克雷白氏肺炎桿菌CG43第三型線毛用於表面抗原呈現與其表現調控

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國立交通大學

生物科技研究所

碩士論文

克雷白氏肺炎桿菌CG43第三型線毛用於表面抗原呈現與

其表現調控

Application of type 3 fimbriae of Klebsiella pneumoniae

CG43 in epitope display and its expressional regulation

研究生: 楊其駿 (9528505) Student: Chi-Zyun Yang 指導教授: 彭慧玲博士 Adviser: Hwei-Ling Peng, Ph.D

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中文摘要

為了可以表現克雷白氏肺炎桿菌第三型線毛的重組質體 pmrkABCD 來呈列第二型登革熱病毒的套膜蛋白第三區塊 （DEN2EIII），首先，我們以此包含主要抗原決定位的核酸為模版，以聚合脢連鎖反應分別增幅可轉譯成約 20 個胺基酸片段長度的核 酸，再將這五段核酸序列分別插入 pmrkABCD 質體中 MrkA A25 或 D27 位置，再將選殖的重組質體包括 pA25-1，pD27-1，pD27-2， pD27-3，pD27-4，pD27-5 分別轉殖到大腸桿菌 JM109。接著，以西方墨點法分別以 MrkA 和 DEN2EDIII 多株抗體偵測的結果顯示：雖有不同程度的表現差異，這些重組質體皆可表現帶有 DEN2EDIII 抗原的重組線毛單體，而此重組的單體蛋白也可組裝成多體的線毛結構；免疫螢光顯微鏡檢分析也確認這些重組的線毛可表現在細菌表面。初步純化結果顯示線毛表現的培養條件仍需改進，只有提升線毛量作為將來免疫小鼠的疫苗來源，才能進一步評估其免疫效果。大多數的克雷白氏肺炎桿菌臨床分離株帶有可轉錄第三型線毛的基因組，顯示此線毛在克雷白氏肺炎桿菌附著宿主細胞造成感染過程中可能扮演重要的角色。至今，細菌如何調控第三型線毛的表現，仍不清楚。之前的實驗結果顯示，破壞可以調控細菌莢膜產生的調控 基因 rcsB，會增加第一型纖毛的表現，相反的會降低 MrkA 蛋白的表

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現。我們以帶有 PmrkA-LacZ 報導系統的 CG43S3Z01 的 rcsB 缺損株為

跳躍子接受株，以 X-Gal 培養盤篩選顏色變化，在大約 70000 突變株中挑出八株會改變顏色的突變珠；其中 6 株會增加 LacZ 活性，2 株會降低 LacZ 活性。核酸定序分析結果顯示兩株增加 LacZ 活性的插 入點分別是 glycosidase 和 lacI 基因，此二基因與 LacZ 活性調控有關， 與 MrkA 蛋白調控無關。而兩株降低 LacZ 活性的插入點分別是 carbon starvation protein A 和會受到滲透壓影響的 yehZ 基因，此結果確認此 系統的選擇效果，但是跳躍子突變效率仍需提高。

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Abstract

We report the use of the type 3 fimbriae, encoded by mrkABCD, from Klebsiella pneumoniae CG43 to display the domain III of envelope protein (E) from type 2 dengue virus, DEN2EDIII. The DNA containing the DEN2EDIII, which has been shown as a major antigenic determinant, was used as a template for PCR amplification to generate 5 DNA

segments (~60 bp) encoding different parts of the DEN2EDIII. The PCR products were cloned into the A25 or D27 site on MrkA reside in the plasmid pmrkABCD. The resulting plasmids including pA25-1, pD27-1, pD27-2, pD27-3, pD27-4 and pD27-5 were then transformed

separatelyinto E. coli JM109. The analysis of these recombinant type 3 fimbriae assessed with western blotting hybridization against anti-MrkA or anti-DEN2EDIII antibody confirmed the expression of the

recombinant proteins, although with different levels. And the

recombinant subunits could assemble into polymeric fimbriae from monomers. The analysis of immunofluorescence microscopy using

anti-MrkA or anti-DEN2EDIII antibody also demonstrated the expression of the recombinant type 3 fimbriae on the surface of the transformed bacteria. However, culture condition and purification method have to be improved to obtain sufficient amount of the recombinant fimbriae. The subsequent use as the vaccine antigen for mouse immunization and the efficacy could then be evaluated.

The fact that most Klebsiella pneumoniae clinical isolates carry type 3 fimbriae gene clusters and hence play an important role as a major adhesin for the bacterial attachment to the host cell to establish an

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infection has been speculated. Nevertheless, how the expression of the type 3 fimbriae controlled remains unknown. Herein, a Tn5 mediated mutagenesis was employed to K. pneumoniae CG43Z01rcsB- strain

carrying Pmrk-lacZ. Eight out of approximately 70000 Tn5-insertion mutants

showed alteration of LacZ activity and were isolated. Six mutants

exhibited an increased LacZ activity and two mutants carried a decreased LacZ activity. After the inserted were regions isolated and the sequences determined, it was revealed that the disrupted genes include the gene encoding a putative glycosidase and lacI for the mutants with increasing LacZ activity, and the gene encoding carbon starvation protein and an osmolarity-inducible protein YehZ. The results demonstrated the

feasibility of the reporter-selection and the efficiency of Tn-mutagenesis have to be improved, however.

