Accepted Manuscript

Role of the interplay between quorum sensing regulator VqsR and the Pseudomonas
quinolone signal in mediating carbapenem tolerance in Pseudomonas aeruginosa

Darija Viducic, Keiji Murakami, Takashi Amoh, Tsuneko Ono, Yoichiro Miyake

PII: S0923-2508(17)30042-6
DOI: 10.1016/j.resmic.2017.02.007
Reference: RESMIC 3575

To appear in: Research in Microbiology

Received Date: 13 September 2016

Revised Date: 12 February 2017
Accepted Date: 14 February 2017

Please cite this article as: D. Viducic, K. Murakami, T. Amoh, T. Ono, Y. Miyake, Role of the interplay
between quorum sensing regulator VqsR and the Pseudomonas quinolone signal in mediating
carbapenem tolerance in Pseudomonas aeruginosa, Research in Microbiologoy (2017), doi: 10.1016/
j.resmic.2017.02.007.

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 ACCEPTED MANUSCRIPT
1 For publication

2 Role of the interplay between quorum sensing regulator VqsR and the

3 Pseudomonas quinolone signal in mediating carbapenem tolerance in

4 Pseudomonas aeruginosa

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5

6 Darija Viducica,b,*, Keiji Murakamia , Takashi Amoha, Tsuneko Onob, Yoichiro Miyakea

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a
7 Department of Oral Microbiology, Institute of Biomedical Sciences, Tokushima University Graduate

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8 School, Kuramoto-cho 3-18-15, Tokushima 770-8504, Japan
b
9 Department of Molecular Microbiology, Institute of Health Biosciences, Tokushima University Graduate

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10 School, Kuramoto-cho 3-18-15, Tokushima 770-8509, Japan
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12 Darija Viducic (darija.viducic@gmail.com) *Correspondence and reprints

13 Keiji Murakami (kmurakami@tokushima-u.ac.jp)
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14 Takashi Amoh (c301351002@tokushima-u.ac.jp)

15 Tsuneko Ono (tono08@mac.com)
16 Yoichiro Miyake (miyake@tokushima-u.ac.jp)
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18 Abstract
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19 Pseudomonas aeruginosa coordinates its response to environmental conditions through

20 activation of a quorum sensing (QS) system. In this study, we investigated the regulatory interaction
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21 between the QS transcriptional regulator VqsR and the Pseudomonas quinolone signal (PQS)
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22 through integration of sigma factor RpoS, and we addressed whether one of the pathways
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23 controlling carbapenem tolerance can be attributed to VqsR. We demonstrate that vqsR expression

24 at the transcriptional level is regulated by pqsA, pqsR, and pqsE. Assessment of the transcriptional

25 expression of vqsR, lasI, rhlI, and qscR in ∆pqsA and ∆pqsA∆rpoS mutants provided insight into

26 pqsA- and rpoS-dependent regulation of vqsR and vqsR-controlled genes. Exogenously

27 supplemented PQS reversed expression of vqsR and vqsR-controlled genes in the ∆pqsA mutant to

28 wild-type levels, but failed to increase expression levels of lasI and qscR in the ∆pqsA∆rpoS

29 mutant to levels observed in wild-type PAO1. The ∆vqsR mutant showed reduced survival when

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30 challenged with carbapenems compared to wild-type PAO1. Introduction of a pqsA mutation into

31 the ∆vqsR mutant completely abolished its carbapenem-sensitive phenotype.

32 We conclude that a regulatory link between PQS and vqsR exists, and that RpoS is important

33 in their interaction. We also demonstrate that VqsR affects carbapenem tolerance.

34 Keywords: Pseudomonas aeruginosa; Quorum sensing; Antibiotics; Tolerance

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1. Introduction

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40 Pseudomonas aeruginosa is a major cause of infections in immunocompromised patients,

41 especially those suffering from cystic fibrosis, cancer or burn wounds. The pathogenic potential of
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42 P.aeruginosa is largely attributed to secretion of a wide range of cell-associated and extracellular

43 virulence factors [1]. Production of virulence factors is coordinated through activation of a cell-
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44 density-dependent signaling system known as quorum sensing (QS) [2]. P. aeruginosa possesses
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45 two N-acyl-homoserine lactone (AHL) QS systems, namely, the las and rhl systems. Intercellular
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46 signals for the las and rhl QS systems are N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-

47 HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), that are coinducers for transcriptional
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48 activators LasR and RhlR, respectively [3]. Activated LasR regulates expression of the rhlR gene,
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49 suggesting that las and rhl constitute a hierarchical system in which the las system exerts control

50 over the rhl system [2]. The las and rhl systems positively regulate production of virulence factors

51 such as elastase (LasA and LasB), exotoxin A, alkaline protease, rhamnolipids, hydrogen cyanide

52 (HCN), pyocyanin, and the cytotoxin lectin LecA [4]. In addition to the las and rhl systems, P.

53 aeruginosa possesses another QS system called the Pseudomonas quinolone signal (PQS) [5], a

54 compound related to 4-quinolone antibiotics that serves as a co-inducer for the LysR-type

55 transcriptional activator PqsR (also referred to as MvfR) [6,7]. Mutations in the pqsABCDE operon
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56 and pqsR gene inhibit production of PQS, pyocyanin and other QS-controlled virulence factors

57 [8,9]. The fifth gene of the pqs operon, pqsE, is co-regulated together with the pqsABCD genes, and

58 belongs to the family of enzymes known as metallo-β-lactamase, with a stable Fe2+/Fe3+center in

59 the active site [10]. PqsE is not required for PQS synthesis, functions independently of PqsR and

60 PQS and is connected to the rhl QS system in a regulatory manner [11]. Among the many

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61 characterized transcriptional regulators that affect expression of QS systems, a fourth LuxR

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62 homolog in P. aeruginosa, VqsR, is a virulence and QS regulator; VqsR has been shown to regulate

63 QS systems by directly controlling expression of QscR, a LasR-RhlR homolog [12,13]. VqsR is

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64 regulated by LasR [14]. A mutation in vqsR abolishes production of N-acyl-homoserine lactone,

65 decreases production of virulence factors and downregulates expression of genes belonging to the

66 the PQS system [15].

