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Title
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PecS regulates the urate-responsive expression of type 1 fimbriae in Klebsiella
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pneumoniae CG43
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Short title
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PecS regulation of Klebsiella pneumoniae type 1 fimbriae
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Contents category
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Cell and Molecular Biology of Microbes
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Authors and Affiliations
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Zhe-Chong Wang1, Chia-Jui Liu1, Ying-Jung Huang2, Yu-Seng Wang1, and
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Hwei-Ling Peng1*
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1
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Technology,
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Hematology-Oncology, Chang Gung Memorial Hospital, Tao Yuan, Taiwan.
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* Corresponding author.
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Mailing address: Department of Biological Science and Technology, National Chiao
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Tung University, 75 Po Ai Street, Hsin Chu, 30068, Taiwan, Republic of China. Tel:
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886-3-5712121
Department of Biological Science and Technology, School of Biological Science and
National
ext.
Chiao
56916;
Tung
University,
Fax:
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Hsin
886-3-5729288;
Chu,
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Division
E-mail
of
address:
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[email protected]
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Words in summary: 248
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Words in main text: 5285
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Numbr of tables/figures: 10
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ABSTRACT
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In the Klebsiella pneumoniae CG43 genome, the divergently transcribed genes
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respectively coding for PecS, the MarR-type transcription factor, and PecM, the drug
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metabolite transporter, are located inbetween the type 1 and type 3 fimbrial gene
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clusters. The intergenic sequence pecO between pecS and pecM contains 3 putative
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PecS binding sites and a CpxR box. Electrophoretic mobility shift assay revealed that
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the recombinant PecS and CpxR could specifically bind to the pecO sequence, and the
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specific interaction of PecS and pecO could be attenuated by urate. The expression of
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pecS and pecM are negatively regulated by CpxAR and PecS, and are inducible by
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exogenous urate in the absence of cpxAR. Compared to CG43S3cpxAR, the derived
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mutant CG43S3cpxARpecS or CG43S3cpxARpecSpecM exerts similar level of
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sensitivity to H2O2 or to paraquat, but a higher level of mannose sensitive yeast
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agglutination activity and FimA production. The promoter activity and transcript
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levels of fimA in CG43S3cpxAR are also increased by deleting pecS. However, no
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binding activity between PecS and the fimA promoter could be observed. Nevertheless,
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PecS deletion can reduce the expression of the global regulator HNS and release the
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negative effect of HNS on FimA expression. In CG43S3cpxAR, the expression of
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FimA as well as PecS is inducible by urate, while the urate-induced FimA expression
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is inhibited by the deletion of pecS. Taken together, we propose that K. pneumoniae
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PecS indirectly and negatively regulates the expression of type 1 fimbriae, and the
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regulation is urate inducible in the absence of CpxAR.
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INTRODUCTION
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The nosocomial pathogen, Klebsiella pneumoniae, causes suppurative lesions,
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septicemia, and urinary and respiratory tract infections in immunocompromised
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patients (Han 1995; Schelenz, et al. 2007). In Taiwan, the incidence of Klebsiella liver
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abscesses (KLAs) in patients with diabetes, malignancy, renal disease, or pneumonia
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has been steadily increasing (Fung, et al. 2002). Recently, KLAs have also been
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reported in Western and other Asian countries (Pope, et al. 2011). Although the
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pathogenic mechanism of KLA remains unknown, several virulence traits including
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K1 capsular polysaccharides (Fung, et al. 2002), magA (Chuang, et al. 2006), iron
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acquisition loci on pLVPK (Tang, et al. 2010), as well as type 1 and type 3 fimbriae
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(Struve, et al. 2009; Stahlhut, et al. 2012) have been implicated with a role in the
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pathogenesis.
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Fimbriae mediate the attachment of bacteria to biotic or abiotic surfaces, and are
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also involved in infection and biofilm formation (Van Houdt and Michiels 2005;
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Nuccio and Baumler 2007). In K. pneumoniae isolates, type 1 and type 3 fimbrial
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operons are physically linked (Struve, et al. 2009; Wang, et al. 2013). The expression
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of type 1 fimbriae is phase-variable, and is mediated by the invertible fimS, located
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upstream of fimA. The fimS switch, which alternates bacteria between type 1
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fimbriated and non-fimbriated states, is controlled by site-specific recombinases FimB
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and FimE (McClain, et al. 1991). The interplay of DNA binding proteins H-NS, IHF
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and Lrp also influences the fimS orientation (Corcoran and Dorman 2009). Originally
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characterized in Klebsiella strains, type 3 fimbriae provide the bacteria adhering
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ability to epithelial cells of the respiratory and urinary tracts (Hornick, et al. 1992;
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Tarkkanen, et al. 1997; Jagnow and Clegg 2003). A determinant role of type 3
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fimbriae in the biofilm formation has also been repeatedly demonstrated (Di Martino,
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et al. 2003; Struve, et al. 2009; Schroll, et al. 2010). The expression of type 3 fimbriae
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is regulated by the second messenger c-di-GMP, and the corresponding regulators
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MrkH, MrkI and MrkJ (Johnson and Clegg 2010; Wilksch, et al. 2011; Wu, et al.
