+57.021465 Da) and lysine acetylation (+42.010571 Da) were set as 201


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variable modifications. Proteins were identified by at least two peptides applying a stringent 202

SEQUEST filter. Sequest identifications required deltaCn scores of greater than 0.10 and XCorr 203

scores of greater than 2.2, 3.3 and 3.8 for doubly, triply and quadruply charged peptides. 204

Acetylated peptides that passed these filter criteria were examined manually and accepted only 205

when b- or y- ions confirmed the acetylation site. 206

207

Quantification of acetylated peptides. To compare the abundance of acetylated peptides in 208

different biological samples, we used Scaffold (Sequest v. 2.7 rev. 11, Thermo Electron including 209

Scaffold_3_00_05, Proteome Software Inc., Portland, OR) to perform spectral counting analysis. 210

After fragmentation of a specific peptide, Scaffold can quantify protein amounts from different 211

samples according to the number of MS/MS fragment ion spectra. If a peptide was abundant in 212

the overview MS scan, then many MS/MS fragment ion spectra were generated for this peptide. 213

The number of these spectra is considered to the spectral count for that peptide. To compare the 214

abundance of any given acetylated peptide between samples, we calculated the ratio of spectral 215

counts of one sample versus the other. In-gel tryptic digests for the MS analysis are not suited to 216

added peptide standards; therefore, we applied a semi-quantitative approach. We processed and 217

measured all samples to be compared with the same methods, at the same time, in parallel, and 218

loaded equal amounts of tryptic digest to the LC column. Each comparison was performed on 219

multiple replicates. 220

221

In vitro acetylation reactions. Two different methods were used to detect in vitro acetylated 222

proteins using AcCoA as the acetyl group donor: detection by anti-acetylated lysine Western 223

immunoblot and detection of AcCoA hydrolysis. 224


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In vitro acetylation followed by anti-acetyllysine Western immunoblot analysis. Combinations of 226

20 µM RcsB, 0.4 mM AcCoA, and 2 µM YfiQ were incubated at 37°C for 1 hour in 50 mM Tris 227

HCl (pH 8), 10% glycerol, 1 mM DTT, 10 mM sodium butyrate, and 0.1 mM EDTA, as described 228

previously (34). (For the Western immunoblot analysis, see below) 229

230

In vitro acetylation followed by measurement of AcCoA hydrolysis. This biochemical in vitro 231

assay detects acetylation by measuring the hydrolysis of AcCoA. Biochemical assays were 232

performed as described previously (35) with some modifications. First, reactions were performed 233

in the presence of 150 mM NaCl at 25°C for 1 hr to prevent protein precipitation during the 234

reaction. Additionally, after stopping the reactions with guanidine HCl, samples were transferred 235

to Nanosep 10K MWCO centrifugation devices (Pall Life Sciences, VWR) and centrifuged at 236

17,000 x g for 10 minutes to separate CoA and the protein. The Ellman’s reagent was then added 237

to the flow-through and monitored as described (35). This biochemical in vitro acetylation assay 238

was performed in the absence of DTT because this reducing agent generates false positive signals. 239

Reactions reached completion within one hour, and a saturating concentration of RcsB was 240

determined to be 0.15 mM. High concentrations of YfiQ (100 μg) were necessary to detect 241

activity, possibly due to competition for binding between AcCoA and the His6 tag (35). 242

243

In vitro phosphorylation reactions. In vitro phosphorylation was carried out by incubating lithium 244

potassium AcP (Sigma) with purified His-tagged RcsB at 30°C for 30 min in buffer (40 mM Tris-245

HCl pH 8.0, 10 mM MgCl2, 40 mM KCl and 1 mM DTT), as described previously (36). 246

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Detection of phosphorylated RcsB. Similar to a previously reported procedure (36), 248

phosphorylated RcsB from in vitro phosphorylation was separated from non-phosphorylated 249

RcsB using zinc(II) Phos-TagTM SDS-PAGE (10% acrylamide (37.5:1), 350 mM Tris, pH 6.8, 250

0.1% SDS, 75 μM Phos-Tag (NARD Institute LTD), and 150 μM Zn(NO3)2). Purified protein 251

was detected by staining the gel with SimplyBlueTM (Invitrogen). 252

253

Western immunoblot analysis. Purified proteins were separated by SDS-PAGE. Proteins were 254

transferred onto a methanol-treated PVDF membrane at 25-30 V at 4°C for 16 hours. The blot 255

was blocked with 5% (w/v) milk prepared in PBS for 1 hour at room temperature. The blot was 256

washed with PBST for 5 minutes. A rabbit polyclonal antibody raised against an acetylated 257

lysine-containing peptide (Cell Signaling 9441) was used at a 1:500 dilution in 5% milk in PBS at 258

4°C overnight. The blot was washed 3 times for 5 minutes each with PBST and then incubated 259

with HRP-conjugated goat anti-rabbit secondary antibody (Cell Signaling 7074S) at a 1:1000 260

dilution in 5% milk in PBS for 1 hour at room temperature. The blot was washed 3 times for 5 261

minutes each with PBST and exposed using 20X LumiGLOR Reagent and Peroxide (Cell 262

Signaling 7003). 263

264

Motility assays. Three independent colonies representing three biological replicates were used to 265

inoculate 5 mL of TB and grown aerobically with agitation at room temperature. These overnight 266

cultures were diluted to a final OD600 of 0.1 in TB. 5 μl were inoculated onto the surface of 267

tryptone motility plates [TB supplemented with 0.25% (w/v) agar] and incubated at 28°C and the 268

diameter measured over time. The values represent the means with standard deviations. The 269

experiment included at least three replicates and was repeated at least once. 270


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271

Results 272

A novel RcsB-dependent behavior requires the phosphoryl acceptor site Asp-56 273

To determine how acetylation might regulate RcsB, we monitored RcsB activity using the 274

canonical RcsB-dependent promoter rprA (PrprA) fused transcriptionally to lacZ. This PrprA-275

lacZ fusion was carried on prophage λ (8), which we inserted into its attachment site in the E. coli 276

K-12 BW25113 chromosome. We grew the resultant λPrprA-lacZ lysogen (strain AJW3759, 277

Table 1) at 37°C in buffered tryptone broth (TB7), monitored β-galactosidase activity over time, 278

and observed that rprA transcription peaked during exponential phase (Figure 2A-B), a behavior 279

that required the RR RcsB (Figure 2B). 280

To further characterize this novel RcsB-dependent behavior, we asked whether PrprA 281

activity, like other RcsB-regulated behaviors, depended on the phosphoryl acceptor residue (Asp-282

56) in the REC domain of RcsB. From a single copy plasmid, we expressed WT rcsB (pLIH002, 283

Table 1) or the mutant rcsB-D56A (pLIH003, Table 1) in an rcsB null mutant of the λPrprA-lacZ 284

lysogen (strain AJW4884) and found that WT rcsB complemented exponential phase PrprA 285

activity, while the rcsB-D56A allele did not (Figure 2C). Since the rcsB-D56A allele carried by 286

the pLIH003 inhibited the activity of endogenous WT RcsB (Figure 2D), we conclude that RcsB-287