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67 Bacteria have developed diverse mechanisms to overcome the effect of antimicrobials;
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68 among these is the antibiotic tolerance mechanism in which bacteria abolish the killing effects of

69 antimicrobial agents without a change in the minimal inhibitory concentration (MIC) of the agent;
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70 these bacteria persist in the presence of antimicrobials [16]. The isolation of hip mutants of
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71 Escherichia coli that display 1,000-fold-higher persistence against killing by antibiotics than the

72 wild-type strain suggests that persisters or non-growing tolerant cells are essentially responsible for
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73 the resistance/tolerance of biofilms and stationary phase cells to therapeutic intervention [17,18].
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74 Various stress mechanisms, such as the stringent response regulator ppGpp and alternative sigma
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75 factors RpoS and RpoN, have all been reported to modulate the effects of antimicrobials in P.

76 aeruginosa [19-22]. To coordinate the response to challenging environmental conditions such as the

77 presence of antimicrobials, P. aeruginosa employs QS. It has been demonstrated that PQS plays an

78 important role in the response to antibiotic stress conditions through its pro- and antioxidant

79 activities [23,24]. Furthermore, we recently reported that RpoN employs PQS and PqsE to respond

80 to carbapenems [25].

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81 In this study, we sought to further understand the transcriptional link between PQS and vqsR

82 and the importance of sigma factor RpoS in their interaction. The findings of this study demonstrate

83 that pqs signaling positively affects expression of vqsR. Furthermore, our results show that vqsR

84 plays a role in carbapenem tolerance, and underscore the importance of PQS in antibiotic stress.

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90 2. Materials and methods

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91 2.1. Bacterial strains and culture conditions
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92 Bacterial strains, plasmids, and primers used and generated in this study are shown in Table

93 1. Bacteria were routinely cultured at 37 °C in Luria Bertani medium (LB) or on L-agar plates
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94 supplemented with 10% sucrose when necessary. Vogel-Bonner minimal medium (VBMM) [26]
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95 was used in mating experiments. The following antibiotics for plasmid selection and propagation

96 were added as required: ampicillin (100 µg/ml), gentamicin (20 µg/ml) and tetracycline (10 µg/ml)
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97 (for E. coli), and carbenicillin (300 µg/ml), gentamicin (100 µg/ml), and tetracycline (100 µg/ml)

98 (for P. aeruginosa).
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99 2.2. Reagents

100 Biapenem was purchased from Meiji Seika Pharma Co., Ltd (Yokohama, Japan), and

101 doripenem was purchased from Shionogi & Co., Ltd (Osaka, Japan); both were used at a

102 concentration of 32 µg/ml. Synthetic PQS was obtained from the Toyama Chemical Co., Ltd.

103 (Tokyo, Japan) and was solubilized in methanol to achieve a stock concentration of 20 mM.

104 2.3. Generation of mutant strains

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105 Unmarked deletions of vqsR, pqsE and pqsA genes were constructed in P. aeruginosa PAO1

106 [27] as described previously [25] using the pEX18Gm suicide vector via the sacB-based selection

107 method [28,29]. The ∆pqsR mutant PAO1 was constructed by amplifying a 591-bp EcoRI-BamHI

108 pqsR upstream fragment using the primers pqsR-up-F and pqsR-up-R, and was linked to a 578-bp

109 BamHI-HindIII pqsR downstream fragment that was amplified using primers pqsR-down-F and

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110 pqsR-down-R. The resulting PCR products were digested and cloned in suicide plasmid

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111 pEX18Gm to give pEX18Gm-∆pqsR, which was then used to construct a ∆pqsR mutant. In-frame

112 deletion of rpoS was obtained via splicing by an overlap extension PCR strategy [30]. Briefly, the

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113 primers were designed to amplify approximately 500-bp fragments located upstream and

114 downstream of rpoS from the P. aeruginosa PAO1 genomic DNA. Next, the two DNA fragments

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containing a 20-bp overlap were fused together and the final product was produced using a third
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116 round of PCR. The resulting fragment was cloned into pCR2.1-TOPO (Thermo Fisher Scientific,
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117 MA, USA), digested with EcoRI/BamHI and inserted into EcoRI/BamHI-digested pEX18-Gm to

118 create pEX18Gm-∆rpoS. To construct plasmid pEX18Gm-∆mexGHI-opmD, a fragment upstream

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119 of the mexG regulatory region and downstream of the opmD regulatory region was amplified by
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120 PCR, and the products were cloned into EcoRI-BamHI-digested pEX18Gm using an In-Fusion HD

121 Cloning kit (TaKaRa Bio Inc. Shiga, Japan). The resulting plasmids carrying the gene of interest
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122 were mobilized in P. aeruginosa PAO1 from E. coli S17-1 λpir [31], and integrants were selected
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123 on VBMM medium containing gentamicin. Mutants were selected by plating of integrants on
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124 medium containing 10% sucrose to remove the vector sequence from the chromosome. Deletions

125 were confirmed by PCR and sequence analysis. The ∆vqsR∆pqsA and ∆pqsA∆rpoS mutants were

126 constructed by introduction of the pEX18Gm-∆vqsR and pEX18Gm-∆rpoS deletion constructs,

127 respectively, into a P. aeruginosa PAO1 ∆pqsA mutant.

