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1 Title 2 PecS regulates the urate-responsive expression of type 1 fimbriae in Klebsiella 3 pneumoniae CG43 4 5 Short title 6 PecS regulation of Klebsiella pneumoniae type 1 fimbriae 7 8 Contents category 9 Cell and Molecular Biology of Microbes 10 11 Authors and Affiliations 12 Zhe-Chong Wang1, Chia-Jui Liu1, Ying-Jung Huang2, Yu-Seng Wang1, and 13 Hwei-Ling Peng1* 14 1 15 Technology, 16 Hematology-Oncology, Chang Gung Memorial Hospital, Tao Yuan, Taiwan. 17 * Corresponding author. 18 Mailing address: Department of Biological Science and Technology, National Chiao 19 Tung University, 75 Po Ai Street, Hsin Chu, 30068, Taiwan, Republic of China. Tel: 20 886-3-5712121 Department of Biological Science and Technology, School of Biological Science and National ext. Chiao 56916; Tung University, Fax: 1 Hsin 886-3-5729288; Chu, 2 Division E-mail of address: 21 [email protected] 22 23 Words in summary: 248 24 Words in main text: 5285 25 Numbr of tables/figures: 10 2 26 ABSTRACT 27 In the Klebsiella pneumoniae CG43 genome, the divergently transcribed genes 28 respectively coding for PecS, the MarR-type transcription factor, and PecM, the drug 29 metabolite transporter, are located inbetween the type 1 and type 3 fimbrial gene 30 clusters. The intergenic sequence pecO between pecS and pecM contains 3 putative 31 PecS binding sites and a CpxR box. Electrophoretic mobility shift assay revealed that 32 the recombinant PecS and CpxR could specifically bind to the pecO sequence, and the 33 specific interaction of PecS and pecO could be attenuated by urate. The expression of 34 pecS and pecM are negatively regulated by CpxAR and PecS, and are inducible by 35 exogenous urate in the absence of cpxAR. Compared to CG43S3cpxAR, the derived 36 mutant CG43S3cpxARpecS or CG43S3cpxARpecSpecM exerts similar level of 37 sensitivity to H2O2 or to paraquat, but a higher level of mannose sensitive yeast 38 agglutination activity and FimA production. The promoter activity and transcript 39 levels of fimA in CG43S3cpxAR are also increased by deleting pecS. However, no 40 binding activity between PecS and the fimA promoter could be observed. Nevertheless, 41 PecS deletion can reduce the expression of the global regulator HNS and release the 42 negative effect of HNS on FimA expression. In CG43S3cpxAR, the expression of 43 FimA as well as PecS is inducible by urate, while the urate-induced FimA expression 44 is inhibited by the deletion of pecS. Taken together, we propose that K. pneumoniae 3 45 PecS indirectly and negatively regulates the expression of type 1 fimbriae, and the 46 regulation is urate inducible in the absence of CpxAR. 47 4 48 INTRODUCTION 49 The nosocomial pathogen, Klebsiella pneumoniae, causes suppurative lesions, 50 septicemia, and urinary and respiratory tract infections in immunocompromised 51 patients (Han 1995; Schelenz, et al. 2007). In Taiwan, the incidence of Klebsiella liver 52 abscesses (KLAs) in patients with diabetes, malignancy, renal disease, or pneumonia 53 has been steadily increasing (Fung, et al. 2002). Recently, KLAs have also been 54 reported in Western and other Asian countries (Pope, et al. 2011). Although the 55 pathogenic mechanism of KLA remains unknown, several virulence traits including 56 K1 capsular polysaccharides (Fung, et al. 2002), magA (Chuang, et al. 2006), iron 57 acquisition loci on pLVPK (Tang, et al. 2010), as well as type 1 and type 3 fimbriae 58 (Struve, et al. 2009; Stahlhut, et al. 2012) have been implicated with a role in the 59 pathogenesis. 60 Fimbriae mediate the attachment of bacteria to biotic or abiotic surfaces, and are 61 also involved in infection and biofilm formation (Van Houdt and Michiels 2005; 62 Nuccio and Baumler 2007). In K. pneumoniae isolates, type 1 and type 3 fimbrial 63 operons are physically linked (Struve, et al. 2009; Wang, et al. 2013). The expression 64 of type 1 fimbriae is phase-variable, and is mediated by the invertible fimS, located 65 upstream of fimA. The fimS switch, which alternates bacteria between type 1 66 fimbriated and non-fimbriated states, is controlled by site-specific recombinases FimB 5 67 and FimE (McClain, et al. 1991). The interplay of DNA binding proteins H-NS, IHF 68 and Lrp also influences the fimS orientation (Corcoran and Dorman 2009). Originally 69 characterized in Klebsiella strains, type 3 fimbriae provide the bacteria adhering 70 ability to epithelial cells of the respiratory and urinary tracts (Hornick, et al. 1992; 71 Tarkkanen, et al. 1997; Jagnow and Clegg 2003). A determinant role of type 3 72 fimbriae in the biofilm formation has also been repeatedly demonstrated (Di Martino, 73 et al. 2003; Struve, et al. 2009; Schroll, et al. 2010). The expression of type 3 fimbriae 74 is regulated by the second messenger c-di-GMP, and the corresponding regulators 75 MrkH, MrkI and MrkJ (Johnson and Clegg 2010; Wilksch, et al. 2011; Wu, et al. 76 2012). 