JB Accepts, published online ahead of print on 16 March ... · 3/13/2012 · 157 BioDev plasmid minipreps purification system B. DNA was recovered from the gel 158 with the QIAquick

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The RNA chaperone Hfq regulates antibiotic biosynthesis in the 1

rhizobacterium Pseudomonas aeruginosa M18 2

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Guohao Wang1, Xianqing Huang1, *, Sainan Li, Jiaofang Huang, Xue Wei, Yaqian Li, 4

Yuquan Xu† 5

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State Key Laboratory of Microbial Metabolism, and School of Life Sciences & 7

Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 8

200240, P. R. China 9

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Running title: Hfq regulates antibiotic biosynthesis in Pseudomonas 11

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1 These authors contributed equally to this work. 15

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For correspondence: 17

*E-mail: [email protected], Tel./Fax: (+86) 21 34204347 18

†E-mail: [email protected], Tel./Fax: (+86) 21 34204854 19

Postal mail: College of Life Sciences and Biotechnology, Shanghai Jiao Tong 20

University, 800 Dongchuan Road, Shanghai 200240, P. R. China 21

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00029-12 JB Accepts, published online ahead of print on 16 March 2012

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Abstract 23

The rhizosphere microbe P. aeruginosa M18 shows strong antifungal activities, 24

mainly due to the biosynthesis of antibiotics like pyoluteorin (Plt) and 25

phenazine-1-carboxylic acid (PCA). The ubiquitous RNA chaperon Hfq regulates 26

bacteria virulence and stress tolerance through global post-transcriptional regulation. 27

Here, we explored the molecular mechanism by which Hfq controls antibiotic 28

biosynthesis in P. aeruginosa M18. The robust downregulation of Plt biosynthesis by 29

Hfq was mediated exclusively by the post-transcriptional downregulation of the plt 30

transcriptional activator PltR. Hfq post-transcriptionally repressed phzM expression 31

and consequently reduced the conversion of PCA to pyocyanin. However, Hfq 32

positively controlled the phz2 operon and PCA biosynthesis through both 33

QscR-mediated transcriptional regulation at the promoter and an unknown regulation 34

at the operator. Also, Hfq was shown to directly bind at the mRNA 5úntranslated 35

leaders of pltR, qscR, and phzM. These three negatively regulated target genes of Hfq 36

shared a similar secondary structure with a short single-stranded AU-rich spacer (a 37

potential Hfq-binding motif) linking two stem-loops. Taken together, these results 38

indicate that Hfq, potentially in collaboration with unknown sRNAs, tightly controls 39

antibiotic biosynthesis through both direct post-transcriptional inhibition and indirect 40

transcriptional regulation.41


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Introduction 42

Pseudomonas aeruginosa is a Gram-negative, rod-shaped, and polar-flagella 43

bacteria that inhabits diverse environments in soil, water, animals, and plants (38). 44

This remarkable ecological diversity has greatly enriched the number of active 45

Pseudomonas spp. secondary metabolites, including antibiotics, virulence factors, 46

and siderphores, which contribute to symbiosis, competition, virulence, or biocontrol 47

(2, 14, 32). For any given Pseudomonas strain, the secondary metabolite profile 48

depends on both the environmental fluctuations encountered during evolution and on 49

its genome features and corresponding regulatory mechanisms. Like other bacteria, 50

Pseudomonas spp. including P. aeruginosa has also evolved multiple molecular 51

mechanisms that regulate products of secondary metabolism in response to diverse 52

environmental pressures and ecological competition (15, 24, 41, 49). 53

The P. aeruginosa strain M18 isolated from rhizosphere was reported to 54

simultaneously produce phenazine-1-carboxylic acid (PCA) and pyoluteorin (Plt), 55

which can efficiently inhibit soil-borne phytopathogenic fungi (22, 53). Our previous 56

comparative studies have shown that the predominant phenazine produced by M18 is 57

PCA, while the clinically isolated P. aeruginosa strain PAO1 predominately 58

accumulates pyocyanin (PYO), a derivative of PCA (19). PYO functions as an 59

important virulence factor (25). There is also evidence that PYO is not required for 60

the fungicide action of P. aeruginosa (12). PCA has been shown to suppress root 61

disease in Pseudomonas strains producing only PCA but not PYO, such as P. 62

fluorescens 2-79 (17) and P. chlororaphis 30-84 (31). The differential production of 63


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these two phenazines by strains PAO1 and M18 is mainly due to 64

temperature-dependent expression of phzM, a gene encoding an 65

adenosylmethionine-dependent methyltransferase (19). The phzM gene, together 66

with the flavin-dependent hydroxylase encoding gene phzS, is necessary for 67

conversion of PCA to PYO (32). 68

In P. aeruginosa, two copies of the phz gene cluster (phzABCDEFG) and a 69

number of modifying genes (such as phzM and phzS) are responsible for the 70

biosynthesis of PCA and its derivatives (29, 32). The other antibiotic produced by 71

the strain M18 is Plt, which is biosynthesized via a hybrid polyketide 72

synthase-nonribosomal peptide synthetase pathway. The plt gene cluster is composed 73

of the structural operon pltLABCDEFG and the pltM gene, the ATP-binding cassette 74

(ABC) transport operon pltHIJKNO, and two regulatory genes, pltR and pltZ, 75

divergently transcribed from these two operons in both P. fluorescence Pf-5 and P. 76

aeruginosa M18 (5, 20-22, 35). Our previous studies have shown that Plt 77

biosynthesis is strongly downregulated by three typical quorum-sensing (QS) 78

systems (Las, Rhl, and PQS) in the strain M18 (8, 30, 54). The activation of Plt 79

biosynthesis and expression of the plt gene cluster is dependent on both the 80

two-component signal transduction system GacS/GacA (11) and the Plt biosynthetic 81

pathway-specific transcriptional activator PltR, which in turn must be translationally 82

activated by GacA (21). The biosynthesis of PCA drastically increased in the Gac- or 83

Las-inactivated mutant (8, 11), while was almost entirely inhibited by inactivation of 84

the PQS or Rhl system in strain M18 (30, 54). In addition, the PCA biosynthesis was 85


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negatively regulated by the LuxR-type regulatory protein QscR in P. aeruginosa 86

M18 (50) and in other P. aeruginosa strains (9, 27). These data lead us to speculate 87

that a unique and complex regulation network is involved in the differential control 88

of secondary metabolites, including Plt and PCA biosynthesis, in P. aeruginosa M18, 89

but this remains to be determined. 90

In addition to two-component signal transduction and cell density-dependent 91

quorum-sensing, the small non-coding RNA (sRNA) is another important class of 92

regulator utilized by bacteria to coordinate expression of secondary metabolite genes 93

in response to environmental and metabolic stresses (41, 51). The Hfq protein is a 94

conserved RNA chaperon protein first characterized as a host factor (HF-1) for 95

phage Qβ RNA replication, and subsequently shown to be widely distributed in the 96

bacteria kingdom with multiple homologues in the annotated genomic database (3). 97

As a bacterial homologue of the eukaryotic and archaeal Sm/LSm proteins, Hfq is 98

known largely for its global post-transcriptional regulation by binding AU-rich 99

sequences of target mRNA and facilitating pairing between sRNAs and mRNAs (3). 100

Most Hfq homologues are known to function as homo-hexamers with two 101

independent RNA-binding motifs (3). The hfq mutants display pleiotropic 102

phenotypes in E. coli, Salmonella, Sinorhizobium meliloti, and Staphylococcus 103

aureus, including decreased growth rate, increased sensitivity to various 104

environmental stressors, and attenuated virulence (7, 13). 105

The P. aeruginosa Hfq homologue (82 residues), which shares 92% identity 106

with the E. coli Hfq (102 residues) in the N-terminus, can functionally substitute for 107


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the E.coli Hfq to translationally activate rpoS mRNA expression in E. coli (43). The 108

P. aeruginosa hfq mutant alters the production of elastase, catalase, and pyocyanin, 109

and even causes significant attenuation of virulence (42). Hfq also moderately 110

stimulated translation of the QS response regulators RhlR and QscR during the 111

bacterial stationary phase (44). In addition, Hfq has been confirmed to bind to and 112

stabilize the sRNA RsmY, which in turn binds to the translational repressor RsmA 113

(44). However, the molecular mechanisms of Hfq regulation of secondary 114

metabolism, including antibiotic biosynthesis, remain to be determined. 115

This study was motivated by the observation that cell cultures from the hfq 116

mutant exhibited a significantly deepened blue color in comparison to the parent 117

strain M18. It has been reported that blue-green pigment produced by P. aeruginosa 118

is mainly composed of PYO (blue) and pyoverdine (green) (33). The possibility of 119

elucidating the unique gene regulatory network controlling secondary metabolism in 120

P. aeruginosa M18 prompted a further investigation of the Hfq-mediated global 121

regulation on the biosynthesis of PYO (blue pigment) and other antibiotics, 122

including PCA and Plt. 123

In this study, we show that the hfq mutant displayed significantly reduced PCA 124

production and drastically increased PYO and Plt production. The Hfq protein 125

strongly inhibited phzM expression at the post-transcriptional level, resulting in a 126

significant decrease in the conversion of PCA to PYO. The strong down-regulation 127

of Plt biosynthesis by Hfq was exclusively mediated by the plt transcriptional 128

activator PltR. However, phz2 operon expression and PCA biosynthesis were 129


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strongly up-regulated by Hfq, with two target sequences located at the promoter 130

region (for receiving the QscR-mediated regulation) and the operator region. 131

Moreover, Hfq was also shown to directly bind to the phzM, qscR, and pltR mRNA 132

leaders. A new model is proposed to summarize Hfq-dependent pleiotropic 133

regulation of antibiotic biosynthesis. 134


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Materials and Methods 135

Bacterial strains, plasmids, primers, and growth conditions 136

Bacterial strains, plasmids, and primers used in this study are listed in Table S1 137

and S2. E. coli was routinely grown in LB medium at 37 °C (37). P. aeruginosa and 138

its derivatives were grown at 28 °C in King's medium B (KMB) (23) or pigment 139

producing medium (PPM) (28). When required, 20 μg ml-1 X-Gal was used for 140

blue/white colony screening, 5% sucrose for counter-selecting the suicide plasmid 141

pEX18Tc, 4 mg ml–1 ONPG (ortho-nitrophenyl-β-D-galactopyranoside) in 100 mM 142

phosphate buffer (pH 7.0) for β-galactosidase assays, and 0.5 mM IPTG for 143

blue/white colony screening and promoter induction. For P. aeruginosa and its 144

derivative strains, antibiotics were added at the following final concentrations (μg 145

ml-1): gentamicin (Gm) 45, kanamycin (Km) 50, spectinomycin (Sp) 100, 146

tetracycline (Tc) 120, and chloramphenicol (Cm) 200. For E. coli, Km 50, Gm 10, 147

