Editorial Manager(tm) for PLoS ONE Manuscript Draft Manuscript Number: Title: Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa Short Title: Antibiotic-Resistant Pseudomonas SCV Article Type: Research Article Section/Category: Other Keywords: Pseudomonas aeruginosa SCV aminoglycosides PQS PhoP-PhoQ MexXY Corresponding Author: Pierre Cornelis Corresponding Author's Institution: Vrije Universiteit Brussel First Author: Qing Wei Order of Authors: Qing Wei;Saeed Tarighi;Andreas Dötsch;Susanne Haüssler;Mathias Muesken;Victoria Wright;Miguel Cámara;Paul Williams;Steven Haenen;Bart Boerjan;Annelies Bogaerts;Evy Vierstraete;Peter Verleyen;Liliane Schoofs;Ronnie Willaert;Valérie De Groote;Jan Michiels;Aurélie Crabbé;Pierre Cornelis Abstract: Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10-5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous overexpression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS and were strongly down-regulated in PAO-SCV. A proteome analysis confirmed higher expression of the two-component response regulator PhoP in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. However, no evidence was found for a compensatory mutation explaining the emergence of one analyzed revertant, suggesting epigenetic changes. However, high expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic downregulation of the Pseudomonas quinolone signal quorum sensing system. The phenotypic change is reversible and its origin is probably epigenetic. Suggested Reviewers: Leo Eberl University of Zürich leberl@botinst.unizh.ch Leo Eberl is a specialist of quorum sensing regulation, whom advice on the manuscript could be highly valuable because QS is down-regulated in our SCV. Robert Hancock University of British Columbia bob@cmdr.ubc.ca Bob Hancock has published many interesting articles about antibiotic resistance in Pseudomonas aeruginosa, and, in particular the role played by the PhoP-PhoQ system which seems to central in our case. Opposed Reviewers: Cover Letter Dear Editor, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 We would like to submit the following manuscript to PloSOne, entitled “Phenotypic and GenomeWide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa”. As you know SCVs are frequently isolated from tissues infected with P. aeruginosa, especially in the case of CF patients lungs chronically colonized by this bacterium. In this work we describe the isolation of one in vitro selected SCV after culturing in the presence of gentamycin. This SCV shows resistance to different aminoglycosides, and to cefotaxime but is prone to reversion resulting in loss of the resistance to the antibiotic. Phenotypic, metabolic, proteomic, transcriptomic, and genomic data were here combined to show that the resistance to aminoglycosides is multi-layered, arising primarily from mutations in the two-component system PhoP-PhoQ and in the MexS regulator, inducing the overexpression of the MexAB-OprM efflux pump and the aminoglycoside efflux pump MexXY-OprM, together with genes involved in the modification of LPS. One interesting observation is the down-regulation of the genes involved in the production of the Pseudomonas quinolone signal (PQS) molecule, resulting in decreased production of virulence factors and lowered pathogenicity. Finally, a genome sequence analysis revealed that the mutations are present in both the SCV and the revertant, suggesting epigenetic changes which could lead to the apparition or the reversionof the SCV morphotype. Sincerely yours, Pierre Cornelis Corresponding author *Manuscript Click here to download Manuscript: SCV-PloS-One.doc 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant 2 Small Colony Variant (SCV) of Pseudomonas aeruginosa 3 4 5 6 Qing Wei1, Saeed Tarighi1, Andreas Dötsch2, Susanne Häussler2,3, Mathias Müsken2,3, Victoria J. Wright4, Miguel Cámara4, Paul Williams4, Steven Haenen5, Bart Boerjan5, Annelies Bogaerts5, Evy Vierstraete5, Peter Verleyen5, Liliane Schoofs5, Ronnie Willaert6, 7 Valérie N. De Groote7, Jan Michiels7, Aurélie Crabbé8, and Pierre Cornelis1* 8 9 10 11 1 Laboratory of Microbial Interactions, 6Structural Biology Brussels, Department of Molecular and Cellular Interactions, Flanders Institute for Biotechnology (VIB), Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium 12 13 2 Chronic Pseudomonas Infections, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany 14 15 16 3 Twincore, Center for Experimental and Clinical Infection Research, a joint venture of the Helmholtz Center for Infection Research and the Medical School Hannover, Hannover, Germany 17 18 4 School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham NG72RD, UK 19 20 5 Functional Genomics and Proteomics, Faculty of Sciences, K.U.Leuven, Naamsestraat 59, B3000 Leuven, Belgium 21 22 7 Centre of Microbial and Plant Genetics, K.U. Leuven, Kasteelpark Arenberg 20 box 2460, B3001 Heverlee, Belgium 23 24 8 The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, 1001 South McAllister Avenue, Tempe, Arizona 85287, U.S.A. 25 26 Short title: Antibiotic-Resistant Pseudomonas SCV 27 Keywords: Pseudomonas aeruginosa, SCV, aminoglycosides, PQS, PhoP-PhoQ, MexXY 28 *Corresponding author: 29 Pierre Cornelis; Tel: +32 2 6291906; Fax: +32 2 6291902; E-mail: pcornel@vub.ac.be 30 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 31 ABSTRACT 32 Background 33 Small colony variants (SCVs) are slow-growing bacteria, which often show increased 34 resistance to antibiotics and cause latent or recurrent infections. It is therefore important to 35 understand the mechanisms at the basis of this phenotypic switch. 36 Methodology/Principal findings 37 One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the 38 cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of 39 large colonies with a frequency of 10-5 on media without antibiotics while it was stably 40 maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective 41 motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal 42 (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic 43 resistance mechanism, including simultaneous over-expression of two drug efflux pumps 44 (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP- 45 PhoQ two-component system. Conversely, the genes for the synthesis of PQS and were 46 strongly down-regulated in PAO-SCV. A proteome analysis confirmed higher expression of 47 the two-component response regulator PhoP in PAO-SCV. Finally, genomic analysis revealed 48 the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 49 repressor of the mexXY and mexAB-oprM genes. However, no evidence was found for a 50 compensatory mutation explaining the emergence of one analyzed revertant, suggesting 51 epigenetic changes. However, high expression of phoP and phoQ was confirmed for the SCV 52 variant while the revertant showed expression levels reduced to wild-type levels. 