128 2.4. Construction of chromosomal vqsR–lacZ transcriptional reporter

129 The vqsR promoter region was amplified using the primer pairs vqsR1-F and vqsR1-R

130 (Table 1). The amplified fragment was digested with EcoRI-BamHI and cloned in EcoRI-BamHI-

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131 digested mini-CTX-lacZ [32]. The construct containing the promoter region of vqsR fused to the

132 lacZ gene was integrated as a single copy into the attB site in the PAO1 chromosome using site-

133 specific integration and subsequent backbone excision using an FLP recombinase encoded on the

134 pFLP2 plasmid [29].

135 2.5. RNA isolation, reverse transcription, and quantitative real-time PCR (qRT-PCR) analysis

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136 Strains were grown overnight in 10 ml of LB medium at 37 °C for 16 h, and the cells were

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137 then washed and used to inoculate 10-ml subcultures in LB medium to an OD595 of 0.01. Cultures

138 were incubated at 37 °C and sampled at the 24 h time point. For the PQS induction assay, the cells

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139 were supplemented with PQS at a concentration of 100 µM at 6 h time points and allowed to grow

140 until the 24 h time point. Total RNA was isolated from bacterial cells using the Trizol reagent

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(Invitrogen) and was treated with Turbo DNase (Ambion, Austin, TX) according to the
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142 manufacturer's instructions. cDNA was generated from 1 µg of DNase-treated RNA using the
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143 Transcriptor First Strand cDNA synthesis kit (Roche Applied Science). qRT-PCR reactions were

144 carried out in a Light Cycler (Roche Molecular Biochemicals) using the LightCycler FastStart DNA
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145 Master PLUS SYBR Green I kit (Roche Applied Science) according to specifications of the
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146 supplier. Data were analyzed using LightCycler software (version 3.5). All qRT-PCR analysis was

147 performed in triplicate on RNA from three independent cultures, and quantification of rpsL gene
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148 expression was used to correct for differences in the amount of starting material. qRT-PCR

149 primers for each gene of interest are listed in Table 1.

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2.6. β-Galactosidase assay

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151 Cells from overnight cultures grown in LB medium were washed and resuspended in fresh

152 LB medium to an OD595 of 0.01. Samples (100-200 µl) were collected throughout the growth cycle,

153 and β-galactosidase specific activities were determined as previously described [33].

154 2.7. Antibiotic susceptibility testing

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155 A broth microdilution method [34] was used to determine the MIC of each agent, with the

156 following modification: an inoculum suspension with a density of 106 colony forming units (CFU)

157 per milliliter (CFU/ml) of exponentially growing bacterial cells was used. The cell cultures were

158 then incubated with antimicrobial agent overnight (16-18 h) at 37 °C. The MIC was defined as the

159 lowest concentration of antimicrobial agent that completely inhibited growth of the organism.

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160 2.8. Time-kill assays

Time-kill studies were performed with a final inoculum of approximately 108 CFU/ml

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162 stationary-phase cells grown for 16 h in a final volume of 10 ml. Before the start of the experiment,

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163 cells were washed once and resuspended in LB medium, and then grown with antibiotics in a shaker

164 at 37 °C for 24 h. Samples were collected at several time points, tenfold serial dilutions were

165
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prepared with 0.85% NaCl and colony counts were determined by plating 100 µl onto LB-agar in
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166 duplicate. After 24 h of incubation at 37 °C, the plates with 30-300 colonies were used for CFU
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167 counts. Microbial killing was assessed at defined time points by counting colonies and calculating

168 the percent survival relative to untreated cells at time zero. Data were collected from at least five
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169 independent experiments.

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170 3. Results

171 3.1. pqs Signaling is transcriptionally linked to vqsR, and rpoS participates in this interaction
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172 Previous reports have demonstrated the significance of VqsR in regulation of las and rhl
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173 QS systems through direct regulation of transcriptional regulator QscR [12,13]. Since a regulatory
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174 effect of pqs signaling on vqsR has not been reported, we set out to investigate the transcriptional

175 link between the elements of pqs signaling and transcriptional regulator VqsR in more detail. We

176 began by examining the expression of vqsR throughout the growth cycle in the strain with a

177 mutation in the pqsA gene, the first gene in the PQS biosynthesis operon. PQS is a third signal

178 molecule in addition to the 3-oxo-C12-HSL and C4-HSL produced by P. aeruginosa [5,8] and is

179 synthesized through the activity of several putative enzymes encoded by the pqsABCDE and phnAB

180 operons, and pqsH, in addition to the LysR-type regulator PqsR [9]. We constructed pvqsR-lacZ

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181 transcriptional fusion as a single copy on the wild-type PAO1 and the ∆pqsA mutant chromosome.

182 For the β-galactosidase activity assay, overnight cell cultures were diluted to an OD595 of 0.01 and

183 sampled every 3 h for 12 h, with a final measurement taken at 24 h. The results demonstrated that at

184 the 24 h time point, the vqsR transcriptional levels in the ∆pqsA mutant were 2.7-fold lower than

185 those in the wild-type PAO1 (Fig. 1A). The regulatory network of PQS synthesis involves

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186 activation of transcriptional regulator PqsR, which further binds PQS and affects the expression of

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187 the pqsABCDE operon [7]. It is known that pqsE, the fifth gene in the pqsABCDE operon, is not

188 required for 2-alkyl-4-quinolone (AQ) biosynthesis [6,9], but instead serves as a “PQS signal

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189 response protein” that is implicated in regulation of PQS-associated virulence factors such as

190 pyocyanin, elastase and rhamnolipids [8,9,11]. To further address the transcriptional link between

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vqsR and elements of pqs signaling, we proceeded with the experiments to characterize how
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192 mutations in pqsR and pqsE affect the expression of vqsR. We constructed fusion strains with
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193 pvqsR-lacZ as a single copy on the ∆pqsR mutant and the ∆pqsE mutant chromosomes, and