Tc 15, and Cm 40 were used. 148

DNA manipulations 149

All molecular biological methods not described in detail were performed using 150

standard methods (37). Taq, LA Taq (TaKaRa), and KOD plus DNA polymerase 151

(Toyobo), RNA reverse transcriptase, restriction endonucleases, DNA ligase, DNA 152

molecular mass markers (MBI Fermentas), and other associated products were used 153

as recommended by the manufacturers. Genomic DNA was extracted with the EZ-10 154

spin column genomic DNA isolation kit supplied by Bio Basic Inc. Plasmid DNA 155

was purified using the TaKaRa miniBEST plasmid purification kit Ver 2.0 and the 156


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BioDev plasmid minipreps purification system B. DNA was recovered from the gel 157

with the QIAquick Gel extraction kit (Qiagen) and the AxyPrep DNA gel extraction 158

kit (Axygen). DNA was synthesized and sequenced by Invitrogen Biotechnology 159

Corporation and Beijing HuaDa Genomics Institute. 160

Bioinformatics analysis of DNA and RNA sequence 161

A database search for similar nucleotide sequences was carried out with NCBI 162

BLAST. Putative promoters were predicted with NNPP (Promoter Prediction by 163

Neural Network) (36). MEME (http://meme.nbcr.net/) was utilized to search 164

consensus sequence motifs (1). The maximum number of motifs to find is 3. The 165

default distribution of motif occurrences is assumed to be zero or one occurrence of 166

per sequence. The optimum width of each motif ranged from 2 to 15 nucleotides. 167

RNA secondary structures were predicted at 28 °C with Mfold 3.2. In cases where 168

multiple structures were predicted, the most stable structure was chosen (57). 169

IntaRNA (v1.2.5) (28 °C, 10 continuous bases pairing, and other defaulting 170

parameters) (39) was used to search for potential sRNAs interacting with the phzM, 171

qscR, and pltR mRNAs. 172

Inactivation and complementation of the hfq gene in P. aeruginosa M18 173

The P. aeruginosa M18 hfq gene was deleted and replaced with a gentamicin 174

resistance cassette by in vitro mutation (Fig. 1A) and in vivo homologous 175

recombination. Two fragments (479 bp and 468 bp) flanking the hfq ORF were 176

amplified with the KOD plus DNA polymerase and two pairs of primers Ph1 and 177

Ph2 or Ph3 and Ph4 (Table S2). Both the 3énd of the upstream fragment and the 178


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5énd of the downstream fragment carried the same restriction site, PstI, for the 179

insertion of the gentamicin resistance cassette. These two flanking PCR fragments 180

were simultaneously cloned into the EcoRI-HindIII digested pEX18Tc to generate 181

the recombinant plasmid pEX-H1H2. The 825 bp gentamicin resistance cassette 182

from pUCGm was then inserted into the PstI site of pEX-H1H2. The resulting 183

plasmid, pEX-H1H2-Gm, was transformed from E. coli SM10 into P. aeruginosa 184

M18 by biparental mating. Transconjugants were selected on LB plates containing 185

Sp (to counter-select E. coli SM10), Gm, and 15% sucrose. After a second 186

crossing-over, the Gm-resistant, Tc-sensitive, and sucrose-resistant recombinants 187

with a deletion in hfq were obtained. The hfq mutant, designed as M18hfq, was 188

confirmed by PCR and sequencing with primers Ph1 and Ph4. 189

To complement the hfq mutant M18hfq, the recombinant expression plasmid 190

pBBR-hfq was constructed as follows. The 636 bp fragment, which carrys the entire 191

249 bp hfq coding region and its own promoter, was amplified from the M18 192

genomic DNA with the KOD plus DNA polymerase and the primers PhfqB1 and 193

PhfqB2, and then cloned into the Pseudomonas-E. coli shuttle vector pBBR1MCS at 194

XhoI and HindIII sites. 195

Quantification of antibiotic production 196

Cells from fresh overnight cultures of P. aeruginosa M18 and its derivative 197

strains were inoculated into 100 ml KMB in a 500 ml Erlenmeyer flask at the final 198

concentration of OD600 = 0.05, and then cultured at 28 °C with shaking at 200 rpm. 199

PCA and Plt were respectively extracted with chloroform (pH 4.0) and ethyl acetate, 200


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and quantified with HPLC as previously described (22). Quantification of PYO 201

production was carried out at pH 7.0 in chloroform as previously described (10). 202

In this study, each experiment was independently repeated at least three times, 203

using triplicate parallel samples within each experiment. Unless stated otherwise, 204

each value represents the mean and standard deviation (SD) of three replicates. 205

Student's t test in Microsoft Excel was used to evaluate the significance of 206

difference. 207

Construction of lacZ reporter gene fusions 208

To investigate the regulatory functions of Hfq on relevant target operons or 209

genes, including phz1, phz2, phzM, plt, qscR, and pltR, the lacZ reporter gene was 210

fused to the promoters and/or operator regions of these genes, creating the lacZ 211

translational (indicating the total regulatory level), transcriptional, and 5ÚTR-lacZ 212

fusion vectors (Table S1). Generally, the first several codons, operator, and promoter 213

region of these genes were used for constructing the translational lacZ fusions in the 214

vector pME6015 in which the promoter and first eight codons of the E. coli lacZ 215

gene had been deleted. The transcriptional lacZ fusions were constructed by cloning 216

the promoter region (upstream of the TSS) into the plasmid pME6522 carrying the 217

promoterless lacZ gene and its own RBS. Likewise, the 5ÚTR-lacZ fusions were 218

constructed by introducing the entire or partial operator region (+1 to ATG) into the 219

plasmid pME9533, which harbours the tac promoter and a replaceable operator 220

region upstream of lacZ. 221

For constructing the phzM and phz2 fusions with lacZ (Table S1), a series of 222


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gradually shorten fragments upstream of phzM (Fig. 2A) and phz2 (Fig. 4A) were 223

cloned into the EcoRI/PstI-digested plasmids pME6015, pME6522, and the 224

KpnI/PstI-digested plasmid pME9533 to generate two arrays of phzM-lacZ and 225

phz2-lacZ translational, transcriptional, and 5ÚTR-lacZ fusions. The pltLp-lacZ 226

transcriptional fusion plasmid (p6522-pltLp) containing the pltL promoter region 227

(-85 to +1) was constructed based on the EcoRI/PstI-digested plasmid pME6522. 228

Similarly, the PCR fragment covering the 46 bp pltL operator (+1 to ATG) and first 229

nine codons was cloned into the KpnI/PstI-digested pME9533, producing the pltLo 230

(5ÚTR)-lacZ fusion plasmid p9533-pltL. By cloning the PCR fragment containing 231

the 63 bp operator region (+1 to ATG) and the first nine codons of pltR into the 232

KpnI/PstI-digested plasmid pME9533, we constructed the pltRo-lacZ fusion plasmid 233

p9533-pltRo. The qscRo-lacZ fusion (p9533-qscRo) was constructed by cloning the 234

PCR fragment covering 545 bp upstream of translational start site and first 9 codons 235

of qscR into pME9533. The qscR´-´lacZ translational fusion plasmid (p6015-qscR) 236

harbouring the qscR operator and promoter region (-217 to +572 relative the putative 237

+1) was constructed based on the EcoRI/PstI-digested plasmid pME6015. The 238

qscRp-lacZ transcriptional fusion plasmid (p6522-qscRp) containing the qscR 239

promoter region (-126 to +1) was constructed based on the EcoRI/PstI-digested 240

plasmid pME6522. 241

Site-directed mutagenesis of the AT-rich motif of the phzM and pltR leader 242

regions 243

The lacZ reporter plasmids, in which the AT-rich motif (encoding a potential 244


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AU-rich Hfq-binding motif) of phzM leader region was changed by site-directed 245

mutagenesis, were constructed to assess the importance of this AT-rich motif for the 246

Hfq regulation on phzM expression. The phzM leader mutant fragment (+130 to 247

+297) containing 4-nt substitution (A142G, T144C, T146C, and T148C) in the 248

AT-rich motif (Fig. 2B) was PCR amplified with the mutant forward primer 249

PphzMo-M1-1 and the reverse primer PphzMo-M1-2 (Table S2), and subsequently 250

cloned into the KpnI/PstI-digested pME9533 vector. The resulting plasmid 251

pPMO-2-M1 was verified by sequencing, and the 4-nt mutant phzM leader fragment 252

was shown to be located downstream of the tac promoter carried by the plasmid 253

itself. In the same way, the phzM leader mutant fragment (+130 to +297) carrying a 254

2-nt substitution (T143C and T148C) (Fig. 2B) was recombined into the 255

KpnI/PstI-digested pME9533, yielding the plasmid pPMO-2-M2. The plasmid 256

p9533-pltRo-M1 was constructed by cloning the 63 bp mutant operator region (+1 to 257

ATG) of pltR, which carries a 5-nt replacement (A29G, U31C, U33C, U35C, and 258

A37G) in the AT-rich spacer (Fig. 8A), into the KpnI/PstI-digested plasmid 259

pME9533. 260

β-Galactosidase assays 261

P. aeruginosa M18 and its derivative strains carrying the lacZ reporter plasmid 262

(Table S1) were inoculated from the overnight culture into 500-ml Erlenmeyer flasks 263

containing 100 ml KMB to a final concentration of OD600 = 0.05, and then cultured 264

at 28 °C with shaking at 200 rpm. Cells were harvested at different time points for 265

assaying the β-galactosidase specific activities, according to the method of Miller 266


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(34) and as described by Huang et al. (22). 267

Expression and purification of the P. aeruginosa M18 Hfq protein 268

A 269 bp DNA fragment containing the 249 bp entire hfq ORF was PCR 269

amplified with the proof-reading enzyme KOD, and then cloned into the 270

NdeI-HindIII sites of plasmid pET28a. The resulting hfq recombinant expression 271

plasmid pET28a-hfq (Table S1) was confirmed by sequencing and transformed into 272

E.coli BL21 (DE3) cells. Cell cultures were incubated at 37 °C in LB medium to an 273

OD600 of 0.8, induced with 0.5 mM IPTG, and subsequently grown at 16 °C for 20 274

hours. The cells were then harvested by centrifugation, and resuspended in 30 ml 275

nickel A buffer (20 mM imidazole, 300 mM NaCl, 25 mM Tris-HCl at pH 8.0), 276

supplemented with 1μg ml-1 leupeptin, 1 μg ml-1 aprotinin, and 50 μM PMSF 277

(phenylmethylsulfonyl fluoride). Cell re-suspension solution was slowly shaken at 278

4 °C for 30 minutes, and cells were lysed using an ultrasonic cell disruptor. 279

Subsequently, the cell lysate was loaded onto a Ni-NTA agarose column (GE 280

healthcare). The column was washed two times with 5 ml nickel buffer, and then Hfq 281

protein was eluted with the elution buffer containing 500 mM imidazole. After the 282

removal of imidazole by HPLC (AKTA), the purified Hfq protein was stored in a 283

buffer containing 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM DTT, and 1 mM 284