77 Inbetween the gene clusters fimBEACDFGHK and mrkABCDF, the 4.6 kb of 78 DNA contains homologues of pecM and pecS, a putative high-affinity nickel 79 transporter encoding gene and 2 orfs (Struve, et al. 2009). PecS, which belongs to the 80 multiple antibiotic resistance regulator MarR family of transcriptional regulators, 81 negatively controls the expression of several virulence factors in Dickeya dadantii 82 (Erwinia chrysthenthemi) (Reverchon, et al. 1994; Ellison and Miller 2006; 83 Hommais, et al. 2008; Struve, et al. 2009). PecM is a transmembrane protein of the 84 drug/metabolite transporter (DMT) family. In D. dadantii, PecM acts as an efflux 85 pump to excrete indigoidine for defense against reactive oxygen species (Rouanet and 6 86 Nasser 2001; Zakataeva, et al. 2006). 87 In D. dadantii, Agrobacterium tumefaciens, and Streptomyces coelicolor, the 88 PecS protein exerts a negative autoregulation by directly binding on pecO, the 89 intergenic sequence between the pecS and pecM genes (Praillet, Reverchon and 90 Nasser 1997; Perera and Grove 2010). In addition, the binding activity of PecS to 91 pecO could be attenuated by exogenous urate in A. tumefaciens and S. coelicolor 92 (Perera and Grove 2010, 2011; Huang, Mackel, et al. 2013). Urates are secreted for 93 the prevention of extended tissue damage when a plant is invaded by 94 phytopathogens (Alamillo and Garcia-Olmedo 2001; Averyanov 2009). The plant 95 pathogens may use urate as a signal to release the PecS-mediated suppression which 96 in turn increases the virulent gene expression for an effective colonization 97 (Hommais, et al. 2008; Perera and Grove 2010; Mhedbi-Hajri, et al. 2011). On the 98 other hand, S. coelicolor PecS is proposed as the oxidative stress response activator 99 since urate production is associated with the generation of reactive oxygen species 100 (Huang, et al. 2013). 101 We show here in K. pneumoniae CG43, expression of the divergently transcribed 102 pecS and pecM are downregulated by PecS and the envelope stress responsive two 103 component system CpxAR. Although no apparent role in the oxidative stress response, 104 a negative effect of PecS on the expression of type 1 fimbriae is identified. Moreover, 7 105 urate is shown to be an inducer for the expression of not only pecS and pecM, but also 106 type 1 fimbriae. 107 8 108 METHODS 109 Bacterial strains, plasmid, primer and growth conditions. Table 1 lists the 110 bacterial strains and plasmids used in this study, and Table 2 lists the primers. 111 Bacteria were grown in 4 ml Luria-Bertani (LB) broth, which was shaken 37C at 200 112 rpm, unless otherwise indicated. The antibiotics used included ampicillin (100 113 g/mL), kanamycin (25 g/mL), streptomycin (500 g/mL), and chloramphenicol (35 114 g/mL). 115 116 Plasmid construction for the expression of PecS and CpxR. The coding regions of 117 pecS and cpxR were PCR-amplified with primer pairs WCC107/WCC108 and 118 WCC148/WCC149, respectively, from the CG43S3 genome. The amplified DNA was 119 individually cloned into cloning vector yT&A (Yeastern Biotech Co., Ltd.), and the 120 resulting recombinant plasmids named pyT-pecS and pyT-cpxR respectively. For 121 protein expression and purification, the coding regions from pyT-pecS and pyT-cpxR 122 were subcloned into pQE-81L-Kan (Qiagen) producing the plasmids pQE81LK-pecS 123 and pQE81LK-cpxR. The PecS site-directed mutation plasmid pQE81LK-pecSD61S 124 was generated using PCR-based mutagenesis with the plasmid pQE81LK-pecS as 125 template and WCC211 and WCC212 primer pair to substitute the aspartic acid at 126 residue 61 of PecS with serine. 9 127 128 Expression and purification of the recombinant proteins. The plasmids 129 pQE81LK-cpxR, 130 transformed into E. coli JM109, and protein production was induced with 0.5 mM 131 IPTG for 5 h at 37 C. The overexpressed protein was then purified from the soluble 132 fraction of the cell lysate by affinity chromatography using His-Bind resin essentially 133 according to the QIAexpress expression system protocol (Qiagen). The purified CpxR 134 and PecS proteins were dialyzed against Tris-buffered saline (pH 7.4) containing 10% 135 glycerol at 4 C overnight, followed by condensation with PEG 20000. The protein 136 purity was determined using SDS-PAGE. pQE81LK-pecS and pQE81LK-pecSD61S were individually 137 138 DNA electrophoretic mobility shift assay (EMSA). The pecO and the putative 139 promoter fragment of fimA were PCR-amplified using biotin-labeled primer pairs 140 WCC154/WCC155 141 WCC153/WCC155 and WCC58/WCC64. The DNA binding reaction was performed 142 in a 20 L binding buffer, and the mixture resolved using 5% native polyacrylamide 143 gel electrophoresis. After being transferred onto a biodyne B Nylon membrane, the 144 biotin-labeled DNA was detected using a LightShift chemiluminescent EMSA kit 145 (Pierce). The interaction buffer, for PecS and pecO or for PecS and PfimA, contained and WCC58/WCC59, 10 or non-labeled primer pairs 146 0.5 M Tris (pH 8.0), 50 mM NaCl, 0.06% BRIJ58, 20 g/mL BSA, 0.05 mg/mL of 147 sheared salmon sperm DNA and 1.5% glycerol (Perera and Grove 2010). To analyze 148 the urate effect, urate (Sigma-Aldrich) was firstly dissolved in 1 M NaOH and then 149 added to the reaction buffer. For the interaction between CpxR and pecO, the binding 150 buffer contained 20 mM Tris-HCl pH 7.0, 30 mM acetyl phosphate, 125 mM KCl, 10 151 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.25 mg/mL BSA and 0.05 mg/mL of 152 sheared salmon sperm DNA (Liu, et al. 2011). 