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2012).
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Inbetween the gene clusters fimBEACDFGHK and mrkABCDF, the 4.6 kb of
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DNA contains homologues of pecM and pecS, a putative high-affinity nickel
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transporter encoding gene and 2 orfs (Struve, et al. 2009). PecS, which belongs to the
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multiple antibiotic resistance regulator MarR family of transcriptional regulators,
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negatively controls the expression of several virulence factors in Dickeya dadantii
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(Erwinia chrysthenthemi) (Reverchon, et al. 1994; Ellison and Miller 2006;
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Hommais, et al. 2008; Struve, et al. 2009). PecM is a transmembrane protein of the
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drug/metabolite transporter (DMT) family. In D. dadantii, PecM acts as an efflux
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pump to excrete indigoidine for defense against reactive oxygen species (Rouanet and
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Nasser 2001; Zakataeva, et al. 2006).
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In D. dadantii, Agrobacterium tumefaciens, and Streptomyces coelicolor, the
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PecS protein exerts a negative autoregulation by directly binding on pecO, the
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intergenic sequence between the pecS and pecM genes (Praillet, Reverchon and
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Nasser 1997; Perera and Grove 2010). In addition, the binding activity of PecS to
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pecO could be attenuated by exogenous urate in A. tumefaciens and S. coelicolor
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(Perera and Grove 2010, 2011; Huang, Mackel, et al. 2013). Urates are secreted for
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the prevention of extended tissue damage when a plant is invaded by
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phytopathogens (Alamillo and Garcia-Olmedo 2001; Averyanov 2009). The plant
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pathogens may use urate as a signal to release the PecS-mediated suppression which
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in turn increases the virulent gene expression for an effective colonization
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(Hommais, et al. 2008; Perera and Grove 2010; Mhedbi-Hajri, et al. 2011). On the
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other hand, S. coelicolor PecS is proposed as the oxidative stress response activator
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since urate production is associated with the generation of reactive oxygen species
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(Huang, et al. 2013).
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We show here in K. pneumoniae CG43, expression of the divergently transcribed
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pecS and pecM are downregulated by PecS and the envelope stress responsive two
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component system CpxAR. Although no apparent role in the oxidative stress response,
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a negative effect of PecS on the expression of type 1 fimbriae is identified. Moreover,
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urate is shown to be an inducer for the expression of not only pecS and pecM, but also
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type 1 fimbriae.
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METHODS
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Bacterial strains, plasmid, primer and growth conditions. Table 1 lists the
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bacterial strains and plasmids used in this study, and Table 2 lists the primers.
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Bacteria were grown in 4 ml Luria-Bertani (LB) broth, which was shaken 37C at 200
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rpm, unless otherwise indicated. The antibiotics used included ampicillin (100
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g/mL), kanamycin (25 g/mL), streptomycin (500 g/mL), and chloramphenicol (35
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g/mL).
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Plasmid construction for the expression of PecS and CpxR. The coding regions of
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pecS and cpxR were PCR-amplified with primer pairs WCC107/WCC108 and
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WCC148/WCC149, respectively, from the CG43S3 genome. The amplified DNA was
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individually cloned into cloning vector yT&A (Yeastern Biotech Co., Ltd.), and the
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resulting recombinant plasmids named pyT-pecS and pyT-cpxR respectively. For
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protein expression and purification, the coding regions from pyT-pecS and pyT-cpxR
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were subcloned into pQE-81L-Kan (Qiagen) producing the plasmids pQE81LK-pecS
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and pQE81LK-cpxR. The PecS site-directed mutation plasmid pQE81LK-pecSD61S
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was generated using PCR-based mutagenesis with the plasmid pQE81LK-pecS as
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template and WCC211 and WCC212 primer pair to substitute the aspartic acid at
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residue 61 of PecS with serine.