53 Conclusions 54 By combining data coming from phenotypic, gene expression and proteome analysis, we 55 could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial 56 including overexpression of efflux mechanisms, LPS modification and is accompanied by a 57 drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system. The 58 phenotypic change is reversible and its origin is probably epigenetic. 59 60 INTRODUCTION 61 Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium found in diverse 62 ecological habitats such as soils, marshes and coastal marine waters. As an opportunistic 63 pathogen, P. aeruginosa is able to infect humans, animals and plants [1,2,3]. P. aeruginosa is 64 a primary nosocomial diseases causative agent and represents the major cause of morbidity 65 and mortality in patients with cystic fibrosis (CF). P. aeruginosa produces a large panel of 66 secreted virulence factors like the phenazine pyocyanin, the siderophore pyoverdine, elastase, 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 67 and toxins. It is also characterized by its high level of drug resistance involving the formation 68 of antibiotic-resistant biofilms resulting from the emergence of phenotypic variants [2,3]. 69 During the course of infection, P. aeruginosa can efficiently adopt diverse strategies to evade 70 antimicrobial stresses and the host immune system defenses, making it impossible to eradicate 71 this bacterium permanently from CF lungs [2,4]. Important phenotypic variations can occur 72 during chronic colonization, such as conversion to mucoidy [5], the emergence of persister 73 cells after antibiotics treatment [6,7] or the occurrence of small colony variants with higher 74 resistance to antibiotics [8,9,10,11,12]. Compared to wild-type P. aeruginosa, SCVs show 75 increased antibiotic resistance, enhanced biofilm formation, reversion to wild-type-like 76 morphotypes, reduced motility, and slow and auto-aggregative growth behavior [13,14]. 77 SCVs have been isolated from CF lungs or sputum [4,8,9,12], laboratory-grown biofilms 78 [11,12,14], in vitro selection upon antibiotic exposure [15,16] or as a consequence of gene 79 inactivation [17,18]. Clinically, P. aeruginosa SCVs have already been proven to associate 80 with chronic infections behaving as persisters in pathogenesis of CF patients and making 81 almost impossible for clinicians to eradicate the infections [8,19,20]. The intracellular second 82 messenger cyclic-di-GMP (c-di-GMP) [21] has been recently shown to be involved in SCV 83 phenotype switching in terms of biofilm formation, reduced motility, and exopolysaccharide 84 (EPS) production [18,22,23,24,25,26]. The “phenotypic variant regulator”, PvrR, containing a 85 conserved EAL domain of phosphodiesterase (PDE) involved in the hydrolysis of c-di-GMP, 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 86 has been identified to control the phenotypic switch from an antibiotic resistant and auto- 87 aggregative rough SCV (RSCV) of P. aeruginosa strain PA14 to wild-type-like antibiotics 88 susceptible revertants [15]. Another characteristic driven by the elevated level of c-di-GMP 89 in SCVs is the contribution of two EPS-encoding loci in some P. aeruginosa strains (PA2231- 90 PA2245 for psl and PA3058-PA3064 for pel) to auto-aggregation and hyper adherence 91 phenotypes characterized by increased Congo Red dye binding [27,28,29]. Although 92 antibiotics resistance of P. aeruginosa has been connected to biofilm formation and linked to 93 phenotypic variation [15], the mechanisms underlying the extremely high antibiotic resistance 94 of SCVs has not been reported extensively due to the unavailability, in some cases, of the WT 95 counterpart for comparison. 96 In this study, we present the identification of a novel, reversion-prone, P. aeruginosa SCV 97 with distinct features, including resistance to various antibiotics, defective motility, and 98 absence of production of the quorum sensing PQS signal molecule. Using a combination of 99 genomic, transcriptomic, proteomic and phenotypic approaches, we provide the first evidence 100 of concerted mechanisms harnessed by this P. aeruginosa SCV leading to antibiotic resistance 101 as well as down-regulation of acute virulence genes, probably involving the PhoP PhoQ two 102 component system. 103 104 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 105 Results 106 Phenotypic characterization of a gentamicin-resistant P. aeruginosa PAO1-SCV and large 107 colony pseudo-revertants 108 Following sub-culturing P. aeruginosa PAO1 (ATCC 15692) in the presence of high- 109 concentration of gentamicin (200 μg ml-1, Gm), we isolated a Gm-resistant SCV designated 110 PAO-SCV, which formed small (ca. 1/5 of the wild-type diameter), smooth colonies after 111 three days of incubation at 37°C on LB agar plates (Figure 1A). PAO-SCV grown in liquid 112 LB also showed a delayed entry in exponential phase compared to the wild-type (Figure 1B). 113 PAO-SCV showed high level of resistance towards gentamicin and cefotaxime (Table 1 and 114 Figure 2). The persistence fraction of PAO-SCV after treatment with the fluoroquinolone 115 antibiotic ofloxacin was approximately 2-fold higher compared to the PAO1 wild-type strain 116 (Figure S1). 117 In the absence of Gm large colonies variants tended to appear, characterized by rough 118 contours, at a frequency of 10-5 (Figure 1A and Figure 2) on agar plates. The frequency of 119 reversion varied between 1.3 10-5 to 8.7 10-5 depending on the medium used (LB or CAA) or 120 the incubation temperature (25°C or 37°C). Importantly, no large colonies appeared when the 121 PAO-SCV was grown in the presence of Gm since the cells from large colonies regained full 122 Gm and cefotaxime sensitivity (Fig. 2). Given its unstable character, PAO-SCV was kept on 123 LB plates supplemented with Gm (200 µg ml-1) to avoid the emergence of pseudo-revertants. 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 124 However, during experiments described below no antibiotic was added (unless mentioned in 125 the text) in order to avoid Gm-induced changes independent of those caused by the SCV 126 phenotype. At the end of experiments cell suspensions were diluted and the number of large 127 colonies counted. When their number was less than 1/105 the experiment was considered to be 128 valid. 129 PQS production is strongly decreased in PAO-SCV 130 We observed that the small colony variant showed reduced production of some known 131 quorum sensing-dependent virulence factors (pyocyanin, pyoverdine, elastase, and a total 132 absence of motility [Fig. S2]). Likewise, the PAO-SCV showed strongly reduced virulence 133 using both plants (Belgian endive) and Drosophila as hosts (Fig. S3). This prompted us to 134 look at the production of quorum sensing signal molecules themselves, including N-3- 135 (oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) for the LasR–LasI system and N- 136 butyryl-L-homoserine lactone (C4-HSL) for the RhlR–RhlI system [30,31]. Finally, we also 137 checked the production of 4-quinolones such as 2-heptyl-4-quinolone (HHQ) and 2-heptyl-3- 138 hydroxy-4-quinolone (PQS) [32]. The levels of 3-oxo-C12-HSL and C4-HSL in the cell 139 culture supernatants were similar for the wild-type, PAO-SCV and the pseudo-revertant 140 (results not shown). However, in PAO-SCV a strong decrease in the production of both HHQ 141 and PQS was observed as compared to that of wild-type while the wild type level was 142 restored in the pseudo-revertant (Figure 3 and results not shown). 