EDTA. 285

In vitro synthesis of the unlabeled and biotin-labeled RNAs 286

The RNAs for EMSAs were synthesized in vitro on Applied Biosystems 394 287

automated DNA/RNA synthesizers using cyanoethyldiisopropylamino 288


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phosphoramidite chemistry and/or labeled in 3′-end with biotin by TaKaRa 289

Biotechnology Co, .Ltd (Dalian, China). The RNA products were purified by anion 290

exchange HPLC. Seven RNA fragments are the phzM (+131 – +190 nt relative to 291

+1), phzM-M1 (carrying a 4-nt substitution), phz2-1 (+50 – +89 nt relative to +1), 292

phz2-2 (+92 – +131 nt relative to +1), qscR (-48 – -97 relative to ATG ), pltR (+9 – 293

+62 nt relative to +1), and pltR-M1 (carrying a 5 nt substitution) mRNA 5′ UTRs. 294

Electrophoretic mobility shift assay (EMSA) of RNA-protein interaction 295

EMSA analysis was carried out with the Thermo Scientific LightShift 296

Chemiluminescent RNA EMSA Kit. The biotin-labeled RNA probes (1 nM) were 297

incubated with increasing amounts of the purified Hfq protein in 20 μl binding buffer 298

(100 mM HEPES at pH 7.3, 200 mM KCl, 10 mM MgCl2, 10 mM DTT). For 299

competition assays, a 1000- or 1500-fold molar excess of unlabeled RNAs were 300

added into the binding reaction mix as competitors. After the addition of probe, 301

EMSA reaction mixtures were incubated at room temperature for 30 minutes. After 302

incubation, 10 μl samples were mixed with 2.5 μl of 5× loading buffer (50% glycerol, 303

0.1% bromophenol blue), and then loaded on a native 6% polyacrylamide gel. 304

Electrophoresis was performed in 0.5× TBE buffer at 100 V for 40 minutes. The 305

separated protein-RNA conjugates were electrophoretically transferred from the gel 306

to a positively charged nylon membrane (Ambion) by semi-dry transfer apparatus 307

(Tanon). The transferred Hfq-RNA complexes and free RNAs were cross-linked to 308

the membrane by a 320 nm UV-light cross-linking instrument. Finally, the 309

biotin-labeled bands were detected by Thermo Scientific Chemiluminescent Nucleic 310


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Acid Detection Module. The stabilized streptavidin-horseradish peroxidase (HRP) 311

conjugate was utilized to probe for the biotin-labeled RNAs transferred to nylon 312

membrane. Then, an enhanced luminol-based substrate was catalyzed by HRP with 313

optimized blocking and wash step that together produce high sensitivity light. 314

Subsequently, the membrane with light was placed to a film cassette and exposed to 315

X-ray film for 20-30 seconds. 316

Nucleotide sequence accession number 317

P. aeruginosa M18 complete genome was deposited in the Genbank (Genbank 318

accession no. CP002496). The locus tags of hfq, phzM, qscR, phz2, phz1, and pltR 319

are respectively PAM18_5055, PAM18_0728, PAM18_3144, PAM18_3143 to 3137, 320

PAM18_0727 to 0721, and PAM18_2384.321


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Results 322

Identification and inactivation of the hfq gene in P. aeruginosa M18 323

PCR amplification and sequencing analysis indicated that the hfq gene of P. 324

aeruginosa M18 shares 99% sequence identity with P. aeruginosa PAO1 and 57% 325

sequence identity with E.coli. The 249 bp M18 hfq gene encodes a 9.103 kDa protein. 326

The hfq gene has two flanking genes, the miaA gene encoding a tRNA 327

δ(2)-isopentenylpyrophosphate transferase and the PAM18_5054 ORF encoding a 328

putative GTP-binding protein, both organized in the same direction in all P. 329

aeruginosa strains. 330

To investigate the regulatory activities of Hfq on antibiotic biosynthesis in P. 331

aeruginosa M18, we replaced the hfq gene with the gentamicin resistant cassette 332

(Fig. 1A) through homologous recombination. The resultant hfq deletion mutant was 333

validated by PCR and sequencing. Moreover, the hfq deletion was confirmed by 334

semi-quantitative RT-PCR to have no polar effect on the expression of flanking 335

genes miaA and PAM18_5054 (Fig. 1B). 336

Impaired growth of P. aeruginosa M18 by the hfq deletion 337

The hfq deletion gave rise to a serious impairment in P. aeruginosa M18 growth 338

in PPM medium as evidenced by a significant decline of OD600 from 4.0 in wild type 339

M18 cultures to 1.0 in M18hfq (Fig. 1D). The PPM medium, in which glucose is 340

utilized as a major carbon source, is generally used for PCA production (23). This 341

impaired growth of the hfq mutant in PPM was restored to the wild type growth level 342

by ectopic expression of exogenous hfq via transformation of the plasmid pBBR-hfq 343


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(Fig. 1D), which was constructed by cloning the entire hfq ORF and its own 344

promoter region into the Pseudomonas-E. coli shuttle vector pBBR1MCS. Due to 345

the poor growth of the hfq mutant in PPM media, P. aeruginosa M18 and its 346

derivative strains were generally grown in KMB media in which the main carbon 347

source is glycerol. When grown in KMB agar plates, the hfq mutant took twice as 348

long to form colonies compared to the wild type strain M18. In KMB liquid media, 349

the hfq mutant exhibited an obvious decrease in cell growth compared to the parent 350

strain M18 (Fig.1E–G). Likewise, the decreased growth of the hfq mutant was 351

restored to the wild type level in KMB media by transformation of the plasmid 352

pBBR-hfq (Fig. S1D). 353

Strong and differential regulation of Hfq on antibiotic biosynthesis 354

P. aeruginosa M18, M18hfq, and their derivative strains were inoculated on 355

KMB plates, incubated at 28 °C for 60 hours, and changes in pigment production 356

were observed. As shown in Fig. 1C, the loss of hfq function gave rise to a deeper 357

blue pigment, implying that the hfq mutant likely led to the overproduction of the 358

blue pigment PYO. Furthermore, the introduction of an exogenous hfq gene derived 359

from the plasmid pBBR-hfq almost completely reversed this colour change in the hfq 360

mutant cultures relative to the wild type strain (Fig. 1C). The same change in 361

pigment production resulting from the hfq deletion was also observed in KMB liquid 362

media (data not shown). 363

To further determine whether the deeper blue colour of the hfq mutant was 364

caused by the overproduction of the blue pigment PYO, we quantitatively analyzed 365


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the production of PYO, PCA, and Plt in KMB media. As expected, the hfq deletion 366

mutant M18hfq displayed a significant enhancement of PYO production compared 367

to the M18 parent strain. The PYO production by M18hfq strain reached a peak of 368

18.7 μg ml-1 after 60 h of growth, while the wild type M18 strain accumulated only 369

1.5 μg ml-1 PYO within the same culture period (Fig. 1E). At the same time, we also 370

observed that the accumulation of PCA was significantly reduced in the hfq deletion 371

mutants (Fig. 1F). Based on these results, we speculated that the reduced PCA 372

probably resulted from conversion of PCA to PYO in the hfq mutant. Of course, we 373

can not exclude the possibility that the biosynthesis of PCA itself was also reduced 374

by the hfq deletion. The decreased amount of PCA (about 24 μg ml-1, Fig. 1F) was 375

still higher than the increased amount of PYO produced by strain M18hfq (about 17 376

μg ml-1, Fig. 1E). In addition, the hfq deletion led to a nearly seven-fold increase in 377

Plt production, and the peak of Plt production rose sharply from 33.8 μg ml-1 in the 378

wild type M18 strain to 240.8 μg ml-1 in the M18hfq strain (Fig. 1G). These data 379

suggest that Hfq strongly downregulates the biosynthesis of PYO and Plt, and 380

significantly upregulates the biosynthesis of PCA in P. aeruginosa M18. 381

To further verify that the inhibition of PCA production and the overproduction 382

of PYO and Plt is exclusively caused by the hfq deletion in the strain M18hfq, the 383

hfq expression plasmid pBBR-hfq and the corresponding empty plasmid 384

pBBR1MCS (as the control) were used to transform the M18 strain and the hfq 385

deletion mutant M18hfq, and antibiotic production was quantified in KMB media. 386

Compared to the empty plasmid pBBR1MCS, hfq overexpression deriving from 387


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pBBR-hfq resulted in a significant increase in PCA production (Fig. S1A), but a 388

significant reduction in Plt production (Fig. S1B) in both the wild-type strain M18 389

and its hfq deletion mutant. PYO production of M18hfq was restored to the wild type 390

level by the exogenous hfq expression resulting from pBBR-hfq (Fig. S1C). 391

However, no obvious influence of hfq overexpression on in PYO production was 392

detected between in M18/pBBR-hfq as compared with M18/pBBR1MCS (Fig. S1C), 393

but this can be accounted for by the fact that the PYO level produced by the M18 394

strain was originally very low. These results confirmed the regulation of antibiotic 395

biosynthesis by the RNA chaperon Hfq in P. aeruginosa M18. 396

Hfq downregulated phzM expression at the post-transcriptional stage and 397

consequently inhibited the conversion of PCA to PYO 398

Deletion of the global post-transcriptional regulator hfq substantially increased 399

PYO production, suggesting that Hfq downregulates the PYO biosynthetic genes. 400

We first examined the expression of phzM, a gene involved in the first step of 401

conversion of PCA to PYO. To this end, a series of phzM-lacZ fusion plasmids (Fig. 402

2A and Table S1) were constructed and transformed into the wild type M18 and hfq 403

mutant M18hfq. The β-galactosidase (LacZ) activities of these transformed bacteria 404

were then measured in KMB media to compare phzM expression in the presence and 405

absence of hfq (Fig. 2A). The LacZ activities expressed from the phzMo (5´406

UTR)-lacZ fusion plasmids pPMO-1 and pPMO-2 were markedly elevated in the hfq 407

mutant M18hfq compared to the parental strain M18. Although LacZ expression was 408

relatively low, the hfq deletion resulted in an obvious increase in phzM´-´lacZ 409


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translational fusion expression (pPML). In contrast, the wild type strain M18 and the 410

hfq mutant M18hfq did not show a significant difference in LacZ expressed from the 411

phzMp-lacZ transcriptional fusion plasmid pPMC or from the phzMo-lacZ fusion 412

plasmids, pPMO-3 and pPMO-4. Based on these results, the target regulatory 413

element of Hfq was localised to the phzM 5´-UTR from +130 to +190 bp 414

downstream of the phzM transcriptional start site (TSS) (Fig. 2A), implying that Hfq 415

exerts negative control on phzM expression and consequently inhibits the conversion 416

of PCA to PYO, most likely at the post-transcriptional level. The results of the 417

real-time RT-PCR analysis also suggested that the accumulation of phzM mRNA in 418

the early stationary phase was significantly enhanced in the hfq mutant compared to 419

the wild type strain (Fig. S2). 420

A multiple alignment was carried out by the MEME software between the P. 421

aeruginosa M18 phzM leader region from +130 to +190 bp and the target regulatory 422

regions containing their respective potential AU-rich Hfq-binding motifs (3) within 423