D56A is expressed and that exponential phase rprA transcription requires Asp-56 of RcsB and 288

thus active, phosphorylated RcsB. Since both the Rcs phosphorelay and AcP are reported to 289

activate RcsB (11,37,38), we tested if these phosphoryl sources could activate PrprA activity. 290

Because the sensor protein RcsC has been reported to function as either a net kinase or a net 291

phosphatase (11,37,38), we assessed the influence of RcsC on rprA promoter activity. First, we 292

deleted rcsC and found that PrprA activity diminished relative to the WT parent (Figure 2E), 293


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indicating that RcsC activates PrprA transcription. We then overexpressed RcsC, which is 294

reported to decrease phospho-RcsB-dependent mucoidy (11, Figure S1), and observed inhibition. 295

In contrast, RcsF overexpression, reported to increase mucoidy (11, Figure S1), activated rprA 296

transcription (Figure 2F). We conclude that endogenous RcsC functions as a net kinase in this 297

genetic background grown under these conditions. We further propose that the equilibrium 298

between RcsC kinase and phosphatase activities is sensitive to RcsC concentration. 299

300

AckA influences exponential phase rprA transcription. 301

AcP has been shown genetically to activate the Asp-56-dependent RcsB activity required 302

for mucoidy and for inhibition of flagellar biogenesis (11). AcP also has been shown both 303

biochemically and genetically to donate phosphoryl groups to RRs (38). We therefore assessed 304

the role of AcP in activating RcsB at the rprA promoter. To test whether AcP can act as a 305

phosphoryl group donor for RcsB, we used Phos-TagTM, a dinuclear metal complex (i.e. 1,3-306

bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II)) that has affinity for 307

phosphomonoester dianions, such as the one found in a phosphorylated RR. When included in an 308

SDS-PAGE, Phos-TagTM slows the migration of the phosphorylated protein, allowing it to be 309

distinguished from the non-phosphorylated protein by mobility shift. When purified RcsB was 310

incubated with AcP, we observed a shifted band that was sensitive to heat (Figure 3), indicative 311

of a heat-labile aspartyl phosphate. We conclude that this shifted band is composed of 312

phosphorylated RcsB and that RcsB can catalyze its own phosphorylation using AcP as the 313

phosphodonor. 314

Since AcP can donate its phosphoryl group to RcsB in vitro, we asked whether AcP 315

activates rprA transcription in vivo. The enzyme phosphotransacetylase (Pta) converts AcCoA to 316


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AcP, while acetate kinase (AckA) converts AcP to acetate (Figure 4A). Deletion of both ackA 317

and pta results in the lack of AcP (10). In contrast, deletion of ackA alone causes accumulation of 318

AcP (10). If AcP activated the observed Asp-56-dependent rprA transcription, then PrprA activity 319

would be expected to decrease in the ackA pta double mutant and increase in the ackA null 320

mutant. As predicted, the ackA pta double mutant displayed a small but reproducible reduction in 321

rprA transcription whether or not rcsC was intact (Figure 2E). These results support the 322

hypothesis that both AcP and RcsC contribute to RcsB phosphorylation. Contrary to expectations, 323

however, rprA transcription was reduced in the ackA mutant rather than increased (Figure 2E 324

and 4B). This latter result suggests that the accumulation of some metabolic intermediate in the 325

ackA mutant inhibits RcsB activity. We reasoned that this inhibitory mechanism might involve 326

protein acetylation because we have observed that a deletion of ackA changes the acetylation 327

profile of RNA polymerase (36) and enhances global E. coli protein acetylation (B. Zemaitaitis 328

and A. J. Wolfe, unpublished results). We thus considered the possibility that protein acetylation 329

was involved in the reduced rprA transcription exhibited by the ackA mutant. 330

331

The deacetylase CobB activates PrprA activity and regulates RcsB acetylation in vivo 332

Consistent with the hypothesis that acetylation may regulate rprA transcription, others 333

have reported that RcsB could be acetylated in vitro, that this acetylation reduces RcsB DNA 334

binding affinity, and that this acetylation can be reversed by CobB (14). We therefore tested if 335

protein acetylation inhibited rprA transcription. In the absence of cobB, which encodes the only 336

known E. coli deacetylase, there was a strong reduction in rprA transcription compared to that of 337

its WT parent, similar to that of the ackA mutant (Figure 4B). Similar results were obtained when 338

WT cells were grown in the presence of nicotinamide, an inhibitor of CobB (39) (Figure 4C). We 339


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conclude that CobB enhances rprA transcription, likely by deacetylating some protein that inhibits 340

rprA transcription in its acetylated form. 341

Because deletion or inhibition of CobB reduced PrprA activity (Figure 4B and C) and 342

since CobB deacetylates Lys-180Ac of RcsB in vitro (14), we asked whether RcsB was acetylated 343

in vivo and whether CobB regulated RcsB deacetylation in vivo. We first attempted to detect 344

acetylated endogenous RcsB, but failed because the concentration of RcsB was too low. We 345

therefore used pHis6rcsB (31) to overexpress RcsB in WT cells and in its isogenic cobB mutant 346

and grew the resultant transformants in TB7 supplemented with and without NAM. These results 347

have been corroborated by quantitative mass spectrometric analysis of cell-free lysates prepared 348

from WT cells and cobB mutants that do not overexpress RcsB (B. Schilling, B. Gibson, and A. J. 349

Wolfe, unpublished data). For each strain/condition, we performed four biological replicates in 350

parallel and prepared cell lysates. We excised RcsB protein from each SDS polyacrylamide gel 351

and evaluated the acetylation status using Orbitrap Velos LC-MS/MS analysis. To ensure 352

comparable protein abundances for tryptic digestion and MS analysis, RcsB protein obtained from 353

all strains were processed in parallel and similar protein concentrations were loaded onto each 354

SDS-polyacrylamide gel. This allowed us to use the Scaffold software to perform a semi-355

quantitative comparison of abundances (given as spectral counts, SC; (40)) for each detected 356

peptide that contained an acetylated lysine (Table 2, Tables S1-S3). 357

RcsB is acetylated in vivo. In four parallel bioreplicates of the untreated WT strain, 358

Orbitrap Velos mass spectrometry reproducibly detected acetylation of four lysine residues: Lys-359

72, Lys-125, Lys-128 and Lys-154. Each acetylated lysine was identified by at least 1 SC in at 360

least 2 replicates, with an average of 4.0 total SC over all four replicates (Table 2, Table S1A-D). 361

CobB affects RcsB acetylation in vivo. In the cobB mutant, mass spectrometry 362


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reproducibly detected acetylation of 8 lysine residues: the four listed above plus Lys-118, Lys-363

140, Lys-149, and Lys-180 (Table 2, Tables S2A-D). In the parent strain treated with NAM, 364

mass spectrometry also reproducibly detected acetylation of 8 lysine residues: the first seven 365

identified in the cobB mutant and Lys-173 instead of Lys-180 (Table 2, Tables S3A-D). 366

Furthermore, the acetylated peptides were more abundant in the cobB mutant (average total SC of 367

12.5) and in the NAM-treated parent strain (average total SC of 10.5) than in the parent control 368

(average total 4.0 SC). However, these increases in SC were not distributed evenly over all 369

acetylated lysines, suggesting that CobB does not regulate all acetylated RcsB lysines similarly. 370