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 143 Comparison of proteome profiles of PAO-SCV and wild-type P. aeruginosa 144 Because profound phenotypic changes were detected in PAO-SCV, we decided to compare 145 the proteomes of PAO-SCV and wild-type cells (Figure 4). After protein identification with 146 MALDI-TOF MS analysis, we found at least 24 differentially expressed proteins, whereby 16 147 proteins were less abundant and 8 more abundant in PAO-SCV (Table 2). The proteins 148 showing differential abundance are involved in amino acid biosynthesis and metabolism, 149 motility, transport of small molecules and transcriptional regulation. According to this 150 analysis, the two-component response regulator PhoP is one of the most prominently induced 151 proteins in PAO-SCV. Another finding is the over-expression of the major outer membrane 152 protein OprF in PAO-SCV, which is the P. aeruginosa major non-specific porin allowing 153 diffusion of various solutes, such as nitrates or nitrites under anaerobic conditions or small 154 oligosaccharides with a molecular weight up to 1519 Da [33,34]. We also found decreased 155 expression of the anaerobiosis-induced outer membrane porin OprE, which, similarly to 156 OprD, was predicted to be involved in outer membrane permeability of the β-lactam antibiotic 157 imipenem and basic amino acids [35,36]. 158 Genome-wide transcriptional profile of PAO-SCV and PAO1 159 Since some of the differentially produced proteins could already give clue to the changes 160 occurring in the SCV mutant, we decided to further investigate which global changes in gene 161 expression could account for this phenotypic variation. The gene transcription profiles of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 162 PAO-SCV and WT strains were compared in early and late stationary-phase of growth, 163 corresponding to incubation times of 20 and 40 h respectively, using P. aeruginosa 164 Affymetrix GeneChips. The results are presented using Venn diagrams and pie charts for 165 simplicity, facilitating the understanding and interpretation of the overall genome 166 transcriptional profile [37]. The tables showing the complete lists of differentially expressed 167 genes are shown as supplementary material (Tables S1 to S5). As shown in Figure 5 and in 168 supplementary Tables S1-S5), during stationary phase, a total of 642 genes representing 169 approximately 12% of the entire genome displayed a differential expression pattern in PAO- 170 SCV compared to that of wild type PAO1 (P value < 0.05, Student’s t-test). Among these 642 171 genes, 466 were up-regulated (≈ 73% of differentially regulated genes, from 2- to 26-fold, see 172 Table S3) and 176 were down-regulated (≈ 27% of differentially regulated genes, from 2- to 173 16-fold, see Table S4). Interestingly, remarkable differences were observed for up-regulated 174 genes (Figure 5B), among which 356 genes were found to be highly expressed during late 175 stationary phase while only 164 genes were up-regulated during early stationary phase as 176 compared to the wild-type. Genes involved in amino acid biosynthesis and metabolism 177 showed an increased transcription level in both early and late stationary phase of growth of 178 PAO-SCV (Table S2). Genes involved in antibiotic resistance and genes coding for 179 membrane proteins were highly expressed in the SCV mutant in early stationary phase. 180 Conversely, some genes involved in the production of secreted factors and those related to 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 181 phage, transposon and plasmids were expressed at a lower level in PAO-SCV compared to the 182 wild-type. 183 Figure 6A shows that some of the genes known to be involved in antibiotic resistance are up- 184 regulated in the PAO-SCV. These could be classified into four different functional groups, 185 linked to four distinct resistance mechanisms (See lists of selected genes in Table S3). Among 186 these are efflux pump systems genes known to contribute to resistance to aminoglycosides, 187 including mexAB-oprM and mexXY and their respective mexR and mexZ regulatory genes 188 [38]. The observed higher expression of these efflux pumps is in agreement with the results 189 showing a higher resistance to all aminoglycosides and to the cephalosporin antibiotic 190 cefotaxime (Figure 2 and Table 1). Interestingly, expression of another resistance-nodulation- 191 cell division (RND) efflux pump, MexGHI-OpmD, is reduced in PAO-SCV in late stationary 192 phase. This efflux system has been shown to be important for PQS-mediated signaling, 193 pyocyanin production, and is thought to be a general phenazine transporter, including 194 pyocyanin [39,40,41]. Again, this observation is in line with the reduced production of 195 pyocyanin by PAO-SCV and the quasi-absence of HHQ and PQS in culture supernatants 196 (Figure 3). Among PAO-SCV up-regulated genes are those involved in LPS modification, 197 including migA (PA0705) encoding a glycosyl transferase, and the gene cluster PA3552- 198 PA3559 (arnBCADTEF-PA3559, Figure 6B), which are homologues of the pmrHFIJLKM 199 genes of Salmonella enterica involved in lipid A modification [42,43,44]. Interestingly, phoP- 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 200 phoQ, together with the upstream porin protein gene oprH was markedly up-regulated 201 throughout the stationary phase in PAO-SCV, forming the third functional group and making 202 the link with the overexpression of migA and arnBCADTEF-PA3559 (Figure 6C). As already 203 mentioned, higher levels of the transcriptional regulator PhoP were also detected by 2D- 204 PAGE analysis. The PhoP-PhoQ system is known to be involved in aminoglycoside 205 resistance in P. aeruginosa [45]. 206 A fourth functional group of genes markedly up-regulated in PAO-SCV included those 207 encoding membrane proteins, transcriptional regulators and transporters of small molecules 208 (Figure 6D). More specifically, several genes encoding outer membrane proteins are up- 209 regulated in PAO-SCV: the previously mentioned oprH, oprD, PA1198 (encoding a 210 lipoprotein), oprQ, opdQ, opdP, and the lipoprotein gene omlA. OprQ, and OpdP belong to 211 the OprD family and have been proposed to contribute to the transport of arginine [46]. In this 212 context, it is interesting to note that the genes PA5152 (ABC transporter, ATP binding 213 component), and, to a large extent, PA5153 (periplasmic binding protein), probably involved 214 in the transport of arginine, are also up-regulated. 