1

Role of FimW, FimY, and FimZ in regulating the expression of type I 1

fimbriae in Salmonella enterica serovar Typhimurium 2

3

Supreet Saini§, Jeffrey A. Pearl§, and Christopher V. Rao* 4

5

Department of Chemical and Biomolecular Engineering 6

University of Illinois at Urbana-Champaign 7

Urbana, Illinois, United States, 61801 8

9

10

Running Title: Regulation of type I fimbriation in S. typhimurium 11

Key words: Type I fimbriae, FimW, FimY, FimZ, Gene regulation 12

13

§ Authors contributed equally to this work 14

15

* Corresponding author. Department of Chemical and Biomolecular Engineering, 16

University of Illinois at Urbana-Champaign, 600 S. Mathews Ave, Urbana, IL, United 17

States, 61810. Phone: (217) 244-2247. Fax: (217) 333-5052. Email: 18

chris@scs.uiuc.edu. 19

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01694-08 JB Accepts, published online ahead of print on 13 February 2009

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Abstract 20

Type I fimbriae in Salmonella enterica serovar Typhimurium are surface appendages 21

that facilitate binding to eukaryotic cells. Expression of the fim gene cluster is known to 22

be regulated by three proteins – FimW, FimY, and FimZ – and a tRNA encoded by 23

fimU. In this work, we investigated how these proteins and tRNA coordinately regulate 24

fim gene expression. Our results indicate that FimY and FimZ independently activate 25

the PfimA promoter which controls the expression of the fim structural genes. FimY and 26

FimZ were also found to strongly activate each other’s expression and weakly activate 27

their own expression. FimW was found to negatively regulate fim gene expression by 28

repressing transcription from the PfimY promoter, independent of FimY or FimZ. 29

Moreover, FimW and FimY interact within a negative feedback loop as FimY was found 30

to activate the PfimW promoter. In the case of fimU, expression of this gene was not 31

found to be regulated by FimW, FimY, or FimZ. We also explored the effect of fim gene 32

expression on Salmonella Pathogenecity Island 1 (SPI1). Our results indicate that FimZ 33

alone is able to enhance the expression of hilE, a known repressor of SPI1 gene 34

expression. Based on our results, we were able to propose an integrated model for the 35

fim gene circuit. As this model involves a combination of positive and negative 36

feedback, we hypothesized that the response of this circuit may be bistable and thus a 37

possible mechanism for phase variation. However, we found that the response was 38

continuous and not bistable. 39

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Introduction 40

Type I fimbriae in Salmonella enterica serovar Typhimurium (S. typhimurium) are 41

proteinaceous surface appendages that carry adhesions specific for mannosylated 42

glycoproteins (9). Type I fimbriae are involved in S. typhimurium pathogenicity by 43

facilitating the binding to and invasion of intestinal epithelial cells (44). In orally 44

inoculated mice, a wild-type strain has been shown to cause more infections and deaths 45

than a fim- mutant strain (19). A fim- mutant has also been shown to exhibit several-fold 46

weaker binding to HEp-2 and HeLa cells, and the defect in binding could be restored by 47

complementing the fim system on a plasmid (4). Apart from type I fimbriae, mutations in 48

different Salmonella fimbrial systems - lpf, pef, and agf - have all been also shown to 49

greatly reduce virulence in mice (48). These systems appear to work synergistically in 50

order to facilitate colonization of the ileum (5). In S. typhimurium, the fim gene cluster 51

possesses all of the genes necessary for type I fimbrial production. This gene cluster is 52

composed of six structural genes, three regulators, and a tRNA specific for rare arginine 53

codons (AGA and AGG). The structural genes - fimA, fimI, fimC, fimD, fimH, and fimF - 54

are all expressed in one transcript from the PfimA promoter (27, 37-39). The regulators - 55

fimZ, fimY, and fimW - are all expressed from independent promoters (45, 47, 49). The 56

tRNA encoded by fimU is located at one end of the cluster and is required for the 57

effective translation of the regulatory genes that all carry rare arginine codons (43). 58

Type I fimbriation is environmentally regulated with fim gene expression favored in 59

static liquid media, whereas growth on solid media inhibits expression (17). Moreover, 60

S. typhimurium cultures in fimbriae-inducing conditions contain cells in both fimbriated 61

and non-fimbriated states (36). While the regulation of fim gene expression has been 62