For example, the average SC for peptides containing acetylated Lys-72 or Lys-125 did not differ 371

significantly between strains/conditions; in contrast, we reproducibly detected acetylation of 3 372

residues (Lys-118, Lys-140, and Lys-149) in the cobB mutant and the NAM-treated parent that 373

were not detected in the untreated parent (Table 2). We conclude that RcsB is acetylated in vivo 374

and that CobB may promote deacetylation of certain RcsB lysines in vivo. 375

376

AckA impacts the acetylation status of RcsB in vivo 377

If RcsB acetylation is responsible for the weak rprA transcription in the ackA mutant, then 378

the RcsB acetylation pattern in the ackA mutant should differ from RcsB from WT cells and, 379

perhaps, resemble that of the cobB mutant. We therefore overexpressed RcsB in the WT strain 380

and its isogenic ackA and cobB mutants, separated the proteins by SDS-PAGE, and subjected the 381

tryptic digests to mass spectrometry as described above. Once again, we performed replicate 382

experiments in parallel, and processed the same amount of protein from all samples so that we 383

could compare the spectral counts for each acetylated peptide in each mutant relative to WT. 384

When comparing the results of this experiment (Table 3) to those of the previously described 385


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experiment (Table 2), we noticed a general increase in the numbers of detected acetylated lysine 386

residues and their spectral counts. We attribute this difference primarily to the larger amounts of 387

RcsB analyzed in the second experiment, which was performed a year later, and to the use of a 388

different liquid chromatography (LC) column prior to Orbitrap Velos MS. 389

The general pattern however, was reproducible: acetylated peptides were more abundant 390

in the cobB mutant (average total SC of 14.5) than in the parent control (average total 8.5 SC) 391

(Table 3), supporting the conclusion that CobB may deacetylate RcsB in vivo. For the parent, we 392

reproducibly detected acetylation of 5 lysine residues, each identified by 1-2 SC per replicate 393

(Table 3, Tables S4A-B). For the cobB mutant, we observed reproducible acetylation of 7 394

lysines, each identified by 1-4 SC per replicate (Table 3, Tables S5A-B). Moreover, some 395

acetylated peptides were reproducibly more abundant than the same peptides from the parent, 396

suggesting that CobB deacetylates these sites in vivo. 397

As predicted, the acetylation profile of RcsB isolated from the ackA mutant (Table 3, 398

Tables S6A-B) generally resembled that of the cobB mutant (Table 3, Tables S5A-B). Of the 7 399

acetylated RcsB lysines from the cobB mutant, 6 lysines were reproducibly acetylated in the ackA 400

mutant. Several acetylated lysines (Lys-118, Lys-128, and Lys-154) were detected more often in 401

both mutants than in their WT parent. Like RcsB from the cobB mutant (average total 14.5 SC), 402

acetylated peptides from the ackA mutant were two-fold more abundant (average total 17 SC) 403

than the same peptides from WT cells (average total 8.5 SC). We conclude that disruption of ackA 404

promotes acetylation of some RcsB lysines in vivo. We further propose that the reduction in rprA 405

transcription in the ackA and cobB mutants results from the same mechanism, an increase in RcsB 406

acetylation. 407

408


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YfiQ regulates PrprA activity independently of RcsB acetylation 409

Because S. enterica Pat and its E. coli homolog YfiQ were reported to acetylate E. coli 410

RcsB in vitro and inhibit its ability to bind DNA (14), we asked if the acetyltransferase YfiQ 411

affects PrprA activity and found that it does: deletion of yfiQ inhibited rprA transcription (Figure 412

5). We therefore asked whether YfiQ affects RcsB acetylation in vivo. However, the acetylation 413

profile of RcsB isolated from the yfiQ mutant did not differ much from that of its parent (Table 3, 414

Tables S7A-B). While not identical, the lysine acetylation sites were quite similar. Most notably, 415

the overall SC for the yfiQ mutant and its parent were identical (average total 8.5 SC) and there 416

was no substantial change in the SC for individual acetylated lysines. Thus, the acetyltransferase 417

YfiQ appears to have no effect on the acetylation status of RcsB in vivo in this growth condition. 418

We conclude that YfiQ enhances rprA transcription by acetylating a protein other than RcsB. 419

420

In vitro acetylation of RcsB 421

Since our in vivo observations argue that YfiQ does not regulate the acetylation status of 422

RcsB (Table 3, Tables S7A-B), but the published in vitro results show that YfiQ can acetylate 423

RcsB (14), we investigated the effect of purified YfiQ on the acetylation status of purified RcsB. 424

We first purified RcsB using gel filtration chromatography, finding that the 23.7 kDa RcsB 425

purified from WT E. coli cells resolved into two peaks with estimated molecular mass values of 426

100 kDa (high molecular weight or peak 1 (RcsB-1)) and 60 kDa (low molecular weight or peak 2 427

(RcsB-2)) (Figure S2A). Dynamic light scattering revealed that monodisperse solutions of RcsB-428

1 and RcsB-2 have globular species with exclusion radii of 8.9 nm and 7.2 nm, respectively 429

(Figures S2B and S2C). Taken together, these results are consistent with the formation in 430

solution of both tetramers (RcsB-1) and dimers (RcsB-2). 431


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Anti-acetyllysine immunoblot analysis detected acetylation of purified YfiQ (Figure 6A). 432

It also detected acetylation of RcsB-1 and RcsB-2, but this acetylation was only visible after a 433

long exposure (Figure S3). This suggests that YfiQ and a small fraction of both RcsB oligomers 434

were acetylated under the conditions used for purification. Indeed, Orbitrap Velos mass 435

spectrometric analysis (33,41) detected 10 acetylated lysines in RcsB-1 (Table 4, Table S8A), 9 436

acetylated lysines in RcsB-2 (Table 4, Table S8D), and 8 acetylated lysines in YfiQ (Tables 437

S9A-C). The average total SC of acetylated peptides for both RcsB-1 (22 SC) and RcsB-2 (20 438

SC) were similar. The distribution of acetylated RcsB peptides also was similar, but not identical. 439

For both RcsB oligomers, incubation with AcCoA dramatically increased acetylation 440

(Figure 6A, compare lanes 5 and 6 to lanes 3 and 4) and increased the abundance of acetylated 441

RcsB peptides by about two-fold (Table 4, Tables S8B and E). The distribution of acetylated 442

lysines differed somewhat between the two oligomers. This difference suggests the possibility 443

that the oligomeric structure could influence the acetylation pattern. For YfiQ, incubation with 444

AcCoA also detected increased acetylation of YfiQ (Figure 6A, compare lane 2 to lane 1, Tables 445

S9D and E), suggesting that YfiQ might autoacetylate. 446

To determine whether RcsB can be a substrate of YfiQ, we used a biochemical assay that 447

detects AcCoA hydrolysis (35). When RcsB and AcCoA were present at saturating 448

concentrations, the activity of YfiQ towards either RcsB-1 or RcsB-2 was nearly identical 449

(Figure 6B). For both oligomers, AcCoA hydrolysis increased as the concentration of RcsB was 450

increased; incubation of 0.02 mM YfiQ with 15 times the concentration of RcsB (0.30 mM) 451

hydrolyzed more AcCoA than in the presence of equimolar amounts of RcsB (0.02 mM). This 452

behavior indicates that RcsB acts as a substrate of YfiQ. We also found that YfiQ hydrolyzed 453