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v 致謝在這不連續的兩年當中，感謝彭老師的耐心教導。在我有瓶頸時，不厭其煩地幫助。不論是實驗上的問題，抑或是寫論文做簡報，老師也都給了我許多的建議。這些幫助，都讓我在往後的研究生涯中能有很大的助益。也很感謝老師在我出國回來後的繼續教導。我也感謝丸子學姐一路上耐心的指導我，並且提供我許多建議與幫助，讓我可以順利地進行我的研究。健誠學長、小新學長與靜柔學姐也都幫忙我注意實驗上、報告上以及生活上的小細節。哲充也常與我討論實驗，讓我發現我沒注意到的地方，一些需要體力的事也常常麻煩他。佩君與家華的開朗，為苦悶的實驗生活帶來很多的歡樂。她們也常幫忙注意提醒我，讓我不至因為我的不小心的個性而耽誤。志桓、雅雯、顗峰、小珊、還有朝陽學長也都幫忙我很多。豪君、品瑄以及崴云也實驗生活提供不少樂趣。我也感謝我的父母一直以來不斷守護我，忍受我的固執與任性。還有許許多多的人在我生活上不斷鼓勵我、幫助我、關心我。我很珍惜這一段日子。

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Abbreviation

cfu Colony-forming unit

DENIIDIII Dengue virus serotype II domain III

ELISA The Enzyme-Linked ImmunoSorbent Assay FITC Fluorescein isothiocyanate

g The acceleration due to gravity at the earth’s surface IgG Immunoglobulin G kb Kilobase(s) kDa Kilodalton(s) g Microgram ml Milliliter l Microliter mm Millimeter mM Millimolar M Micromolar

MR/K Mannose resistance Klebsiella-like hemagglutinins nm Nanometer

OD Optical Density

PAGE Polyacrylamide gel electrophoresis PBS Phosphate-Buffer saline

PCR Polymerase chain reaction SDS Sodium dodecyl sulfate

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Introduction

Klebsiellap neumoniae, an Enterobacteriaceae gram negative bacterium, is recognized as an important opportunistic pathogen that attacks immunocompromised patients. It is a common cause of urinary tract infections, respiratory tract infections and septicaemia especially in immunocompromised individuals who are hospitalized and suffer from severe underlying diseases, such as chronic pulmonary obstruction or diabetes mellitus (Chen, Hsueh et al. 2000). In recent years, the cases of diabetes mellitus patients that suffer from liver abscess (LA) caused by highly virulent K. pneumoniae strains have increasingly occurred.

Several virulence factors of K. pneuniornae have been identified, including acidic polysaccharide capsule (CPS), lipopolysaccharides, iron acquisition systems and several distinct types of adherence factors (Nassif and Sansonetti 1986). The virulence traits for KP-PLA (pyogenic liver abscess)have recently been reported, which include K1, K2, magA, rmpA (regulator of mucoid phenotype), uge (UDP-glucose4-epimerase), wabG, and iron acquisition systems encoded by iuc and iro, kfu (an iron uptake system), and TonB (Izquierdo, Coderch et al. 2003; Regue, Hita et al. 2004; Hsieh, Lin et al. 2008). The capsular serotype K1 or K2 has been shown to be more prevalent in strains causing liver abscess than the ones causing bacteremia(Yeh, Kurup et al. 2007). The magA gene located in the K1 cps gene cluster has been reported in 98.1% to 83.3% of K. pneumoniae strains isolated from patients with LA and was significantly more prevalent than the bacteremic strains. Mutations resulting the loss of K1 CPS became avirulent in mouse model (Chuang, Fang et al. 2006).

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The prevalence of rmpA in LA strains was also higher than that in bacteremic strains. The iron uptake system encoding genes have been shown to be highly up-regulated in response to sera. Nevertheless, mutation in all of the 3 loci (irp2, iuc, and iroA) is necessary to decrease the virulence (Huang, Liao et al. 2009). The tonB mutant has been shown to be a potential vaccine candidate because it can induce a significant protective immune response (Huang, Liao et al. 2009).

Adherence factors which enable bacterial binding to specific host cells play important roles in establishing an infection. Fimbriae, one of the adherence factors, are long, thread-like appendages on bacterial surface. They are found in as many as 500 copies per cell (Klemm and Schembri 2000). Each fimbrial fiber is a polymer composed of hundreds of structural subunits called pilin. In the infection process, the adhesin which is located on the tip of fimbriae determines the specific binding to the host cell.

Most clinical K. pneumoniae isolates express two types of fimbrial adhesins, type 1 and type 3 fimbriae (Gerlach, Clegg et al. 1989). Type 1 fimbriae found in a majority of enterobacterial species are about 7 nm wide and 1 m long. Activity of type 1 fimbriae was determined by their ability to mediate mannose-sensitive agglutination of guinea pig or fowl erythrocytes, and hence referred as mannose-sensitive hemagglutinins (MSHA). They have been shown to be required for bacterial attachment to mannose units of the glycoprotein receptor on the surface of urinary epithelium cells(Buchanan, Falkow et al. 1985). A number of studies

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have shown that type 1 fimbriae play a significant role in the ability of Escherichia coli infection of the urinary tract(Connell, Agace et al. 1996). Recently, type 1 fimbriae were also established as an important virulence factor in K. pneumoniae urinary tract infection (Struve, Bojer et al. 2009).

Type 1 fimbriae, expressed by fimACDFGHIK gene cluster, are composed primarily of the structural subunit FimA, with minor amounts of three ancillary subunits, FimF, FimG, and the mannose-specific

adhesin FimH. FimH adhesin is an allosteric protein that mediates the catch bond mechanism of adhesion where the binding is increased under increased shear stress. The fimC and fimD genes respectively encode a fimbrial chaperone and usher protein.The function of fimI gene product is unknown, but this product has been found to be essential for type 1

fimbria biosynthesis in E. coli (Valenski, Harris et al. 2003). The fimK gene located directly downstream of fimH is only present in K.

pneumoniae but not in E. coli. The fimK gene product has previously been shown to be involved in type 1 fimbria expression (Rosen, Pinkner et al. 2008). Regulation of the type 1 fimbria expression in E. coli is very complex, and several regulatory factors that act by altering the expression of fimB and fimE have been described. The recombinases FimB and FimE regulate the phase switch of type 1 fimbriae. FimE turns the switch from “ON” position to “OFF” position, while FimB can turn the switch in either direction(Klemm 1986).