153 154 Construction of the gene deletion mutants and the gene complement strain. 155 Specific gene deletion was introduced to the chromosome of K. pneumoniae CG43S3 156 by using an allelic-exchange strategy essentially as described (Lai, et al. 2003). In 157 brief, the DNA fragments of 1 kb flanking both ends of cpxAR, pecS and pecM gene 158 were amplified using PCR with the primer sets WCC138/ WCC139 and WCC140/ 159 WCC141, WCC111/ WCC112 and WCC113/WCC114, and WCC117/WCC118 and 160 WCC119/WCC114, respectively. The two amplified DNA fragments were cloned into 161 suicide vector pKAS46 (Skorupski and Taylor 1996). The resulting plasmid was 162 transformed into E. coli S17-1pir and then mobilized by conjugation to the 163 streptomycin-resistant strain, K. pneumoniae CG43S3. Several kanamycin-resistant 164 transconjugants, with the plasmid integrated into the chromosome through 11 165 homologous recombination, were selected from M9 agar plates supplemented with 166 kanamycin and propagated in 2 mL of LB broth overnight. A small aliquot of the 167 culture was plated on LB agar containing 500g/mL of streptomycin. The 168 streptomycin-resistant and kanamycin-sensitive colonies were isolated, and the 169 specific gene deletion of cpxAR, pecS and pecM were verified with PCR analysis 170 using primer sets WCC142/WCC143, WCC115/WCC116 and WCC120/ WCC121, 171 respectively. For complementation analysis, the DNA region containing pecS was 172 amplified using PCR with primer set WCC111/WCC114, and the DNA fragment 173 cloned into pKAS46, and transferred to pecS gene deletion mutant by conjugation. 174 175 Measurement of promoter activity. The putative promoter regions of pecS, pecM, 176 fimA, and fimB were PCR-amplified using primers WCC144/ WCC145, WCC146/ 177 WCC147, WCC156/ WCC157 and WCC158/ WCC159. The amplicons were then 178 cloned into placZ15 (Lin, et al. 2006) to generate PpecS-lacZ, PpecM-lacZ, PfimA-lacZ and 179 PfimB-lacZ. The promoter-reporter plasmids were individually mobilized into K. 180 pneumoniae CG43S3lacZ strains through conjugation from E. coli S17-1 pir. The 181 β-galactosidase activity was measured for the bacteria grown to exponential phase 182 with OD600 of 0.6~0.7. The promoter activity was expressed as Miller units. Each 183 sample was assayed in triplicate, and at least 3 independent experiments were 12 184 conducted. The data were calculated from 3 independent experiments, and are shown 185 as the means and standard deviations from the 9 samples (Lin, et al. 2006). As 186 described in (Wilkinson and Grove 2004), the pH of the modified LB broth, which 187 has been supplemented with different concentrations of urate (dissolved in 1 M 188 NaOH), was adjusted to 7.5 with HCl. NaOH and HCl are also added to modify the 189 no-urate control LB. The bacteria were grown in the modified LB broth to late 190 exponential phase with OD600 of 0.9~1 before the promoter activity measurement. 191 192 Quantitative RT-PCR (qRT-PCR). Total RNAs were isolated from early 193 exponential phase (OD600 of 0.4) K. pneumoniae CG43S3 and the derived strains, 194 which had been refreshed from the overnight cultures, using an RNeasy Midi column 195 (Qiagen) according to the manufacturer’s instruction. Purified RNA was 196 DNase-treated with RNase-free DNase I (MoBioPlus) to eliminate DNA 197 contamination, and the cDNAs were then synthesized using a random hexamer primer 198 form RevertAidTM H Minus First-strand cDNA synthesis kit (Fermentas, Canada). 199 PCR was performed using an ABI Prism 7000 Detection system according to 200 manufacturer’s instructions, and products were detected using SYBR Green PCR 201 Master Mix (Roche, Germany). The 23S rRNA level was used for the total RNA 202 normalization. Analysis was performed in triplicate in a reaction volume of 25 L 13 203 containing 12.5 L SYBR Green PCR Master Mix, 300 nM primer pair, 9.5 L 204 distilled H2O, and 1 L cDNA. Samples were heated for 10 min at 95C and 205 amplified for 40 cycles of 15 s at 95C and 60 s at 60C. Quantification was 206 performed using the 2-△△Ct Method (Wang, et al. 2013). 207 208 PecS antisera preparation. The pecS coding sequence was amplified using PCR 209 from the K. pneumoniae CG43S3 genome, and ligated into the expression vector 210 pQE-81L-Kan (Qiagen). The plasmid pQE81LK-pecS was transformed into E. coli 211 JM109, and the gene expression of the recombinant protein His6-PecS was induced 212 with 0.5 mM isopropyl--D-thiogalactopyranoside (IPTG) for 5 h at 37 C. The 213 soluble His6-PecS protein was purified using a nickel column (Novagen, Madison, WI, 214 USA). One milligram of the purified protein emulsified with 500 l of complete 215 Freund’s adjuvant was used to immunize New Zealand white rabbits weighing 2.0 ± 216 0.5 kg by intramuscular injection. The rabbits were boosted three times at 2-week 217 intervals with 500 g purified PecS recombinant protein. The PecS antisera was 218 obtained by intracardiac puncture 8 weeks later. 