5´-UTRs of six other reported Hfq target genes (Fig. S3). The result shows that a 10 424

bp AT-rich motif was located at +141 to +150 bp downstream of the TSS of phzM 425

gene (Fig.S3). The Mfold software was used for predicting the RNA secondary 426

structure of the phzM mRNA leader harbouring the candidate regulatory region of 427

Hfq. As shown in Fig. 2B, the short single-stranded AU-rich sequence (potential 428

Hfq-binding motif) was found between two stem loops. To further explore whether 429

this AU-rich motif is required for Hfq binding and Hfq-mediated post-transcriptional 430

regulation, two mutants of this AT-rich motif in the plasmid pPMO-2 were 431


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constructed by PCR. The mutants contained either 4 nt substitutions (A142G, T144C, 432

T146C, and T148C) or 2 nt substitutions (T143C and T148C). The strains M18 and 433

M18hfq transformed with pPMO-2 or its AT-rich motif mutants (pPMO-2-M1 and 434

M2) were assayed for LacZ activity in KMB media (Fig.2C). Indeed, negative 435

regulation of Hfq on the phzMo-2-lacZ fusion (pPMO-2) expression was reduced or 436

even entirely eliminated by the two AU-rich motif mutants. This result further 437

suggested that the AU-rich motif within the phzM mRNA 5´-UTR plays a key role in 438

post-transcriptional repression by Hfq. It is also noteworthy that the 4 nt 439

substitutions (M1) in the AT-rich motif brought about a significant increase in LacZ 440

expression (pPMO-2-M1) in the wild type strain M18 (Fig.2C). This further 441

demonstrates that the AU-rich motif in the phzM 5´-UTR is critical for the 442

Hfq-mediated inhibition of phzM expression. In addition, a relatively minor decline 443

in the lacZ fusion expression from pPMO-2-M2 was observed owing to the 2-bp 444

substitution in the AT-rich motif in the wild type strain M18 by an unknown 445

mechanism (Fig. 2C). 446

Hfq directly binds to the phzM mRNA 5' leader between +130 to +190 nt 447

To explore whether Hfq directly binds to the phzM mRNA 5ÚTR, we 448

expressed and purified the Hfq protein (Fig. 3A), and carried out an EMSA analysis 449

to assess the binding activity of Hfq to the 60 nt biotin-labeled 5ÚTR covering +130 450

to +190 downstream of the phzM TSS that also carries a potential Hfq-binding 451

AU-rich motif. It need to be mentioned that the presence of a His6-tag in the 452

recombinant 9.1 kDa Hfq protein caused a larger delay in gel mobility on 453


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SDS-PAGE (Fig. 3A). This phenomenon frequently occurs in other recombinant 454

proteins with His6-tags. As shown in Fig. 3B, when 1 nM biotin-labeled phzM 455

mRNA leader was incubated with increasing amounts of Hfq (0 to 24 μM) in a 20 μl 456

reaction system, four bands (C1 to C4) were clearly observed, corresponding to four 457

Hfq-phzM complexes. As presented in Lanes 2 to 9 of Fig. 3B, with increasing Hfq 458

protein, the Hfq-phzM complex bands with lower mobility gradually increased in 459

intensity, and while the complex bands with higher mobility gradually decreased in 460

intensity and eventually disappeared above 20 μM Hfq. Concomitant with the 461

increased Hfq binding, the biotin-labeled free phzM mRNA gradually decreased, and 462

finally, was almost completely bound by 24 μM Hfq protein. In addition, the phzM 463

mRNA itself was also observed to form 2 to 3 oligomers, which were sequentially 464

reduced in number and intensity as the Hfq concentration was increased, eventually 465

disappearing completely above 20 μM (Fig. 3B, Lanes 1 to 7). A 1500-fold molar 466

excess of unlabeled phzM mRNA almost entirely displaced the biotin-labeled phzM 467

mRNA from Hfq (Fig. 3B, Lane 10), thus demonstrating the specificity of the 468

Hfq–phzM mRNA interaction. Also, we tested whether a 4-nt replacement in the 469

AU-rich spacer (Fig.2B) affects the binding affinity between the phzM mRNA leader 470

and the Hfq protein. As expected, the mutated phzM mRNA leader displayed 471

significantly weakened binding affinity with Hfq as compared with the wild-type 472

phzM (Fig. 3C). A higher proportion of 1 nM mutated phzM remained unbound by 473

even 24 μM Hfq (Fig. 3C). In contrast, 1 nM wild-type phzM began to be strongly 474

bound by only 0.8 μM Hfq, and finally, was almost entirely bound by 24 μM Hfq 475


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(Fig. 3B). In summary, the results depicted in Fig. 3B and 3C strongly suggests that 476

the P. aeruginosa M18 Hfq protein can directly bind to the phzM mRNA 5' UTR 477

between +130 to +190, a region that contains a typical Hfq-binding AU-rich motif. 478

Hfq contributed to the transcriptional and post-transcriptional upregulation of 479

phz2 expression 480

In P. aeruginosa M18, two sets of phenazine biosynthetic gene clusters, 481

phzA1-G1 and phzA2-G2, share over 98% identity in amino acid coding sequences, 482

but possess distinct upstream 5' non-coding sequences, including promoter and 483

regulatory elements, implying distinct regulatory mechanisms. The preceding results 484

(Fig. 1E and F) imply that the total decrease in PCA production of the hfq deletion 485

mutant may be due mainly to the enhanced conversion of PCA to PYO, and may also 486

be owing partly to inhibition of the biosynthesis of PCA itself. The following 487

experiments were designed to determine which of the two phz operons is under the 488

control of Hfq, and to further investigate the underlying regulatory mechanism of 489

Hfq-mediated upregulation of PCA biosynthetic genes. The phz1'-'lacZ and 490

phz2'-'lacZ translational fusion plasmids (pMP1L and pMP2L), which carry the phz1 491

fragment from -340 to +361 bp and the phz2 fragment from -344 to +227 bp , were 492

utilized to assess the regulation of these two gene clusters. These two lacZ gene 493

reporter plasmids were introduced into strains M18 and M18hfq, and the LacZ 494

activities of the four strains were measured in KMB media (Fig. 4A and B). The 495

phz2'-'lacZ translational fusion expression from pMP2L was reduced by 96% in the 496

hfq deletion mutant (29.7 Miller units of LacZ activity) compared to that in the M18 497


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strain (691 Miller units) (Fig. 4A), while no significant difference in the total 498

translational phz1'-'lacZ fusion expression (pMP1L) was detected between wild type 499

strain and M18hfq (Fig. 4B). From these results, it appears that Hfq may 500

preferentially upregulate the expression of phz2 operon and the fraction of PCA 501

produced through phz2 operon activation, while much of the other fraction of PCA 502

biosynthesized by the phz1 operon is likely converted into PYO through the 503

promoted phzM expression following Hfq deletion. In addition, qRT-PCR analysis 504

was performed to quantify the accumulation of phz2 mRNA in the early stationary 505

phase of growth in M18 and M18hfq. The results depicted in Fig. S2 suggest that the 506

hfq deletion resulted in a remarkable decrease in phz2 mRNA level in M18hfq 507

compared to M18. 508

To further define the target regulatory elements of Hfq within the phz2 operon 5' 509

UTR, we constructed a set of phz2-lacZ fusion plasmids and compared LacZ 510

expression between M18 and M18hfq in KMB media (Fig. 4A). The hfq deletion 511

caused a significant decrease in LacZ activity expressed from the phz2po-2-lacZ 512

fusion plasmid p6522-phz2po-2, the transcriptional fusion plasmid p6522-phz2p-5, 513

and the 5ÚTR-lacZ fusion plasmid p9533-phz2o-6. In contrast, there was no 514

obvious difference in the LacZ activity between M18 and M18hfq stains transformed 515

with the transcriptional fusion plasmids p6522-phz2p-3, p6522-phz2p-4, or the 5´516

UTR-lacZ fusion plasmid p9533-phz2o-7 (Fig. 4A). Based on these data, it can be 517

concluded that there are two key regulatory fragments mediating positive regulation 518

by Hfq located from -78 to +1 bp upstream and from +1 to +113 bp downstream of 519


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the phz2 transcript TSS, suggesting that Hfq upregulates phz2 expression both at the 520

transcriptional and post-transcriptional levels. 521

The 5' UTR (+1 to +113 nt) of phz2 mRNA was not found to contain an 522

AU-rich potential Hfq-binding motif in a single-stranded spacer region between two 523

stem-loops (Fig. S4A). Two AU-high fragments (+50 to + 90 nt and +90 to +130 nt) 524

did not show measurable binding with the Hfq protein (Fig. S4B and C), suggesting 525

that the phz2 mRNA is probably not a direct target of Hfq regulation, and that other 526

transcriptional or posttranscriptional regulators are more likely to mediate the 527

positive regulation by Hfq on the +1 to +113 nt operator region of the phz2 operon. 528

QscR mediated the transcriptional upregulation of Hfq on the phz2 expression 529

It has been widely reported that Hfq, a sRNA chaperon protein, regulates 530

expression of target genes typically at the post-transcriptional level (3). The result 531

that the phz2 expression was upregulated by Hfq at the transcriptional level suggests 532

that transcriptional regulation by Hfq may be mediated indirectly through a 533

transcriptional regulator controlling phz2 expression. The LuxR family 534

transcriptional regulator QscR has been reported to negatively control phz expression 535

(9, 50) and phz2 transcription (27), and so is one candidate mediating the positive 536

regulation of phz2 transcription by Hfq. To test this hypothesis, the qscR-lacZ 537

translational, transcriptional, and 5 ´ UTR-lacZ fusion expression respectively 538

deriving from the plasmids p6015-qscR, p6522-qscRp, and p9533-qscRo, was 539

assessed in M18 and M18hfq strains growing in KMB media (Fig. 5A–C). The hfq 540

deletion brought about a remarkable enhancement in the expression of qscR′-′lacZ 541


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translational fusion (Fig. 5A) and qscRo-lacZ fusion (Fig. 5C) throughout the 542

experimental period compared to the M18 strain. A slight increase was also observed 543

in the qscRp-lacZ transcriptional fusion expression in the hfq mutant strain as 544

compared to the wild type strain (Fig. 5B), implying that Hfq may indirectly exert a 545

low level of transcriptional downregulation on the qscR gene expression. These 546

results suggest that qscR expression was downregulated by Hfq mainly at the 547

post-transcriptional level. The qRT-PCR results also indicated a substantial increase 548

in the accumulation of qscR mRNA at the early stationary phase in the hfq mutant 549

compared to the parent strain (Fig. S2). 550

The phz2p-5-lacZ transcriptional fusion expression from the plasmid 551

p6522-phz2p-5, which contains the promoter region (-78 to +1) of the phz2 operon 552

(Fig. 4A), was assayed in the strain M18 and the qscR mutant M18qscR to confirm 553

the negative regulation of phz2 transcription by QscR (Fig. 5E). The phz2p-5-lacZ 554

transcriptional fusion expression was significantly enhanced, although not being a 555

great increase, in the qscR mutant as compared to the wild type strain (Fig. 5E). 556