AcCoA (Figure S4), adding support to the supposition that YfiQ might autoacetylate. 454


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To identify the RcsB lysines acetylated in the presence of YfiQ, we used mass 455

spectrometric analysis (33,41). Following incubation of either RcsB oligomer with YfiQ and 456

AcCoA, we observed acetylation of Lys-180, consistent with the previous report that YfiQ 457

acetylates Lys-180 in vitro (14), but we also observed YfiQ-dependent acetylation of several 458

other RcsB lysines: Lys-125, Lys-127, Lys-149 and Lys-186 (Table 4, Tables S8C and F). 459

Surprisingly, incubation with both AcCoA and YfiQ did not dramatically change the abundance 460

of acetylated lysines relative to incubation with AcCoA alone (Table 4, Tables S8B and E). 461

Anti-acetyl-lysine immunoblot analysis yielded a similar result (Figure 6A, compare lanes 9 and 462

10 to lanes 5 and 6). Instead, the pattern of acetylation changed. For some residues, the level of 463

acetylation increased relative to their status after incubation with AcCoA alone. For other 464

residues, the acetylation decreased (Table 4). For example, both RcsB-1 and RcsB-2 exhibited 465

increased SC for acetylated Lys-125, Lys-180, and Lys-186. In contrast, the SC for acetylated 466

Lys-140 of RcsB-1 decreased by more than five-fold (Table 4). While these in vitro results show 467

that YfiQ can affect the acetylation status of multiple RcsB lysines, our in vivo results showing 468

that YfiQ does not affect RcsB acetylation (Table 3, Tables S7A-B) argue against a direct role 469

for YfiQ in RcsB acetylation, at least under the tested conditions. 470

471

Analysing the function of acetylated RcsB lysines in rprA transcription 472

In the absence of ackA or cobB, rprA transcription was inhibited and RcsB acetylation 473

increased. One RcsB lysine (Lys-154) that was reproducibly more acetylated in the mutants 474

relative to the WT parent (Tables 2 and 3) was found to be critical for RcsB activity. To assess 475

the effect of acetylation on RcsB function, we applied an approach commonly used in studies of 476

acetylation in eukaryotes (42), converting this AckA- and/or CobB-sensitive lysine to a glutamine 477


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(which mimics a neutral, acetylated lysine) or to an arginine (which mimics a positive, non-478

acetylatable lysine). We introduced this rcsB variant (carried by pVEC) into the rcsB null strain 479

(AJW4884) and monitored PrprA activity. The acetylated and non-acetylated mimics of Lys-154 480

exhibited opposite behaviors. The mimic of acetylated Lys-154 (RcsB-K154Q) exhibited a strong 481

reduction in PrprA activity, a behavior that was indistinguishable from that of the vector control 482

(Figure 7A), the non-phosphorylatable mutant RcsB-D56A, and the RcsB-K154A mutant (data 483

not shown). This contrasted with the behavior of the non-acetylated mimic (RcsB-K154R), which 484

more closely resembled that of the WT protein (Figure 7A). We next tested whether the inability 485

of RcsB-K154Q to support PrprA activity was due to a lack of expression or a loss of function. To 486

do so, we took advantage of the report that RcsB inhibits migration through semi-solid agar by 487

repressing transcription of flhDC, which encodes the master regulator of flagella biosynthesis 488

(11,43). We asked if expression of the rcsB mutants could inhibit migration of the highly motile 489

strain AJW3331. Indeed, each mutant rcsB allele (D56A, K154A, K154Q, and K154R) inhibited 490

migration to a level similar to that of the WT allele (Figure 7B). We conclude that the mutants 491

are expressed and that they retain function. We therefore propose that the positive charge of Lys-492

154 is critical to RcsB-dependent activation of rprA transcription and that acetylation of Lys-154 493

may regulate RcsB activity. 494

495

Discussion 496

Given the diversity of the bacterial proteins that have been detected as acetylated (44-46), 497

there is great potential for this post-translational modification to impact many facets of bacterial 498

physiology. However, the study of bacterial protein acetylation is young and many fundamental 499

questions remain unanswered. In this report, we described genetic and biochemical analyses of 500


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RcsB acetylation that have led to four key findings. First, we provided the first evidence that in 501

vivo acetylation of a bacterial transcription factor could regulate its activity. Second, we verified 502

that RcsB is a substrate of CobB (14) and showed that it regulates the acetylation status of RcsB 503

in vivo. Third, we showed that rprA transcription is activated by YfiQ, but likely not by direct 504

acetylation of RcsB. Since YfiQ is the only known protein acetyltransferase in E. coli, this result 505

suggests that other mechanisms of acetylation might exist in E. coli. Fourth, we showed that the 506

Pta-AckA pathway influences protein acetylation. 507

508

Both phosphorylation and acetylation regulate rprA transcription. 509

Majdalani et al. reported that PrprA activity and rprA RNA levels depend on RcsB. As 510

cultures transition into stationary phase, rprA RNA levels increase 8-fold, while PrprA activity 511

increases only 2.5-fold. Thus, these authors proposed that RprA is stabilized during the transition, 512

allowing for accumulation in stationary phase. The net result is increased expression of the 513

alternative sigma factor σS (8,47). We have extended this model, showing that PrprA activity, and 514

thus RprA synthesis, can peak during exponential growth in buffered tryptone broth (Figure 2A-515

B). While the physiological impact of this novel behavior remains obscure, it has given us a 516

unique opportunity to investigate the effect that Nε-lysine acetylation exerts on the function of a 517

phosphorylated transcription factor. 518

Like other reported RcsB-activated behaviors, this exponential burst of rprA transcription 519

requires the phospho-acceptor site (Asp-56) of RcsB (Figure 2C). Like many of these behaviors, 520

this novel exponential phase rprA transcription required the kinase activity of the bifunctional 521

RcsC (Figure 2E). It also involved AcP as reported previously for two other RcsB-regulated 522

promoters: cps and flhD (11). In this study, we showed that purified RcsB can become 523


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phosphorylated when incubated with AcP (Figure 3). 524

If AcP donates its phosphoryl group to RcsB, then why is phospho-RcsB-dependent rprA 525

transcription inhibited in the ackA mutant? The answer appears to be acetylation. Escalante-526

Semerena and colleagues previously reported that RcsB could become acetylated on Lys-180 in 527

vitro by YfiQ or Pat, the S. enterica YfiQ homolog, and deacetylated by CobB (14). We have 528

confirmed this report and extended its significance. We confirmed that YfiQ acetylates RcsB in 529

vitro (Table 4 and Figure 6). We further showed that RcsB acetylation occurs in vivo on multiple 530

lysines (Tables 2 and 3) and that both CobB and AckA affect RcsB acetylation in vivo (Figure 531

4). We also provided genetic evidence that acetylation of a specific lysine may exert an effect on 532