Type 3 fimbriae are 2 to 4 nm wide and 0.5 to 2 m long fimbriae (Hornick, Thommandru et al. 1995). They are characterized by their

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ability to agglutinate tannic acid treated, but not native, erythrocytes in a mannose resistant manner, and hence they are referred as mannose resistant Klebsiella-like (MR/K) hemagglutinin (Duguid 1959; Gerlach, Clegg et al. 1989). Besides Klebsiella species, type 3 fimbriae are common in the isolates of Enterobacter, Serratia, Proteus and

Providencia (Old and Scott 1981; Old and Adegbola 1982; Korhonen, Tarkka et al. 1983; Buchanan, Falkow et al. 1985; Old and Adegbola 1985). Historically, type 3 fimbriae have not been associated with E. coil; however, two recent independent studies have reported the expression of type 3 fimbriae in E. coil strains (Ong, Ulett et al. 2008). In vitro studies have revealed that type 3 fimbriae mediate adhesion to different

structures in human kidney and lung tissue, epithelial cells from human urine sediments as well as endothelial and bladder epithelial cell lines (Struve, Bojer et al. 2009). Furthermore, type 3 fimbriae of K.

pneunioniae have been shown to influence the biofilm formation (Struve, Bojer et al. 2009). Their involvement of biofilm formation and binding to different host structures suggested an important role in determining

bacterial virulence.

Type 3 fimbriae are encoded by the mrk gene cluster, which includes the mrkA gene encoding the major fimbrial subunit and mrkD encoding the fimbrial adhesin responsible for MR/K hemagglutination. It has been reported that the N terminal domain of MrkD adhesin is responsible for receptor binding (Tarkkanen, Allen et al. 1990). MrkD adhesin has also been shown to mediate adhesion to collagen; however, the exact identity of the MrkD receptor remains elusive (Hornick, Thommandru et al. 1995).

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MrkC is an outer membrane usher protein, which anchors the fimbriae to the bacterial cell surface. MrkB, a periplasmic chaperone, acts to stabilize fimbrial subunits and carries them to the outer membrane usher

protein(Choudhury, Thompson et al. 1999). Downstream of mrkD is mrkF of which the encoding product has been reported to affect stability of the intact fimbrial appendages on bacterial surface (Huang, Liao et al. 2009).

Assembly of type 3 fimbriae is via the chaperone-usher pathway, in which the chaperone-mediated extraction of subunits from the inner cytoplasmic membrane (IM) is coupled with their folding into an assembly-competent state(Sauer, Barnhart et al. 2000). The

immunoglobulin-like chaperones protect the nascent-folded subunits from premature oligomerization in the periplasmic space by directly capping. These preassembly complex protected by the chaperone was then

delivered to the outer membrane (OM) assembly site that is comprised of the usher.

Analysis of the gene loci upstream of the mrkABCDF revealed genes homologous to pecM and pecS, which encode regulators for the

expression of virulence genes in Erwinia chrysanthemi (Reverchon, Nasser et al. 1994). As shown in Fig. 1, the two divergently transcribed genes were named phgM and phgS respectively (黃盈蓉 2006). The genes downstream to the mrkABCEF are genes KP4551, KP4552, and KP4554 encoding c-di-GMP binding domain protein (PilZ domain protein), transcription factor with an HTH domain, and c-di-GMP

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phosphodiesterase (EAL) domain protein (Fig. 2), respectively. Whether the regulators are involved in the control of the expression of type 3 fimbriae remains unknown.

Fimbriae which expressed on bacterial surface have been evaluated as a target protein display system for medicine selection or vaccine development. Fimbriae are particularly attractive candidates for epitope expression and several of the properties also hold promise for the

feasibility of vaccine development: first, fimbriae are present in extremely high numbers at the cell surface; second, they are as strong immunogens, and thirdly, they can be easily purified(Connell, Agace et al. 1996). Until now, hepatitis B virus and foot-and-mouth disease virus epitopes have been successfully expressed on type 1 fimbriae and P pili of E. coli (Hedegaard and Klemm 1989; Bakker, van Zijderveld et al. 1990).

Dengue virus (DV) is an arthropod-borne human pathogen that causes a serious public health threat in tropical and subtropical regions of the world. DV has four serotypes (DEN-1 to DEN-4) that cause diseases ranging from mild dengue fever to severe symptoms such as dengue hemorrhagic fever and dengue shock syndrome (Chambers, Hahn et al. 1990). The virion of DEN contains three structural proteins: a 12 kDa nucleocapsid or core protein (C), 8 kDa membrane protein (M), and 53 kDa envelope protein (E), as well as seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The X-ray structures reveal that the E protein is composed of three domains, domain I, II, and III. The

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domain III, an immunoglobin(Ig)-like domain, is considered as a location that bind with host cell receptor(Konishi, Kosugi et al. 2006). The recent report employing the domain III-specific monoclonal antibody has

demonstrated a neutralization activity to block the viral infection(Modis, Ogata et al. 2005).

In this thesis, two studies are carried out. The first one is to “Set up a DEN2-EIII display system using type 3 fimbriae”. We plan to generate recombinant type 3 fimbriae by introducing DEN2-EIII amino acid sequences to MrkA pilin and then purify the recombinant fimbriae to immunize mouse to evaluate the possibility as vaccine antigen. The

second aim is to “Identify possible regulator(s) involved in controlling the expression of type 3 fimbriae”. We use Tn5 transposon, which can

randomly insert into to the chromosome of the K. pneumoniae harboring the PmrkA-LacZ reporter plasmid (Fig. 3). The assay takes advantage of the

fact that-galactosidase activity could be monitored by the changes of white/blue color on the selective medium containing X-gal.

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Materials and Methods

Strains, plasmids and growth conditions

The bacteria strains, plasmids and primers used in this study are listed in Tables 1, 2, and 3. Bacteria were incubated at 37°Cin Luria Broth (LB) medium or the medium supplemented with antibiotic ampicillin (100 g/ml), kanamycin (25 g/ml) or chloramphinicol (35

g/ml). The bacterial growth was determined by measuring the optical density at 600 nm (OD600).