Similarly, the phz2'-'lacZ translational fusion (pMP2L) expression, which may 557

reflect the total regulatory level of the phz2 operon, was also significantly enhanced 558

by the qscR mutation (Fig. 5D). Not unexpectedly, no significant difference in the 559

phz2o-6-lacZ fusion expression from the plasmid p9533-phz2o-6 was observed 560

between the M18 strain and the qscR mutant (Fig. 5F). In addition, the qscR 561

mutation did not bring about obvious influence on the expression of phz1p-lacZ 562

transcriptional fusion (pMP1C) (Fig. 5G). These data strongly suggests that QscR 563


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downregulated the transcription of its neighboring phz2 operon in M18. Furthermore, 564

an alignment analysis revealed a lux box, a characteristic motif of the LuxR regulon, 565

located from -56 to -37 bp upstream of the TSS of phz2 operon (Fig. 6A). Taken 566

together, we conclude that the transcriptional upregulation of phz2 expression by Hfq 567

was mediated mainly by the transcriptional regulator QscR. 568

The secondary structure prediction of the qscR mRNA leader showed an 569

AU-rich single-stranded sequence linking two stem-loops (Fig. 6B), which is similar 570

to the two RNA secondary structures in the mRNA leaders of phzM (Fig. 2B) and 571

pltR (Fig. 8A), two other negative regulated targets of Hfq. EMSA was carried out to 572

assess the binding affinity of Hfq to the qscR mRNA 5ÚTR, which ranges from - 48 573

to - 97 nt upstream of the translation start site AUG of qscR and contains the 574

AU-rich region (Fig. 6B and C). The Hfq protein showed strong binding activity 575

with the qscR 5ÚTR (Fig. 6C). 576

577

Hfq negatively regulated the expression of Plt biosynthetic genes exclusively 578

through the plt transcriptional activator PltR 579

The above results (Fig. 1G) clearly demonstrated that the RNA chaperon 580

protein Hfq strongly inhibited pyoluteorin biosynthesis in P. aeruginosa M18. The 581

actual molecular mechanism by which Hfq negatively controlled Plt biosynthesis 582

remains unknown, however. To identify the target regulatory region of Hfq within 583

the intergenic region between divergently-transcribed pltLABCDEFG and pltR, the 584

pltLA'-'lacZ translational (pMEAZ), pltLp-lacZ transcriptional (p6522-pltLp), and 585


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pltLo-lacZ (p9533-pltLo) fusion plasmids were introduced separately into the strains 586

M18 and M18hfq, and the LacZ activities were measured in KMB media. As shown 587

in Fig. 7C, no significant difference was detected in the pltLo-lacZ fusion expression 588

between the strains M18 and M18hfq. However, the hfq mutation brought about a 589

notable increase in the total translational pltLA'-'lacZ fusion expression (Fig. 7A). 590

More significantly, a two- to three-fold increase in the pltLp-lacZ transcriptional 591

fusion expression was observed in the M18hfq strain compared to the wild type 592

strain (Fig. 7B). Consistent with the pattern of LacZ expression, the result from 593

qRT-PCR analysis showed that the hfq deletion caused a drastic increase of pltA 594

mRNA accumulation in early stationary growth phase of the strain M18hfq 595

compared to the wild type strain (Fig. S2). Based on these results, it is reasonable to 596

deduce that Hfq is likely to exert an indirect negative regulation on pltLABCDEFG 597

transcription in P. aeruginosa M18. 598

Like QscR-mediated transcriptional regulation of phz2 expression, it could also 599

be logically assumed that the transcriptional repression on pltLABCDEFG 600

expression by Hfq is mediated by an intermediary transcriptional regulator. The 601

LysR-type pathway-specific transcriptional activator PltR of the divergently 602

transcribed plt operon (22, 35) is just such a candidate. Indeed, PltR has been 603

reported to play an important role in Gac/Rsm signal transduction and in the Las/Rhl 604

QS global regulatory network, especially the GacS/GacA two-component regulatory 605

system, involved in Plt biosynthesis in P. aeruginosa M18 (21). To confirm the 606

hypothesis that Hfq indirectly downregulated plt operon transcription through PltR, 607


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the translational (pltR'-'lacZ), transcriptional (pltRp-lacZ), and (pltRo-lacZ) fusion 608

plasmids were separately transformed into the wild type strain M18 and the hfq 609

mutant strain M18hfq, and β-galactosidase assays were performed (Fig. 7D–F). The 610

pltRp-lacZ transcriptional fusion expression (pRZCF) was not significantly different 611

between the strains M18 and M18hfq (Fig. 7E). However, the LacZ activity, which 612

was expressed from both the pltR'-'lacZ translational fusion (pRZLF) and the 613

pltRo-lacZ fusion (p9533-pltRo) was significantly enhanced in the hfq mutant (Fig. 614

7D and F). Consistent with these results, the accumulation of pltR mRNA in M18hfq 615

was significantly elevated by about two-fold in the strain M18 (Fig. S2). In summary, 616

the RNA chaperon, Hfq, strongly downregulated pltR expression at the 617

post-transcriptional level and consequently controlled the transcription of the 618

pltLABCDEFG operon. 619

Hfq directly bound to the 5' UTR of pltR mRNA 620

Similar to the negative regulation of PYO production and phzM expression by 621

the Hfq protein, Plt production by the plt operon was subject to strong negative 622

regulation by Hfq. The only difference is that the latter regulation was mediated 623

exclusively by the plt transcriptional activator PltR. On the basis of this similar 624

negative regulation by Hfq on phzM and pltR, the pltR mRNA 5' UTR was assumed 625

to share a similar RNA secondary structure with the phzM mRNA leader. As shown 626

in Fig. 8A, the 63 nt pltR mRNA leader (+1 to ATG) indeed forms an almost 627

identical RNA secondary structure where a 9 bp single-stranded AU-rich linker (a 628

potential Hfq binding motif) is flanked by two stem-loop structures. This type of 629


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mRNA secondary structure is likely to be a typical characteristic of Hfq regulons. 630

We thus questioned whether a 5-nt replacement in the AU-rich spacer of pltR mRNA 631

leader affects the regulatory effect of Hfq on pltR. As shown in Fig. 8B, the LacZ 632

expression from the plasmid p9533-pltRo-M1 did not show significant difference 633

between the wild-type strain M18 and its hfq mutant strain M18hfq, demonstrating 634

that the regulatory effect of Hfq on pltR was diminished by the mutated AU-rich 635

spacer. 636

EMSA analysis was performed to assess the direct interaction of Hfq with the 637

pltR mRNA leader (Fig. 8C). The retarded bands representing the Hfq-pltR 638

complexes were clearly observed in the EMSA gel, revealing the strong binding 639

activity between the Hfq protein and the pltR mRNA. The number and density of 640

Hfq-pltR complexes gradually increased as the Hfq concentration was raised from 0 641

to 5.6 μM. When Hfq was above 8.0 μM, stronger binding between Hfq and pltR 642

was observed, and gels showed slower Hfq-pltR complex mobility and fewer 643

complexes (Fig. 8C). A 1500-fold excess of unlabeled pltR (Fig. 8C, last lane) 644

almost entirely displaced the biotin-labeled pltR from the Hfq-RNA complex. Also, 645

the data in Fig. 8D shows that the mutated pltR mRNA leader with a 5-nt substitution 646

(Fig. 8A) was still able to be bound by Hfq, but with significantly reduced binding 647

affinity. A higher proportion of 1 nM mutated pltR mRNA leader remained unbound 648

by Hfq at any concentration up to 28 μM (Fig. 8D). In contrast, 1 nM wild-type pltR 649

mRNA leader could be entirely bound by only 12 μM Hfq (Fig. 8C, Lane 7). These 650

results strongly suggested that Hfq directly bound to the pltR mRNA leader, and 651


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consequently inhibited pltR mRNA expression. 652

653


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Discussion 654

There is growing evidence that the RNA chaperone Hfq functions as an 655

important global post-transcriptional regulator in many aspects of cell physiology 656

and metabolism, including virulence and stress responses (3, 7). Here, we 657

investigated the molecular mechanisms by which Hfq regulated antibiotic 658

biosynthesis in the rhizosphere-isolated P. aeruginosa strain M18. The hfq deletion 659

mutant M18hfq exhibited several significant phenotypes, including reduced PCA 660

production, elevated PYO and Plt production, and impaired growth (almost entirely 661

inhibited growth in PPM media). As summarized in a proposed model in Fig. 9, the 662

differential and strong regulation of Hfq on antibiotic biosynthesis was shown to 663

occur at both the direct post-transcriptional level and at the indirect transcriptional 664

level mediated by other regulators. Hfq post-transcriptionally downregulated phzM 665

expression and consequently inhibited the conversion of PCA to PYO. Hfq was also 666

shown to post-transcriptionally inhibit the mRNA expression of pltR and qscR, 667

thereby indirectly exerting strong negative regulation on the plt transcription and 668

positive regulation on the phz2 transcription. In addition, our previous studies have 669

suggested that Plt can auto-induce the expression of its own biosynthetic operon 670

pltLABCDEFG and is probably exported from the cell by the ABC (ATP-binding 671

cassette) transport system PltHIJKNO (20). All three important QS systems, Las, Rhl, 672

and PQS, are involved in strong and differential regulation of antibiotic biosynthesis 673

in P. aeruginosa M18 (8, 30, 54). Furthermore, phzM and its regulatory genes lasI 674

and ptsP are expressed in a temperature-dependent manner (19). 675


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Hfq has predominantly emerged as an RNA chaperon and post-transcriptional 676

regulator (3). Although a few researches have shown that Hfq can independently 677

regulate gene expression by impacting polyadenylation or translation of mRNAs (7, 678

16, 26) or directly regulate target genes expression at levels other than mRNA (45, 679

46), we tend to assume that Hfq, potentially in cooperation with other unknown cis- 680

or trans- encoded sRNAs, post-transcriptionally downregulated the phzM, qscR, and 681

pltR expression. Therefore, we have attempted to screen the sRNA candidates 682

controlling antibiotic biosynthesis of P. aeruginosa M18 from those reported or 683

predicted homologous Pseudomonas sRNAs. 42 homologous Pseudomonas sRNAs 684

(20 detected by RT-PCR or northern blotting and 22 by predicted) (41) were 685

respectively searched by intaRNA (v1.2.5) (39) for short stretches of 10 nucleotides 686

complementary to the phzM, qscR, and pltR mRNA leaders. 12 (4 and 8), 9 (3 and 6), 687

and 4 (1 and 3) sRNAs, including CrcZ, PhrY, PhrW, RgsA, and SsrS et al., was 688

respectively predicted to probably targeting the phzM, qscR, and pltR mRNA leaders 689

(Table S3). It needs further research to ascertain whether these sRNAs along with 690