RcsB activity (Figure 7). 533

534

What is the effect of acetylation on RcsB? 535

We propose that the positive charge at position 154 is required for RcsB-dependent rprA 536

transcription and that acetylation of Lys-154 would inhibit RcsB activity, possibly by interfering 537

with the binding of RcsB to its DNA site. In the absence of ackA or cobB, rprA transcription 538

diminished (Figure 4B) and acetylation of Lys-154 increased substantially (Table 3). 539

Furthermore, a genetic mimic of acetylated Lys-154 (RcsB-K154Q) appeared inactive at the rprA 540

promoter, while a genetic mimic of non-acetylated Lys-154 (RcsB-K154R) remained active 541

(Figure 7A). Our proposal is supported by a previous report on the structural analysis of Erwinia 542

amylovora RcsB DNA-binding domain (48). The authors reported that Lys-153 of E. amylovora 543

RcsB (homologous to Lys-154 in E. coli RcsB), positioned in the supporting helix a7 of the 544

DNA-binding HTH motif, is capable of interacting with DNA. Because the RcsB-K154Q and 545

RcsB-K154R mutants retained the ability to inhibit migration on semi-solid agar plates (Figure 546


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7B), which requires the binding of RcsB to the flhDC promoter, acetylation could impact some 547

later event in the initiation of rprA transcription, such as the recruitment of RNA polymerase. 548

However, while the inhibition of migration provides evidence that the mutant proteins are 549

functional and that RcsB does not require Lys-154 to bind at the flhDC promoter, it does not 550

directly address the role of Lys-154 at the rprA promoter. This is because the flhDC promoter is 551

quite complex and the binding of the mutant RcsB proteins could be aided by one of the many 552

proteins that also regulate flhDC. 553

554

CobB regulates RcsB acetylation. 555

CobB enhanced PrprA activity (Figure 4B and 4C), suggesting that CobB activates by 556

deacetylating some acetylated protein. Given that we found that RcsB is hyperacetylated in the 557

cobB mutant (Tables 2 and 3), we propose that acetylation of RcsB is responsible for reducing 558

PrprA activity and that CobB regulates the deacetylation of certain RcsB lysines in vivo. While 559

certain RcsB lysines (e.g. Lys-118 and Lys-154) appeared to be sensitive to CobB (i.e., they 560

exhibited increased acetylation in the absence of CobB activity (Tables 2 and 3), others (e.g., 561

Lys-72 and Lys-125) appeared to be insensitive to CobB, suggesting that some acetylated lysines 562

are not deacetylated or that some presently unknown deacetylase or other deacetylation 563

mechanism exists. 564

565

Does AckA influence protein acetylation? 566

We hypothesize that AckA is a regulator of protein acetylation. We base this hypothesis 567

on the following observations. First, we showed that RcsB is hyperacetylated when isolated from 568

the ackA mutant and that its acetylation profile was similar to that of RcsB isolated from the cobB 569


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mutant (Table 3). Second, we previously reported that the acetylation pattern of RNA polymerase 570

isolated from an ackA mutant grown in the presence of glucose is substantially different from the 571

pattern obtained with the WT parent (36). Indeed, preliminary analyses show that ackA mutants 572

exhibit a dramatic increase in global protein acetylation that is eliminated when pta is also deleted 573

(B. Zemaitaitis and A. J. Wolfe, unpublished results). Efforts to understand the underlying 574

mechanism are underway. 575

576

Does a lysine acetyltransferase regulate RcsB? 577

The mechanism by which RcsB becomes acetylated in vivo remains unknown. Deletion of 578

yfiQ diminished rprA transcription (Figure 5), suggesting that YfiQ is an activator of rprA 579

transcription. However, YfiQ is unlikely to act directly upon RcsB, as deletion of yfiQ did not 580

exert a major effect on the acetylation status of RcsB (Table 3). Further support for the 581

proposition that YfiQ is not the physiological acetyltransferase for RcsB acetylation comes from 582

the observation that deletion of yfiQ enhances migration in semi-solid agar rather than the 583

inhibition predicted if the main role of YfiQ were to acetylate Lys-180 and thus inhibit binding of 584

RcsB to its DNA site (J. Escalante-Semerena, personal communication). 585

If YfiQ does not acetylate RcsB in vivo, two scenarios are possible: (1) the existence of 586

another protein acetyltransferease or (2) a mechanism that does not involve an acetyltransferase. 587

The ability of purified RcsB to become acetylated when incubated with AcCoA alone is 588

suggestive of an alternative mechanism of acetylation (Figure 6 and Table 4). It is unlikely that 589

purified RcsB was contaminated by an acetyltransferase, as in-solution mass spectrometric 590

analysis did not detect a contaminating acetyltransferase (data not shown). An alternative is that 591

RcsB autoacetylates in vivo with AcCoA as the acetyl donor. This mechanism is not 592


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unprecedented, as others have reported the autoacetylation of several other proteins (49-52), 593

including proteins in the mitochondrion, where little is known about potential mitochondrial 594

lysine acetyltransferase(s) and the conditions are favorable for AcCoA-dependent lysine 595

acetylation, i.e., alkaline pH (~8.0) and high AcCoA concentration (~5 mM) (53-55). 596

597

Why is RcsB regulated by both aspartyl phosphorylation and Nε-lysine acetylation? 598

Since RcsB regulates over 100 different genes, involved in many aspects of bacterial 599

physiology, it is likely that acetylation affects other RcsB-regulated promoters. Regulating RcsB 600

with a simple phosphorylation-dependent “on-off” switch may not provide enough sophisticated 601

control of gene expression. Indeed, this study shows that RcsB can be post-translationally 602

modified in vivo on eight different residues (i.e., phosphorylated on Asp-56 and acetylated on at 603

least 7 lysines), raising the opportunity for 256 different isoforms. The existence of multiple RcsB 604

isoforms could increase functional diversity, which could dictate promoter specificity or affinity 605

for the diverse RcsB interacting proteins (5). Furthermore, multiple and different modifications 606

could interact (56). In bacteria, such an interaction is not unprecedented. In Mycoplasma 607

pneumoniae, serine/threonine/tyrosine phosphorylation and Nε-lysine acetylation are common co-608

modifiers of the same proteins, and phosphorylation and acetylation are proposed to regulate each 609

other (57). Like RcsB, CheY is a RR that can be co-regulated by phosphorylation and acetylation 610

and these modifications of CheY inhibit each other (58-65). Additionally, we show that RcsB can 611

form dimers or tetramers (Figure S2), a behavior that has been described for some other RRs (66-612

68). 613

614

Concluding Remarks 615


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In summary, we propose that RcsB can become acetylated on multiple lysines in vivo. We 616

further propose that one of these acetylations (Lys-154) inhibits the ability of RcsB to activate 617

rprA transcription in vivo. Much remains to be learned, including the mechanism(s) by which 618

RcsB becomes acetylated, how acetylation inhibits RcsB function and whether acetylation 619

interacts with phosphorylation, and how the Pta-AckA pathway regulates protein acetylation. 620

621

Acknowledgments 622

We thank Sandy Thao and Jorge Escalante-Semerena for allowing us access to unpublished data. 623

We thank Susan Gottesman, Robert Blumenthal, Kaymeuang Cam, and Jorge Escalante-624

Semerena for their generous donations of phage, plasmids or strains. We thank Dhaval Nanavati 625

for his mass spectrometric identification of acetylated RplB. We thank all members of both the 626