215 The transcriptome analysis not only provided insights into the PAO-SCV mechanisms 216 involved in aminoglycoside-resistance, but also explained some of the prominent phenotypic 217 changes. As shown in Figure 7A, transcript levels of the pqsABCDE genes as well as for the 218 two neighboring anthranilate synthase genes phnA and phnB were strongly reduced in PAO- 12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 219 SCV, in line with the results presented in Figure 3 showing a strong decrease in HHQ and 220 PQS production. As a result of the down-regulation of PQS genes (pqsA-E, pqsH, phnAB), 221 genes such as lasA (coding for elastase), phzC2-G2, phzB1, phzS (for pyocyanin 222 biosynthesis), hcnC (for HCN production) and rhlA (for rhamnolipids synthesis) were also 223 down-regulated. Lower rhamnolipid production could also partly explain the observed 224 decreased swarming motility and the absence of channels in PAO-SCV biofilms [47,48]. In 225 line with the absence of changes in AHLs production, the transcription of lasI and rhlI coding 226 for the 3-oxo-C12-HSL and C4-HSL synthases was unchanged. 227 Another interesting finding is the differential expression of genes associated with biofilm 228 formation. We found that in PAO-SCV, flagellar synthesis genes expression was reduced 229 compared to wild-type PAO1 which was also confirmed by proteomic analysis (see Figure 5 230 and Table 2). This result is in line with the total absence of motility of PAO-SCV (Figure S2). 231 The third interesting functional group is formed by phage-related genes including phage those 232 involved in Pf1 phage production and the PA0616-PA0647 cluster, the expression of which 233 was greatly reduced in PAO-SCV compared to wild-type (Figure 7C). 234 Validation of microarray results via Quantitative RT PCR 235 Quantitative real time PCR was used to measure the level of transcripts of the phoP and phoQ 236 genes in wild type, SCV, and one pseudo-revertant. As shown in Figure 8, the level of phoP 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 237 and phoQ transcription was increased in the SCV while the levels were similar for wild-type 238 and the revertant large colony variant. 239 Whole genome analysis of P. aeruginosa SCV 240 The genome of the originally selected pseudo-revertant (REV) of PAO-SCV was fully 241 sequenced using the Illumina Genome Analyzer. The choice to re-sequence the revertant only 242 was justified by the fact that it should contain all mutations present in three strains (PAO1, 243 PAO-SCV, revertant), and single nucleotide polymorphisms (SNPs) could be easily checked 244 for their presence in the genomes of PAO-SCV and its clonal wild-type using a combination 245 of PCR amplification and Sanger sequencing. A limited list of sequence variations in relation 246 to the PAO1 sequence was found (Table 3 and Table S6 for full list), most of which were 247 already detected when we re-sequenced these regions in our own PAO1 lab strain and in the 248 PAO1 strain of Chronic Pseudomonas Infection Group in Helmholtz Infection Research 249 Centre. Finally, seven changes remained that were unique to REV after elimination of the 250 mutations also found in PAO1 wild-type. Through PCR amplification of these regions and re- 251 sequencing via the Sanger method these differences in sequence were confirmed to be present 252 in both REV and SCV, which was surprising since the phenotypic differences between SCV 253 and REV are very dramatic and expected to be reflected by differences in genetic background. 254 Interestingly, changes were identified in phoPQ and mexZ, in line with the results of the 255 transcriptional analysis. Specifically, the phoP gene contains a SNP which confers a histidine 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 256 (H) to arginine (R) change while, remarkably, phoQ harbors an in-frame 39-bp deletion in its 257 coding sequence, deleting a 13 amino acids sequence RLLRSEHKQRERY between residues 258 226 and 239. In PAO-SCV the mexZ gene was inactivated by the introduction of a stop codon, 259 which could explain the over-expression of the MexXY pump involved in aminoglycosides 260 and fluoroquinolone resistance. 261 Discussion 262 The SCV phenotype observed in this study is reminiscent of the observations made by Tarighi 263 et al. who found that knocking out the the ppgL gene (PA4204) of P. aeruginosa caused a 264 SCV phenotype with the apparition of large colony variants [17]. In this particular case the 265 SCV phenotype was thought to be due to the accumulation of a toxic intermediate: 266 gluconolactone [17]. This observation suggests that SCV phenotypes can be the results of 267 exposures to different stresses. In P. aeruginosa, several mechanisms of aminoglycoside 268 resistance have been described: resistance through efflux systems, by alteration of porins or 269 outer membrane properties (including LPS modification), resistance through chromosomal 270 mutations of regulatory genes, and resistance through enzymatic drug modification, including 271 both intrinsic and acquired resistance [49,50,51,52,53,54]. P. aeruginosa can use these 272 mechanisms in combination, to reach high-level of resistance to certain antibiotics, which is 273 precisely what we observed in this study since we found an overexpression of two efflux 274 systems (MexXY-OprM, MexAB-OprM, increased expression of the arnBCADTEF-PA3559 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 275 LPS modification genes, of the porins OprH, OprF, and decreased expression of OprE. The 276 tripartite efflux pump MexXY-OprM is known to be a major contributor to aminoglycoside 277 resistance in P. aeruginosa [55,56,57,58] as well as the efflux of the drug tigecycline [59]. In 278 addition, MexAB-OprM was also proven to confer resistance to β-lactams, fluoroquinolones 279 [50] and to contribute to aminoglycosides resistance [60]. Both efflux pumps share the same 280 efflux porin, OprM [61]. In P. aeruginosa, the PhoP-PhoQ two-component regulatory system 281 is known to be induced upon Mg2+ starvation to up-regulate the production of the outer- 282 membrane protein OprH and to increase the resistance to the polycationic antibiotic 283 polymyxin B [62]. In addition, PhoP-PhoQ is also involved in resistance to antimicrobial 284 cationic peptides and aminoglycoside antibiotics [45], in good agreement with the higher 285 resistance of PAO-SCV to this class of antibiotics. The arnBCADTEF-PA3559 genes could 286 also be involved in conferring a higher resistance to aminoglycosides. Intriguingly, PA3559 287 encodes a UDP-glucose dehydrogenase that is induced by low concentrations of Mg2+ [43] 288 and its expression depends on the PmrA-PmrB two-component regulatory system, which is 289 itself regulated by the PhoP-PhoQ two-component system [63,64,65,66]. The general porin 290 OprF, which is overexpressed in the SCV, has been recently shown to participate in resistance 291 mechanisms to a broad spectrum of antibiotics such as β-lactams, cephalosporins, and 292 fluoroquinolones [67], supporting the role of OprF as an intrinsic antibiotic resistance 293 contributor as well as partially explaining the cefotaxime resistance revealed by