Construction of the recombinant plasmids

The recombinant plasmids pD27-1, pD27-2, pD27-3, pD27-4, pD27-5, and pA25-2 clones were constructed by PCR cloning using pGEM-T Easy-D2E as the template, and the 5 amplified fragments cloned respectively into yT&A cloning vector. The PmeI was then used to cleave the clone and the PmeI fragment, containing the target DNA was respectively subcloned into A25 and D27 site of pMrkABCD (徐幸瑜 2007).

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Bacteria were incubated at 37°C in Luria Broth (LB) overnight and collected by centrifugation for 1 min (8,000 rpm). The pellet was

resuspended in 150 l protein lysis buffer (50 mM Tris-HC1 pH 8.0, 1 mM EDTA, 100 mM NaC1), added with 15 l of 4% SDS loading dye (100 mM Tris-HCl pH 8.0, 4% SDS, 0.2% bromophenol blue, 200 mM

-mercaptoethanol, 20% glycerol), and then heated at 95°C for 30 min. The cell extracts were resolved using 13.5% sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Protein expression for fimbriae polymer pattern

Bacteria were incubated at 37°C in Luria Broth (LB) overnight and collected by centrifugation for 1 min (8,000 rpm). The pellet was

resuspended in 150 l protein lysis buffer (50 mM Tris-HC1 pH 8.0, 1 mM EDTA, 100 mM NaC1), added with 15 l of 4% SDS loading dye (100 mM Tris-HCl pH 8.0, 4% SDS, 0.2% bromophenol blue, 200 mM

-mercaptoethanol, 20% glycerol). The cell extracts were resolved using 13.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE).

Purification of type 3 fimbriae

The fimbriae purification process was carried out with a minor modification of the described methods (Feutrier, Kay et al. 1986;

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Girardeau, Der Vartanian et al. 1988; Sakellaris, Balding et al. 1996; Zalewska, Piatek et al. 2003). The recombinant E. coli JM109 [D27], E. coli JM109 [D27-1], E. coli JM109 [D27-2], E. coli JM109 [D27-3], E. coli JM109 [D27-4], E. coli JM109 [D27-5], E. coli JM109 [A29-2], and JM109[pmrkABCD] were cultured at 37°Cfor 24 h in 24 ml LB broth, and the bacteria were collected by centrifugation for 3 min (8,000 rpm, 4°C) and were suspended in 1 ml phosphate-buffered saline (PBS). The bacterial suspension was heated at 65°C for 2 h and homogenized in blender for 20 min at ambient temperature. Finally, 30% saturated ammonium sulfate at 4°C was added and the mixture stored at 4°C

overnight. The fimbriae containing pellet was collected by centrifugation (20,000 g, 90 min) and suspended in 40 l PBS.

Western blotting analysis

The fimbrial proteins were resolved by 13.5% (for fimbriae

monomer pattern) or 8% (for fimbriae polymer pattern) SDS-PAGE, and electrophoretically transferred to polyvinylidene difluoride membranes (ImmobilonTM-P, Millipore). Subsequently, the membranes were soaked in 5% skim milk in 1 x phosphate-buffered saline (PBS) at 4°C overnight. After washing 3 times with 1 x PBS, the membrane was incubated with the antiserum of a 10000-fold diluted anti-MrkA or 5000-fold diluted anti-DEN2EDIII at room temperature for 2 h. Followed by incubation with a 5000-fold diluted alkaline phosphatase-conjugated anti-rabbit immunoglobulin at room temperature for 2 h, three washes were applied

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and the bound antibodies were detected by using the chromogenic

reagents BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (Nitro blue tetrazolium).

Immunofluorescence microscopy

Bacteria cultured in LB at 37°C for 16 h were collected, washed twice by phosphate buffer saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3), and were dissolved in 1 ml of sterile water. Ten microliters of the bacterialsuspension were loaded on glass slide and the bacteria thermally-fixed. Subsequently, an appropriate concentration of diluted antibody (1/300 of anti-MrkA or 1/5 dilution of anti-DEN2DIII) was added and incubated at room temperature 1 h, and then washed twice by PBS. An appropriate dilution of secondary

antibody (1/100 of the FITC conjugated anti-rabbit IgG) was then added and the slide incubated at room temperature for 1 h in dark. After washed twice with PBS, a drop of 10% glycerol phosphate buffer was added on the slide. Finally, the slide was covered with coverslip, fixed with nail polish, and placed under the fluorescent microscope for observation.

Preparation of genomic DNA

The bacteria cultured at 37°C in LB overnight were collected by centrifugation for 3 min (8,000 rpm). The pellet was resuspended in 800

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EDTA, 40 mM Tris-HCl pH 8.0, 0.2% Triton X-100) and heated at 37℃ for 30 min. Proteinase K solution 100 g/ml was then added and heated at 50°C overnight. The solution was put on ice for 10 min and 250l saturated NaCl solution was added, and the carefully mixed by shaking for 10 min. After centrifugation for 20 min (13000 rpm), 500 l of the supernatant was added with 1000 l 99% alcohol in an eppendorf tube. The precipitate collected by centrifugation for 10 min (13000 rpm). After gently rinsed with 75% alcohol, the pellet was dried and resuspended in sterile water.

Transposon Mutagenesis

Transposon mutagenesis with a mini-Tn5(Km) was performed by biparental mating (de Lorenzo, Herrero et al. 1990). The recipient (K. pneumoniae CG43Z01 rcsB- [P-mrkA-LacZ]), donor (E. coli S17-1 λ pir

[pUTmini-Tn5Km2]), were grown overnight in LB with the appropriate antibiotics. After incubation of the recipient and donor at 37°C for 30 min, 0.5 ml of the recipient was mixed with 1 ml of the donor. Cells were

collected by centrifugation, suspended in 100 l of saline solution, and spotted on an NC membrane on LB plate. After 16 h incubation at 37°C, cells were scraped off the NC membrane and resuspended in 5 ml of LB, and serial dilutions were plated on selective minimal medium, which was M9 with kanamycin and chloramphenicol.