219 220 Western blot analysis. Aliquots of the total cellular lysates were resolved through 221 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the 14 222 proteins were electrophoretically transferred onto a polyvinylidene difluoride (PVDF) 223 membrane (Millipore, Billerica, MA, USA). After incubation with 5% skim milk at 224 room temperature for 1 h, the membranes were washed 3 times using phosphate 225 buffered saline with Tween 20 (PBST), and were then incubated with an anti-GAPDH 226 (GeneTex Inc.), anti-FimA (Wang, et al. 2013), anti-MrkA (Wang, et al. 2013) or 227 anti-PecS antiserum at room temperature for 2 h. Again, the membranes were washed 228 3 times with 1X PBST, and subjected to incubation with a 1:5000 dilution of the 229 secondary antibody, alkaline phosphatase-conjugated anti-rabbit immunoglobulin G 230 (Millipore, AP132A), at room temperature for 1 h. Finally, the blots were rewashed, 231 and the secondary antibodies bound on the PVDF membrane were detected using 232 chromogenic reagents 5-bromo-4-chloro-3-indolyl phosphate and nitro blue 233 tetrazolium. Bacteria were inoculated into the modified LB broth and grown at 37C 234 for 20 h before the western blotting analysis for urate effect on the expression of type 235 1 fimbriae and PecS. 236 237 H2O2 and paraquat survival assessment. Overnight-grown bacteria diluted 1:100 in 238 LB broth was incubated at 37 C to OD600 of 0.6-0.7. An aliquot (1 ml) of the 239 bacteria was collected by centrifugation, resuspended in 1 ml 0.85% saline with 10 240 mM H2O2 and 500 M paraquat respectively, and then subjected to 37 C incubation 15 241 for 40 min. After the stress treatment, the bacteria solution was diluted serially to 10-6 242 and 5 l of each sample was dropped onto an LB agar plate and incubated at 37 C 243 overnight. 244 245 Yeast-cell agglutination assay. The agglutination of yeast Saccharomyces cerevisiae 246 AH109 was conducted as described in (Wang, et al. 2013). Bacteria (109 cfu/mL) 247 were suspended in PBS with or without 5% mannose, and then mixed with yeast 248 suspended in PBS (10 mg/mL) into each well of a 24-well microtiter plate (Orange 249 Scientific, Catalogue# 4430300). The degree of clumping was assessed by 250 observation. 251 252 Biofilm formation analysis. Bacteria diluted 1:100 in LB broth supplemented with 253 appropriate antibiotics were inoculated into each well of a 96-well microtitre dish 254 (Orange Scientific) and statically incubated at 37 C for 24 h. Planktonic cells were 255 removed and the cells washed once with distilled water to remove unattached cells. 256 Crystal violet (0.1%, w/v, Sigma) was used to stain the attached cells for 30 min. 257 Unattached dye was removed by washing three times with distilled water, and the 258 stained biomass was solubilized in 1% (w/v) SDS. A595 was determined and relative 259 bacterial biofilm-forming activities were calculated. 260 16 261 RESULTS 262 The genomic organization of pecS and pecM genes is conserved in several K. 263 pneumoniae strains 264 Figure 1 shows that the gene organization of the divergently transcribed pecS and 265 pecM clustered with type 1 and type 3 fimbriae coding genes could be identified in 266 the clinical isolate K. pneumoniae CG43, NTUH-K2044 (Wu, et al. 2009) and 267 MGH78578 (Liao, et al. 2011), and the plant endophyte K. pneumoniae 342 (Fouts, et 268 al. 2008). K. pneumoniae PecS contains the characteristic N-terminal helical segment 269 and the four residues W17, D61, W68, R94 implicated in urate binding (Perera and 270 Grove 2010), and shares a sequence identity of 43%, 47% and 40%, respectively, with 271 the PecS of S. coelicolor, A. tumefaciens, and D. dadantii. The residues for the ligand 272 binding pocket of HucR and DNA binding helix are also conserved (Perera and Grove 273 2010). K. pneumoniae PecM sequence is similar to that of D. dadantii with identity of 274 43%, of A. tumefaciens with identity of 45%, and of S. coelicolor with identity of 275 46%. Moreover, the 10 transmembrane spanners are also present. 276 Urate is able to attenuate the specific binding of PecS to pecO sequence 277 As shown in Fig. 2a, K. pneumoniae CG43 pecO also contains the putative PecS 278 binding sites identified for the pecO of A. tumefaciens and S. coelicolor (Perera and 279 Grove 2010; Huang, Mackel, et al. 2013). Besides, a putative CpxR binding sequence, 17 280 GTAAA-N4~8-GTAAA (Yamamoto and Ishihama 2006), could be identified. We then 281 performed EMSA to determine if K. pneumoniae PecS binds specifically to pecO only. 282 As shown in Fig. 2b, 1 nM PecS could bind to the biotin labeled pecO, pecO*. The 283 PecS-pecO*complex formation is inhibited by excess of non-labeled pecO implying a 284 specific interaction between PecS and pecO. In addition, adding urate to the reaction 285 diminishes the PecS-pecO* complex formation. To confirm a role of urate in the 286 binding of PecS to pecO, PecSD61S, a site-directed mutant with one of the urate 287 binding residues changed from aspartate to serine has been generated. The lower 288 panel of Fig. 2b shows that the PecSD61S-pecO*complex