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Expression and purification of the recombinant proteins. The plasmids
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pQE81LK-cpxR,
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transformed into E. coli JM109, and protein production was induced with 0.5 mM
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IPTG for 5 h at 37 C. The overexpressed protein was then purified from the soluble
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fraction of the cell lysate by affinity chromatography using His-Bind resin essentially
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according to the QIAexpress expression system protocol (Qiagen). The purified CpxR
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and PecS proteins were dialyzed against Tris-buffered saline (pH 7.4) containing 10%
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glycerol at 4 C overnight, followed by condensation with PEG 20000. The protein
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purity was determined using SDS-PAGE.
pQE81LK-pecS
and
pQE81LK-pecSD61S
were
individually
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DNA electrophoretic mobility shift assay (EMSA). The pecO and the putative
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promoter fragment of fimA were PCR-amplified using biotin-labeled primer pairs
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WCC154/WCC155
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WCC153/WCC155 and WCC58/WCC64. The DNA binding reaction was performed
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in a 20 L binding buffer, and the mixture resolved using 5% native polyacrylamide
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gel electrophoresis. After being transferred onto a biodyne B Nylon membrane, the
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biotin-labeled DNA was detected using a LightShift chemiluminescent EMSA kit
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(Pierce). The interaction buffer, for PecS and pecO or for PecS and PfimA, contained
and
WCC58/WCC59,
10
or
non-labeled
primer
pairs
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0.5 M Tris (pH 8.0), 50 mM NaCl, 0.06% BRIJ58, 20 g/mL BSA, 0.05 mg/mL of
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sheared salmon sperm DNA and 1.5% glycerol (Perera and Grove 2010). To analyze
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the urate effect, urate (Sigma-Aldrich) was firstly dissolved in 1 M NaOH and then
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added to the reaction buffer. For the interaction between CpxR and pecO, the binding
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buffer contained 20 mM Tris-HCl pH 7.0, 30 mM acetyl phosphate, 125 mM KCl, 10
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mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.25 mg/mL BSA and 0.05 mg/mL of
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sheared salmon sperm DNA (Liu, et al. 2011).
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Construction of the gene deletion mutants and the gene complement strain.
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Specific gene deletion was introduced to the chromosome of K. pneumoniae CG43S3
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by using an allelic-exchange strategy essentially as described (Lai, et al. 2003). In
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brief, the DNA fragments of 1 kb flanking both ends of cpxAR, pecS and pecM gene
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were amplified using PCR with the primer sets WCC138/ WCC139 and WCC140/
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WCC141, WCC111/ WCC112 and WCC113/WCC114, and WCC117/WCC118 and
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WCC119/WCC114, respectively. The two amplified DNA fragments were cloned into
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suicide vector pKAS46 (Skorupski and Taylor 1996). The resulting plasmid was
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transformed into E. coli S17-1pir and then mobilized by conjugation to the
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streptomycin-resistant strain, K. pneumoniae CG43S3. Several kanamycin-resistant
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transconjugants, with the plasmid integrated into the chromosome through
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homologous recombination, were selected from M9 agar plates supplemented with
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kanamycin and propagated in 2 mL of LB broth overnight. A small aliquot of the
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culture was plated on LB agar containing 500g/mL of streptomycin. The
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streptomycin-resistant and kanamycin-sensitive colonies were isolated, and the
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specific gene deletion of cpxAR, pecS and pecM were verified with PCR analysis
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using primer sets WCC142/WCC143, WCC115/WCC116 and WCC120/ WCC121,
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respectively. For complementation analysis, the DNA region containing pecS was
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amplified using PCR with primer set WCC111/WCC114, and the DNA fragment
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cloned into pKAS46, and transferred to pecS gene deletion mutant by conjugation.
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Measurement of promoter activity. The putative promoter regions of pecS, pecM,
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fimA, and fimB were PCR-amplified using primers WCC144/ WCC145, WCC146/
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WCC147, WCC156/ WCC157 and WCC158/ WCC159. The amplicons were then
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cloned into placZ15 (Lin, et al. 2006) to generate PpecS-lacZ, PpecM-lacZ, PfimA-lacZ and
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PfimB-lacZ. The promoter-reporter plasmids were individually mobilized into K.