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studied extensively in Escherichia coli, far less is known about the regulation in S. 63

typhimurium (1, 28). In particular, despite homology between the structural genes for 64

type I fimbriae in E. coli and S. typhimurium, their expression is regulated in completely 65

different manners. No homologs of E. coli regulators, FimB and FimE, are present in S. 66

typhimurium (25, 29). Also, the S. typhimurium PfimA promoter is inactive in E. coli, 67

indicating that the PfimA promoter is regulated by different factors in these two organisms 68

(49). In S. typhimurium, the expression of the structural genes is regulated by three 69

transcription factors: FimY, FimZ, and FimW (45, 47, 49). Both FimZ and FimY are 70

essential for expression of the structural genes from the PfimA promoter (49). In 71

particular, deletion of either the fimY or fimZ gene reduces expression from the PfimA 72

promoter and prevents S. typhimurium from making type I fimbriae. FimZ has been 73

shown to bind the PfimA promoter and promote transcription (13, 49). FimY, on the other 74

hand, is thought to facilitate activation of the PfimA promoter as direct binding has not 75

been observed (45). FimW is a negative regulator of fim gene expression (46). FimW 76

has also been suggested to auto regulate its expression as enhanced PfimW activity has 77

been observed in ∆fimW mutant. In DNA-binding assays, FimW was not observed to 78

bind any of the fim promoters. However, FimW was found to interact with FimZ in a 79

LexA-based two-hybrid system in E. coli (46). Thus, a possible mechanism for FimW-80

mediated repression may be that it binds FimZ and prevents it from activating 81

transcription. However, analysis for FimW’s amino acid sequence predicts that it has a 82

DNA-binding domain. Moreover, it is related to a broad range of prokaryotic 83

transcription factors, with its closest relatives being BpdT from Rhodococcus and an 84

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uncharacterized response regulator, TodD, from Pseudomonas putida (30, 31). Thus, 85

FimW may also act by an alternate mechanism involving DNA binding. 86

In addition to these transcription factors, the fimU tRNA also plays a role in fim gene 87

expression (43). All three regulators - FimZ, FimY, and FimW - contain a number of the 88

rare arginine codons, AGA and AGG, recognized by the fimU tRNA. In the case of 89

FimY, ∆fimU mutants have been shown to be non-fimbriated due to inefficient 90

translation of fimY mRNA. This translational regulation results from FimY having three 91

rare arginine codons within its first 14 amino acids. The phenotypic effect of ∆fimU 92

mutation could, however, be overcome by expressing fimU from a plasmid or by 93

changing these three rare arginine codons in fimY to ones more efficiently translated. 94

As a pathogen, S. typhimurium invades host cells by a process in which effector 95

proteins are injected into the target cells with the help of the Salmonella Pathogenicity 96

Island 1 (SPI1) type III secretion system (T3SS) (12, 14). SPI1 gene expression is 97

regulated by a number of proteins, with the critical activator being HilA (2). Expression 98

of hilA, in turn, is regulated by three AraC-like transcriptional activators: hilC, hilD, and 99

rtsA (20, 22, 23, 33, 41, 42). HilD activity is controlled by HilE; this protein binds HilD 100

and is thought to prevent it from activating the PhilA promoter (6, 8). FimY and FimZ 101

have been previously shown to regulate SPI1 gene expression by repressing hilA 102

expression through their activation of the PhilE promoter (7). 103

In this work, we investigated the gene circuit regulating fim expression. Using 104

genetic approaches, we found that FimZ and FimY activate each other’s expression, 105

and that each protein can independently activate the PfimA promoter. Moreover, FimZ 106

and FimY were found to be weak auto-activators. Our data also suggests that FimW-107

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mediated repression occurs at the level of fimY transcription. With regards to fimU, we 108

found that none of the fim regulatory genes had any effect on its transcription. As the 109

fim gene circuit involves a combination of positive and negative feedback, we tested 110

whether induction was bistable. However, we found the cell population responded 111

homogeneously when induced. Finally, we looked at the link between the fim and SPI1 112

gene circuits and found that the PhilE promoter is activated solely by FimZ. Collectively, 113

these results allow us to propose an integrated model for the regulation of the fim gene 114

circuit in S. typhimurium. 115

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Materials and Methods 116

General Techniques and Growth Conditions. All culture experiments were 117

performed in Luria-Bertani (LB) broth (tryptone: 10 g/l, yeast extract: 5 g/l, and NaCl: 10 118

g/l) at 37˚C unless otherwise noted. Antibiotics were used at the following 119

concentrations: ampicillin at 100 µg/ml, chloramphenicol at 20 µg/ml, and kanamycin at 120