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In order to analyze the Tn-insertion sites, chromosomal DNA of the mutants containing the mini-Tn5 Km2 were isolated and then subjected to restrict enzyme PstI digestion. The restricted fragments were then ligated to the PstI site of pUC19 and the ligation mixture used to transform to E. coli JM109. The transformants were selected using the LB plates

supplemented with both kanamycin and ampicillin. Finally, the

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Results

Part I

Setup a DEN2EDIII display system using type 3 fimbriae

1. Construction of the DEN2EDIII-MrkA clones

It has been shown that the insertion of a small peptide in the residue A25 or D27 of MrkA (Fig.4) had no apparent effect on the expression of the recombinant fimbriae (徐幸瑜 2007). Whether the insertion of

DEN2EDIII affects the expression of the fimbriae was investigated. The recent report employing the monoclonal antibody specific to DEN2E domain III has demonstrated a neutralization activity(Abd-Jamil, Cheah et al. 2008). In order to use type 3 fimbriae as an expression

system for the vaccine development, selection of an epitope-containing peptide is important. It is hence the DEN2EDIII was selected as a foreign sequence to be displayed by the type 3 fimbriae expression system which has previously been set up in our laboratory (徐幸瑜 2007).

It has been reported that the major subunit of type 1 fimbriae can accommodate the size of the protein fragments ranging between 10-30 amino acids(Klemm and Schembri 2000). As shown in Fig. 5A, the ~ 100 aa DEN2EDIII divided into 5 fragments of about 20 amino acids (Fig. 5B) was cloned by PCR. The PCR products were cloned into yt&A, and then subcloned using the restriction enzyme PmeI to the plasmid pA25 and pD27, respectively. Six recombinant plasmids were obtained and named

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pA25-2, pD27-1, pD27-2, pD27-3, pD27-4 and pD27-5.

As shown in Table 4, sequence analysis of these recombinant clones revealed additional amino sequences, which derived from the in-frame construct on the designed primer sequences added to the DEN2EDIII fragments. The resulting clones A25-2, D27-1and D27-2 contained 29 aa; 28 aa for D27-3; 30 aa for D27-4 and D27-5. Notably, the D27-3

contained some alterations of the DEN2EDIII fragment 3 sequence.

2. Assessments of the expression of the recombinant DEN2EDIII-MrkA fimbriae

In order to investigate whether the insertion of DEN2EDIII fragments affect the expression of the fimbriae, total lysates from the recombinant E. coli JM109 containing A25-2, D27-1, D27-2, D27-3, D27-4 or D27-5 were collected and the proteins analyzed by SDS-PAGE and western blotting analysis. Since the recombinant MrkA of the

displayed vector pD27 carries 5 extra aa (徐幸瑜 2007), higher

molecular weight than ~ 23 kDa of the MrkA was expected (lanes 2 and 3 of Fig. 6A). Except the recombinant protein of D27-4, the molecular weight of the other recombinant MrkA also appeared to be in larger size than the native MrkA. As shown in Fig. 6A, extra band with

approximately 2 kDa larger, which is probably corresponding to the signal peptide-containing recombinant MrkA, were observed.

Expression of the recombinant MrkA was subsequently demonstrated by western blotting analysis with anti-MrkA antibody. As shown in Fig.

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6B, the arrowhead-labeled bands in Fig. 6A could be hybridized with the anti-MrkA antibody indicating expression of the recombinant MrkA. The insertion of the DEN2EDIII fragments had no apparent effect on the

expression.Whether the DEN2EDIII fragments were properly expressed was also investigated. As shown in Fig. 6C, western blotting analysis using anti-DEN2EDIII antibody showed only the recombinant clones carrying MrkA-DEN2EDIII could be detected using anti- DEN2EDIII antibody.The detection for D27-3 and D27-5 (lanes 7 and 9, respectively) exhibited higher intensity than those of the others implying a high level expression of the DEN2EDIII fragments.

3. Analysis of the expression of the recombinant fimbriae

Polymer pattern analysis for the recombinant clones was carried out to study if the recombinant DEN2EDIII-MrkA monomer could be

assembled properly onto the bacterial surface. As shown in Fig. 7A, approximately same amount of the total proteins were resolved on the SDS-polyacryamide gel without heat treatment.The western blotting analysis with anti-MrkA antibody revealed different levels of polymer formation, and the polymeric fimbriae of D27-4 appeared to be better formed than the others (Fig. 7B).

4. Analysis of expression of the recombinant fimbriae with IFM

IFM analysisprobed with anti-MrkA or anti-DEN2EDIII antibody was also applied to confirm the expression of the recombinant fimbriae

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on the bacterial surface. As shown in Fig.8A, all the recombinant fimbriae carrying with the recombinant MrkA could be observed indicating

expression on the cell surface. The analysis using anti-DEN2EDIII as shown in Fig. 8B, expression of the recombinant fimbriae was confirmed. Although the inserted DEN2EDIII fragments contain only about 25

amino acids, the florescent signals were apparent implying proper expression of the DEN2EDIII peptides.

5. Purification of the recombinant fimbriae

In order to use the fimbriae to induce effective animal immune

reaction, a quick and simple purification method is essential. To avoid the process of ultracentrifugation, we’ve tried to modify the purification

procedures according to the methods (Feutrier, Kay et al. 1986; Girardeau, Der Vartanian et al. 1988; Sakellaris, Balding et al. 1996; Zalewska,

Piatek et al. 2003). Analysis of the purified fimbriae as shown in Fig. 9A revealed that most of the recombinant fimbriae were in short oligomeric form. The western blotting analysis indicated that the purified

recombinant fimbriae from the D27-4 strain had similar polymeric pattern, although with lower amount, as those from pmrkABCE or pD27-4 (Fig. 9B). The barely detectable bandings of the other recombinant fimbriae indicated a poor purification result.