Wolfe and Visick labs for critical and open discussions. We specifically thank Sylvia A. Reimann 627

for her help and support, Bruno P. Lima for his help with Phos-Tag, Bozena Zemaitaitis for her 628

analyses of global acetylation in E. coli, and Richard M. Schultz for his modeling of RcsB 629

structure. This work was supported by NIH grant GM066130 and Loyola University Chicago 630

Potts Foundation award LU11200 awarded to A.J.W and by Deutsche Forschungsgemeinschaft 631

project AN746/2-1 awarded to H.A. It also was supported with Federal funds from the National 632

Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health 633

and Human Services, under Contracts No. HHSN272200700058C and HHSN272201200026C to 634

W.F.A. 635


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59. Barak, R., Abouhamad, W. N., and Eisenbach, M. (1998) J Bacteriol 180, 985-988 744 60. Ramakrishnan, R., Schuster, M., and Bourret, R. B. (1998) Proc Natl Acad Sci U S A 95, 745

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molecular biology 376, 1260-1271 749 63. Barak, R., and Eisenbach, M. (2004) Journal of molecular biology 342, 375-381 750 64. Barak, R., Prasad, K., Shainskaya, A., Wolfe, A. J., and Eisenbach, M. (2004) Journal of 751

molecular biology 342, 383-401 752 65. Liarzi, O., Barak, R., Bronner, V., Dines, M., Sagi, Y., Shainskaya, A., and Eisenbach, M. 753

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Tomita, M., Wanner, B. L., and Mori, H. (2006) Molecular systems biology 2, 2006 0008 767 72. Zuber, M., Hoover, T. A., and Court, D. L. (1995) Journal of bacteriology 177, 4238-4244 768 73. Amann, E., Ochs, B., and Abel, K. J. (1988) Gene 69, 301-315 769 74. Clarke, D. J., Joyce, S. A., Toutain, C. M., Jacq, A., and Holland, I. B. (2002) Journal of 770

bacteriology 184, 1204-1208 771 772 773


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Table 1. Bacterial strains, bacteriophage, plasmids, and primers used in this study. 774

Strain, phage, plasmid, or primer

Relevant Characteristic Source/Reference

Strains

BW25113 F- λ- Δ(araD-araB)567 Δ(rhaD-rhaB)568 ΔlacZ4787 rrnB3 rph-1 hsdR514

(69)

AJW678 thi-1 thr-1(Am) leuB6 metF159(Am) rpsL136 ΔlacX74 (70) AJW3759 BW25113 λΦ(PrprA142-lacZ) λ: λrprA142 BW25113 AJW3331 AJW678 λΦ(PrprA142-lacZ) λ: λrprA142 AJW678

AJW4884 AJW3759 ΔrcsB::FRT P1: JW2205 (71) AJW3759, then removed antibiotic marker

AJW3976 AJW3759 ΔrcsC::tet P1: MZ63 (72) AJW3759 AJW3981 AJW3759 ΔackA::FRT-kan-FRT P1: JW2293 (71) AJW3759

JE8659 ΔcobB::cat Escalante-Semerena (U. of Georgia)

AJW5011 AJW3759 ΔcobB::cat P1: JE8659 AJW3759 AJW3797 AJW3759 ΔyfiQ:: FRT-kan-FRT P1: JW2568 (71) AJW3759

EPI300TM-T1R F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15lacX74 recA1 endA1 araD139 (ara-leu)7697 galU galK rpsL nupG trfA tonA dhfr

EpicentreR Biotechnologies

BL21 (DE3) Magic a derivative of BL21 cells carrying a plasmid encoding rare tRNAs (29) Phage

λrprA142 rprA142-lacZ (8) Plasmids

pVEC Single-copy, lacZ null derivative of pCC1 (EpicentreR), control for pLIH002 and pLIH003 CmR

(27)

pLIH001 pVEC derivative with kanamycin cassette removed This study pLIH002 pLIH001 carrying rcsB(WT) CmR This study pLIH003 pLIH001 carrying rcsBD56A allele CmR This study pLIH004 pLIH001 carrying rcsBK154Q allele CmR This study


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pLIH005 pLIH001 carrying rcsBK154R allele CmR This study pLIH006 pLIH001 carrying rcsBK154A allele CmR This study pTrc99a Vector control for pPSG980 ApR (73) pPSG980 pTrc99a carrying Φ(PlacUV5–rcsC) ApR (7,74) pMH300 pTrc99a carrying Φ(PlacUV5–rcsF) ApR (37) pCA24n Control plasmid CmR (28) pCA24n-rcsB pCA24n expressing 6xHis-RcsB from an IPTG-inducible promoter CmR (28)

pHis6rcsB pIM10 vector expressing 6xHis-RcsB from an IPTG-inducible promoter, KnR SpR

(31)

Primers (5’-3’) rcsBFBamHI GGATCCAGGAAGGTAGCCTATTACATGAAC This study rcsBRBamHI GGATCCTTAGTCTTTATCTGCCGGACTTAAGG This study pCC1RBZ GTAAAACGACGGCCAGTGAATTG This study

rcsBK154Q ACAAGCGTCTCTCGCCACAGGAGAGTGAAGTTCTGCG, AAACAG

This study

rcsBK154A TGACAAGCGTCTCTCGCCAGCAGAGAGTGAAGTTCTGC, AAAGCA

This study

rcsBK154R ACAAGCGTCTCTCGCCAAGAGAGAGTGAAGTTCTG, AAAAGA

This study

rcsBD56A CATGTGTTGATTACCGCTCTCTCCATGCCTGGC, GATGCT This study

775


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Table 2. Spectral counts of lysine acetylated RcsB peptides isolated from WT cells, cobB mutants, and WT cells exposed to 776

nicotinamide (NAM) 1,4 777

778 Average SC2 SC Ratio2

Acetylated lysine

WT cobB WT + NAM

cobB/WT NAM/WT

72 1.0 0.8 1.0 0.8 1.0 118 0.0 3.0 2.3 >3.03 >2.3 125 1.5 2.0 1.5 1.3 1.0 128 0.5 0.8 1.0 1.5 2.0 140 0.0 2.0 0.8 >2.0 >0.8 149 0.0 1.5 1.5 >1.5 >1.5 154 1.0 2.0 2.0 2.0 2.0 173 0.0 0.0 0.5 NA >0.5 180 0.0 0.5 0.0 >0.5 NA

Total SC 4.0 12.5 10.5 3.1 2.6

779 1Average spectral counts (SC) of detected lysine acetylation sites in the wild type (WT), its isogenic cobB mutant (cobB) and in the 780

WT treated with NAM (WT+NAM). 781

2Using data obtained from Scaffold 3 proteome software, the spectral counts of four biological replicates for each strain/condition 782

were counted manually, were averaged over the four replicates, and were compared as ratios of the mutant or the treated WT relative 783

to the untreated WT. 784

3When the SC of a peptide with an acetylated lysine in the cobB mutant or in the NAM-treated WT is greater than the SC of the same 785


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peptide in the untreated WT control, the ratio is underlined. A ratio of >n refers to any acetylated peptide that was not detected in WT 786