phenotypic 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 294 assays. In PAO-SCV we observed a down-regulation of phage genes compared to the wild- 295 type. The expression of phage genes has been shown to reciprocally associate with biofilm 296 formation and antibiotic resistance as evidenced by Whiteley and colleagues [68]. These 297 authors found that phages genes were up-regulated in mature biofilms as compared to 298 planktonic cultures while they were shown to be down-regulated in biofilms exposed to the 299 aminoglycoside tobramycin. A puzzling observation is the strong down-regulation in PAO- 300 SCV of PQS biosynthesis genes and of the PQS-related efflux pump MexGHI-OpmD [39], 301 which is confirmed by the near absence of PQS production presented in Figure 3. It has been 302 suggested that high production of PQS, which results in an autolysis phenotype, could be 303 explained by the induction of prophages [22], which fits with the results presented here. 304 Collectively, our whole genome expression analysis of PAO-SCV versus wild-type PAO1 305 allowed us to get a good correlation between phenotypic traits (antibiotic resistance, PQS and 306 virulence factors production), proteomic and gene expression data. However, we failed to 307 pinpoint the mutation(s) leading to the phenotypic conversion to the SCV state since all of the 308 mutations observed in the revertant were also present in the SCV genome. One possible 309 explanation is a phenotypic switch without mutation, like the recently described bi-stable 310 phenotypic switch due to the LysR regulator BexR [69]. 311 Experimental procedures 312 Bacterial strains and culture conditions 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 313 P. aeruginosa PAO1 Strain (ATTC 15692) and its gentamicin-resistant mutant PAO-SCV 314 were used in this study. P. aeruginosa strains were grown at 37°C in Luria-Bertani (LB) broth 315 or on LB agar plates, iron poor casamino acids (CAA) medium (Difco Laboratories) or 316 Pseudomonas agar medium (Difco Laboratories). The antibiotics gentamicin (Gm) at 200 µg 317 ml-1 and spectinomycin (Sp) at 50 µg ml-1 were used when necessary. Growth rate of three 318 replicates for each strain was monitored spectrophotometrically (Bioscreen C, Thermo 319 Labsystems). 320 Motility assay 321 Swarming, swimming and twitching motility were determined as previously described [70]. 322 To investigate swarming motility, 4 µl of overnight cultures of P. aeruginosa grown in LB (1 323 x 109 cells) were placed in the center of 0.4% agar LB or CAA plates while swimming 324 motility was evaluated using 0.3% agar LB or CAA plates. For twitching motility, LB or 325 CAA plates containing 1.5% agar were inoculated with a toothpick by stabbing the plates. 326 The plates were then incubated at 37°C. In the case of twitching, after incubation the LB- or 327 CAA-agar media was removed from the plates and plates were stained with 1% crystal violet 328 (Merck) in 33% acetic acid for a minimum of 20 min. Spreading of bacteria from the 329 inoculation point was measured and pictures were taken. Three independent experiments were 330 performed. 331 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 332 Detection and analysis of signal molecules 333 Rapid detection of N-acyl homoserine lactones (AHLs) in filter sterilized (0.2 µm pore-size 334 filters) culture supernatants was done using AHL reporter plate bioassays by either E. coli 335 JM109 carrying the plasmid pSB401 for the detection of N-(butanoyl)-L-homoserine lactone 336 (C4-HSL) [71] or E. coli MH155 [72] for the detection of N-(3-oxododecanoyl)-L- 337 homoserine lactone (3-oxo-C12-HSL). For accurate AHL quantification, 100 ml of acidified 338 filter-sterilized culture supernatants were extracted with equal volumes of dichloromethane. 339 The organic phase was removed and dried by evaporation in vacuum. Extracts were re- 340 dissolved in 1 ml of 50% acetonitrile. Thin-layer chromatography (TLC) plates Silica gel 60 341 F254 (Merck) and RP-18 F245 (Merck) were used for detection of 3-oxo-C12-HSL and C4- 342 HSL, respectively. Twenty µl of extracted AHLs were fractionated on TLC plates. After 343 development in a solvent mixture of methanol/water (60:40, vol/vol), the plates were dried 344 and overlaid with 50 ml of soft top LB-agar mixed with 1 ml of overnight culture of an E. coli 345 MH155 strain harboring the reporter plasmid pUCP22NotI-PlasB::gfp(ASV)Plac ::lasR (to 346 detect 3-oxo-C12-HSL) or 1 ml of overnight culture of E. coli JM109 pSB401 (to detect C4 347 and C6-HSL). After 18 h incubation at 37°C the production of 3-oxo-C12-HSL was detected 348 under UV by visualization of green fluorescent spots and production of C4 and C6 was visible 349 by the production of light. 350 The alkyl-hydroxy quinolones PQS and HHQ were extracted from 10 ml of early stationary 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 351 phase filtered supernatants by adding equal volumes of acidified ethyl acetate. The organic 352 phase was dried and the residue re-suspended in 50 ml methanol. Ten µl samples of this 353 extract were spotted onto normal phase silica 60 F254 (Merck) TLC plates, pre-treated by 354 soaking in 5% K2HPO4 for 30 min and activated at 100°C for 1 h. Extracts were separated 355 using a dichloromethane:methanol (95:5, vol/vol) solvent system until the solvent front 356 reached the top of the plate. PQS was visualised under UV light and specific detection was 357 done using soft top LB-agar including 1ml of overnight culture of a P. aeruginosa lecA::lux 358 ∆pqsA strain as bioreporter [73]. Bioluminescence was detected and quantified with a Bio 359 Imaging System (Syngene). Amounts of C4-HSL, C6-HSL, 3-oxo-C12-HSL and PQS were 360 determined by measuring the diameter of the spots. 361 Persistence assay 362 The persistence assay was performed essentially as described previously [6]. Shortly, cultures 363 were grown overnight at 37°C in 100 ml LB medium in erlenmeyer flasks. One mL of a 364 stationary phase culture was treated with 10 µL of ofloxacin at a final concentration of 5 µg 365 mL-1; a control treatment was performed with sterile water. Both treatments were performed 366 at 37 °C, shaking at 200 rpm, during five hours, after which the number of colony forming 367 units were determined by plate counts. The persister fraction is defined as the number of 368 surviving cells after treatment with ofloxacin, divided by the number of cells after the control 369 treatment. The relative persister fraction for each strain is the persister fraction of the strain 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 370 divided by that of the wild-type. The mean relative persister fraction is calculated as the 371 inverse logarithm of the mean of the logarithmic values of these relative persister fractions of 372 separate experiments. Each experiment was independently repeated at least four times. The 373 mean relative persister fractions are displayed with the bars representing the 25 th and 75th 374 percentiles as shown in Figure S1. Each experiment was repeated three times. 