194 monitored expression of vqsR throughout the growth cycle. Although ∆pqsR and ∆pqsE mutants
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195 and wild-type PAO1 had identical growth rates, the vqsR expression profile was significantly
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196 different (Fig. 1B, 1C). Measurement of β-galactosidase activity throughout the growth kinetics

197 revealed low expression levels of vqsR in the ∆pqsR and ∆pqsE mutants, with the most pronounced
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198 difference observed at the 24-h time point, for which vqsR levels in the ∆pqsR and ∆pqsE mutants

199 were 3.2- and 2.7-fold lower than those of the wild-type PAO1, respectively. To verify data
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200 obtained in the β-galactosidase activity assay, we performed qRT-PCR analysis of vqsR expression

201 in the wild-type PAO1 and mutant strains after growth in LB medium for 24 h (Fig. 1D). The

202 obtained data closely correlated with data from the β-galactosidase assay, demonstrating

203 downregulation of vqsR expression in the mutant strains. In addition to results of the previous

204 study, which demonstrated indirect regulation of PQS by VqsR [13], here our results indicated that

205 vqsR expression is dependent on the pqsA, pqsR and pqsE genes, confirming the interdependence of

206 VqsR and PQS in this regulation.

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207 The intermediary regulators through which PQS regulates VqsR, and vice-versa, remain

208 unknown. Given that the sigma factor RpoS modulates expression of a wide variety of QS-

209 controlled genes [35], we investigated the regulatory network between PQS and VqsR and whether

210 RpoS might be important in this interaction. To do this, we performed qRT-PCR analysis of the

211 expression of vqsR and the vqsR-controlled genes lasI, rhlI, qscR in wild-type PAO1 and the

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212 ∆pqsA, ∆rpoS and ∆pqsA∆rpoS mutants grown in LB medium for 24 h (Fig. 2A). The transcript

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213 levels of vqsR in the ∆pqsA mutant confirmed data obtained using the β-galactosidase assay,

214 showing 3-fold lower levels of vqsR. In the ∆pqsA mutant, we also observed downregulation of rhlI

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215 (1.7-fold) and qscR (4-fold), with the most prominent downregulation of lasI (10-fold) compared to

216 wild-type PAO1. The ∆rpoS mutant expressed vqsR levels comparable to those observed for wild-

217
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type PAO1. The expression of lasI was downregulated 1.75-fold, rhlI by 5.4-fold and qscR was
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218 upregulated 1.5-fold by the mutation in the rpoS gene. The ∆pqsA∆rpoS mutant demonstrated even
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219 more pronounced differences than the ∆pqsA and ∆rpoS mutants, with lasI downregulation of 85-

220 fold, rhlI of 13.3-fold, qscR of 5.8-fold, and vqsR of 13.7-fold compared with the wild-type PAO1.
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221 To analyze whether the observed expression of lasI, rhlI, qscR and vqsR could be restored through
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222 the PQS-dependent pathway, we supplemented cells with 100 µM PQS once the cells entered the

223 late logarithmic phase of growth to circumvent possible growth defects when using high
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224 concentrations of PQS. We performed transcriptional analysis after 24 h of growth (Fig. 2B). The

225 provision of exogenous PQS to wild-type PAO1 induced lasI, rhlI and vqsR expression 3-, 1.3, and
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226 2-fold, respectively, and had no effect on the expression of qscR. In the ∆pqsA mutant, PQS

227 positively affected expression of all genes (Fig. 2B). Notably, PQS supplementation to the ∆rpoS

228 mutant had no impact on lasI, rhlI and qscR transcription; however, it led to a decrease in vqsR

229 transcript levels relative to the wild-type PAO1 (Fig. 2B). The expression levels in the ∆pqsA∆rpoS

230 mutant were similar to those observed in the ∆pqsA mutant, with the exception of lasI and qscR

231 levels, which remained 3.1- and 2-fold lower, respectively, than those in the wild-type PAO1 (Fig.

232 2B). The results demonstrate the following: i) RpoS does not directly regulate the expression of

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233 vqsR, and exogenous PQS requires RpoS to activate VqsR; and ii) exogenously supplemented PQS

234 does not circumvent the requirement for both pqsA and rpoS in the expression of lasI and qscR.

235 3.2. Expression of vqsR affects tolerance to carbapenems, and a mutation in pqsA alters the

236 carbapenem-sensitive phenotype of the ∆vqsR mutant

237 Using observations from transcriptional studies that underscore the transcriptional link

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238 between PQS and vqsR,, and in line with results of previous studies suggesting a direct role for

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239 PQS in regulating the response to antibiotic stress [19,23-25], we further investigated a potential

240 role for VqsR in promoting formation of carbapenem-tolerant persisters. It had been proposed that

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241 the level of persister cells increases in the stationary phase (1%) and P. aeruginosa increases the

242 amount of persisters in response to the QS system by activating global transcriptional regulators and

243
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a stringent response [36,37]. Carbapenems are the primary antimicrobial agents currently used in
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244 therapy for infections caused by P. aeruginosa, mainly due to their resistance to β-lactamase
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245 AmpC. To inhibit cell wall synthesis, carbapenems require interaction with penicillin-binding

246 proteins (PBPs) involved in the formation of peptidoglycan in the bacterial cell wall [38]. We
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247 postulated that both PQS and VqsR—which regulate the MexGHI-opmD efflux pump [39,13]—and
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248 the role of PQS in membrane-altering activity [40] might interfere with the carbapenem mode of

249 action. VqsR and PQS are both activated and maximally produced during the stationary phase of
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250 growth; therefore, for the time-kill assays, we used stationary phase cells. We first determined
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251 MICs for wild-type PAO1, the ∆vqsR mutant, the ∆pqsA mutant and the ∆vqsR∆pqsA mutant using
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252 the standard microdilution assay. No significant differences were observed in MICs between the

253 wild-type PAO1 and mutant strains for the antibiotics tested (MIC for biapenem and doripenem: 0.5

254 µg/ml). The carbapenems were used at a concentration of 32 µg/ml because antibiotic-tolerant cells

255 are capable of tolerating antibiotic concentrations above the MIC. The MIC is defined as the

256 minimum concentration required to inhibit cell growth; therefore, to assess both the rate and extent

257 of killing, time-dependent killing assays were used.