Hfq control antibiotic biosynthesis in the M18 strain. In addition, the presence of 691

multiple promoters in the longer intergenic region will prompt us to further assess 692

whether there exist any cis-encoded sRNAs in the opposite direction of phzM, qscR, 693

and pltR genes. 694

We also questioned whether two important sRNAs, RsmY and RsmZ, are 695

involved in Hfq-mediated regulation of antibiotic biosynthesis in P. aeruginosa M18. 696

These two sRNAs have been extensively studied to be involved in the control of 697


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secondary metabolism and quorum sensing in P. florescence CHAO and P. 698

aeruginosa (24, 40). They can sequester the translational repressor RsmA, which in 699

turn blocks ribosome binding at the RBS of target mRNAs (18, 47). Although Hfq 700

has been reported to can bind to and stabilize RsmY (44), there appears to be no 701

direct evidence that Hfq regulates the expression of other target genes in 702

collaboration with these two sRNAs. In P. aeruginosa M18, the rsmY mutation led to 703

an almost complete inhibition of Plt biosynthesis and a mild increase in PCA 704

biosynthesis, while the rsmZ mutation did not show a significant influence on Plt and 705

PCA biosynthesis (data not shown). However, Hfq was shown to exert both strong 706

negative regulation on Plt biosynthesis and significant positive regulation on PCA 707

biosynthesis in M18. Therefore, it can be reasonably speculated that Hfq and 708

RsmY/RsmZ control antibiotic biosynthesis through two distinct regulatory 709

pathways and mechanisms in P. aeruginosa M18. In addition, in P. aeruginosa, two 710

Hfq-dependent sRNAs, PrrF1 and PrrF2, have been characterized that are involved 711

in iron homeostasis (52), and two other Hfq-dependent sRNAs, PhrS and PhrD, in 712

PqsR-mediated quorum sensing (40). PhrS, expression of which is induced under 713

low oxygen conditions, activates PqsR expression and thus elevates PYO and PQS 714

levels (40). However, PYO production was significantly enhanced in the hfq mutant 715

M18hfq. Therefore, whether the P. aeruginosa M18 Hfq protein regulated PYO 716

biosynthesis with cooperation with the PhrS sRNA remains unknown. 717

Hfq and/or Hfq-dependent sRNA has often been reported to perform two 718

opposing functions in a target mRNA-specific way. For example, the Hfq-dependent 719


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sRNA, DsrA, not only activates the translation of RpoS but also represses the 720

translation of H-NS (4). It can be proposed that whether one target mRNA is 721

positively or negatively regulated by Hfq likely depends on the secondary structure 722

of the target mRNA leader. As shown in this study, a similar secondary structure with 723

a short single-stranded AU-rich spacer (a potential Hfq-binding motif) linking two 724

stem-loops occurs in the mRNA leaders of three negative Hfq regulons, pltR, phzM, 725

and qscR. Therefore, this typical RNA secondary structure could be a common 726

characteristic of mRNAs subject to negative regulation by Hfq. The similar RNA 727

structure has also been reported in other Hfq negative regulons, such as the rpoS 728

mRNA repressed by the Hfq-dependent OxyS sRNA (55) and the auto-repressed hfq 729

mRNA (48) in E.coli. Interestingly, similar RNA structures were also found in other 730

Hfq-dependent sRNAs such as OxyS, which was shown to repress fhlA and rpoS 731

translation (56). In collaboration with an unidentified sRNA, Hfq may repress their 732

target mRNAs through several possible mechanisms, including stabilizing the 733

inhibitory stem-loop structures, blocking ribosome binding, and antisense 734

degradation. 735

The ubiquitously expressed protein Hfq has been increasingly reported to 736

functions as a central regulator for adjusting global gene expression profiles to be 737

responsible for the fitness, virulence, and stress or host response in most bacteria (6, 738

7). Transcriptomic results have indicated that Hfq directly or indirectly regulates 739

almost 20% of all Salmonella genes and 15% of all P. aeruginosa genes (7). Multiple 740

transcriptional or post-transcriptional regulators mediate bacterial transcriptomic 741


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regulation by Hfq. Such a global cascade regulatory network might be thought of as 742

an economical and energy-saving regulatory strategy in bacteria. P. aeruginosa M18, 743

an effective biocontrol strain isolated from the rhizosphere of sweet melon, probably 744

has evolved a unique profile of secondary metabolites including antibiotics during 745

long-term inhabitation in rhizosphere soil. This can be partly illustrated by our 746

previous results that the temperature-dependent and strain-specific manners of the 747

M18 strain contributed to the production of the strong antifungal compound PCA 748

rather than the virulence factor PYO (19). Hfq may provide a new pathway by which 749

P. aeruginosa M18 can inhibit PYO biosynthesis and concomitantly promote PCA 750

biosynthesis. 751

In summary, our results demonstrate that the Hfq chaperone protein is a critical 752

regulator of P. aeruginosa M18 secondary metabolism at both the 753

post-transcriptional and transcriptional levels (the latter mediated by PltR and QscR). 754

Further studies will focus on identifying the potential sRNAs involved in the 755

Hfq-dependent regulation of antibiotic biosynthesis by high-throughput methods, 756

such as deep-sequencing, tilting array, and Hfq-RNA CoIP. These studies might help 757

unravel the Hfq-dependent genomic expression profile, reconstruct the 758

Hfq-dependent global regulatory network of secondary metabolism including 759

antibiotic biosynthesis, and determine the molecular mechanism by which Hfq 760

controls the growth of cells and the utilization of carbon in P. aeruginosa M18.761


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Acknowledgements 762

We thank Dieter Haas for generously providing the plasmids in this study. We thank Geng Wu 763

for providing the protein purification equipment. This work was supported by grants from the 764

National Natural Science Foundation of China (No. 30800009), the National High-Tech 765

Research and Development Program (863 Program) of China (No. 2007AA02Z215), the 766

National Key Basic Research Program (973 Program) of China (No. 2009CB118906), the Key 767

Project of the Shanghai Committee of Science and Technology (No. 08391911900), and the 768

Shanghai Leading Academic Discipline Project (No. B203). 769

770

References 771

1. Bailey, T. L., N. Williams, C. Misleh, and W. W. Li. 2006. MEME: discovering and analyzing 772

DNA and protein sequence motifs. Nucleic. Acids. Res. 34:W369-73. 773

2. Bender, C., V. Rangaswamy, and J. Loper. 1999. Polyketide production by plant-associated 774

Pseudomonads. Annu. Rev. Phytopathol. 37:175-196. 775

3. Brennan, R. G., and T. M. Link. 2007. Hfq structure, function and ligand binding. Curr. Opin. 776

Microbiol. 10:125-33. 777

4. Brescia, C. C., P. J. Mikulecky, A. L. Feig, and D. D. Sledjeski. 2003. Identification of the 778

Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure. RNA 779

9:33-43. 780

5. Brodhagen, M., I. Paulsen, and J. E. Loper. 2005. Reciprocal regulation of pyoluteorin 781

production with membrane transporter gene expression in Pseudomonas fluorescens Pf-5. Appl. 782

Environ. Microbiol. 71:6900-6909. 783

6. Chambers, J. R., and K. S. Bender. 2011. The RNA chaperone Hfq is important for growth and 784

stress tolerance in Francisella novicida. PLoS One 6:e19797. 785

7. Chao, Y., and J. Vogel. 2010. The role of Hfq in bacterial pathogens. Curr. Opin. Microbiol. 786

13:24-33. 787

8. Chen, Y., X. Wang, X. Huang, X. Zhang, and Y. Xu. 2008. Las-like quorum-sensing system 788

negatively regulates both pyoluteorin and phenazine-1-carboxylic acid production in 789

Pseudomonas sp. M18. Sci. China C Life Sci. 51:174-81. 790

9. Chugani, S. A., M. Whiteley, K. M. Lee, D. D'Argenio, C. Manoil, and E. P. Greenberg. 2001. 791

QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa. 792

Proc. Natl. Acad. Sci. U.S.A. 98:2752-7. 793

10. Essar, D. W., L. Eberly, A. Hadero, and I. P. Crawford. 1990. Identification and 794

characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: 795


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

39

interchangeability of the two anthranilate synthases and evolutionary implications. J. Bacteriol. 796

172:884-900. 797

11. Ge, Y., X. Huang, S. Wang, X. Zhang, and Y. Xu. 2004. Phenazine-1-carboxylic acid is 798

negatively regulated and pyoluteorin positively regulated by gacA in Pseudomonas sp. M18. 799

FEMS Microbiol. Lett. 237:41-47. 800

12. Gibson, J., A. Sood, and D. A. Hogan. 2009. Pseudomonas aeruginosa-Candida albicans 801

interactions: localization and fungal toxicity of a phenazine derivative. Appl. Environ. Microbiol. 802

75:504-13. 803

13. Gripenland, J., S. Netterling, E. Loh, T. Tiensuu, A. Toledo-Arana, and J. Johansson. 2010. 804

RNAs: regulators of bacterial virulence. Nat. Rev. Microbiol. 8:857-66. 805

14. Gross, H., and J. E. Loper. 2009. Genomics of secondary metabolite production by 806

Pseudomonas spp. Nat. Prod. Rep. 26:1408-46. 807

15. Haas, D., and C. Keel. 2003. Regulation of antibiotic production in root-colonizing Pseudomonas 808

spp. and relevance for biological control of plant disease. Ann. Rev. Phytopathol. 41:117-153. 809

16. Hajnsdorf, E., and P. Regnier. 2000. Host factor Hfq of Escherichia coli stimulates elongation of 810

poly(A) tails by poly(A) polymerase I. Proc. Natl. Acad. Sci. U.S.A. 97:1501-5. 811

17. Hamdan, H., D. M. Weller, and L. S. Thomashow. 1991. Relative importance of fluorescent 812

siderophores and other factors in biological control of Gaeumannomyces graminis var. tritici by 813

Pseudomonas fluorescens 2-79 and M4-80R. Appl. Environ. Microbiol. 57:3270-7. 814

18. Heeb, S., C. Blumer, and D. Haas. 2002. Regulatory RNA as mediator in 815

GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens 816

CHA0. J. Bacteriol. 184:1046-56. 817

19. Huang, J. F., Y. Q. Xu, H. Y. Zhang, Y. Q. Li, X. Q. Huang, B. Ren, and X. H. Zhang. 2009. 818

Temperature-dependent expression of phzM and Its regulatory genes lasI and ptsP in rhizosphere 819

Isolate Pseudomonas sp. strain M18. Appl. Environ. Microbiol. 75:6568-6580. 820

20. Huang, X., A. Yan, X. Zhang, and Y. Xu. 2006. Identification and characterization of a putative 821

ABC transporter PltHIJKN required for pyoluteorin production in Pseudomonas sp. M18. Gene 822

376:68-78. 823

21. Huang, X., X. Zhang, and Y. Xu. 2008. PltR expression modulated by the global regulators 824

GacA, RsmA, LasI and RhlI in Pseudomonas sp. M18. Res. Microbiol. 159:128-136. 825

22. Huang, X., D. Zhu, Y. Ge, H. Hu, X. Zhang, and Y. Xu. 2004. Identification and 826

characterization of pltZ, a gene involved in the repression of pyoluteorin biosynthesis in 827