(SC=0), but detected in the cobB mutant or the NAM-treated WT (SC=n). 787

4The spectral counts of the detected lysine acetylation sites in each replicate and the peptide scores and mass deviations as well as the 788

complete MS/MS spectra and fragment ion series can be found in Tables S1A-D, S2A-D, and S3A-D, where A-D denote the four 789

bioreplicates. 790

791


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Table 3. Spectral counts of lysine acetylated RcsB peptides isolated from WT cells or yfiQ, cobB, or ackA mutant cells 1,4 792

793 Average SC2 SC Ratio2

Acetylated lysine

WT cobB ackA yfiQ cobB/WT ackA/WT yfiQ/WT

63 1.0 1.0 1.5 0.0 1.0 1.5 0.0 72 2.0 0.5 4.5 2.0 0.33 2.33 1.0

118 1.5 3.5 4.5 2.5 2.4 3.0 1.7 125 1.5 1.5 2.0 1.5 1.0 1.4 1.0 128 0.5 2.0 1.0 0.0 4.0 2.0 0.0 140 0.0 1.5 0.5 0.0 >1.5 >0.5 0.0 149 0.0 0.5 0.0 0.0 >0.5 0.0 0.0 154 0.5 3.0 2.0 1.0 6.0 4.0 2.0 173 1.5 0.0 0.0 1.5 0.0 0.0 1.0 180 0.0 1.0 1.0 0.0 >1.0 >1.0 0.0

Total SC 8.5 14.5 17.0 8.5 1.7 2.0 1.0 794 1Average spectral counts (SC) of detected lysine acetylation sites in the wild type (WT), and its isogenic cobB, ackA and yfiQ mutants. 795

2Using data obtained from Scaffold 3 proteome software, the spectral counts of two biological replicates for each strain were counted 796

manually, were averaged over the two replicates, and were compared as ratios of the mutants relative to the untreated WT. 797

3When the SC of a peptide with an acetylated lysine in a mutant is less than the SC of the same peptide in the WT control, the ratio is 798

italicized. When the SC of a peptide with an acetylated lysine in a mutant is greater than the SC of the same peptide in the WT control, 799

the ratio is underlined. A ratio of >n refers to any acetylated peptide that was not detected in WT (SC=0), but detected in a mutant 800

(SC=n). 801


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complete MS/MS spectra and fragment ion series can be found in Tables S4A-B, S5A-B, S6A-B and S7A-B, where A-B denote the 803

two bioreplicates. 804

805


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Table 4. Spectral counts of acetylated peptides of RcsB1,4 806

807

Acetylated

lysine

RcsB-1 RcsB-2 Average SC2 SC Ratio2 Average SC SC Ratio

Control AcCoA AcCoA+

YfiQ AcCoA /control

AcCoA+YfiQ /AcCoA

control AcCoA AcCoA+

YfiQ AcCoA /control

AcCoA+YfiQ /AcCoA

63 2.0 1.0 0.0 0.53 0.0 0.0 0.0 0.0 NA NA 72 2.0 2.0 1.5 1.0 0.8 0.5 2.5 1.5 5.0 0.6 118 5.5 7.0 10.0 1.3 1.4 6.0 8.5 10.5 1.4 1.2 125 2.0 4.0 13.5 2.03 3.4 3.0 4.0 9.0 1.3 2.3 127 0.0 1.0 2.0 >1.0 2.0 0.0 1.0 2.0 >1.0 2.0 128 3.0 4.0 6.0 1.3 1.5 1.5 4.5 7.0 3.0 1.6 140 0.5 19.0 3.0 38.0 0.2 0.0 3.0 3.5 >3.0 1.2 149 1.0 2.0 5.0 2.0 2.5 2.5 5.0 5.0 2.0 1.0 154 3.5 8.0 4.5 2.3 0.6 3.0 6.5 5.0 2.2 0.9 173 2.0 2.0 2.5 1.0 1.3 1.0 2.5 2.0 2.5 0.8 180 0.5 1.0 6.0 2.0 6.0 1.5 2.5 6.5 1.6 2.3 186 0.0 1.0 3.0 >1.0 3.0 1.0 0.5 2.5 0.5 5.0

Total SC 22 52 57 2.4 1.1 20 40.5 54.5 2.0 1.3 808 1Average spectral counts (SC) of detected lysine acetylation sites in purified RcsB (control), and purified RcsB incubated with AcCoA 809

(AcCoA) or purified RcsB incubated with AcCoA plus YfiQ (AcCoA+YfiQ). 810

2Using data obtained from Scaffold 3 proteome software, the spectral counts of two replicates for each condition were counted 811

manually, were averaged over the two replicates, and were compared as the ratio of SC detected for RcsB incubated with AcCoA or 812

with AcCoA and YfiQ relative to SC detected with purified RcsB alone. 813


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3When the SC of a peptide with an acetylated lysine following AcCoA incubation is substantially more than the SC of the same 814

peptide in the absence of AcCoA or if the SC of a peptide with an acetylated lysine following incubation with both AcCoA and YfiQ 815

is substantially more than the SC of the same peptide in the presence of AcCoA alone, the ratio is underlined. If the ratio is 816

substantially less, then the ratio is italicized. A ratio of >n refers to any acetylated peptide that was not detected in the absence of 817

AcCoA (SC=0), but detected in the presence of AcCoA (SC=n). 818


complete MS/MS spectra and fragment ion series can be found in Tables S10A-F, where A-C respectively denote RcsB-1 alone, 820

RcsB-1 incubated with AcCoA, and RcsB-1 incubated with both AcCoA and YfiQ, and D-F respectively denote RcsB-2 alone, RcsB-821

2 incubated with AcCoA, and RcsB-2 incubated with both AcCoA and YfiQ. 822


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823

824

Figure 1. Schematic diagrams of the Rcs phosphorelay. 825

A) Model for Rcs kinase signaling. In the presence of an activating environmental stimulus, 826

RcsC acts as a net kinase, using ATP as a phosphoryl group donor, this phosphoryl group is 827

transferred from RcsC to the Hpt RcsD, which donates the phosphoryl group and activates RcsB. 828

B) Model for Rcs phosphatase signaling. Under conditions that stimulate RcsC net phosphatase 829

activity, acetyl-phosphate (AcP) is an alternative phosphoryl group donor for RcsB. The RcsCD 830

pathway can remove phosphoryl groups from phosphorylated RcsB to generate inorganic 831

phosphate. 832

833 834


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2

Prp

rA A

ctiv

ity (M

iller

units

)

A B

0

2000

4000

6000

0 1 2 3 4 5 6 7 8 9 10

lag exponential stationary

Time (hours)

Prp

rA A

ctiv

ity (M

iller

units

)

A600

C

05000

100001500020000

0.03 0.3 3

WT/pVEC

WT/prcsBD56A WT/prcsBWT

A600

Prp

rA A

ctiv

ity (M

iller

units

)

Prp

rA A

ctiv

ity (M

iller

units

)

D rcsB/pVEC

rcsB/prcsBD56A rcsB/prcsBWT

F

Prp

rA A

ctiv

ity (M

iller

units

)

05000

100001500020000

0.03 0.3 3A600

WT/pTrc99a WT/prcsC WT/prcsF

02000400060008000

10000

0.03 0.3 3

Prp

rA A

ctiv

ity (M

iller

units

)