375 Biofilm formation assay 376 The protocol of O'Toole et al. [74] was followed with some minor modifications. Briefly, 377 bacterial strains were grown aerobically in LB broth at 37°C for 24 h. Cells diluted to an 378 absorbance of 0.5 at 600 nm were then sub-cultured (1:100), in LB and CAA broth in 379 triplicates in a final volume of 2 ml in 96- or 24-well polystyrene plates (BD Bioscience) and 380 incubated aerobically at 30°C without shaking for 48 h. The culture was removed from the 381 wells and plates washed twice with distilled water. The biofilm was then stained with 1% 382 crystal violet (1% crystal violet in 33% acetic acid) and incubated at room temperature for 30 383 min. Crystal violet was removed from the wells and plates were washed twice with phosphate 384 buffered saline (PBS) (0.1 M, pH 7.2). Plates were dried and crystal violet stain was dissolved 385 using 70% ethanol of which absorbance was determined at 590 nm. The average of three 386 measurements per strain was determined and expressed as biomass production characteristic 387 of biofilm formation. The same procedures were performed with abiotic materials including 388 borosilcate glass, polyvinyl chloride plastics and polypropylene plastics. The biofilm 389 formation was also analyzed in 96-well microtiter plates with an image-based approach [75]. 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 390 Briefly, bacterial strains were grown overnight in LB broth at 37°C. Cultures were diluted to 391 an absorbance of 0.02 at 600 nm and a final volume of 100 μl was transferred in triplicate into 392 the wells of the 96-well μClear microplate (Greiner Bio-One). The plate was covered with an 393 air-permeable BREATHseal cover foil (Greiner Bio-One) and placed into a humid incubator 394 at 37°C. After 24 h incubation, the bacterial cultures were stained with the LIVE/DEAD 395 BacLight Bacterial Viability Kit (Molecular Probes, Invitrogen). Fifty μl of a diluted staining 396 solution (1:500) was added into each well resulting in a final concentration of 1.4 μM SYTO9 397 and 8.3 μM PI (propidium iodide). After 48 h incubation structured biofilm were observed. 398 Microscopy was performed after 72 h using an Olympus Fluoview 1000 system equipped 399 with an x40/NA (numerical aperture) 0.90 air lens. Total biovolumes of the image stacks of 400 each strain were calculated using the tool PHLIP [76]. 3D-visualization of the biofilms was 401 performed with the software IMARIS (version 5.7.2, Bitplane). 402 Proteome analysis by two-dimensional (2D) gel electrophoresis 403 P. aeruginosa cells were harvested in early stationary phase by centrifugation (4,000 g, 10 404 min, 4°C) and washed three times with Tris-HCl buffer (pH 8.0). To prepare extracts of 405 cellular proteins, bacterial cells were washed twice in PBS buffer (pH 8.0) and re-suspended 406 in a solution containing 40 ml of 2.5 mM Tris-HCl (pH 8.0), one tablet of protease inhibitor 407 (Sigma), 80 µl of 0.5 M Na2EDTA, and 400 µl of DNase at 10 mg ml-1. After lysis of the cells 408 by sonication with a Branson Sonifier 250, each suspension was centrifuged (2,500 g, 15 min, 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 409 4°C) to remove the cell debris and unbroken cells. The supernatant was then subjected to a 410 second centrifugation (50,000 g, 40 min, 4°C) to remove the insoluble components, and the 411 protein concentration in the resulting supernatant was determined by a Bradford Protein assay 412 (Bio-Rad). For the preparation of extracellular protein extracts, the supernatants obtained after 413 centrifugation of the bacterial cultures (6,000 g, 15 min, 4°C) were passed through a 0.2 µm- 414 pore-size filter. Deoxycholic acid (sodium salt) was added to a final concentration of 0.2 mg 415 ml-1. After 30 min of incubation on ice, the proteins were precipitated by addition of 6% 416 (wt/vol) trichloroacetic acid and incubated at 4°C for 2 hr. After centrifugation (18,000 g, 30 417 min, 4°C) the precipitated proteins were re-suspended in distilled water, and eight volumes of 418 cold acetone (-20°C) were added. After incubation at -20°C for 2 h, the mixture was 419 centrifuged (3,500 g, 20 min, 4°C), and the pellet was allowed to dry for 5 min before it was 420 dissolved in an appropriate amount of solubilisation buffer. After centrifugation (50,000 g, 40 421 min, 4°C) to remove the insoluble components, the protein concentration of the remaining 422 supernatant was determined. Protein extracts were either used immediately for 2-D gel 423 electrophoresis or stored at -80°C. Isoelectric focusing was performed with the IPGphor 424 system and Immobiline DryStrip gel strips (GE Healthcare). Equal quantities of solubilised 425 proteins from the different P. aeruginosa strains were diluted to obtain a final volume of 360 426 µl with solubilisation solution and applied to the Immobiline gel strips by in-gel rehydration. 427 Linear immobilized pH gradients (pH 4 to 7) were used. Thirty to 50 µg of protein was 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 428 applied for analytical gels (silver staining), and 200 to 500 µg of protein was loaded for 429 Coomassie staining was used. After rehydration under silicone oil for 10 hr, the proteins were 430 focused for a total of 120 kV/h at 20°C. The proteins were reduced by equilibration of the 431 strips in equilibration solution (6 M urea, 30% glycerol, 2% [wt/vol] sodium dodecyl sulfate, 432 and 1% [wt/vol] dithiothreitol in 0.05 M Tris-HCl (pH 8.8) for 15 min and then 433 carbamidomethylated in the same solution containing 260 mM iodoacetamide for 15 min. The 434 strips were transferred to 12% acrylamide gradient gels and electrophoresis was performed 435 overnight at 125 V at 10°C. Gels were stained with Coomassie brilliant blue solution and 436 spots of interest were then further analyzed through peptide mass fingerprinting according to 437 the described protocol [77]. Peptides examined on a MALDI-TOF mass spectrometer 438 (Bruker) and analyzed by MASCOT (Matrix Science) were used to identify proteins from 439 peptide identifications using the NCBInr database. 440 Microarray and quantitative real time PCR analysis 441 Cultures were grown in triplicate until early (24 h) and late (48 h) stationary phase, 442 respectively, allowing three biological replicates per condition. Total RNA was obtained from 443 the cultures of early and late stationary phase by first treating the cells with RNAprotect 444 Bacteria Reagent (Qiagen) as recommended by the manufacturer. Cells were then lysed and 445 total RNA was extracted using the RNeasy Midi Kit (Qiagen), on-column DNase digestion 446 was performed using the RNase-free DNase Set (Qiagen) according to the manufactures 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 447 instructions. RNA integrity was assessed using the 2100 bioanalyzer (Agilent Technologies 448 Inc.). cDNA synthesis, fragmentation and labeling were performed according to the supplier 449 protocol for the P. aeruginosa Genechip genome array (Affymetrix) at 50°C. Washing and 450 staining of the arrays was performed according to the manufacturer’s instructions using a 451 fluidics station 400 (Affymetrix). Slides were scanned using the 2500A GeneArray Scanner 452 (Agilent Technologies Inc.) and Affymetrix MAS 5.0. Data analysis was performed using 453 GeneSpring GX (Agilent Technologies Inc.) in which the scaled data was further normalized 454 by per Chip and per Gene median normalizations. Filtering of genes was performed to find 455 genes that had changed in expression by a magnitude of 2-fold (P value less than 0.05, 456 Student’s t-test). 