starts to form with the 289 addition of 0.25 nM PecSD61S. However, more complex forms of PecSD61S-pecO* are 290 found when compared to PecS-pecO*. The binding activity of PecSD61S-pecO* could 291 also be inhibited by an excess of non-labeled pecO but not by adding urate. This 292 supports that D61 of PecS plays a critical role in the urate binding reaction and the 293 mutation probably increases its binding affinity to pecO. 294 Phosphorylation of CpxR is required for its binding to pecO DNA 295 To investigate if the CpxR box on pecO plays a regulatory role, we also performed 296 EMSA for a possible interaction between the two component system response 297 regulator CpxR and pecO. As shown in Fig. 2c, CpxR had no DNA binding activity in 298 the absence of acetyl-phosphate (upper panel) while the CpxR-pecO* complex 18 299 formed when acetyl-phosphate was included in the reaction indicating that the 300 phophorylation of CpxR is required for its binding to pecO. As the recombinant 301 protein His6-CpxR reaches 12.5 nM, a binding complex with the biotin-labeled pecO* 302 is formed (lower panel of Fig. 2c). The complex form diminishes with the presence of 303 excess non-labeled pecO, showing a binding specificity between His6-CpxR and 304 pecO. 305 CpxAR and PecS negatively influences the expression of pecS and pecM 306 The specific binding of CpxR to pecO sequence suggests a transcriptional level 307 regulation. As shown in Fig. 3a, the promoter activity of pecS and pecM is increased 308 by the deletion of cpxAR from CG43S3lacZ and the activity of PpecS and PpecM 309 further increased in CG43S3lacZcpxARpecS. The results indicate a possibility of 310 a negative regulation of CpxAR and PecS on the expression of pecS and pecM. The 311 transcriptional level regulation is also supported by the qRT-PCR analysis showing 312 that the levels of pecS and pecM transcripts of CG43S3cpxAR or 313 CG43S3cpxARpecS increased when compared to that of CG43S3 (Fig. 3b). 314 Moreover, deletion of pecM from CG43S3cpxAR increases the pecS and pecM 315 promoter activity (Fig. 3a), the pecS transcript levels (Fig. 3b), and the PecS protein 316 amounts (Fig. 3c). Since PecM is necessary for the DNA-binding activity of PecS in 317 D. dadantii (Praillet, Reverchon, Robert-Baudouy, et al. 1997), the pecM deletion 19 318 effect could be explained by that the removal of pecM reduces the DNA binding 319 activity of PecS thus releases the negative autoregulation, which in turn increases the 320 expression of PecS. 321 PecS and PecM expression are urate-inducible. Urate is a ligand for 322 PecS-dependent regulation in D. dadantii, A. tumefaciens and S. coelicolor. Figure. 4a 323 shows that the promoter activities of pecS and pecM are much higher than those 324 obtained in Fig. 3a. Since 1M NaOH has been added to LB, we speculate that the 325 excess sodium ions (the final concentration of NaOH is 0.06 M) contained in the 326 modified LB may have stimulatory effects on the promoter activity. As shown in the 327 left panel of Fig. 4a, the promoter activity of pecS and pecM in K. pneumoniae 328 CG43S3lacZ are not affected by the exogenous urates. Figure 3c shows that PecS 329 production in K. pneumoniae CG43S3 is barely detectable compared to the cpxAR 330 deletion mutant. Hence the cpxAR deletion mutant is used to determine the urate 331 effect. The right panel of Fig. 4a shows that the promoter activity of pecS and pecM 332 are slightly increased by the addition of 1 mM urate to the culture of CG43S3cpxAR. 333 When the concentration increased to 5 mM, a urate-inducible expression of pecS and 334 pecM could be observed (Fig. 4b). Moreover, the mRNA levels of pecS and pecM are 335 apparently increased by urate in a dose dependent manner (Fig. 4b). 336 Since urate production is associated with the generation of reactive oxygen 20 337 species, an investigation then needs to be carried out to see if the urate-induced PecS 338 and PecM expression are required for the oxidative stress response. We have 339 previously reported a functional role for SodA and YjcC in the oxidative stress 340 response in K. pneumoniae CG43 (Huang, Wang, et al. 2013). As shown in the upper 341 panel of Fig. 4c, CG43S3sodA and CG43S3yjcC exhibit higher levels of sensitivity 342 to paraquat and H2O2 when compared to CG43S3. By contrast, deletion of cpxAR, 343 pecS, or pecM exerts no apparent change of the susceptibility of CG43S3 to 500 M 344 paraquat or 10 mM H2O2 treatment suggesting PecS and PecM as well as CpxAR may 345 have no major role in the oxidative stress response. Furthermore, the exogenous urates 346 exert no apparent effect on CG43S3 responding to the oxidative stresses (lower panel 347 of Fig. 4c). 