Pseudomonas sp. M18. FEMS. Microbiol. Lett. 232:197-202. 828

23. King, E. O., M. K. Ward, and D. E. Raney. 1954. Two simple media for the demonstration of 829

pyocyanin and fluorescin. J. Lab. Clin. Med. 44:301-7. 830

24. Lapouge, K., M. Schubert, F. H. T. Allain, and D. Haas. 2008. Gac/Rsm signal transduction 831

pathway of gamma-proteobacteria: from RNA recognition to regulation of social behaviour. Mol. 832

Microbiol. 67:241-253. 833

25. Lau, G. W., D. J. Hassett, H. Ran, and F. Kong. 2004. The role of pyocyanin in Pseudomonas 834

aeruginosa infection. Trends Mol. Med. 10:599-606. 835

26. Le Derout, J., M. Folichon, F. Briani, G. Deho, P. Regnier, and E. Hajnsdorf. 2003. Hfq 836

affects the length and the frequency of short oligo(A) tails at the 3' end of Escherichia coli rpsO 837

mRNAs. Nucleic. Acids. Res. 31:4017-23. 838

27. Ledgham, F., I. Ventre, C. Soscia, M. Foglino, J. N. Sturgis, and A. Lazdunski. 2003. 839


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

40

Interactions of the quorum sensing regulator QscR: interaction with itself and the other regulators 840

of Pseudomonas aeruginosa LasR and RhlR. Mol. Microbiol. 48:199-210. 841

28. Levitch, M. E., and E. R. Stadtman. 1964. Study of the biosynthesis of phenazine-1-carboxylic 842

acid. Arch. Biochem. Biophys. 106:194-9. 843

29. Li, Y., X. Du, Z. J. Lu, D. Wu, Y. Zhao, B. Ren, J. Huang, X. Huang, and Y. Xu. 2011. 844

Regulatory feedbackloop of two phz gene clusters through 5'-untranslated regions in Pseudomonas 845

sp. M18. PLoS One 6:e19413. 846

30. Lu, J., X. Huang, K. Li, S. Li, M. Zhang, Y. Wang, H. Jiang, and Y. Xu. 2009. LysR family 847

transcriptional regulator PqsR as repressor of pyoluteorin biosynthesis and activator of 848

phenazine-1-carboxylic acid biosynthesis in Pseudomonas sp. M18. J. Biotechnol. 143:1-9. 849

31. Maddula, V. S., E. A. Pierson, and L. S. Pierson, 3rd. 2008. Altering the ratio of phenazines in 850

Pseudomonas chlororaphis (aureofaciens) strain 30-84: effects on biofilm formation and pathogen 851

inhibition. J. Bacteriol. 190:2759-66. 852

32. Mavrodi, D. V., W. Blankenfeldt, and L. S. Thomashow. 2006. Phenazine compounds in 853

fluorescent Pseudomonas spp.: Biosynthesis and regulation. Annu. Rev. Phytopathol. 44:417-445. 854

33. Mavrodi, D. V., R. F. Bonsall, S. M. Delaney, M. J. Soule, G. Phillips, and L. S. Thomashow. 855

2001. Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide 856

from Pseudomonas aeruginosa PAO1. J. Bacteriol. 183:6454-65. 857

34. Miller, J. H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, 858

Cold Spring Harbor, N.Y., USA. 859

35. Nowak-Thompson, B., N. Chaney, J. S. Wing, S. J. Gould, and J. E. Loper. 1999. 860

Characterization of the pyoluteorin biosynthetic gene cluster of Pseudomonas fluorescens Pf-5. J. 861

Bacteriol. 181:2166-74. 862

36. Reese, M. G. 2001. Application of a time-delay neural network to promoter annotation in the 863

Drosophila melanogaster genome. Comput. Chem. 26:51-56. 864

37. Sambrook, J., and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual, 3rd edn. CSH 865

Press, Cold Spring Harbor, N.Y., USA. 866

38. Silby, M. W., C. Winstanley, S. A. Godfrey, S. B. Levy, and R. W. Jackson. 2011. 867

Pseudomonas genomes: diverse and adaptable. FEMS Microbiol. Rev. 35:652-80. 868

39. Smith, C., S. Heyne, A. S. Richter, S. Will, and R. Backofen. 2010. Freiburg RNA Tools: a web 869

server integrating INTARNA, EXPARNA and LOCARNA. Nucleic. Acids Res. 38:W373-7. 870

40. Sonnleitner, E., N. Gonzalez, T. Sorger-Domenigg, S. Heeb, A. S. Richter, R. Backofen, P. 871

Williams, A. Huttenhofer, D. Haas, and U. Blasi. 2011. The small RNA PhrS stimulates 872

synthesis of the Pseudomonas aeruginosa quinolone signal. Mol Microbiol 80:868-885. 873

41. Sonnleitner, E., and D. Haas. 2011. Small RNAs as regulators of primary and secondary 874

metabolism in Pseudomonas species. Appl. Microbiol. Biotechnol. 91:63-79. 875

42. Sonnleitner, E., S. Hagens, F. Rosenau, S. Wilhelm, A. Habel, K. E. Jager, and U. Blasi. 2003. 876

Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1. Microb. Pathog. 35:217-28. 877

43. Sonnleitner, E., I. Moll, and U. Blasi. 2002. Functional replacement of the Escherichia coli hfq 878

gene by the homologue of Pseudomonas aeruginosa. Microbiology 148:883-91. 879

44. Sonnleitner, E., M. Schuster, T. Sorger-Domenigg, E. P. Greenberg, and U. Blasi. 2006. 880

Hfq-dependent alterations of the transcriptome profile and effects on quorum sensing in 881

Pseudomonas aeruginosa. Mol. Microbiol. 59:1542-58. 882

45. Takada, A., M. Wachi, A. Kaidow, M. Takamura, and K. Nagai. 1997. DNA binding properties 883


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

41

of the hfq gene product of Escherichia coli. Biochem. Biophys. Res. Commun. 236:576-9. 884

46. Updegrove, T. B., J. J. Correia, R. Galletto, W. Bujalowski, and R. M. Wartell. 2010. E. coli 885

DNA associated with isolated Hfq interacts with Hfq's distal surface and C-terminal domain. 886

Biochim. Biophys. Acta. 1799:588-96. 887

47. Valverde, C., S. Heeb, C. Keel, and D. Haas. 2003. RsmY, a small regulatory RNA, is required 888

in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas 889

fluorescens CHA0. Mol. Microbiol. 50:1361-79. 890

48. Vecerek, B., I. Moll, and U. Blasi. 2005. Translational autocontrol of the Escherichia coli hfq 891

RNA chaperone gene. RNA 11:976-84. 892

49. Venturi, V. 2006. Regulation of quorum sensing in Pseudomonas. FEMS Microbiol. Rev. 893

30:274-91. 894

50. Wang, Y., X. Huang, H. Hu, X. Zhang, and Y. Xu. 2008. QscR acts as an intermediate in 895

gacA-dependent regulation of PCA biosynthesis in Pseudomonas sp. M18. Curr. Microbiol. 896

56:339-45. 897

51. Waters, L. S., and G. Storz. 2009. Regulatory RNAs in bacteria. Cell 136:615-28. 898

52. Wilderman, P. J., N. A. Sowa, D. J. FitzGerald, P. C. FitzGerald, S. Gottesman, U. A. 899

Ochsner, and M. L. Vasil. 2004. Identification of tandem duplicate regulatory small RNAs in 900

Pseudomonas aeruginosa involved in iron homeostasis. Proc. Natl. Acad. Sci. USA 101:9792-7. 901

53. Wu, D. Q., J. Ye, H. Y. Ou, X. Wei, X. Huang, Y. W. He, and Y. Xu. 2011. Genomic analysis 902

and temperature-dependent transcriptome profiles of the rhizosphere originating strain 903

Pseudomonas aeruginosa M18. BMC Genomics 12:438. 904

54. Yan, A., X. Huang, H. Liu, D. Dong, D. Zhang, X. Zhang, and Y. Xu. 2007. An rhl-like 905

quorum-sensing system negatively regulates pyoluteorin production in Pseudomonas sp. M18. 906

Microbiology 153:16-28. 907

55. Zhang, A., S. Altuvia, A. Tiwari, L. Argaman, R. Hengge-Aronis, and G. Storz. 1998. The 908

OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein. EMBO J. 909

17:6061-8. 910

56. Zhang, A., K. M. Wassarman, J. Ortega, A. C. Steven, and G. Storz. 2002. The Sm-like Hfq 911

protein increases OxyS RNA interaction with target mRNAs. Mol. Cell 9:11-22. 912

57. Zuker, M. 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic. 913

Acids Res. 31:3406-15. 914

915

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Figure legends 917

Figure 1. Regulation of cell growth and antibiotic biosynthesis by Hfq in P. aeruginosa M18. 918

A) The construction map of the P. aeruginosa M18 hfq mutant (M18hfq). The hfq gene in P. 919

aeruginosa M18 was replaced with the gentamicin-resistant cassette by homologous 920

recombination. 921

B) No polar effect of the hfq deletion on the expression of its flanking genes, miaA and 922

PAM18-5054, was detected by semi-quantitative RT-PCR in P. aeruginosa M18. 923

C) The hfq deletion stimulated the blue-pigment production in P. aeruginosa M18. The tested 924

strains were grown on KMB plates for 60 hours. Six tested strains were P. aeruginosa M18, 925

M18hfq, and their four derivative strains carrying the hfq expression plasmid pBBR-hfq or the 926

empty plasmid pBBR1MCS (abbreviated as pBBR) as the control. 927

D) Cell growth assay of P. aeruginosa M18, M18hfq, and their derivative strains carrying 928

pBBR-hfq or the empty plasmid pBBR in PPM media. The hfq deletion caused a significant 929

impairment of M18 growth, which in turn was recovered to the wild-type level by the 930

exogenously expressed Hfq from the plasmid pBBR-hfq. 931

E) PYO production (solid symbols) and cell growth (open symbols) of the strains M18 and its 932

hfq mutant M18hfq in KMB media. PYO production was significantly enhanced and cell growth 933

significantly decreased by the hfq deletion in KMB media. 934

F) PCA production (solid symbols) and cell growth (open symbols) of the strains M18 and 935

M18hfq in KMB media. PCA production of M18hfq was almost entirely reduced in KMB 936

medium. 937

G) Plt production (solid symbols) and cell growth (open symbols) of the strains M18 and 938