457 Bacterial cells were harvested in stationary phase, bacterial RNA was extracted by using 458 RNeasy Midi Kit (QIAGEN). The purity and concentration of the RNA was determined by 459 spectrophotometry (NanoDrop, Thermo Scientific). First-strand cDNA was reverse 460 transcribed from one microgram of total RNA by using First-strand cDNA Synthesis Kit 461 (Amersham Biosciences, GE Healthcare). qRT-PCR was performed in a Bio-Rad (Hercules, 462 CA, USA) iCycler with Bio-Rad iQ SYBR Green Supermix. For all primer sets, the following 463 cycling parameters were used: 94°C for 3 min followed by 40 cycles of 94°C for 30 s, 55°C 464 for 45 s and 72°C for 30 s, followed by 72°C for 7 min. The outer membrane lipoprotein oprI 465 gene was used to normalize gene 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 466 expression[78][78][78][78][78][71][78][77][78][69][78][78][84]. Amplification products 467 were electrophoresed on 0.8% agarose gels. For statistical analysis of relative gene 468 expression, the 2-△△CT method was used [79]. All experiments were carried out in triplicate. 469 Acknowledgements 470 This work was supported by two research grants from the FWO Belgium (Fonds voor 471 Wetenschappelijk Onderzoek Vlaanderen) and a grant from BBSRC UK (BBF0143921). QW 472 has a CSC-VUB scholarship and got a FEMS student fellowship to perform experiments in 473 the lab of Dr. Susanne Haüssler. BB and AB are research assistants and PV is a postdoctoral 474 researcher of the FWO-Vlaanderen. We wish to thank Dr. Jean Paul Pirnay, Daniel De Vos, 475 and Florence Bilocq from the Military hospital in Brussels for the VITEK analysis. We would 476 like to thank De Meuter Sonja and Van Hemelrijck Herman for technical assistance. 477 478 References 479 480 481 482 483 484 485 486 1. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, et al. (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406: 959-964. 2. Lyczak JB, Cannon CL, Pier GB (2002) Lung infections associated with cystic fibrosis. Clin Microbiol Rev 15: 194-222. 3. Lyczak JB, Cannon CL, Pier GB (2000) Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2: 1051-1060. 4. 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The large colony variant shows evidence 705 of autolysis. 706 B: Growth of wild-type (●) and PAO-SCV (○) in LB liquid medium measured in the 707 Bioscreen. 708 709 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 710 Figure 2 711 Sensitivity to gentamicin (A) and cefotaxime (B) of wild type PAO1 (left), PAO-SCV 712 (middle), and one pseudo-revertant (right). The pseudo-revertant and wild-type are sensitive 713 to both antibiotics while PAO-SCV is resistant. Notice the presence of large colonies in the 714 bacterial lawns corresponding to PAO-SCV. 715 Figure 3 716 Production of signal molecules 717 Detection of HHQ and PQS: lane 1: wild-type supernatant from a stationary phase culture in 718 LB, lane 2: same, but from another culture, lane 3 and 4: PAO-SCV supernatant. 719 Figure 4 720 2-D gel electrophoresis of soluble proteins from wild-type and from PAO-SCV grown in LB 721 medium till stationary phase. Spots showing differences are indicated as well as the 722 corresponding PA gene number. See Table 2 for details. 723 Figure 5 724 A: Comparison of up-regulated and down-regulated genes in PAO-SCV in early stationary 725 phase (ESP) and in late stationary phase (LSP). 726 B: Repartition of up- and down-regulated genes in function of genes categories (color coded). 727 728 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 729 Figure 6 730 A: Differentially expressed genes corresponding to efflux systems. The mexXY and mexAB- 731 oprM genes are up-regulated while the mexGHI-opmD genes are down-regulated in PAO- 732 SCV. 733 B: Up-regulation of the arnBCADTEF-PA3559 operon for lipid A modification in PAO-SCV. 734 C: Up-regulation of oprH-phoPQ operon in PAO-SCV. 735 D: Hierarchical clustering of differentially-expressed genes in PAO-SCV corresponding to 736 membrane proteins, transcriptional regulators, and transporters. 737 Figure 7 738 A: Down-regulation of quorum-sensing-regulated genes in PAO-SCV: pqsABCDE-phnAB for 739 the biosynthesis of PQS, the two phenazine biosynthesis operons (phz), the rhamnolipid 740 production rhlA gene, and the hydrogen cyanide production gene (hcn). 741 B: Up-regulation of pyochelin siderophore biosynthesis (pch) and uptake (fptA) genes in 742 PAO-SCV. 743 C: Down-regulation of two phage-related clusters of genes in PAO-SCV. 744 Figure 8 745 Quantitative real time PCR analysis of phoP and phoQ gene expression in wild type PAO1, in 746 PAO-SCV, PAO-SCV grown in the presence of Gm (20 µg ml). 747 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 748 Supplementary figures: 749 Figure S1 750 Mean relative persister fraction of wild-type PAO1 and PAO-SCV after exposure to 751 ofloxacin. See text for details. 752 Figure S2 753 (A) Production of virulence factors pyocyanin, pyoverdine, and elastase; (B) motility of wild 754 type PAO1 and PAO-SCV on swimming (top), swarming (middle), and twitching (bottom) 755 plates; (C) atomic force microscopy images of wild-type and SCV showing the loss of 756 flagella. 757 Figure S3 758 Virulence of wild-type, PAO-SCV and revertant (A) in plants (Cychorium intybus) and (B) of 759 wild-type and PAO-SCV in Drosophila melanogaster larvae. 760 761 762 763 764 765 766 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 767 768 769 770 771 772 ___________________________________________________________________________ Table 1. Minimal inhibitory concentrations (MICs) of different antibiotics against wild type (PAO), PAO-SCV and one selected pseudo-revertant (REV) as measured by the Bio-Merieux VITEK 2 system. 