Part II

Identification of the regulator(s) involved in controlling the expression of type 3 fimbriae

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RcsB is a two-component regulator of capsule synthesis (Stout and Gottesman 1990). It has been recently reported in E. coli that the rcsB deletion decreased the expression of type 1 fimbriae suggesting a positive regulatory role for the expression of type 1 fimbriae (Schwan, Shibata et al. 2007). In contrast, our laboratory has shown that the deletion of rcsB in K. pneumoniae CG43 increased the expression of type 1 fimbriae. However, the expression of MrkA, the major subunit of type 3 fimbriae was reduced (data not shown). Since the regulatory system for the expression of type 3 fimbriae remains unknown, we intend to apply transponson mutagenesis onto the rcsB deletion mutant for a possible suppression of the phenotype. To facilitate the screening process, the promoter reporter construct was employed. It is hence total proteins isolated from K. pneumoniae CG43S3Z01 and CG43S3Z01rcsB-, and CG43S3Z01rcsB-[pLacZ15] and CG43S3Z01rcsB-[pPmrkA-lacZ] were

comparatively analyzed to confirm the rcsB deleting effect. As shown in Fig. 10A, approximately similar amount of the proteins were isolated. The subsequent western blotting analysis using anti-MrkA antibody showed that the rcsB deletion indeed reduced the expression of MrkA of CG43S3Z01rcsB- or CG43S3Z01rcsB-[pPmrkA-lacZ].

1. Screen for the mutants carrying alteration of MrkA promoter activity

As shown in Fig.3, Tn5 mobilized from E. coli to K. pneumoniae CG43S3Z01 rcsB-[pPmrkA-lacZ] and the transconjugants were selected on

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X-gal containing M9-Km plates. Approximately 70,000 mutants were picked onto LB-X-gal plate to further clarify the changes of the colony color. Finally, eight mutants with apparent color changes, two white and six deep blue, were isolated and named M1~M8 individually.

2. Analysis of the transposon insertion site

In order to determine the inserted sequences, chromosomes of the eight mutant were isolated and completely digested by restriction enzyme PstI. As shown in Fig. 3, there is a PstI site outside the transposon kanamycin-resistant gene and hence the nucleotides of K. pneumoniae next to the kanamycin-resistant gene could be included with PstI digestion. The M4 strain grew poorly on the kanamycin/ chloramphinicol- containing medium and hence was not included for the following study. The PstI-digested fragments were cloned into vector pUC19, and the transformants selected on Knanmycin containing plates. As shown in Fig11B, except M3, six recombinant clones namely pM1, pM2, pM5, pM6, pM7, and pM8 were obtained and further confirmed by PstI digestion. After subjected to more insertion enzymes digestion, the restriction pattern of pM1 and pM6 appeared to be identical (Fig. 11C). Finally, the clones were sent to commercial service for sequence determination using the primers M13R and M13F.

The Blast tool (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) was then applied to analyze the determined sequences and the inserted genes by the transposon include a putative glycosidase encoding gene (Fig. 12A), lacI (Fig. 12B), gene encodes RelE/ParE family or carbon

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starvation protein A (Fig. 12C), and yehZ gene (Fig. 12D). YehZ is homologous to OsmF, an osmotically inducible protein (Checroun and Gutierrez 2004).

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Discussion

Part I

Setup a DEN2EDIII display system using the recombinant type 3 fimbriae

Analysis of monomeric form of the recombinant MrkA indicated that the insertion of about 30 amino acids of the DEN2EDIII fragments had no apparent influence on the expression of MrkA. However, unexpected protein size of D27-3 (Fig. 6A, lane 7) and D27-4 (Fig. 6A, lane 8) were found. Analysis of pD27-3 revealed that two DEN2EDIII fragment 3 were inserted into MrkA and hence size of the recombinant MrkA was the largest. This also suggested that the insertion sequence could be up to 47 aa with no apparent effect on the expression of the recombinant MrkA. On the other hand, the insertion of part 4 of DEN2EDIII may alter the structure of MrkA and hence resulted in a smaller protein in D27-4 (lane 8 in Fig. 6A).

The upper band of the two major bands in Fig. 6A is speculated as the one still carrying the signal peptide as a cytosolic form. As shown in the lane 2 and lane 8 in Fig 6A, one major band was observed indicating that most of the MrkA had been transported out to the periplasmic space. The differential transported level is probably due to different exposure of the signal peptide on the recombinant MrkA to be recognized by the signal peptidase.

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antibody, the polymer pattern analysis revealed only the recombinant fimbriae D27-4 could be comparable with that of the wild type carrying pmrkABCD. This is probably due to the smallest size of the monomeric form of D27-4 could be better assembly into polymeric form. As shown in Fig.8, the better detection of D27-3 and D27-5 by IFM analysis indicated the two recombinant epitopes are properly exposed to be detected by anti-DEN2EDIII antibody.

The previous study has shown that the recombinant type 3 fimbriae inserted PERV (Porcine endogenous retrovirus) envelope amino acid sequence can inducePERV envelope antibody in mouse (陳欣瑜 2008). We’ve expected that the purified recombinant fimbriae can induce immune reaction in animals as well. However, the purified fimbriae, even from D27-4 (lane 8 in Fig.9A) with the largest amount, were not enough for the following immunization study. Since the purified fimbriae appeared to be good, alternative way such as improving the cultured condition for stable fimbrial expression might be helpful to obtain enough amounts of the fimbriae for immunization.