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pneumoniae CG43S3lacZ strains through conjugation from E. coli S17-1 pir. The
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β-galactosidase activity was measured for the bacteria grown to exponential phase
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with OD600 of 0.6~0.7. The promoter activity was expressed as Miller units. Each
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sample was assayed in triplicate, and at least 3 independent experiments were
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conducted. The data were calculated from 3 independent experiments, and are shown
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as the means and standard deviations from the 9 samples (Lin, et al. 2006). As
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described in (Wilkinson and Grove 2004), the pH of the modified LB broth, which
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has been supplemented with different concentrations of urate (dissolved in 1 M
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NaOH), was adjusted to 7.5 with HCl. NaOH and HCl are also added to modify the
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no-urate control LB. The bacteria were grown in the modified LB broth to late
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exponential phase with OD600 of 0.9~1 before the promoter activity measurement.
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Quantitative RT-PCR (qRT-PCR). Total RNAs were isolated from early
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exponential phase (OD600 of 0.4) K. pneumoniae CG43S3 and the derived strains,
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which had been refreshed from the overnight cultures, using an RNeasy Midi column
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(Qiagen) according to the manufacturer’s instruction. Purified RNA was
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DNase-treated with RNase-free DNase I (MoBioPlus) to eliminate DNA
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contamination, and the cDNAs were then synthesized using a random hexamer primer
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form RevertAidTM H Minus First-strand cDNA synthesis kit (Fermentas, Canada).
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PCR was performed using an ABI Prism 7000 Detection system according to
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manufacturer’s instructions, and products were detected using SYBR Green PCR
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Master Mix (Roche, Germany). The 23S rRNA level was used for the total RNA
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normalization. Analysis was performed in triplicate in a reaction volume of 25 L
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containing 12.5 L SYBR Green PCR Master Mix, 300 nM primer pair, 9.5 L
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distilled H2O, and 1 L cDNA. Samples were heated for 10 min at 95C and
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amplified for 40 cycles of 15 s at 95C and 60 s at 60C. Quantification was
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performed using the 2-△△Ct Method (Wang, et al. 2013).
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PecS antisera preparation. The pecS coding sequence was amplified using PCR
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from the K. pneumoniae CG43S3 genome, and ligated into the expression vector
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pQE-81L-Kan (Qiagen). The plasmid pQE81LK-pecS was transformed into E. coli
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JM109, and the gene expression of the recombinant protein His6-PecS was induced
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with 0.5 mM isopropyl--D-thiogalactopyranoside (IPTG) for 5 h at 37 C. The
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soluble His6-PecS protein was purified using a nickel column (Novagen, Madison, WI,
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USA). One milligram of the purified protein emulsified with 500 l of complete
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Freund’s adjuvant was used to immunize New Zealand white rabbits weighing 2.0 ±
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0.5 kg by intramuscular injection. The rabbits were boosted three times at 2-week
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intervals with 500 g purified PecS recombinant protein. The PecS antisera was
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obtained by intracardiac puncture 8 weeks later.
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Western blot analysis. Aliquots of the total cellular lysates were resolved through
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sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the
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proteins were electrophoretically transferred onto a polyvinylidene difluoride (PVDF)
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membrane (Millipore, Billerica, MA, USA). After incubation with 5% skim milk at
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room temperature for 1 h, the membranes were washed 3 times using phosphate
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buffered saline with Tween 20 (PBST), and were then incubated with an anti-GAPDH
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(GeneTex Inc.), anti-FimA (Wang, et al. 2013), anti-MrkA (Wang, et al. 2013) or
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anti-PecS antiserum at room temperature for 2 h. Again, the membranes were washed
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3 times with 1X PBST, and subjected to incubation with a 1:5000 dilution of the
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secondary antibody, alkaline phosphatase-conjugated anti-rabbit immunoglobulin G
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(Millipore, AP132A), at room temperature for 1 h. Finally, the blots were rewashed,
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and the secondary antibodies bound on the PVDF membrane were detected using
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chromogenic reagents 5-bromo-4-chloro-3-indolyl phosphate and nitro blue
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tetrazolium. Bacteria were inoculated into the modified LB broth and grown at 37C
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for 20 h before the western blotting analysis for urate effect on the expression of type
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1 fimbriae and PecS.
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H2O2 and paraquat survival assessment. Overnight-grown bacteria diluted 1:100 in
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LB broth was incubated at 37 C to OD600 of 0.6-0.7. An aliquot (1 ml) of the
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bacteria was collected by centrifugation, resuspended in 1 ml 0.85% saline with 10
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mM H2O2 and 500 M paraquat respectively, and then subjected to 37 C incubation
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for 40 min. After the stress treatment, the bacteria solution was diluted serially to 10-6
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and 5 l of each sample was dropped onto an LB agar plate and incubated at 37 C
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overnight.