40 µg/ml. All experiments involving growth of cells carrying pKD46 were performed at 121

30˚C as previously described (15). Loss of the helper plasmid pKD46 was achieved by 122

growth in non-selective conditions on LB agar at 42˚C. Removal of the antibiotic 123

cassette from the FRT-Cm/Kan-FRT insert was obtained by transformation of pCP20 124

into the respective strain and selection on ampicillin at 30˚C. Loss of the helper plasmid 125

pCP20 was obtained by growth at 42˚C under non-selective conditions on LB agar (10). 126

Integrations into the λattB sites of the S. typhimurium and E. coli genomes were done 127

using the helper plasmid, pInt-ts, as described previously (26). Loss of the helper 128

plasmid, pInt-ts, was obtained by growth at 42˚C under non-selective conditions. 129

Primers were purchased from IDT Inc. Enzymes were purchased from New England 130

Biolabs and Fermentas and used according to the manufacturer’s recommendations. 131

Strain and Plasmid Construction. All bacterial strains and plasmids used in this 132

study are described in Tables 1 and 2, respectively. All S. enterica serovar Typhimurium 133

are isogenic derivatives of strain 14028 (American Type Culture Collection, ATCC). The 134

generalized transducing phage of S. typhimurium P22 HT105/1int-201 was used in all 135

transductional crosses (16). 136

The plasmids pKD3 or pKD4 were used as templates to generate scarred FLP 137

recombinant target (FRT) mutants as described previously (14). The ∆fimYZ mutant 138

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was made using primers SS105F and SS105R. The ∆fimZ mutant was made using 139

primers SS105FII and SS105F. The ∆fimY mutant was made using the primers 140

SS105RII and SS105R. The ∆fimW mutant was made using primers SS152F and 141

SS152R. The ∆fimU mutant was made using the primers SS165F and SS165R. All 142

mutations were checked by PCR using primers that bound outside the deleted region. 143

Prior to removal of the antibiotic resistance marker, the constructs resulting from this 144

procedure were moved into a clean wild-type background (14028) by P22 transduction. 145

In order to construct the fluorescent Venus reporter plasmid (35), PCR was used to 146

amplify Venus from pBS7 using primers LC294F and LC296R. The resulting PCR 147

product was used as a template with primers LC295F and LC296R to add three out of 148

frame stop codons and a synthetic Shine-Dalgarno sequence before the Venus start 149

codon. The resultant PCR product was then digested with EcoRI and HindIII and sub-150

cloned into the EcoRI and HindIII cut-sites of pQE80L (Qiagen), yielding pQE80L-151

Venus. The plasmid pQE80L was digested with EcoRI and NheI, and the fragment was 152

cloned into the conditional-replication, integration, and modular (CRIM) plasmid pAH125 153

digested with EcoRI and NheI (26). The resulting CRIM plasmid was called pVenus. 154

Venus transcriptional fusions were made by amplifying the promoter of interest and then 155

cloning these PCR fragments into the multiple cloning site of pVenus. The fimA 156

transcriptional fusion was made using primers SS104F and SS104R. The fimY 157

transcriptional fusion was made using primers SS037F and SS037R. The fimZ 158

transcriptional fusion was made using primers SS103F and SS103R. The fimW 159

transcriptional fusion was made using primers SS154F and SS154R. The fimU 160

transcriptional fusion was made using primers SS162F and SS162R. The hilE 161

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transcriptional fusion was made using primers SS024F and SS024R. The PCR 162

fragments were then digested with KpnI and EcoRI (sequences underlined) and cloned 163

into the multiple cloning site of the pVenus vector. The resulting transcriptional fusions 164

were integrated into the S. typhimurium and E. coli chromosomes at the λattB site using 165

λInt produced from the CRIM helper plasmid pInt-ts, thus creating single-copy 166

transcriptional fusions. In the case of S. typhimurium, the integrated plasmid was moved 167

into different mutant strains by P22 transduction. 168

Expression plasmids for fimY, fimZ, and fimW were made by cloning the respective 169

gene into the multiple cloning site of pPROTet.E (Clontech) under the control of a strong 170

promoter PLTetO1, resulting in plasmids pFimY, pFimZ, and pFimW (34). The plasmid 171

pFimZ was made first by amplifying the fimZ gene using the primers SS106F and 172