Part II

Identification of the regulator(s) involved in controlling the expression of type 3 fimbriae

Fimbriae are an important pathogenic factor for bacteria to attach to the host cell. Regulation to control the expression of the fimbriae is conceivably important. Herein, the random mutagenesis employed had identified several genes which may have a role for the expression of type

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3 fimbriae. The analysis of transposon insertion site of M2 mutant revealed a putative glycosidase. Glycosidase (also called Glycoside hydrolase), catalyzes the hydrolysis of the glycosidic linkage to generate two smaller sugars, has been found in essentially all domains of life. In bacteria and prokaryotes, glycosidase are found both as intracellular and extracellular enzymes that are largely involved in nutrient acquisition (Dwek 1996). The enzyme beta-galactosidase (LacZ) is one of the important occurrences of glycoside hydrolases in bacteria. The white colony of M2 mutant could be resulted from the disruption of the glycosidase gene and hence affected the bacterial digestion of X-gal. The insertion site of M5 was mapped to lacI gene. This could be easily explained by the inability of LacI to repress the LacZ activity and hence a deep blue colony was observed.

Since the available sequences of the M7 clone contained only K. pneumonia genes, the transposon insertion site was speculated. As shown in Fig. 12C, the disrupted genes could be the gene encoding plasmid stabilization system protein of RelE/ParE family or carbon starvation protein A, CstA. RelE/ParE is a family of TA systems, which typically consist of pairs of genes: one for a stable toxin that can cause cell death by disrupting an essential cellular process and the other for a labile antitoxin that can bind the toxin and block activity of the toxin (Anantharaman and Aravind 2003; Engelberg-Kulka, Amitai et al. 2006). The deletion of TA systems has been shown to decrease biofilm formation initially (8 h) on three different surfaces and then increased biofilm formation (24 h) by decreasing biofilm dispersal (Kim, Wang et al. 2009).

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CstA is involved in peptide transport that would assist the cell in escaping carbon starvation (Schultz and Matin 1991). If disruption of the relE/parE gene or cstA could influence the MrkA expression remains to be investigated.

The disrupted gene of M8 is yehZ gene which is located within yehZYXW operon and coding for a putative glycine/betaine/choline transport protein (ABC superfamily). The yehZYXW encoded ABC transporter as an additional element of the global stress response controlled by sigma(s) (Checroun and Gutierrez 2004). The analysis of a yehZ-lacZ transcriptional fusion demonstrated that yehZ is inducible not only by osmolarity, but also upon entry into stationary phase. CstA and YehZ both are related with membrane transporters. Whether they have influences on MrkA expression awaits to be clarified.

While sequencing the other mutant clones, transposase sequence has been identified indicating some unexpected recombination events had occurred. How to improve the transposon delivery system has to be taken into consideration.

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Table 1. Bacteria strains used in this study

Strains Genotypes or relevant properties Reference or source

E. coli

JM109 RecA1 supE44 endA1 hsdR17 gyrA96 RelA1 thiΔ(lac-proAB)

Laboratory stock

S17-1 λ pir Tpr Smr recA, thi, pro,

hsdR-M+[RP4-2-Tc::Mu:KmrTn7](λ pir)

Laboratory stock

K. pneumoniae

CG43 K2 serotype Laboratory stock

CG43S3 ΔrspL, Str Laboratory stock

CG43S3-Z01 CG43S3 ΔlacZ Smr Laboratory stock

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Table 2. Plasmids used and constructed in this study

Plasmids Relevant characteristic Reference or source

yT&A Cloning vector;Apr Yeastern Biotech

pGEMT Cloning vector;Apr Promega

pMrkABC mrkABC gene cluster cloned into pGEMT vector,Apr Laboratory stock pMrkABCD 1 kb fragment amplified using primer pairs, phw03 and MZ006,

and cloned into pMrkABC by AscI and ApaI site, Apr

Laboratory stock

pA25 5 amino acid insered in the 94th amini acid of MrkA of pMrkABCD

Laboratory stock

pD27 5 amino acid insered in the 87th amini acid of MrkA of pMrkABCD

Laboratory stock

pA-2 insert DENIIEDIII part2 in the pA25 PmeI site This study pD-1 insert DENIIEDIII part1 in the pD27 PmeI site This study pD-2 insert DENIIEDIII part2 in the pD27 PmeI site This study pD-3 insert DENIIEDIII part3 in the pD27 PmeI site This study pD-4 insert DENIIEDIII part4 in the pD27 PmeI site This study pD-5 insert DENIIEDIII part5 in the pD27 PmeI site This study

pLacZ15

A derivative of pYC016, containing a promoterless lacZ from K. pneumoniae CG43S3 as the reporter, Cmr

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pMrkA-lacZ primers pmrkA4 and pmrkA5 were used to get 545 bp sequence, BamHI and BglII were used to cut into pLacZ15

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Table 3. Primers used in this study

Primer Sequence 5'-->3' SL1138-3-1 GTTTAAACggaaaggaatgtcatactcta SL1138-3-2 GTTTAAACaaacacaacatggaacaatagtt SL1138-3-3 GTTTAAACagatcccttttgagataatggatttggaa SL1138-3-4 GTTTAAACcagtcaacccaatcgtaacagaa SL1138-3-5 GTTTAAACgagacagctacatcatcataggagta SL1139-3-1 GTTTAAACgtttctgctatttccttcacaactttaaactt SL1139-3-2 GTTTAAACatcttacatggagaaccgtccccttc SL1139-3-3 GTTTAAACacgactgtaatcaggcgacctaaaacatgtct SL1139-3-4 GTTTAAACtctccgaatggaggttctgcttctat SL1139-3-5 GTTTAAACactcctttcttaaaccagttgag

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Table 4.DENIIEDIII inserted sequence and insertion site

Insertion site DENIIEDIII inserted sequence

A25-2 NMFKQTQHGTIVIRVQYEGDGSPCKMFKQNT D27-1 PVFRKGMSYSMCTG KFKVVKEIAETFKQPT D27-2 PVFKQTQHGTIVIRVQYEGDGSPCKMFKQPT D27-3 PVFKQIPFEIMDLEKRHVLGRLITLFKQPT D27-4 PVFKPVNPIVTEKDSPVNIEAE PPFGEFKQPT D27-5 PVFKRDSYIIIGVEPGQLKLNWFKKGMFKQPT

underline: DENIIEDIII amino acid

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Fig. 1. Organization of the fimbriae encoding gene cluster in K. pneumoniae C3091 (http://www.ncbi.nlm.nih.gov/).