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Yeast-cell agglutination assay. The agglutination of yeast Saccharomyces cerevisiae
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AH109 was conducted as described in (Wang, et al. 2013). Bacteria (109 cfu/mL)
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were suspended in PBS with or without 5% mannose, and then mixed with yeast
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suspended in PBS (10 mg/mL) into each well of a 24-well microtiter plate (Orange
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Scientific, Catalogue# 4430300). The degree of clumping was assessed by
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observation.
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Biofilm formation analysis. Bacteria diluted 1:100 in LB broth supplemented with
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appropriate antibiotics were inoculated into each well of a 96-well microtitre dish
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(Orange Scientific) and statically incubated at 37 C for 24 h. Planktonic cells were
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removed and the cells washed once with distilled water to remove unattached cells.
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Crystal violet (0.1%, w/v, Sigma) was used to stain the attached cells for 30 min.
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Unattached dye was removed by washing three times with distilled water, and the
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stained biomass was solubilized in 1% (w/v) SDS. A595 was determined and relative
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bacterial biofilm-forming activities were calculated.
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RESULTS
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The genomic organization of pecS and pecM genes is conserved in several K.
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pneumoniae strains
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Figure 1 shows that the gene organization of the divergently transcribed pecS and
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pecM clustered with type 1 and type 3 fimbriae coding genes could be identified in
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the clinical isolate K. pneumoniae CG43, NTUH-K2044 (Wu, et al. 2009) and
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MGH78578 (Liao, et al. 2011), and the plant endophyte K. pneumoniae 342 (Fouts, et
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al. 2008). K. pneumoniae PecS contains the characteristic N-terminal helical segment
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and the four residues W17, D61, W68, R94 implicated in urate binding (Perera and
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Grove 2010), and shares a sequence identity of 43%, 47% and 40%, respectively, with
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the PecS of S. coelicolor, A. tumefaciens, and D. dadantii. The residues for the ligand
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binding pocket of HucR and DNA binding helix are also conserved (Perera and Grove
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2010). K. pneumoniae PecM sequence is similar to that of D. dadantii with identity of
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43%, of A. tumefaciens with identity of 45%, and of S. coelicolor with identity of
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46%. Moreover, the 10 transmembrane spanners are also present.
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Urate is able to attenuate the specific binding of PecS to pecO sequence
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As shown in Fig. 2a, K. pneumoniae CG43 pecO also contains the putative PecS
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binding sites identified for the pecO of A. tumefaciens and S. coelicolor (Perera and
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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.
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As shown in Fig. 2b, 1 nM PecS could bind to the biotin labeled pecO, pecO*. The
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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
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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 CG43S3lacZ and the activity of PpecS and PpecM
309
further increased in CG43S3lacZcpxARpecS. 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 CG43S3cpxAR or
313
CG43S3cpxARpecS increased when compared to that of CG43S3 (Fig. 3b).
314
Moreover, deletion of pecM from CG43S3cpxAR 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
CG43S3lacZ 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 CG43S3cpxAR.
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, CG43S3sodA and CG43S3yjcC 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 CG43S3constitutively 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
CG43S3cpxAR as well as CG43S3mrkA exerts apparent mannose sensitive yeast
357
agglutination corresponding to type 1fimbriae activity. Deletion of pecS from
358
CG43S3cpxAR further increases the mannose sensitive yeast agglutination activity,
359
while introducing the pecS gene to CG43S3cpxARpecS restores to the
360
agglutination level of CG43S3cpxAR. By contrast, either pecS or pecM deletion has
361
no apparent effect on the biofilm formation of CG43S3 or CG43S3cpxAR (Fig. 5b).
362
Moreover, CG43S3cpxARpecS exhibits an increase in FimA production when
363
compared with CG43S3cpxAR. The increase is no longer observed when the pecS
364
gene is introduced into CG43S3cpxARpecS. On the other hand, deletion of pecS
365
from CG43S3cpxAR 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 CG43S3cpxAR, and introduction of pecS back into CG43S3cpxARpecS
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 CG43S3cpxAR 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 CG43cpxAR 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 CG43S3cpxAR, 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 CG43S3cpxAR (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). Effects of urate, a natural inhibitor of
479
peroxynitrite-mediated toxicity, in the response of Arabidopsis thaliana to the
480
bacterial pathogen Pseudomonas syringae. Plant J. 25:529-540.