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258 To address the role of VqsR in antibiotic tolerance, we performed killing assays for the

259 ∆vqsR mutant and wild-type PAO1 in the presence of 32 µg/ml biapenem and doripenem (Fig. 3A,

260 3B), respectively. The ∆vqsR mutant demonstrated lower tolerance to carbapenems, with a survival

261 rate at 24 h approximately 10-fold-lower than that of the wild-type PAO1. We also performed

262 killing assays in the presence of biapenem (32 µg/ml) for logarithmic phase cells, and observed that

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263 the ∆vqsR mutant had decreased tolerance to biapenem (2.5-fold) relative to the wild-type PAO1

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264 (data not shown). The survival rates of the ∆pqsA mutant in the presence of biapenem and

265 doripenem at 24 h were 6- and 3-fold higher, respectively, than in the wild-type PAO1 (Fig. 4A,

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266 4B). The survival rate for the logarithmic phase cells of the ∆pqsA mutant was slightly higher than

267 for the wild-type PAO1 (data not shown). To test whether loss of PQS production can alter the

268
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sensitive phenotype of the ∆vqsR mutant, a ∆vqsR∆pqsA mutant was constructed. Interestingly,
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269 introducing a pqsA deletion in the ∆vqsR mutant resulted in complete loss of the sensitive
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270 phenotype of the ∆vqsR mutant in the presence of carbapenems (Fig. 4C, 4D). Growth rates of the

271 wild-type PAO1 and mutants were indistinguishable; therefore, the phenotypes of these mutants
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272 were not caused by a reduced growth rate (data not shown). Although this finding suggested that
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273 PQS predominantly defines survival during antibiotic stress, other regulatory mechanisms linking

274 PQS and VqsR are likely at play. The MexGHI-OpmD efflux pump is involved in regulation of
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275 pqs signaling, since its inactivation leads to loss of PQS production and promotes accumulation

276 of the PQS precursor anthranilate, which exerts toxic effects in cells [41]. We were interested in
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277 whether a mutation in the MexGHI-OpmD efflux pump could alter the response to carbapenems. As

278 illustrated in Fig. 5A, the survival rate in the presence of biapenem for the ∆mexGHI-opmD mutant

279 was comparable to that observed in wild-type PAO1, suggesting that MexGHI-OpmD is not

280 directly involved in the response to carbapenems. Since VqsR positively affects expression of

281 mexH [13], and PqsR and PqsE positively regulate expression of mexG [39], we do not exclude the

282 possibility that downregulation of mexG expression in the ∆vqsR mutant, as demonstrated in our

283 transcriptional analysis (Fig. 5B), may also be provoked by downregulation of pqsA expression in

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284 this mutant, and that the MexGHI-OpmD pump may be involved in modulating the response to

285 carbapenems indirectly in the ∆vqsR and ∆pqsA mutants.

286 3.3. Exogenous PQS increases survival of the ∆vqsR mutant in the presence of carbapenems

287 To test whether the addition of exogenous PQS affects formation of carbapenem-tolerant

288 persisters, we studied the effects of PQS on the viability of stationary phase cells for wild-type

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289 PAO1 and the ∆vqsR mutant over 24 h of growth in LB medium supplemented with doripenem at a

addition of PQS at a concentration of 100 µM to

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290 concentration of 32 µg/ml. While stationary

291 phase cells of wild-type PAO1 and the ∆vqsR mutant showed no significant effects on viability,

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292 addition of PQS to stationary phase cells of the ∆vqsR mutant in the presence of doripenem

293 abolished its sensitive phenotype and stimulated a minor increase in survival of the wild-type

294 PAO1 (Fig. 6). This

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suggests that exogenous PQS can reverse the sensitive phenotype of the
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295 ∆vqsR mutant at a concentration that does not affect the viability of cells, possibly by reversing
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296 downregulation of the pqsA gene [12].

297 4. Discussion
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298 The present study sought to better understand the complex network between the elements of
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299 QS, specifically PQS, VqsR, and RpoS, and how these elements might affect the response to

300 antimicrobials such as carbapenems. Transcriptional fusion and qRT-PCR studies have
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301 demonstrated that expression of vqsR during the stationary phase of growth is PQS-dependent.
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302 The initial observation of a transcriptional link between the elements of pqs signaling and vqsR
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303 prompted us to address a potential role for sigma factor RpoS as a participant in the interaction

304 between PQS and vqsR. Available evidence has indicated that VqsR controls the expression of

305 PQS and sigma factor RpoS [12]. However, VqsR has not been demonstrated to be a target of

306 regulation by PQS and RpoS. A model for how PQS modulates expression of vqsR through RpoS

307 and how it may affect carbapenem tolerance accounts for a number of the following observations

308 (Fig. 7). The results have demonstrated that mutations in pqsR, pqsA and pqsE have a negative

309 effect on the expression of vqsR, suggesting that active pqs signaling is required for activation of

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310 vqsR during the stationary phase of growth. The PQS network is positively regulated by the las QS

311 regulatory network, in which LasR positively affects expression of pqsR, which is crucial in

312 regulating expression of the pqsABCDE operon [7]. The las system has been placed at the top of

313 the QS hierarchy and directly controls the rhl system. While recent studies suggest an alternative