SS106R. The PCR product was then digested with EcoRI and KpnI (sequence 173

underlined) and cloned into pPROTet.E. The plasmid pFimY was made by amplifying 174

the fimY gene using the primers SS107F and SS107R. The PCR product was then 175

digested with SalI and BamHI (sequence underlined) and cloned into pPROTet.E. The 176

plasmid pFimW was made by amplifying the fimW gene using the primers SS160F and 177

SS160R. The PCR product was then digested with EcoRI and HindIII (sequence 178

underlined) and cloned into pPROTet.E. In order to mutate the first three arginine rare 179

codons at position 7, 9, and 14 in fimY, primers SS162F and SS107R were used to 180

amplify fimY with the rare arginine codons mutated to consensus arginine codons. The 181

resulting PCR product was used as a template with primers SS167F and SS107R. The 182

amplified product was digested with EcoRI and BamHI and cloned into the multiple 183

cloning site of pPROTet.E. The plasmid is called pFimY*. 184

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In our expression plasmids, in the absence of TetR, the PLTetO-1 promoter is 185

constitutively active. To regulate the expression levels from the PLTetO-1 promoter, the 186

tetR gene was also cloned downstream of the gene target into the plasmids as 187

previously described (40). In this arrangement, in absence of the inducer 188

anhydrotetracycline (aTc), expression from the promoter is inhibited due to TetR. The 189

inhibition, however, is relieved upon addition of 100 ng/ml of aTc, and expression from 190

the PLTetO-1 promoter then takes place. All constructs were sequenced prior to 191

transforming into wild type and mutant strains. The sequences for all the primers used 192

in this study are given in Table 3. 193

Fluorescence Assays. As an indirect measure of gene expression, end-point and 194

dynamic measurement of the fluorescent reporter system were made using a Tecan 195

Safire2 microplate reader. For fluorescence end-point measurements, 1 ml culture was 196

grown at 37˚C overnight and then sub-cultured 1:1000 into fresh media and grown in 197

static conditions for 24 hours at 37˚C. 100 µL of the culture was then transferred into a 198

96 well microplate, and the relative fluorescence and optical density at 600 nm (OD600) 199

measured. The fluorescence readings were normalized with the OD600 absorbance to 200

account for cell density. For time-course measurements, overnight cultures at 37˚C 201

were sub-cultured to an OD of 0.05 into fresh medium and allowed to grow to an OD 202

0.15. 100 µL of the culture was then transferred into a 96 well microplate and overlaid 203

with 25 µL of oil to prevent evaporation. The temperature was maintained at 37˚C, and 204

fluorescence and optical density readings were taken every 5 minutes. All experiments 205

were done in triplicate and average values with the standard deviations reported. 206

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Single-cell measurements were done similarly by growing the cells in non-inducing 207

conditions with vigorous shaking at 37˚C. Overnight cultures were sub-cultured to an 208

OD of 0.05 into fresh media (LB) and grown in inducing conditions of high oxygen and 209

no shaking at 37˚C. Samples were collected at different time points by spinning the cells 210

down, resuspending in phosphate buffered saline (PBS) supplemented with 211

chloramphenicol (34 µg/ml) to stop all translation and arrest cells in their respective 212

state, and finally storing on ice. All flow cytometry experiments were performed on a BD 213

LRS II system (BD Biosciences). Data extraction and analysis for the flow cytometry 214

experiments was done using FCS Express Version 3 (De Novo Software). 215

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Results 217

FimZ and FimY are activators and FimW is a repressor of fim gene expression 218

FimZ and FimY have previously been reported as activators of fim gene expression 219

in S. typhimurium (45, 49). Both have also been reported as essential for fimbriation as 220

deletion of either one results in loss of expression from the PfimA promoter (50). To 221

understand the roles of FimZ and FimY in the fim gene circuit, we measured expression 222

from the PfimA, PfimZ, PfimY, and PfimW promoters in wild type and ∆fimZ, ∆fimY, ∆fimYZ, 223

and ∆fimW mutants (Figure 1). Chromosomally-integrated Venus transcriptional 224

reporters were employed as indirect measures of promoter activities (35). In the cases 225

of all four promoters, activity levels were found to be about two times less active in 226