481
Averyanov A. (2009) Oxidative burst and plant disease resistance. Front Biosci.(Elite
482
Ed) 1:142-152.
483
Chuang YP, Fang CT, Lai SY, Chang SC, Wang JT. (2006). Genetic determinants
484
of capsular serotype K1 of Klebsiella pneumoniae causing primary pyogenic liver
485
abscess. J. Infect. Dis.193:645-654.
486
Corcoran CP, Dorman CJ. (2009). DNA relaxation-dependent phase biasing of the
487
fim genetic switch in Escherichia coli depends on the interplay of H-NS, IHF and
488
LRP. Mol. Microbiol. 74:1071-1082.
489
Di Martino P, Cafferini N, Joly B, Darfeuille-Michaud A. (2003). Klebsiella
490
pneumoniae type 3 pili facilitate adherence and biofilm formation on abiotic surfaces.
491
Res. Microbiol. 154:9-16.
492
Donato GM, Kawula TH. (1999). Phenotypic analysis of random hns mutations
29
493
differentiate DNA-binding activity from properties of fimA promoter inversion
494
modulation and bacterial motility. J. Bacteriol. 181:941-948.
495
Ellison DW, Miller VL. (2006). Regulation of virulence by members of the
496
MarR/SlyA family. Curr. Opin. Microbiol. 9:153-159.
497
Fouts DE, Tyler HL, DeBoy RT, Daugherty S, Ren Q, Badger JH, Durkin AS,
498
Huot H, Shrivastava S, Kothari S, et al. (2008). Complete genome sequence of the
499
N2-fixing broad host range endophyte Klebsiella pneumoniae 342 and virulence
500
predictions verified in mice. PLoS Genet. 4:e1000141.
501
Fung CP, Chang FY, Lee SC, Hu BS, Kuo BI, Liu CY, Ho M, Siu LK. (2002). A
502
global emerging disease of Klebsiella pneumoniae liver abscess: is serotype K1 an
503
important factor for complicated endophthalmitis? Gut 50:420-424.
504
Han SH. (1995). Review of hepatic abscess from Klebsiella pneumoniae. An
505
association with diabetes mellitus and septic endophthalmitis. West J. Med.
506
162:220-224.
507
Hommais F, Oger-Desfeux C, Van Gijsegem F, Castang S, Ligori S, Expert D,
508
Nasser W, Reverchon S. (2008). PecS is a global regulator of the symptomatic phase
509
in the phytopathogenic bacterium Erwinia chrysanthemi 3937. J. Bacteriol.
510
190:7508-7522.
511
Hornick DB, Allen BL, Horn MA, Clegg S. (1992). Adherence to respiratory
30
512
epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3
513
fimbrial gene products. Infect. Immun. 60:1577-1588.
514
Huang CJ, Wang ZC, Huang HY, Huang HD, Peng HL. (2013). YjcC, a
515
c-di-GMP phosphodiesterase protein, regulates the oxidative stress response and
516
virulence of Klebsiella pneumoniae CG43. PLoS One 8:e66740.
517
Huang H, Mackel BJ, Grove A. (2013). Streptomyces coelicolor encodes a
518
urate-responsive transcriptional regulator with homology to PecS from plant
519
pathogens. J. Bacteriol. 195:4954-4965.
520
Hunke S, Keller R, Muller VS. (2012). Signal integration by the Cpx-envelope
521
stress system. FEMS Microbiol. Lett. 326:12-22.
522
Jagnow J, Clegg S. (2003). Klebsiella pneumoniae MrkD-mediated biofilm
523
formation on extracellular matrix- and collagen-coated surfaces. Microbiology
524
149:2397-2405.
525
Johnson JG, Clegg S. (2010). Role of MrkJ, a phosphodiesterase, in type 3 fimbrial
526
expression and biofilm formation in Klebsiella pneumoniae. J. Bacteriol.
527
192:3944-3950.
528
Lai YC, Peng HL, Chang HY. (2003). RmpA2, an activator of capsule biosynthesis
529
in Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the
530
transcriptional level. J. Bacteriol. 185:788-800.
31
531
Liao YC, Huang TW, Chen FC, Charusanti P, Hong JS, Chang HY, Tsai SF,
532
Palsson BO, Hsiung CA. (2011). An experimentally validated genome-scale
533
metabolic reconstruction of Klebsiella pneumoniae MGH 78578, iYL1228. J.
534
Bacteriol. 193:1710-1717.