314 view of the QS regulatory network, providing evidence of a direct regulation of LasR and RhlR by

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315 PqsR during exponential phase [42], in the stationary phase, LasR has an important regulatory role

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316 over pqsR and vqsR [7,14]. A plausible explanation for PqsR-dependent expression of vqsR might

317 come from its positive effect on pqsE that is required for PQS to activate rhl QS [11], and acts on

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318 vqsR through rhl signaling. Our results demonstrated that loss of the alternative sigma factor RpoS

319 does not impact vqsR expression in the stationary phase. In addition, loss of rpoS reduced

320
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expression of rhlI. Despite the lack of effect by RpoS on vqsR expression, our results provide
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321 evidence for the existence of PQS-dependent expression of vqsR through RpoS. Prominent
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322 downregulation of las QS, through loss of both PQS and RpoS, affects expression of vqsR and

323 qscR. LasR serves as a dominant regulator of VqsR [14], and VqsR negatively affects the
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324 expression of QscR [13], which requires lasR-lasI for direct control of gene expression, because its
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325 primary role is to repress lasI [43]. These observations suggest that any perturbation within las QS

326 may consequently affect the downstream regulators under the control of las QS. It has been
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327 demonstrated that exogenous PQS upregulates LasRI, RhlRI and RpoS [8,44], and our results

328 suggested that the positive effect of exogenously supplemented PQS on lasI and rhlI is RpoS-
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329 dependent, since PQS addition failed to enhance lasI and rhlI expression in the ∆rpoS mutant. In

330 support of this observation, our findings in the ∆pqsA∆rpoS mutant have demonstrated that PQS

331 supplementation could not fully restore wild type levels of lasI and qscR. This observation further

332 accentuates the close regulatory association of RpoS and PQS with the las QS, and consequently

333 qscR. Given that QscR responds to LasI-generated AHLs [43], but not to PQS, any effect on the

334 expression of qscR in the presence of PQS is indirectly mediated through las and rhl QS, vqsR or

335 some other unknown intermediary regulators. Nonetheless, lower expression of qscR in the

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336 ∆pqsA∆rpoS mutant in the presence of PQS might be caused by direct repressive effects of vqsR.

337 Interestingly, in the ∆pqsA∆rpoS mutant, the presence of exogenous PQS restored rhlI and vqsR to

338 wild-type levels, suggesting that in the absence of active RpoS, PQS overcomes the rhlI

339 dependency on rpoS and affects expression of vqsR through rhl signaling. Because VqsR

340 indirectly positively regulates the expression of rhl QS [13], exogenous PQS might also affect the

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341 expression of rhlI through VqsR. Although RpoS is not involved in the direct regulation of VqsR,

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342 in the presence of exogenously supplemented PQS, RpoS is required for activation of vqsR by PQS.

343 In this study, we also demonstrated that VqsR positively affects tolerance to carbapenems in

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344 stationary phase P. aeruginosa cells. Given the control of vqsR transcription by the alternative

345 sigma factor RpoN, which is implicated in carbapenem tolerance, and the involvement of VqsR in

346
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QS regulation and QS-dependent virulence factor production, it is plausible to attribute a role to
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347 VqsR as a candidate target for antimicrobials that interfere with QS and the production of virulence
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348 factors [12,15,25]. Furthermore, VqsR affects expression of mexH, a component of the MexGHI-

349 OpmD efflux pump, and it has been established that ∆mexI and ∆opmD mutants display resistance
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350 to kanamycin and spectinomycin, and decreased sensitivity to carbenicillin [41]. Considering the
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351 downregulation of mexG expression in the ∆vqsR mutant, it is plausible that VqsR may also be

352 involved in the response to these classes of antibiotics. Other findings also suggest that VqsM,
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353 which acts upstream of VqsR and binds to the promoter region of nfxB (a transcriptional repressor

354 of the MexCD-OprJ pump), increases susceptibility to kanamycin and tetracycline [45]. These
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355 observations suggest that the interaction with antimicrobials is a common feature of VqsM and

356 VqsR, and that there are potentially overlapping pathways through which VqsM and VqsR

357 modulate the response to antibiotics.

358 Another motivation behind this study was to search for targets that can modulate the

359 response of VqsR to antimicrobials. Our recent findings regarding PQS-mediated tolerance to

360 carbapenems [25] and the data presented here provide insight into the effects of the loss of PQS on

361 antibiotic tolerance in the ∆vqsR mutant. These data provide support for dependent vqsR and pqsA

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362 pathways in antibiotic tolerance, since inactivation of pqsA in the ∆vqsR mutant background

363 completely abolished the vqsR-mediated sensitive phenotype to carbapenems. This observation

364 demonstrates a predominant role for PQS in modulating the response of stationary phase cells in the

365 presence of carbapenems. PQS-associated virulence factor production, including elastase,

366 rhamnolipids and pyocyanin, as well as the role of PQS as a signaling molecule, underscores the

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367 importance of PQS in mediating the response to antibiotic stress [8,44,23,24]. In addition, PQS

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368 promotes alterations in membrane activity associated with outer membrane vesicle (OMV)

369 formation [40]. Therefore, through its interaction with the acyl chains and 4'-phosphate of bacterial

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370 lipopolysaccharide (LPS) [47], PQS may affect some of the pathways for carbapenem delivery and

371 the effective concentration of carbapenem taken up by the cells. In addition, the major outer

372 membrane protein OprF was linked to

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formation of OMVs by modulating production of PQS
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373 [48]. Our results indicate that PQS plays a dual role in defining the response to carbapenems. PQS
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374 can act as an endogenous stress factor that determines survival in the presence of antibiotics [23]. It

375 has been demonstrated that exogenously supplemented PQS positively affects rpoS [8], which may
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376 alleviate the pro-oxidant effects triggered by exogenous PQS and increase survival in the presence
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377 of carbapenem, as demonstrated for the ∆vqsR mutant. In addition to the role of VqsR and PQS in

378 QS regulation and the consequent QS-induced response as a strategy of adaptation to antibiotic
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379 stress, it is noteworthy that both VqsR and PQS are closely associated with the efflux pumps,

380 MexGHI-OpmD and MexEF-OprN [12,48]. Consistent with upregulation of the MexEF-OprN
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381 pump by VqsR during oxidative stress and the role of MexEF-OprN in the export of the 4-

382 hydroxy-2-heptylquinoline (HHQ) signaling molecule, the precursor of the PQS, it remains to be

383 elucidated whether the MexEF-OprN pump may be associated with a response to cell wall stress

384 induced by high concentrations of carbapenem through its influence on modulation of QS and

385 efflux of QS molecules.