348 PecS negatively affects the expression of type 1 fimbriae 349 The clustered gene organization prompts us to investigate whether PecS regulates 350 the expression of type 1 and type 3 fimbriae. Type 1 fimbriae specifically bind to 351 mannosyl proteins and hence the fimbrial activity could be differentiated using 352 mannose as a competitor for the yeast agglutination activity. We have shown 353 previously that K. pneumoniae CG43S3constitutively express type 3 fimbriae while 354 type 1 fimbriae expression is only observed when the type 3 fimbriae major pilin 355 encoding gene mrkA is removed (Wang, et al. 2013). Figure 5a shows that 21 356 CG43S3cpxAR as well as CG43S3mrkA exerts apparent mannose sensitive yeast 357 agglutination corresponding to type 1fimbriae activity. Deletion of pecS from 358 CG43S3cpxAR further increases the mannose sensitive yeast agglutination activity, 359 while introducing the pecS gene to CG43S3cpxARpecS restores to the 360 agglutination level of CG43S3cpxAR. By contrast, either pecS or pecM deletion has 361 no apparent effect on the biofilm formation of CG43S3 or CG43S3cpxAR (Fig. 5b). 362 Moreover, CG43S3cpxARpecS exhibits an increase in FimA production when 363 compared with CG43S3cpxAR. The increase is no longer observed when the pecS 364 gene is introduced into CG43S3cpxARpecS. On the other hand, deletion of pecS 365 from CG43S3cpxAR does not trigger any visible change in MrkA production (Fig. 366 5c). 367 HNS may be the mediator for the PecS-dependent expression of type 1 fimbriae 368 Figure 6a shows that the promoter activity of fimA but not fimB nor fimE is negatively 369 affected by PecS. The mRNA level of fimA is also increased by the deletion of pecS 370 from CG43S3cpxAR, and introduction of pecS back into CG43S3cpxARpecS 371 reduces the FimA transcript level (Fig. 6b). EMSA is subsequently carried out to 372 examine if PecS directly affects fimA expression by binding to its promoter. Figure 6c 373 shows no PecS-PfimA* complex could be identified even with an increased 374 concentration of His6-PecS to 256 nM. These results suggest the possibility that PecS 22 375 376 indirectly regulates the expression of type 1 fimbriae. D. dadantii PecS is a global regulator for the expression of a wide range of genes 377 which includes the DNA binding transcription factor HNS (Hommais et al., 2008). 378 HNS acts as a negative regulator for type 1 fimbriae expression in E. coli (Donato and 379 Kawula 1999). An investigation is carried out to understand if PecS influences type 1 380 fimbriae expression through HNS. As shown in Fig. 6d, removal of the pecS gene 381 from CG43S3cpxAR reduces the mRNA level of hns and the level was restored after 382 introducing pecS-expression plasmid. This implies a positive role of PecS on the 383 expression of HNS. Moreover, increased expression of hns in CG43cpxAR blocks 384 the production of FimA indicating a negative role of HNS on the expression of type 1 385 fimbriae (Fig. 6e). 386 Urate-inducible FimA production is PecS dependent 387 Type 1 fimbriae is the major virulence determinant for the urinary tract infection. 388 Since urate is secreted daily in urine, we go further to study if urate affects type 1 389 fimbriae expression. As shown in Fig. 7a, type 1 fimbriae expression in CG43S3, 390 assessed using the assay of mannose sensitive yeast agglutination or Western blot, 391 could not be induced by the exogenous urates. Only in CG43S3cpxAR, FimA as well 392 as PecS production is enhanced in a urate-dependent manner (Fig. 7b). The FimA 393 transcript levels and PfimA activity are also increased by adding urate to the medium, 23 394 while the urate-dependent expression of fimA is no longer observed when pecS gene is 395 deleted from CG43S3cpxAR (Fig. 7c). 396 24 397 DISCUSSION 398 Klebsiella as well as Erwinia is a member of the Enterobacteriaceae, however, 399 the comparison, on the basis of the intergenic sequence and the sequence homology, 400 reveals that the Klebsiella pecS and pecM locus are more closely related to the soil 401 bacteria Streptomyces and the plant pathogen Agrobacterium. Nevertheless, K. 402 pneumoniae 342 as well as D. dadantii is associated with plant tissue suggesting that 403 K. pneumoniae pecS and pecM are originally acquired from soil microbes. The 404 analysis of the K. pneumoniae CG43 genome revealed 6 additional loci coding for the 405 transcription factors of the MarR family while only PecS belongs to the urate 406 responsive transcriptional regulator (UrtR) subfamily. This supports that K. 407 pneumoniae pecS and pecM loci may have a different evolutionary history from the 408 other six MarR homologs. 409 The urate binding residue D61 of PecS is predicted to be the major determinant 410 for the recognition of the target DNA and PecS dimerization is essential for the 411 specificity (Perera and Grove 2010). Figure 2b shows more complex forms of 412 PecSD61S-pecO* are found when compared to PecS-pecO*. This could be explained 413 by the fact that the mutation reduces the DNA binding specificity, and hence more 414 complex forms are present. As shown in Fig. 3 and Fig. 4, PecM is important for the 415 expression of PecS and its expression is urate-inducible. The DMT family commonly 25 416 transports metabolites, namely amino acids, nucleotides and purine bases from the 417 cytoplasm (Rouanet and Nasser 2001; Zakataeva, et al. 2006; Airich, et al. 2010). 418 How PecM functions as a transporter and if it is selective for pumping out specific 419 small molecules is unknown. 