535
Lin CT, Huang YJ, Chu PH, Hsu JL, Huang CH, Peng HL. (2006). Identification
536
of an HptB-mediated multi-step phosphorelay in Pseudomonas aeruginosa PAO1.
537
Res. Microbiol. 157:169-175.
538
Liu J, Obi IR, Thanikkal EJ, Kieselbach T, Francis MS. (2011). Phosphorylated
539
CpxR restricts production of the RovA global regulator in Yersinia
540
pseudotuberculosis. PLoS One 6:e23314.
541
McClain MS, Blomfield IC, Eisenstein BI. (1991). Roles of fimB and fimE in
542
site-specific DNA inversion associated with phase variation of type 1 fimbriae in
543
Escherichia coli. J. Bacteriol. 173:5308-5314.
544
Mhedbi-Hajri N, Malfatti P, Pedron J, Gaubert S, Reverchon S, Van Gijsegem F.
545
(2011). PecS is an important player in the regulatory network governing the
546
coordinated expression of virulence genes during the interaction between Dickeya
547
dadantii 3937 and plants. Environ. Microbiol. 13:2901-2914.
548
Nuccio SP, Baumler AJ. (2007). Evolution of the chaperone/usher assembly
549
pathway: fimbrial classification goes Greek. Microbiol. Mol. Biol. Rev. 71:551-575.
32
550
O'Gara JP, Dorman CJ. (2000). Effects of local transcription and H-NS on
551
inversion of the fim switch of Escherichia coli. Mol. Microbiol. 36:457-466.
552
Perera IC, Grove A. (2011). MarR homologs with urate-binding signature. Protein
553
Sci. 20:621-629.
554
Perera IC, Grove A. (2010). Urate is a ligand for the transcriptional regulator PecS. J.
555
Mol. Biol. 402:539-551.
556
Pope JV, Teich DL, Clardy P, McGillicuddy DC. (2011). Klebsiella pneumoniae
557
liver abscess: an emerging problem in North America. J. Emerg. Med. 41:e103-105.
558
Praillet T, Reverchon S, Nasser W. (1997). Mutual control of the PecS/PecM couple,
559
two proteins regulating virulence-factor synthesis in Erwinia chrysanthemi. Mol.
560
Microbiol. 24:803-814.
561
Praillet T, Reverchon S, Robert-Baudouy J, Nasser W. (1997). The PecM protein
562
is necessary for the DNA-binding capacity of the PecS repressor, one of the regulators
563
of virulence-factor synthesis in Erwinia chrysanthemi. FEMS Microbiol. Lett.
564
154:265-270.
565
Reverchon S, Nasser W, Robert-Baudouy J. (1994). pecS: a locus controlling
566
pectinase, cellulase and blue pigment production in Erwinia chrysanthemi. Mol.
567
Microbiol. 11:1127-1139.
568
Rouanet C, Nasser W. (2001). The PecM protein of the phytopathogenic bacterium
33
569
Erwinia chrysanthemi, membrane topology and possible involvement in the efflux of
570
the blue pigment indigoidine. J. Mol. Microbiol. Biotechnol. 3:309-318.
571
Schelenz S, Bramham K, Goldsmith D. (2007). Septic arthritis due to extended
572
spectrum beta lactamase producing Klebsiella pneumoniae. Joint Bone Spine
573
74:275-278.
574
Schroll C, Barken KB, Krogfelt KA, Struve C. (2010). Role of type 1 and type 3
575
fimbriae in Klebsiella pneumoniae biofilm formation. BMC Microbiol. 10:179.
576
Skorupski K, Taylor RK. (1996). Positive selection vectors for allelic exchange.
577
Gene 169:47-52.
578
Stahlhut SG, Struve C, Krogfelt KA, Reisner A. (2012). Biofilm formation of
579
Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae.
580
FEMS Immunol. Med. Microbiol. 65:350-359.
581
Struve C, Bojer M, Krogfelt KA. (2009). Identification of a conserved chromosomal
582
region encoding Klebsiella pneumoniae type 1 and type 3 fimbriae and assessment of
583
the role of fimbriae in pathogenicity. Infect. Immun. 77:5016-5024.
584
Tang HL, Chiang MK, Liou WJ, Chen YT, Peng HL, Chiou CS, Liu KS, Lu MC,
585
Tung KC, Lai YC. (2010). Correlation between Klebsiella pneumoniae carrying
586
pLVPK-derived loci and abscess formation. Eur. J. Clin. Microbiol. Infect. Dis.