386 Collectively, the findings of this study provide insight into the existence of transcriptional

387 regulation between PQS and vqsR; it also indicates that sigma factor RpoS is important in this

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388 interaction. We demonstrated that expression of vqsR is correlated with increased survival in the

389 presence of carbapenems in P. aeruginosa. By constructing a ∆vqsR∆pqsA double-deletion mutant,

390 we accentuated the predominant role of PQS over vqsR for survival in the presence of carbapenems.

391 Further studies will seek to understand the mechanism by which these regulatory elements define

392 the response to carbapenems.

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393

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394 Conflict of interest

395 The authors declare no conflict of interest.

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396

397

398
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399 Acknowledgements
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400 D.V. was a recipient of a fellowship from the Fujii-Otsuka Fellowship for International

401 Exchange Program. This work was supported by a Grant-in-Aid for Scientific Research (C) (no.
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402 2646278700) from the Japan Society for the Promotion of Science.
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535

536

537

538

539

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540

541

542

543

544 Figure legends

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545 Fig. 1. Transcriptional analysis of vqsR expression in wild-type PAO1 and mutant strains.

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546 Expression of vqsR-lacZ transcriptional fusion in wild-type PAO1 and the ∆pqsA mutant (A),

547 ∆pqsR mutant (B) and ∆pqsE mutant (C). Cells were grown at 37 °C in LB medium and harvested

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548 at the time points indicated, and β-galactosidase activity was determined. Squares on a dotted line

549 (wild-type PAO1), closed circles on a dotted line (∆pqsA mutant), open circles on a dotted line

550
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(∆pqsR mutant) and open triangles on a dotted line (∆pqsE mutant), represent relative growth at
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551 OD595. D) Expression levels of vqsR in wild-type PAO1 and the ∆pqsA, ∆pqsR and ∆pqsE
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552 mutants. The transcript levels of vqsR in wild-type PAO1 and the mutants grown at 37 °C in LB

553 medium were assessed by qRT-PCR, normalized to rpsL expression, and the levels are expressed
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554 relative to the wild-type PAO1. Each value represents the average of triplicate cultures ± standard
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555 deviation. P values were calculated by Student’s t test; P ≤ 0,05 (*) or ≤ 0,01 (**) versus wild type.

556 Fig. 2. Expression levels of QS genes in wild-type PAO1 and ∆pqsA, ∆rpoS and ∆pqsA∆rpoS
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557 mutants. The transcript levels of lasI, rhlI, qscR, and vqsR in wild-type PAO1 and ∆pqsA, ∆rpoS

558 and ∆pqsA∆rpoS mutants grown in LB medium were assessed by qRT-PCR in the absence (A) or
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559 presence of exogenously supplemented PQS (100 µM) (B). The lasI, rhlI, qscR and vqsR transcript

560 levels were measured by qRT-PCR, normalized to rpsL expression, and the levels are expressed

561 relative to wild-type PAO1. Each value represents the average of triplicate cultures ± standard

562 deviation. P values were calculated by Student’s t test; P ≤ 0,05 (*), ≤ 0,01 (**) or ≤0,001(***)

563 versus wild type (PQS-) (A) or versus wild-type (PQS+) (B).

564 Fig. 3. Effects of vqsR gene inactivation on P. aeruginosa PAO1 tolerance to carbapenems. Time-

565 dependent killing assay for wild-type PAO1 and ∆vqsR mutant in the presence of biapenem (A) and

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566 doripenem (B) at a concentration of 32 µg/ml. CFU values were changed to percentages assuming

567 that survival at time 0 was 100%. Each value represents the average of triplicate cultures ± standard

568 deviation.

569 Fig. 4. Effects of pqsA gene inactivation on P. aeruginosa PAO1 tolerance to carbapenems. Time-

570 dependent killing assay for wild-type PAO1, ∆pqsA mutant and ∆vqsR∆pqsA mutant in the

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571 presence of biapenem (A, C) and doripenem (B, D) at a concentration of 32 µg/ml. CFU values

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572 were changed to percentages, assuming that survival at time 0 was 100%. Each value represents the

573 average of triplicate cultures ± standard deviation.

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574 Fig. 5. Effect of ∆mexGHI-opmD mutant on response to biapenem and expression of mexG in

575 ∆vqsR and ∆pqsA mutants. A) Time-dependent killing assay for wild-type PAO1 and the

576
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∆mexGHI-opmD mutant in the presence of biapenem at a concentration of 32 µg/ml. CFU values
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577 were changed to percentages assuming that survival at time 0 was 100%. B) The transcript levels
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578 of mexG in wild-type PAO1, ∆pqsA and ∆vqsR mutants grown at 37 °C in LB medium were

579 assessed by qRT-PCR, normalized to rpsL expression, and the levels are expressed relative to
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580 wild-type PAO1. Each value represents the average of triplicate cultures ± standard deviation. P
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581 values were calculated by Student’s t test; P ≤ 0,05 (*), ≤ 0,01 (**) or ≤0,001(***) versus wild type.