773 774 775 Antibiotic PAO PAO-SCV REV Piperacillin 8 8 8 Cefotaxime 16 32 16 Ceftazidime 4 4 4 Imipenem 8 8 <=1 Meropenem 4 2 0.5 Gentamicin <=1 >16 8 Ciprofloxacin <0.25 <0.25 <0.25 Levofloxacin 1 0.5 1 36 1 776 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Table 2. Identification of differentially produced proteins in P. aeruginosa PAOSCV by MALDI-TOF MS peptide mass mapping (PMP) Sequence Total Protein Matched pI coverage mass Localization* PA No. Gene identification peptides value (%) (Da) Down in SCV PA0344 PA4273 PA5100 PA2300 PA5171 PA5171 PA4277 PA0291 PA1087 PA1337 PA0956 PA3655 PA4708 PA5505 PA0139 PA3326 Hypothetical protein rplA 50S ribosomal protein L1 hutU Urocanase chiC Chitinase arcA Arginine deiminase arcA Arginine deiminase tufB Elongation factor Tu oprE OprE porin 21 47 50,498 Cyt 6.8 9 80 93,130 Cyt 8.3 34 25 22 51 53 52 61,554 53,066 46,806 Cyt Extr Cyt 6.0 5.2 5.5 25 63 46,675 Cyt 5.5 25 68 43,684 Cyt 5.2 27 60 49,637 OM 8.7 Flagellar hookassociated protein ansB Glutaminaseasparaginase proS Prolyl-tRNA synthetase tsf Elongation factor Ts phuT Heme-transport protein Probable TonBdependent receptor ahpC Alkyl hydroperoxide reductase subunit C Endopeptidase Clp chain P 20 53 47,020 Extr 6.0 22 60 38,620 Per 6.7 22 37 65,536 Cyt 6.1 27 77 30,691 Cyt 5.2 19 68 31,019 Per 6.9 20 69 28,048 OM 7.8 18 66 20,643 Cyt 5.9 18 70 22,128 Cyt 5.4 19 31 58,860 Peri 6.2 18 45 42,347 Extr 6.4 23 70 39,858 Peri 5.6 flgL Up in SCV PA4502 PA0852 PA1074 Probable binding protein component of ABC transporter cbpD Chitin-binding protein CbpD precursor braC Branched-chain amino acid transport 37 protein BraC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 777 17 778 18 19 779 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 PA3655 PA0395 PA4495 PA1179 PA1777 Elongation factor Ts pilT Twitching motility protein PilT Hypothetical protein phoP Twocomponent response regulator PhoP oprF OprF tsf 27 83 30,691 Cyt 5.2 16 56 41,644 OM 7.2 16 55 24,921 Peri 5.8 18 71 25,748 Cyt 5.3 17 75 23,270 OM 4.8 * Cyt= cytoplasm, Peri= periplasm, IM= inner membrane, OM= outer membrane, Extr= extracellular 38 780 1 2 3 4 Locusa 5 6 7PA0748 8 9 10 11PA1179 12 13 PA1180 14 15 16PA1385 17 18PA2020 19 20 igPA2046 21 22PA2141 23 24 781 25 26 782 27 783 28 29 784 30 31 785 32 786 33 34 787 35 788 36 37 789 38 39 790 40 791 41 42 792 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Table 3. List of SNPs in P. aeruginosa SCV revertant Gene name mmsR phoP phoQ mexZ Product Still frameshift probable transcriptional regulator Two-component response regulator PhoP Two-component sensor PhoQ Probable glycosyl transferase Probable transcriptional regulator Intergenic region Hypothetical protein a Positionb Nucleotide Ref. basec SNP base Type 816532 425 G C Single insertion 1277728 41 A G SNP 1279044 683 1505156 552 C T SNP 2213076 400 C T SNP 2239547 280 T G 2356684 517 C - SNP Single deletion 39 bp deletion Protein effectd Position [AA]e SCVf Frameshift 142 + H->R exchange 14 + 228 + 184 ND 134 + In frame deletion Silent mutation Nonsense mutation ND Frameshift Gene locus, gene name and product description are extracted from Pseudomonas Genome Database (http://www.pseudomonas.com). b Chromosomal position (nt). c Ref. base, reference base. d Change of protein sequence. e Position relative to first amino acid of the protein. f Found in SCV as well after Sanger sequencing ND, not determined. 173 + Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image Table Click here to download Table: Table 1.doc ___________________________________________________________________________ Table 1. Minimal inhibitory concentrations (MICs) of different antibiotics against wild type (PAO), PAO-SCV and one selected pseudo-revertant (REV) as measured by the Bio-Merieux VITEK 2 system. Antibiotic PAO PAO-SCV REV Piperacillin 8 8 8 Cefotaxime 16 32 16 Ceftazidime 4 4 4 Imipenem 8 8 <=1 Meropenem 4 2 0.5 Gentamicin <=1 >16 8 Ciprofloxacin <0.25 <0.25 <0.25 Levofloxacin 1 0.5 1 Table Click here to download Table: Table 2.doc Table 2. Identification of differentially produced proteins in P. aeruginosa PAOSCV by MALDI-TOF MS peptide mass mapping (PMP) Sequence Total Protein Matched pI coverage mass Localization* PA No. Gene identification peptides value (%) (Da) Down in SCV PA0344 PA4273 PA5100 PA2300 PA5171 PA5171 PA4277 PA0291 PA1087 PA1337 PA0956 PA3655 PA4708 PA5505 PA0139 PA3326 Hypothetical protein rplA 50S ribosomal protein L1 hutU Urocanase chiC Chitinase arcA Arginine deiminase arcA Arginine deiminase tufB Elongation factor Tu oprE OprE porin 21 47 50,498 Cyt 6.8 9 80 93,130 Cyt 8.3 34 25 22 51 53 52 61,554 53,066 46,806 Cyt Extr Cyt 6.0 5.2 5.5 25 63 46,675 Cyt 5.5 25 68 43,684 Cyt 5.2 27 60 49,637 OM 8.7 Flagellar hookassociated protein ansB Glutaminaseasparaginase proS Prolyl-tRNA synthetase tsf Elongation factor Ts phuT Heme-transport protein Probable TonBdependent receptor ahpC Alkyl hydroperoxide reductase subunit C Endopeptidase Clp chain P 20 53 47,020 Extr 6.0 22 60 38,620 Per 6.7 22 37 65,536 Cyt 6.1 27 77 30,691 Cyt 5.2 19 68 31,019 Per 6.9 20 69 28,048 OM 7.8 18 66 20,643 Cyt 5.9 18 70 22,128 Cyt 5.4 19 31 58,860 Peri 6.2 18 45 42,347 Extr 6.4 23 70 39,858 Peri 5.6 flgL Up in SCV PA4502 PA0852 PA1074 Probable binding protein component of ABC transporter cbpD Chitin-binding protein CbpD precursor braC Branched-chain amino acid transport protein BraC PA3655 PA0395 PA4495 PA1179 PA1777 Elongation factor Ts pilT Twitching motility protein PilT Hypothetical protein phoP Twocomponent response regulator PhoP oprF OprF tsf 27 83 30,691 Cyt 5.2 16 56 41,644 OM 7.2 16 55 24,921 Peri 5.8 18 71 25,748 Cyt 5.3 17 75 23,270 OM 4.8 * Cyt= cytoplasm, Peri= periplasm, IM= inner membrane, OM= outer membrane, Extr= extracellular Table Click here to download Table: Table 3.doc Table 3. List of SNPs in P. aeruginosa SCV revertant Locusa Gene name PA0748 mmsR PA1179 phoP PA1180 phoQ PA1385 PA2020 igPA2046 PA2141 mexZ Product Still frameshift probable transcriptional regulator Two-component response regulator PhoP Two-component sensor PhoQ Probable glycosyl transferase Probable transcriptional regulator Intergenic region Positionb Nucleotide Ref. basec SNP base Type 816532 425 G C Single insertion 1277728 41 A G SNP 1279044 683 1505156 552 C T SNP 2213076 400 C T SNP 2239547 280 T G 39 bp deletion Protein effectd Position [AA]e SCVf Frameshift 142 + H->R exchange 14 + 228 + 184 ND 134 + In frame deletion Silent mutation Nonsense mutation SNP Single Hypothetical protein 2356684 517 C Frameshift deletion a Gene locus, gene name and product description are extracted from Pseudomonas Genome Database (http://www.pseudomonas.com). b Chromosomal position (nt). c Ref. base, reference base. d Change of protein sequence. e Position relative to first amino acid of the protein. f Found in SCV as well after Sanger sequencing ND, not determined. ND 173 + Supporting Information Click here to download Supporting Information: Figure S1.tif Supporting Information Click here to download Supporting Information: Figure S2.tif Supporting Information Click here to download Supporting Information: Figure S3.TIF Supporting Information Click here to download Supporting Information: Table S1.doc Supporting Information Click here to download Supporting Information: Table S2.doc Supporting Information Click here to download Supporting Information: Table S3.doc Supporting Information Click here to download Supporting Information: Table S4.doc Supporting Information Click here to download Supporting Information: TableS 5.doc Supporting Information Click here to download Supporting Information: TableS 6.doc Supporting Information Click here to download Supporting Information: Table S 7.doc