587
29:689-698.
34
588
Tarkkanen AM, Virkola R, Clegg S, Korhonen TK. (1997). Binding of the type 3
589
fimbriae of Klebsiella pneumoniae to human endothelial and urinary bladder cells.
590
Infect. Immun. 65:1546-1549.
591
Van Houdt R, Michiels CW. (2005). Role of bacterial cell surface structures in
592
Escherichia coli biofilm formation. Res. Microbiol. 156:626-633.
593
Vogt SL, Raivio TL. (2012). Just scratching the surface: an expanding view of the
594
Cpx envelope stress response. FEMS Microbiol. Lett. 326:2-11.
595
Wang ZC, Huang CJ, Huang YJ, Wu CC, Peng HL. (2013). FimK regulation on
596
the expression of type 1 fimbriae in Klebsiella pneumoniae CG43S3. Microbiology
597
159:1402-1415.
598
Wilkinson SP, Grove A. (2004). HucR, a novel uric acid-responsive member of the
599
MarR family of transcriptional regulators from Deinococcus radiodurans. J. Biol.
600
Chem. 279:51442-51450.
601
Wilksch JJ, Yang J, Clements A, Gabbe JL, Short KR, Cao H, Cavaliere R,
602
James CE, Whitchurch CB, Schembri MA, et al. (2011). MrkH, a novel
603
c-di-GMP-dependent transcriptional activator, controls Klebsiella pneumoniae
604
biofilm formation by regulating type 3 fimbriae expression. PLoS Pathog 7:e1002204.
605
Wu CC, Lin CT, Cheng WY, Huang CJ, Wang ZC, Peng HL. (2012).
606
Fur-dependent MrkHI regulation of type 3 fimbriae in Klebsiella pneumoniae CG43.
35
607
Microbiology 158:1045-1056.
608
Wu KM, Li LH, Yan JJ, Tsao N, Liao TL, Tsai HC, Fung CP, Chen HJ, Liu YM,
609
Wang JT, et al. (2009). Genome sequencing and comparative analysis of Klebsiella
610
pneumoniae NTUH-K2044, a strain causing liver abscess and meningitis. J. Bacteriol.
611
191:4492-4501.
612
Yamamoto K, Ishihama A. (2006). Characterization of copper-inducible promoters
613
regulated by CpxA/CpxR in Escherichia coli. Biosci. Biotechnol. Biochem.
614
70:1688-1695.
615
Zakataeva NP, Kutukova EA, Gronskii SV, Troshin PV, Livshits VA, Aleshin
616
VV. (2006). [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, CG43S3cpxAR,
645
CG43S3cpxARpecM or CG43S3cpxARpecS 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 CG43S3lacZ (left panel) or CG43S3lacZcpxAR
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 CG43S3cpxAR 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 CG43S3sodA and
663
CG43S3yjcC (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 CG43S3cpxAR 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 CG43S3cpxAR, CG43S3cpxARpecS or
674
CG43S3cpxARpecS::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.
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pneumoniae CG43S3cpxAR, CG43S3cpxARpecS and CG43S3cpxARpecS::
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pecS were assessed with qRT-PCR. (e) Increase hns expression reduces the FimA
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production as assessed using western blot analysis.
39
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Fig. 7. Urate effect on the expression of FimA. (a) Analysis of the urate effect on type
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1 fimbriae expression in K. pneumoniae CG43S3 using the analysis of mannose
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sensitive yeast agglutination (left panel) and Western blot (right panel) (b) Western
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blot analysis of the urate effect on the expression of PecS and FimA. (c) Analysis of
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the urate effect on the expression of FimA using qRT-PCR (left panel) and promoter
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activity assay (right panel). Bacteria were grown in the modified LB supplied without
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or with 0.2-5 mM urate at 37C with agitation.
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Fig. 8. Model of the CpxAR-dependent PecSM regulation on type 1 fimbriae
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expression. Upon stimulating by unknown external signal, the phosphorylated CpxR
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binds to the CpxR box in the intergenic region of pecS and pecM and inhibits their
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transcription. On the other hand, the negative regulation of CpxAR on type 3 fimbriae
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is possibly an indirect control through influencing the expression of the regulators
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MrkJ, MrkH or MrkI. Only in the presence of urates and the absence of cpxAR, the
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PecSM-mediated repression is released and type 1 fimbriae express. HNS is probably
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the mediator for the PecSM-dependent repression and urate-inducible expression of
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type 1 fimbriae.
698
699
40