∆fimZ, ∆fimY, and ∆fimYZ mutants relative to wild type. For all four promoters, note that 227

no further reduction in promoter activity was observed in the double mutant. In a ∆fimW 228

mutant, the activities of all four promoters were approximately two times higher than 229

wild-type levels. While these results agree with previously published data regarding the 230

fim system in S. typhimurium, they still do not tell us how FimW, FimY, and FimZ 231

individually contribute to PfimA activation. 232

233

FimY and FimZ are strong activators of each other’s expression and weak 234

activators of their own expression. 235

To determine the relative effect of FimY and FimZ on fim gene expression, PfimY and 236

PfimZ promoter activities were measured in a ∆fimYZ mutant where either FimZ or FimY 237

was expressed from a strong, aTc-inducible promoter on a plasmid (see Materials and 238

Methods). Using this system, we found that expressing FimZ in the ∆fimYZ mutant led 239

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to a more than ten-fold increase in PfimY activity (Figure 2a). Likewise, expressing FimY 240

in the ∆fimYZ mutant led to about a ten-fold increase in PfimZ levels. In addition to their 241

ability to activate each other’s promoters, FimY and FimZ were found to increase 242

expression from their own promoters roughly three fold. 243

Even though E. coli makes type I fimbriae, the S. typhimurium fim promoters by 244

themselves are inactive in this organism. Therefore, we performed an identical set of 245

experiments in E. coli using the S. typhimurium proteins and promoters. Overall, the 246

results were identical to those in E. coli (Figure 2b). In particular, FimZ expression led 247

to a more than ten-fold increase in PfimY promoter activity and FimY expression led to a 248

ten-fold increase in PfimZ activity. Both FimZ and FimY were again found to weakly 249

activate expression from their own promoters. The goal of these experiments was to 250

remove the affect of any S. typhimurium specific regulatory mechanisms, thus allowing 251

us to more confidently conclude that the observed results are due to direct interactions. 252

Collectively, these results show that FimY and FimZ strongly activate each other’s 253

expression and weakly activate their own expression. This cross regulation also 254

explains why both FimY and FimZ are required for strong PfimA promoter activity, as the 255

expression of each is dependent on the other. 256

257

FimZ and FimY can independently activate expression from PfimA promoter 258

Next, we looked at how FimZ and FimY independently effected PfimA expression. To 259

investigate this problem, we measured PfimA promoter activity in a ∆fimYZ mutant where 260

either FimY or FimZ was expressed using the aTc-inducible system. FimZ expression 261

was found to strongly activate (>15 fold) the PfimA promoter whereas FimY could only 262

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weakly activate (>2 fold) it (Figure 3). We also performed these experiments in E. coli 263

with similar results (data not shown). Based on these results, we conclude that FimZ 264

and FimY can both independently activate the PfimA promoter. In the case of FimY, weak 265

activation of the PfimA promoter is likely due to its strong dependence on the fimU tRNA 266

(see below) (43). 267

268

FimY activates the PfimW promoter and FimW represses the PfimY promoter 269

FimW has previously been observed to repress fim gene expression (46). Consistent 270

with these results, we observed that PfimA, PfimW, PfimY, and PfimZ promoter activities were 271

all elevated in a ∆fimW mutant (Figure 1). To understand the mechanism of FimW-272

mediated repression, we first sought to identify the proteins that regulate expression 273

from the PfimW promoter. To answer this question, we measured expression from the 274

PfimW promoter in a ∆fimYZ mutant where FimW, FimY, and FimZ were independently 275

expressed using the aTc-inducible system. In the case of FimW and FimZ, expression 276

had no effect on PfimW promoter activity (data not shown). However, in the case of FimY, 277

we observed a significant increase in PfimW promoter activity (1052 +/- 381 RFU/OD 278

[uninduced] versus 14718 +/- 1032 RFU/OD [induced]). Similar results were also 279

obtained when these experiments were performed in E. coli (data not shown). To 280

identify the regulatory targets of FimW, we measured the expression of the PfimA, PfimZ, 281

and PfimY promoters in a ∆fimW ∆fimYZ mutant where FimW was expressed using the 282

aTc-inducible system. In the case of the PfimA and PfimZ promoters, we found that FimW 283

expression had no effect. However, in the case of the PfimY promoter, FimW expression 284

led to about a three-fold decrease in PfimY activity (PfimY: 7462 +/- 319 RFU/OD 285