420 The two component system CpxAR is a global regulatory system required for the 421 regulation of the bacterial envelope stress response. The Cpx regulon contains 422 approximately equal numbers of upregulated and downregulated genes, including 423 those for the pilus assembly, efflux pump biogenesis, adherence, antibiotics resistance, 424 virulence, and biofilm development (Vogt and Raivio 2012). The deletion of cpxAR 425 increased not only the expression of pecS and pecM (Fig. 3), but also the mannose 426 sensitivity yeast agglutination activity (Fig. 5a), FimA and MrkA production (Fig. 5c). 427 This implies that the negative role of CpxAR in regulating the expression of type 1 428 fimbriae is probably mediated by the urate-inducible PecS and PecM system. As 429 shown in Fig. 7b, that the urate-inducible expression of FimA is no longer observed 430 by removing the pecS gene further supports the possibility. 431 The conserved genomic organization of fim-pecS-pecM-mrk identified in 432 different K. pneumoniae strains implies a preserved and coordinated functional 433 pathway. Figure 7 shows that PecS only exerted regulatory effect on the expression of 434 type 1 fimbriae, the major determinant for the urinary tract infections in the absence 26 435 of CpxAR. The expression of CpxAR system depends strongly on the environmental 436 pH, and the expression is induced by alkaline pH (Hunke, et al. 2012). We speculate 437 that the CpxAR system may be repressed after bacteria comes into contact with urine, 438 which is slightly acidic (pH 6.5), thereby releasing the repression for pecS and pecM. 439 Besides, FimA as well as PecS exerts a dose dependent expression responding to urate. 440 We speculate that the excess urate in human urine may increase the PecS production 441 but also release the repression of PecS on type 1 fimbriae and hence the FimA 442 production is increased. 443 In D. dadantii, expression of H-NS decreased with the deletion of the pecS gene 444 (Hommais, et al. 2008). E. coli H-NS binds to fimS-IRL which influences the 445 site-specific recombination and thus decreases the fimA promoter activity (O'Gara and 446 Dorman 2000; Corcoran and Dorman 2009). We have shown that the expression of 447 hns is increased by PecS (Fig. 6d), while the FimA production is reduced by 448 increasing hns expression (Fig. 6e). Analysis of the 5’ upstream noncoding sequence 449 of hns reveals 3 putative palindromic-like sequences 450 (CGA-N-W-TCGTA)T-AT(TACGANNNCG) that was predicted as the binding site 451 for D. dadantii PecS (Rouanet, et al., 2004). We propose that, in the presence of 452 excess urates and the absence of CpxAR, PecS is no longer able to bind the promoter 453 sequence of hns which in turn reduces the expression of HNS, and consequently the 27 454 repression of HNS on the expression of type 1 fimbriae is released. However, whether 455 the fact that PecS directly affects the H-NS expression would thereby modulate the 456 fimS switch needs further investigation. 457 We conclude here with a model in Fig. 8, which illustrates how the 458 phosphorylated CpxR, activated upon sensing a not yet identified signal, negatively 459 influences the expression of pecS and pecM, PecS negatively affects its own 460 expression and the auto-repression could be attenuated by exogenous urate but 461 activated by PecM. In the absence of cpxAR and the presence of urate, the expression 462 of pecS is required for the urate-responsive type 1 fimbriae expression that is 463 mediated by the global regulator HNS. Moreover, the negative regulation of CpxAR 464 on type 3 fimbriae is possibly an indirect control through influencing the expression 465 of the regulators MrkJ, MrkH or MrkI. 466 467 ACKNOWLEDGEMENT 468 This work was supported by grants from the National Science Council 469 (NSC100-2320-B-009-003-MY3) and Ministry of Science and Technology (MOST 470 103-2320-B-009-004), Taiwan, ROC. We also thank Professor Lin CT from School of 471 Chinese Medicine, China Medical University, Taiwan, ROC for providing us the HNS 472 expression plasmid. 473 28 474 REFERENCES 475 Airich LG, Tsyrenzhapova IS, Vorontsova OV, Feofanov AV, Doroshenko VG, 476 Mashko SV. (2010). Membrane topology analysis of the Escherichia coli aromatic 477 amino acid efflux protein YddG. J. Mol. Microbiol. Biotechnol. 19:189-197. 478 Alamillo JM, Garcia-Olmedo F. (2001). 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[Export of metabolites by the proteins of the DMT and RhtB families and 617 its possible role in intercellular communication]. Mikrobiologiia 75:509-520. 618 619 620 621 622 623 624 625 36 626 Figure Legends 627 Fig. 1. The organization of pecS and pecM locus-containing gene clusters in K. 628 pneumoniae strain CG43, NTUH-K2044, MGH 78578, and 342. The flanking genes 629 of pecS and pecM were annotated according to the released genome of NCBI by 630 BLASTX analysis. NT: nickel transporter, CL: citrate lyase, HP: hypothetical protein. 