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M18hfq in KMB media. The hfq deletion resulted in a marked increase of Plt production in KMB 939

medium. 940

941

Figure 2. Negative regulation of phzM expression by Hfq and identification of a target 942

regulatory sequence within the phzM operon leader. 943

A) β-Galactosidase activities expressed from a series of phzM-lacZ translational, transcriptional, 944

and 5ÚTR fusions were assayed in P. aeruginosa M18 and its hfq mutant M18hfq after 15 hours 945

of growth in KMB media. The hfq deletion gave rise to a significant increase (shown with 946

asterisks) in expression of the phzM´-´lacZ translational fusion (pPML), the phzMo-lacZ fusions 947

pPMO-1, and pPMO-2. Inhibitory region for Hfq was shown to be located at +130 – +190 948

downstream of the phzM transcriptional start site (TSS). 949

B) Secondary structure of the +116 – +200 (relative to the phzM TSS) region of phzM mRNA 950

leader (predicted by Mfold). The shaded letters show an AU-rich spacer (a potential Hfq-binding 951

motif) linking two stem-loop structures. Two mutants of phzM mRNA leader, M1 and M2, which 952

carried a 4-nt or 2-nt replacement in the AU-rich motif, were used for EMSA analysis，and their 953

corresponding DNAs were utilized to construct the lacZ reporter plasmids (pPMO-2-M1 and 954

pPMO-2-M2). 955

C) Influence of a 4-nt (M1) or 2-nt replacement (M2) in the AU-rich motif on the Hfq regulation 956

of phzM. β-Galactosidase activities, resulting from the phzMo-2-lacZ fusion reporter plasmid 957

pPMO-2, pPMO-2-M1, or pPMO-2-M2, were determined in the wild-type M18 strain and the 958

hfq mutant M18hfq strain in KMB media. 959

960


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Figure 3. Hfq directly binds to the phzM mRNA 5ÚTR. 961

A) Expression and purification of the Hfq protein. SDS-PAGE analysis of the overall protein 962

profile (Left) and the purified Hfq protein (Right) from the cell culture of E.coli BL21 carrying 963

the hfq overexpression plasmid pET28a-hfq. M, protein molecular weight marker; Control, 964

without IPTG induction; Hfq (Left), with IPTG induction. 965

B) Direct binding of Hfq to the phzM mRNA 5ÚTR (+131 – +190) assessed by EMSA. 1 nM 966

biotin-labeled phzM mRNA 5ÚTR was incubated with increasing amounts (0 to 24 μM) of Hfq 967

protein as described in Experimental procedures. Hfq-phzM complexes were shown as C1 to C4. 968

For competition reactions, 1.5 μM unlabeled phzM was included in the binding reaction in molar 969

excess as indicated in Lane 10. 970

C) Influence of a 4-nt replacement (Fig. 2B) within the AU-rich spacer on the binding affinity 971

between the phzM mRNA leader (+131 – +190) and the Hfq protein. 1 nM biotin-labeled 972

phzM-M1 was incubated with increasing amounts (0 to 24 μM) of Hfq protein as described in 973

Experimental procedures. Hfq-phzM-M1 complexes are designed as C1 to C3. 974

975

Figure 4. Inhibition of the phz2 expression by the hfq deletion and identification of two 976

target regulatory sequences in the phz2 operon leader. 977

A) Construction map for a series of phz2-lacZ translational, transcriptional, and 5ÚTR-lacZ 978

fusion plasmids and β-galactosidase activity assay for the M18 or M18hfq strain respectively 979

harbouring these lacZ fusion plasmids after 15 hours of growth. The hfq deletion caused a 980

significant decrease (indicated with asterisks) in the expression of the phz2-lacZ fusions 981

including pMP2L, p6522-phz2po-2, p6522-phz2p-5, and p9533-phz2o-6. Two target regulatory 982


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regions of Hfq on the phz2 operon were respectively located at -78 – +1 bp upstream and +1 – 983

+113 bp downstream of the phz2 TSS. 984

B) Assay for β-galactosidase activities expressed from the phz1´-´lacZ translational fusion 985

(pMP1L) in the strains M18 and M18hfq. The hfq mutant did not alter the phz1´-´lacZ 986

translational fusion expression originating from pMP1L. 987

988

Figure 5. QscR mediated the upregulation of phz2 transcription. 989

A–C) β-Galactosidase activities, which were expressed from the qscR´-´lacZ translational fusion 990

(A, p6015-qscR), qscRp-lacZ transcriptional fusion (B, p6522-qscRp), and qscRo-lacZ fusion (C, 991

p9533-qscRo), were assayed in the strains M18 and M18hfq in KMB media. The hfq mutation 992

caused a significant increase in the expression of qscR´-´lacZ translational (A) and qscRo-lacZ 993

(C) fusions. 994

D–F) β-Galactosidase activities, which were expressed from the phz2´-´lacZ translational fusion 995

(D, pMP2L), phz2p-5-lacZ transcriptional fusion (E, p6522-phz2p-5), and phz2o-6-lacZ fusion 996

(F, p9533-phz2o-6), were assayed in the M18 strain and the qscR mutant M18qscR in KMB 997

media. Expression of both the phz2´-´lacZ translational fusion (D) and the phz2p-5-lacZ 998

transcriptional fusion (E) were significantly increased in the qscR mutant M18qscR compared to 999

the wild type M18 strain. 1000

G) β-Galactosidase activities expressed from the phz1p-lacZ transcriptional fusion (pMP1C) 1001

were assayed in the strains M18 and M18qscR in KMB media. QscR did not exert a significant 1002

influence on the phz1p-lacZ transcriptional fusion expression. 1003

1004


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Figure 6. The direct binding of Hfq to the qscR mRNA 5ÚTR 1005

A) DNA sequence analysis suggested that a conserved lux box (indicated by boxed nucleotides), 1006

a potential target motif of LuxR-type regulator including QscR, is located immediately upstream 1007

of the -35 element of the phz2 promoter. The +1 indicates the transcriptional start site for the 1008

phz2 operon. 1009

B) The mRNA leader covering 97 nts upstream of the translational start site of qscR was 1010

predicted by Mfold to display a RNA secondary structure containing an AU-rich single-stranded 1011

spacer (shown with gray shaded nucleotides) linking two stem-loops. 1012

C) Direct binding of Hfq to the qscR mRNA 5ÚTR (- 48 – -97 relative to the qscR AUG) 1013

containing the AU-rich region assessed by EMSA. 1 nM biotin-labeled qscR RNA was incubated 1014

with increasing amounts (0 to 32 μM) of Hfq protein as described in Experimental procedures. 1015

The retarded bands represent the Hfq-qscR complexes. For competition reactions, 1 μM 1016

unlabeled qscR was included in the binding reaction in molar excess as indicated in Lane 12. 1017

1018

Figure 7. Hfq indirectly downregulated plt transcription through post-transcriptional 1019

repression on pltR. 1020

A–C) β-Galactosidase activities, which derive from the pltA´-´lacZ translational fusion (A, 1021

pMEAZ), pltLp-lacZ transcriptional fusion (B, p6522-pltLp), and pltLo-lacZ fusion (C, 1022

p9533-pltLo), were assayed in the wild type strain M18 (●) and the hfq mutant M18hfq (▲) in 1023

KMB media. The hfq deletion significantly enhanced the expression of both the pltA´-´lacZ 1024

translational fusion (A) and the pltLp-lacZ transcriptional fusion (B) in the hfq mutant compared 1025

to the wild type strain M18. However, there was no significant difference in the pltLo-lacZ 1026


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fusion expression (C) between M18 and M18hfq. 1027

D–F) β-Galactosidase activities, which were expressed from the pltR´-´lacZ translational fusion 1028

(D, pRZLF), pltRp-lacZ transcriptional fusion (E, pRZCF), and pltRo-lacZ fusion (F, 1029

p9533-pltRo), were assayed in the wild type strain M18 (●) and the hfq mutant M18hfq (▲) in 1030

KMB media. The hfq mutation resulted in a significant increase in expression of both the 1031

pltR´-´lacZ translational fusion (D) and the pltRo-lacZ fusion (F). However, the hfq mutation had 1032

no obvious impact on the pltRp-lacZ transcriptional fusion expression (E). 1033

1034

Figure 8. Hfq directly bound to the pltR mRNA 5ÚTR. 1035

A) The pltR mRNA 5ÚTR (+1~ATG) was predicted by Mfold to fold into a characteristic 1036

secondary structure with a short AU-rich single-stranded spacer linking two stem-loops. A 1037

mutant of the pltR mRNA 5ÚTR, M1, which carried a 5-nt replacement in the AU-rich spacer, 1038

was used for EMSA analysis, and its corresponding DNA was utilized to construct the reporter 1039

plasmid p9533-pltRo-M1. 1040

B) Influence of a 5-nt replacement within the AU-rich spacer on the Hfq regulation of pltR. 1041

β-Galactosidase activities, resulting from the pltRo-lacZ fusion reporter plasmid p9533-pltRo or 1042

p9533-pltRo-M1, were determined in the strains M18 and M18hfq in KMB. Regulatory effect of 1043

Hfq on pltR was diminished by a 5-nt replacement within the AU-rich spacer. 1044

C) Direct binding of Hfq to the pltR mRNA 5ÚTR. 1 nM biotin-labeled pltR was incubated with 1045

increasing amounts (0 to 32 μM) of Hfq protein as described in Experimental procedures. 1046

Hfq-pltR complexes are designed as C1 to C5. For competition reactions, 1.5 μM unlabeled pltR 1047

was included in the binding reaction in molar excess as indicated in Lane 13. 1048


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D) A 5-nt replacement (M1) in the AU-rich spacer significantly reduced the binding affinity 1049

between the pltR mRNA 5ÚTR and Hfq. 1 nM biotin-labeled pltR-M1 was incubated with 1050

increasing amounts (0 to 28 μM) of Hfq protein as described in Experimental procedures. 1051

Hfq-pltR-M1 complexes are designed as C1 to C3. 1052

1053

Figure 9. A schematic model outlining the regulation of antibiotic biosynthesis by Hfq in the 1054

rhizobacterium P. aeruginosa M18. Thick arrows, positive regulation; thick lines with flatted 1055

end, negative regulation; thin arrows, biogenesis and transport. Three regulatory pathways of Hfq 1056

respectively are: (1) the negative regulatory pathway of Hfq on Plt production and its 1057

biosynthetic gene expression exclusively mediated by PltR; (2) the positive regulatory pathway 1058

of Hfq on PCA production and the phz2 operon expression; (3) the negative regulatory pathway 1059

of Hfq on PYO production and the phzM expression. Ellipses indicate the Hfq-dependent 1060

mediators of transcriptional regulation. In addition, the plt operon is transcriptionally activated 1061

by the LysR-type regulator PltR and autoinduced by Plt itself. The ABC (ATP-binding cassette) 1062

transport system PltHIJKNO is probably responsible for exporting Plt out of the cell (20). Three 1063

important QS systems, Las, Rhl, and PQS, strongly repressed Plt biosynthesis in P. aeruginosa 1064

M18. The PCA biosynthesis was almost entirely inhibited by the Rhl or PQS inactivated mutant 1065

and was strongly repressed by the Las system (8, 30, 54). Furthermore, phzM and its regulatory 1066

genes lasI and ptsP were expressed in a temperature-dependent manner (19). 1067


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Documents

JB Accepts, published online ahead of print on 16 March ... · 3/13/2012 · 157 BioDev plasmid minipreps purification system B. DNA was recovered from the gel 158 with the QIAquick