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[uninduced] versus 2781 +/- 188 RFU/OD [induced]). Based on these results, we 286

conclude that FimY activates expression from the PfimW promoter and that FimW 287

represses expression from the PfimY promoter. 288

289

The PfimU promoter is not regulated by FimW, FimY, or FimZ 290

Both fimY and fimZ contain rare arginine codons (AGA and AGG) and need fimU, a 291

tRNA specific for rare arginine codons, for effective translation. In a ∆fimU mutant, PfimA 292

activity was less than ten-fold as compared to the wild type levels (wild type: 16723 +/- 293

1173 RFU/OD; ∆fimU: 1389 +/- 261 RFU/OD). Expression of FimY in the ∆fimU mutant 294

using the aTc-inducible system, however, did not increase PfimA activity (988 +/- 319 295

[uninduced] versus 1343 +/- 166 [induced]). Replacing the rare arginine codons in the 296

fimY gene with consensus ones did restore PfimA activity to wild-type levels (817 +/- 73 297

RFU/OD [uninduced] versus 11294 +/- 462 RFU/OD [induced]). These experiments are 298

consistent with previously published results (46), and indicate that fimU is essential for 299

effective fimY translation. 300

As fimU has a strong effect on PfimA promoter activity, we hypothesized that it may 301

be subject to regulation by the other proteins within the circuit. To test this hypothesis, 302

we measured PfimU promoter activity in different regulatory mutants. Contrary to our 303

hypothesis, we did not observe any change in PfimU promoter activity in any mutant (wild 304

type: 26717 +/- 1381 RFU/OD; ∆fimZ: 28991 +/- 2164 RFU/OD; ∆fimY: 25884 +/- 1983 305

RFU/OD; ∆fimYZ: 26516 +/- 1772 RFU/OD; and ∆fimW: 24829 +/- 2073 RFU/OD). 306

Likewise, we did not observe any change in PfimU promoter activity when FimW, FimY, 307

and FimZ were expressed using the aTc-inducible system in wild-type S. typhimurium or 308

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E. coli (data not shown). Based on these results, we conclude that the PfimU promoter is 309

not regulated by any fim protein. 310

311

FimZ alone is able to regulate SPI1 gene expression 312

Previous studies have shown that both FimY and FimZ regulate SPI1 expression 313

through their activation of the PhilE promoter (7). HilE, in turn, is known to bind HilD and 314

repress HilD-mediated activation of PhilA, PhilC, PrtsA, and PhilD promoters (6, 21). To test 315

which protein activates the PhilE promoter, we independently expressed FimY and FimZ 316

in a ∆fimYZ mutant using the aTc-inducible system and then measured expression from 317

the PhilE promoter. Of the two, only FimZ was found to affect PhilE expression: 1089 +/- 318

421 RFU/OD [uninduced] versus 17654 +/- 2234 RFU/OD [induced]. Similar results 319

were also observed in E. coli (data not shown). 320

We note that these results are contrary to those previously reported, where it was 321

shown that both FimY and FimZ were necessary for activation of the PhilE promoter (7). 322

One possible explanation for the discrepancy involves how the two gene were 323

selectively expressed. In the original study, a DNA fragment containing the fimYZ gene 324

cluster was cloned onto a plasmid and expressed using the tetracycline promoter. To 325

study their relative effect, each gene was selectively inactivated using a universal 326

promoter. As part of the PfimY and the whole PfimZ promoter were left intact in their 327

construct, transcriptional inference may have occurred between the various promoters. 328

In our design, we selectively cloned each gene and then expressed it from an inducible 329

promoter, eliminating any potential interfering effects from having the native promoters 330

still present. 331

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Dynamics of fim gene expression. 332

Finally, we wished to investigate the dynamics of fim gene expression. We first 333

measured PfimA promoter activity using a microplate reader in wild type and ∆fimY, 334

∆fimZ, ∆fimYZ, and ∆fimW mutants (Figure 4a). Consistent with our end-point 335

measurements, we found that the PfimA promoter was weakly expressed in the ∆fimY, 336

∆fimZ, and ∆fimYZ mutants. Likewise, expression was enhanced in a ∆fimW mutant. 337

Note that the microplate experiments tell us only about the average response of the 338

population and nothing about how individual cells are behaving. To test whether the 339

cells were responding homogenously, we also performed single-cell measurements of 340