582 Fig. 6. Effect of exogenous PQS on growth and susceptibility to doripenem. The growth curves of
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583 the stationary phase cells of wild-type PAO1 and the ∆vqsR mutant in the presence of PQS (100

584 µM), and time-dependent killing curves of wild-type PAO1 and ∆vqsR mutant for doripenem
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585 (DPM) (32 µg/ml) in the presence of PQS (100 µM) in LB medium at 37 °C. CFU values were

586 changed to percentages, assuming that survival at time 0 was 100%. Each value represents the

587 average of triplicate cultures ± standard deviation.

588 Fig. 7. Schematic diagram defining the regulatory network in P. aeruginosa through which PQS,

589 VqsR and RpoS interact. A detailed explanation of the regulatory network is provided in the text.

590 Solid lines with arrows, dashed lines with blunt ends and dashed lines with arrows are genes that

591 are positively, negatively or indirectly affected, respectively.

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

Strain, plasmid or primer Relevant characteristicsa Reference(s) or source

E. coli DH5α F−endA1 hsdR17 supE44 thi-1 recA1 Laboratory stock

gyrA96 relA1 ∆(lacZYA-argF) U169

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deoR λ(φ80dlacZ∆M15)

S17-1 thi pro hsdR recA Tra+ [31]

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P. aeruginosa

PAO1 wild-type [27]

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∆vqsR PAO1 in-frame deletion of vqsR [25]

∆pqsA PAO1 in-frame deletion of pqsA [25]

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∆pqsE PAO1 in-frame deletion of pqsE [25]
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∆pqsR PAO1 in-frame deletion of pqsR This study

∆rpoS PAO1 in-frame deletion of rpoS This study

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∆vqsR∆pqsA PAO1 in-frame deletion of vqsR and pqsA This study

∆pqsA∆rpoS PAO1 in-frame deletion of pqsA and rpoS This study

D

∆mexGHI-opmD PAO1 in-frame deletion of mexGHI-opmD This study

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PAO1 attB::pvqsR-lacZ pvqsR-lacZ transcriptional fusion This study

integrated at the chromosomal attB site of PAO1

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∆pqsA attB::pvqsR-lacZ pvqsR-lacZ transcriptional fusion This study

integrated at the chromosomal attB site of PAO1∆pqsA

C

∆pqsE attB::pvqsR-lacZ pvqsR-lacZ transcriptional fusion This study

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integrated at the chromosomal attB site of PAO1∆pqsE

∆pqsR attB::pvqsR-lacZ pvqsR-lacZ transcriptional fusion This study

integrated at the chromosomal attB site of PAO1∆pqsR

Plasmids

pCR2.1-TOPO TA cloning vector, Apr Thermo Fisher Scientific

pEX18Gm Broad-host-range gene replacement [28]

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vector; sacB, Gmr

mini-CTX-lacZ Tetr, ori, int, oriT [32]

Ω-FRT-attP-MCS

pFLP2 Source of FLP recombinase, sacB, oriT, rep, Apr [29]

pEX18Gm-∆vqsR vqsR deletion suicide vector [25]

pEX18Gm-∆pqsA pqsA deletion suicide vector [25]

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pEX18Gm-∆pqsE pqsE deletion suicide vector [25]

pEX18Gm-∆pqsR pqsR deletion suicide vector This study

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pEX18Gm-∆rpoS rpoS deletion suicide vector This study

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pEX18Gm-∆mexGHI-opmD mexGHI-opmD deletion suicide vector This study

miniCTX-vqsR vqsR promoter in mini-CTX-lacZ This study

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Primers Sequence (5’-3’)

pqsR-up- F ATAGAATTCCTGGCCGACGGTTGCGAACT
M

pqsR-up-R ATAGGATCCCGCAGCTTCCTGCGCTTTCTC

pqsR-down-F ATAGGATCCAATCGAACCGGAGGCGATGA
D

pqsR-down-R ATAAAGCTTTCGGTCGTTAGACCTACGCGA
TE

rpoS-up-F CATTCAGGTCGGTCAAGCTATCCA

rpoS-up-R TCCGTCACTGTGCCATGTCGTTATCCCTTG

rpoS-down-F CGACATGGCACAGTGACGGAAAACCTTAGA
EP

rpoS-down-R GGAAGTCTGGCCGAACATCACGA

mexG-up-F CGACTCTAGAGGATCAGGTGGGGTCGCCGAGCG
C

mexG-up-R GAAGAAACCGATGAACTGCATGGGTCGTTCCTT
AC

opmD-down-F TTCATCGGTTTCTTCGCCC

opmD-down-R CCATGATTACGAATTGAGCGGACCATCTCGGCC

qRT-PCR primers

vqsR-F GCTGTCGATCGCCACTATCA

vqsR-R TCGGAGTGGGACTTCACGTT

lasI-F AATTGGTCGGCGCGAAGAG

lasI-R ACCAGCGTCTGGATGTCGT

2
 ACCEPTED MANUSCRIPT
rhlI-F TTGCTCTCTGAATCGCTGGAAGG

rhlI-R AAACGGCTGACGACCTCACAC

qscR-F ACCAGCGCACGGTGAAGT

qscR-R CGCCTTGTTGCTGGAGTTG

mexG-F TCGATAACTCGCTCGAAAGCA

mexG-R CATCAGGGCCAGGCAGAT

PT
rpsl-F CGAACTATCAACCAGCTGGTG

rpsl-R GCTGTGCTCTTGCAGGTTGTG

RI
a
Gmr, Tetr, and Apr, resistance to gentamicin, tetracycline, and ampicillin, respectively

SC
Introduced restriction sites are underlined. Sequences introduced for overlap extension PCR are in

italics.

U
AN
M
D
TE
C EP
AC

3
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE
EP
C
AC