A600

E

05000

100001500020000

0.03 0.3 3A600

WT rcsB

WT

ackA pta ackA rcsC ackA pta rcsC

05000

100001500020000

0.03 0.3 3


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838

Figure 2. rprA transcription in exponential phase requires the conserved aspartyl residue 839

56 of RcsB and is regulated by RcsC and AcP. 840

A) PrprA activity of WT BW25113 cells lysogenized with a λPrprA-lacZ transcriptional fusion 841

(strain AJW3759) and aerated at 37°C in TB7. Cells were harvested at multiple points 842

throughout growth to measure A600 and β-galactosidase activity. The values represent the means 843

of triplicate independent cultures with standard deviations. 844

B) PrprA activity of WT cells (AJW3759) and the isogenic rcsB (AJW4884) mutant. The values 845

represent the means of triplicate independent cultures with standard deviations. 846

C) PrprA activity of an rcsB null mutant (AJW4884) transformed with a lacZ null derivative of 847

the single-copy expression plasmid pVEC or with a variant of that vector that encodes the WT 848

rcsB allele (pLIH002) or the rcsBD56A (pLIH003) mutant allele aerated at 37°C in TB7 849

supplemented with 50 µM IPTG. The values represent the means of triplicate independent 850

cultures with standard deviations. 851

D) PrprA activity of WT cells (AJW3759) transformed with pVEC or pVEC encoding WT rcsB 852

(pLIH002) or the rcsBD56A allele (pLIH003) aerated at 37°C in TB7 supplemented with 50 µM 853

IPTG. The values represent the means of triplicate independent cultures with standard deviations. 854

E) PrprA activity of WT cells (AJW3759), ackA (AJW3981), ackA pta (AJW4030), rcsC 855

(AJW3976), and ackA pta rcsC (AJW5187) aerated at 37°C in TB7. The values represent the 856

means of triplicate independent cultures with standard deviations. 857

F) PrprA activity of WT cells transformed with the RcsC expression plasmid prcsC (pPSG980), 858

the RcsF expression plasmid (pMH300), or the vector control (pTrc99a) and aerated at 37°C in 859

TB7 supplemented with 50 µM IPTG to induce rcsC or rcsF expression. The values represent 860


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the means of triplicate independent cultures with standard deviations. 861

862


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863

864 865 866 867 868 869 870

871 872 Figure 3. AcP can act as a phosphoryl group donor to RcsB. 873

Purified His6-RcsB peak 2 was incubated in the absence (lane 1) or presence of 5 mM AcP (lanes 874

2, 3, 4) for 30 minutes at 30°C. A fraction of the reaction was heated at 95°C for 15 minutes to 875

hydrolyze the phosphoryl group (lane 4). The reactions were separated in a Phos-TagTM SDS-PA 876

gel and the gel was stained with coomassie brilliant blue. 877

878 879


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46

880 881

Figure 4. AckA and CobB activate rprA transcription in BW25113 882

A) The Pta-AckA pathway. AcCoA, acetyl coenzyme A; Pta, phosphotransacetylase; Pi, 883


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47

inorganic phosphate; AckA, acetate kinase. Glucose is metabolized to AcCoA. Pta reversibly 884

converts Pi and AcCoA to generate AcP and CoASH. AckA reversibly converts AcP and ADP to 885

ATP and excreted acetate. 886

B) WT cells lysogenized with λPrprA-lacZ (AJW3759) and the isogenic ackA (AJW3981) and 887

cobB (AJW5011) mutants were aerated at 37°C in TB7. Cells were harvested at multiple points 888

throughout growth to measure A600 and β-galactosidase activity. The values represent the means 889

of triplicate independent cultures with standard deviations. 890

C) Peak β-galactosidase activity between A600 0.6 – 0.8 of WT (AJW3759) cells aerated at 37°C 891

in TB7 in the presence or absence of increasing concentrations of nicotinamide (NAM). The 892

values indicate the average of triplicate independent cultures with standard deviations. 893

894

895


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896 897

Figure 5. Analysing the effect of YfiQ at the rprA promoter. 898

λrprA of WT cells (AJW3759) and the isogenic yfiQ null mutant (AJW3797) were grown at 899

37°C in TB7 with shaking. Samples were harvested for A600 and β-galactosidase activity 900

measurements. The values represent the average of triplicate independent cultures and error bars 901

indicate standard deviations. 902

903

904


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49

905 906 Figure 6. In vitro acetylation of RcsB 907

A) In vitro acetylation reactions with RcsB, YfiQ, and AcCoA. 50 µl reactions of 21 µM RcsB-1 908

or -2, 2 µM YfiQ, and 0.4 mM AcCoA were incubated at 37°C for 2 hours. The reactions were 909

quenched with 50 µl 2X SDS-PAGE loading buffer and heated at 95°C for 5 minutes. 15 µl was 910

resolved by 13% SDS-PAGE, proteins were transferred, and acetylated proteins detected by 911

Western immunoblot with anti-acetyl-lysine antibody. Lane 1: YfiQ alone, lane 2: YfiQ + 912


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AcCoA, lane 3: RcsB-1 alone, lane 4: RcsB-2 alone, lane 5: RcsB-1 + AcCoA, lane 6: RcsB-2 + 913

AcCoA, lane 7: YfiQ + RcsB-1, lane 8: YfiQ + RcsB-2, lane 9: YfiQ + RcsB-1 + AcCoA, lane 914

10: YfiQ + RcsB-2 + AcCoA. These results are representative of three independent experiments. 915

The high molecular weight signal in lanes 1, 2, 9 and 10 corresponds to acetylated YfiQ. The 916

acetylated sites were determined using semi-quantitative mass spectrometry (Tables S9A-E). 917

Mass spectrometry also identified the signal visible between the 37-50 kDa markers in lanes 2, 9 918

and 10 as the 43.3 kDa EF-Tu protein and determined the acetylated sites (Table S10). Mass 919

spectrometry identified the signal between the 25-37 kDa markers in lanes 9 and 10 as the 29.9 920

kDa RplB protein and determined its acetylated sites (Table S11). 921

B) Activity of YfiQ using different concentrations of RcsB1 and RcsB2. Reactions were 922

performed using 0.02 mM YfiQ at 25°C for one hour in a 50 μl reaction volume. To initiate the 923

reaction, 0.5 mM AcCoA was used. Activity (nmol/min/mg) is shown as the average of three 924

separate trials. Black bars correspond to 0.02 mM RcsB and red bars to 0.3 mM RcsB. 925

926 927


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928 929 930

931

Figure 7. Analyzing the role of Lys-154 on rprA transcription and migration. 932


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A) PrprA activity of rcsB mutant (AJW4884) transformed with the single copy plasmid pVEC or 933

pVEC carrying WT and mutant rcsB-K154 alleles. Cells were aerated at 37°C in TB7 934

supplemented with 50 µM IPTG. 935

B) Migration analysis of the highly motile WT strain (AJW3331) transformed with pVEC or 936

pVEC carrying WT and mutant rcsB alleles. The histogram shows the last measurement of an 937

8.5 hour time course experiment. These results are representative of the whole experiment. 938

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acetylation of the response regulator rcsb controls ... · 59 encoding rna rpra , which activates...

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