PfimA promoter activity at select times in wild type and a ∆fimW mutant using flow 341

cytometry (Figure 4b). Our results indicate that individual wild-type and ∆fimW mutant 342

cells are responding homogenously with respect to PfimA promoter activity at all times 343

tested. In other words, we did not observe any phase variation or heterogeneity with 344

regards to PfimA promoter activity in our kinetic experiments. 345

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Discussion 347

In this work, we investigated the regulatory gene circuit controlling the expression of 348

type I fimbriae in S. typhimurium. Using genetic approaches, we demonstrated that 349

FimY and FimZ independently activate the PfimA promoter. Of the two, FimZ was found 350

to be the dominant activator. We also found that FimY and FimZ strongly activate each 351

other’s expression and weakly activate their own expression. In addition to these two 352

positive regulators, a third regulator, FimW, is known to repress fim gene expression. 353

We found that FimW negatively regulates fim gene expression by repressing expression 354

from the PfimY promoter. Furthermore, FimW participates in a negative feedback loop as 355

FimY was found to enhance PfimW expression. Interestingly, these results suggest that 356

FimY is both an activator and repressor of fim gene expression, as it can directly 357

activate the PfimZ, PfimY, and PfimA promoters and indirectly repress them by enhancing 358

FimW expression. In addition to these regulators, type I fimbriation is also dependent on 359

expression of rare arginine codon tRNA, fimU. However, our results showed that the 360

PfimU promoter is not regulated by FimY, FimZ, or FimW. The results suggest that fimU 361

does not play a role in the internal regulation of the circuit. Finally, we demonstrated that 362

the previously observed coordinate regulation of SPI1 gene expression by the fim gene 363

circuit (7) occurs through the activation of hilE expression by FimZ. Based on these 364

results, we are able to propose the following model for the fim gene circuit in S. 365

typhimurium (Figure 5). 366

According to our model, induction of the fim circuit begins with activation of the PfimY 367

and PfimZ promoters, resulting in small amounts of fimY and fimZ being expressed. FimY 368

and FimZ then rapidly accumulate in the cell due to the positive feedback loop formed 369

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by the cross-activation of PfimY and PfimZ promoters by these two proteins. Expression of 370

the type I fimbrial structural genes from the PfimA promoter commences when the 371

concentration of FimY and FimZ accumulates within the cell beyond a critical level. 372

These two regulators can independently activate the PfimA promoter; however, their 373

expression is correlated as each activates the others expression. Moreover, FimY and 374

FimZ protein expression levels are controlled by a negative feedback loop involving 375

FimW. In this loop, FimY activates expression of the PfimW promoter, and FimW 376

represses expression from the PfimY promoter. We hypothesize that this negative 377

feedback loop involving FimW prevents runaway expression of FimY and FimZ arising 378

from their participation in an interacting positive feedback. Specifically, we hypothesize 379

that when FimY and FimZ reach their optimum expression levels, the FimW negative 380

feedback loop is activated and halts expression from the PfimY and PfimZ promoters. 381

While our model for the fim circuit explains internal regulation, it still does not explain 382

how the circuit is activated. In particular, we do not know which factors induce the PfimY 383

and PfimZ promoters. We suspect that these factors activate both promoters as each 384

alone exhibits some activity in a ∆fimYZ mutant (Figure 1). In addition to these factors, 385

another open question concerns whether fimU plays a role in regulating circuit 386

dynamics. While it is tempting to speculate that fimU expression is tuned in response to 387

environmental signals and thus affects circuit dynamics, more likely this gene is 388

constitutively expressed like other tRNAs. 389

Our results also indicate that FimW-mediated inhibition of fim gene expression is 390

through repression of the PfimY promoter. Earlier reports suggested that FimW binds 391

FimZ and somehow inhibits FimZ-dependent activation of fim promoters (46). Moreover, 392

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FimW was not found to bind to the PfimW promoter. Based on these results, FimW would 393

appear to repress the PfimY promoter by preventing FimZ from activating it. However, we 394

found that FimW is able to repress the PfimY promoter in the absence of FimZ. Our 395

results would suggest that FimW directly binds the PfimY promoter and represses 396

transcription, irrespective of FimZ. Consistent with our model, FimW has a C-terminal 397