631 632 Fig. 2. (a) The pecO sequence comparison. The 3 putative PecS binding sequences as 633 well as the CpxR box are marked. (b) (c), EMSA of the interaction between the 634 His6-PecS and pecO, and His6-CpxR and pecO, respectively. The biotin-labeled pecO, 635 pecO*, was incubated with an increasing amount of the recombinant PecS and the 636 site-directed mutant PecSD61S (b), and CpxR (c). Binding specificity was investigated 637 by adding 200-fold non-labeled pecO DNA. (b) Different concentrations of urate (1-, 638 5-, and 20-mM) were added as competitor for the binding reaction. (c) The upper and 639 lower panel respectively indicates the result without and with acetyl phosphate added 640 in the reaction. 641 Fig. 3. CpxAR and PecS negatively influence the expression of pecS and pecM (a) 642 The promoter activity of pecS and pecM was assessed by monitoring the expression of 643 -galactosidase on the reporter plasmids PpecS-lacZ and PpecM-lacZ, respectively. (b) 644 The mRNA levels of pecS and pecM of K. pneumoniae CG43S3, CG43S3cpxAR, 645 CG43S3cpxARpecM or CG43S3cpxARpecS were assessed using qRT-PCR. (c) 37 646 Western blot analysis of the PecS production. Total proteins isolated from the bacteria 647 were resolved on SDS-12.5% polyacrylamide gel through electrophoresis, and then 648 transferred onto PVDF, and PecS expression was recognized using anti-PecS 649 antiserum. The quantification data was obtained using ImageJ software. 650 Fig. 4. The pecS and pecM expression are inducible by urate. (a) Urate effects on the 651 promoter activity of pecS and pecM. The promoter activity was respectively assessed 652 by monitoring the expression of -galactosidase on the reporter plasmids PpecS-lacZ 653 and PpecM-lacZ in K. pneumoniae CG43S3lacZ (left panel) or CG43S3lacZcpxAR 654 (right panel), which was grown to the late exponential phase (OD600 of 0.9) in a 655 Luria-Bertani broth supplied with 0.2 mM, 1 mM, or 5 mM of urates. The asterisks 656 denote differences with a statistical significance. (b) The mRNA levels of pecS and 657 pecM gene in K. pneumoniae CG43S3cpxAR were assessed. (c) Paraquat (left panel) 658 and H2O2 (right panel) stress survival analysis. An aliquot of the exponential growth 659 bacteria (OD600 of 0.6-0.7) was collected by centrifugation, resuspended in 0.85% 660 saline with 500 M paraquat or 10 mM H2O2 and then subjected to 37 C incubation 661 for 40 min. The bacteria was then diluted serially and 5 l each was dropped on LB 662 agar plate and incubated at 37 C overnight. The bacteria CG43S3sodA and 663 CG43S3yjcC (Huang, Wang, et al. 2013) are used as control strains for the oxidative 38 664 stress response. The lower panel shows the analysis of urate effect on the stress 665 responses in K. pneumonae CG43S3. 666 Fig. 5. The deletion effect of pecS from K. pneumoniae CG43S3cpxAR on the 667 expression of type 1 and type 3 fimbriae was assessed using (a) Yeast agglutination 668 analysis, (b) Biofilm forming activity, and (c) Western blot analysis for each of the 669 major pilin production. The bacteria were grown with agitation for 20 h. 670 Fig. 6. PecS negatively affects the type 1 fimbriae expression. (a) The promoter 671 activity of fimA, fimB and fimE was assessed using the expression of -galactosidase 672 on the reporter plasmids PfimA-lacZ, PfimB-lacZ and PfimE-lacZ, respectively. (b) The 673 level of fimA mRNA in K. pneumoniae CG43S3cpxAR, CG43S3cpxARpecS or 674 CG43S3cpxARpecS::pecS were individually assessed by using qRT-PCR. The 675 bacteria were grown with agitation to log phase and then collected for analysis. (c) 676 EMSA of the interaction between His6-PecS and biotin-labeled PfimA, PfimA*. The 677 reaction was performed with an increasing amount of the recombinant PecS proteins. 678 pecO* was used as a positive control for PecS binding. (d) The hns mRNA level of K. 679 pneumoniae CG43S3cpxAR, CG43S3cpxARpecS and CG43S3cpxARpecS:: 680 pecS were assessed with qRT-PCR. (e) Increase hns expression reduces the FimA 681 production as assessed using western blot analysis. 39 682 Fig. 7. Urate effect on the expression of FimA. (a) Analysis of the urate effect on type 683 1 fimbriae expression in K. pneumoniae CG43S3 using the analysis of mannose 684 sensitive yeast agglutination (left panel) and Western blot (right panel) (b) Western 685 blot analysis of the urate effect on the expression of PecS and FimA. (c) Analysis of 686 the urate effect on the expression of FimA using qRT-PCR (left panel) and promoter 687 activity assay (right panel). Bacteria were grown in the modified LB supplied without 688 or with 0.2-5 mM urate at 37C with agitation. 689 Fig. 8. Model of the CpxAR-dependent PecSM regulation on type 1 fimbriae 690 expression. Upon stimulating by unknown external signal, the phosphorylated CpxR 691 binds to the CpxR box in the intergenic region of pecS and pecM and inhibits their 692 transcription. On the other hand, the negative regulation of CpxAR on type 3 fimbriae 693 is possibly an indirect control through influencing the expression of the regulators 694 MrkJ, MrkH or MrkI. Only in the presence of urates and the absence of cpxAR, the 695 PecSM-mediated repression is released and type 1 fimbriae express. HNS is probably 696 the mediator for the PecSM-dependent repression and urate-inducible expression of 697 type 1 fimbriae. 698 699 40