LuxR-type helix-turn-helix DNA domain (SM00421) (32). However, at this time we have 398

no direct experimental support for such a mechanism. Moreover, an equally likely 399

hypothesis is that repression by FimW is indirect. Further experiments are clearly 400

required to determine the mechanism of FimW-mediated repression and distinguish 401

between these different putative models. 402

A final unanswered question concerns the role of the positive and negative feedback 403

loops in the fim gene circuit. Our initial hypothesis was that these feedback loops would 404

result in bistability. In particular, interacting positive and negative feedback loops are 405

known to be sufficient ingredients for bistability (24). This bistability could potentially 406

explain the phase variation observed in type I fimbriation during growth in inducing 407

conditions (36). To test whether the fim circuit exhibited bistability, we measured PfimA 408

activity at single-cell resolution as a function of time. Contrary to our initial hypothesis, 409

we did not observe a heterogeneous or switch-like response in induction, the tell tale 410

indicator of bistability. Rather, we observed a continuous or rheostatic-like response in 411

both wild type and a ∆fimW mutant (3). One possibility is that there is a lack of 412

correlation between fim gene expression and the production of type I fimbriae in S. 413

typhimurium (11). Another is that the environment may dictate the response 414

characteristics. For example, under conditions different from those used in our study, 415

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Duguid and coworkers observed subpopulations of cells expressing type I fimbriae, 416

indicative of phase variation (18). With these in mind, we hypothesize that the bacteria 417

exhibit type I fimbriae phase variation under specific environmental conditions and that 418

the regulation of this process involves post-transcriptional mechanisms as well. 419

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Acknowledgements 420

We thank James Slauch and Philip Aldridge for strains and technical advice. This work 421

was partially supported by grants from the National Science Foundation and the 422

National Institutes of Health. 423

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45. Tinker, J. K., and S. Clegg. 2000. Characterization of FimY as a coactivator of 554

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production by the arginine tRNA encoded by fimU in Salmonella enterica serovar 558

Typhimurium. Mol Microbiol 40:757-68. 559

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Bacteriol 183:435-42. 562

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Multiple fimbrial adhesins are required for full virulence of Salmonella 564

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characterization of a fimZ mutant of Salmonella typhimurium. J Bacteriol 567

177:6861-5. 568

50. Yeh, K. S., J. K. Tinker, and S. Clegg. 2002. FimZ binds the Salmonella 569

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FimY. Microbiol Immunol 46:1-10. 571

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576

Table 1: Strains used during this study. 577

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578

Table 2: Plasmids used during this study. 579

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580

Table 3: List of Primers used in the study 581

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583

Figure 1. FimY and FimZ are activators and FimW is repressor of fim gene 584

expression. Comparison of PfimA, PfimY, PfimZ, and PfimW promoter activites in wild type 585

(WT) and ∆fimY, ∆fimZ, ∆fimYZ, and ∆fimW mutants. (Data is average of 3 586

experiments. Each experiment was done in triplicate). 587

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588

Figure 2: FimY and FimZ are strong activators of each other’s expression and 589

also weak auto-activators. Comparison of PfimY and PfimZ promoter activities in a S. 590

typhimurium ∆fimYZ mutant (A) and E. coli (B) where FimY and FimZ are independently 591

expressed from an aTc-inducible promoter on a plasmid. Note that tetR is also 592

expressed from this plasmid in order to achieve aTc-inducible expression. (Data is 593

average of 3 experiments. Each experiment was done in triplicate). 594

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595

Figure 3: FimY and FimZ can independently activate expression from the PfimA 596

promoter. Comparison of PfimA promoter activity in a ∆fimYZ mutant where FimY and 597

FimZ are independently expression from an aTc-inducible promoter on a plasmid. (Data 598

is average of 3 experiments. Each experiment was done in triplicate). 599

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600

Figure 4: Dynamics of PfimA promoter activity. (A) Population-average PfimA activity 601

as a function of time in wild type (WT) and ∆fimY, ∆fimZ, ∆fimYZ, and ∆fimW mutants. 602

(B) Histogram of single-cell PfimA promoter activity at select times in wild type and a 603

∆fimW mutant. Single-cell measurements of promoter activity were obtained using flow 604

cytometry. (Figure 4A: Data is average of single experiment with average of 6 605

independent cultures. The experiment was repeated thrice and identical results 606

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observed. Figure 4B: Population distribution data is from a single experiment. The 607

experiment was repeated thrice and identical results were observed (data not shown)). 608

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609

610

Figure 5: Model for the type I fimbriae gene circuit in S. typhimurium. 611

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615

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