The mrkABCDF is coding for type 3 fimbriae. The gene cluster from fimB to fimK encode the regulatory system, for the expression and assembly, and structural components of type 1 fimbriae. The genes named pecM and pecS are homologs of the genes coding for the regulators for the expression of virulence genes in Erwinia chrysanthem (Reverchon, Nasser et al. 1994).

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Fig. 2. Downstream genes of the type 3 fimbriae gene cluster. The genes KP4551, KP4552, and KP4554 were predicted

byNCBI ( http://www.ncbi.nlm.nih.gov/ ) to encode c-di-GMP binding domain protein (PilZ domain protein), transcription

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Fig. 3. Schematic presentation of the transposon mutagenesis. The plasmid pUTmini-Tn5Km2 was mobilized from E.

coli S17-1 λ pir to K. pneumoniae CG43S3Z01rcsB-, and the mutants selected on M9 agar plates containing kanamycin, chloramphinicol, and X-gal.

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Fig. 4. Amino acid sequences of the major subunit of type 3 fimbriae. The arrows indicate A25 and D27 are the foreign

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(A)

(B)

* : primer

Fig. 5. Amino acid sequences of DEN2EDIII. (A) Multiple-sequence

alignment of domain III region (aa 295 to 395) of four DV serotypes. Identical residues are shaded as dark gray areas, and conserved residues are shaded as light gray areas (Abd-Jamil, Cheah et al. 2008).The 5 fragmented amino acid sequences of are marked with different colors. (B) Sequences of the five peptides, the primers used for the PCR amplification are labeled.

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(A)

(B)

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Fig 6. Analysis of expression of the recombinant fimbriae. E. coli

JM109 carrying each of the recombinant clones were grown overnight at 37°C, and the lysates were collected and heated at 95°C for 30 mins before subjected to the analysis. (A) SDS-13.5% polyacrylamide gel stained with Coomassie blue. (B) and (C) are western blot analysis against anti-MrkA antibody and anti-DENEDIII antibody, respectively. The arrowheads mark the recombinant MrkA. Lanes M, molecular weight marker; 1, E. coli JM109; 2, JM109[pmrkABCD]; 3,

JM109[pD27]; 4, JM109[pA25-2]; 5, JM109[pD27-1]; 6, JM109[pD27-2]; 7, JM109[pD27-3]; 8, JM109[pD27-4]; 9, JM109[pD27-5].

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(A)

(B)

Fig 7. Polymer pattern analysis of the recombinant fimbriae. E. coli

JM109 carrying each of the recombinant clones were grown overnight at 37°C and the total cell lysates collected by centrifugation. (A) SDS–8% polyacrylamide gel stained with Coomassie blue. (B) Western blot analysis by anti-MrkA antibody. Lanes M, Marker; 1, E. coli JM109; 2, JM109[pmrkABCD]; 3, JM109[pD27]; 4, JM109[pA25-2]; 5,

JM109[pD27-1]; 6, JM109[pD27-2]; 7, JM109[pD27-3]; 8, JM109[pD27-4]; 9, JM109[pD27-5].

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(A)

(B)

Fig. 8. Immunofluorescence microscopy (IFM) analysis of the recombinant E. coli displaying with the type 3 fimbriae using the antibody (A)anti-MrkA and (B)anti-DENDIII. (a) JM109, (b) JM109

[pmrkABCD], (c) JM109[pD27], (d) JM109[pA25-2], (e) JM109[pD27-1], (f) JM109[pD27-2], (g) JM109[pD27-3], (h) JM109[pD27-4], (i) JM109[pD27-5].

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(A)

(B)

Fig 9. Analysis of the purified fimbriae. The E. coli JM109 carrying

each of the recombinant plasmids were cultured for 24 h in 24 ml LB broth. The bacteria were collected by centrifugation for 3 min (8,000 rpm, 4°C) and were suspended in 1 ml phosphate-buffered saline (PBS). The bacterial suspension was heated at 65°C 2 h and homogenized in blender for 20 min at ambient temperature. 30% saturate ammonium sulfate was added and stored at 4°Covernight. The pellet was collected by centrifugation (20,000 g, 90 min) and suspended in 40 l PBS. The purified fimbriae were identified by (A) SDS–8% polyacrylamide gel stained with Coomassie blue. (B) Western blot analysis using anti-MrkA

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antibody. Lanes M, Marker; 1, E. coli JM109; 2, JM109[pMrkABCD]; 3, JM109[pD27]; 4, JM109[pA25-2]; 5, JM109[pD27-1]; 6,

JM109[pD27-2]; 7, JM109[pD27-3]; 8, JM109[pD27-4]; 9, JM109[pD27-5].

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(A)

(B)

Fig. 10. Analysis of the expression of the type 3 fimbriae in K.

pneumoniae CG43S3Z01 and K. pneumoniae CG43S3Z01rcsB-. The

bacteria (lanes 1 to 4) were cultured statically in LB for 20 h. The bacteria (lanes 6 to 9) were in shaking culture to OD 0.8. Total cell lysates were heated at 95°C for 30 min before subjected to analysis. (A) SDS–13.5% polyacrylamide gel stained with Coomassie blue;

(B)Western blot analysis using anti-MrkA antibody. The arrowheads mark the recombinant MrkA. Lanes M, Marker; 1 and 6,

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CG43S3Z01[placZ]; 2 and 7, CG43S3Z01rcsB-(placZ);3 and 8,

CG43S3Z01(pmrkA-lacZ); 4 and 9, CG43S3Z01rcsB- (pmrkA-lacZ); 5, E. coli JM109.

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48

(A)

(B)

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Fig. 11. Analysis of the transposon insertion mutant strains. (A) The

mutant strains (M1~M8) with color alteration on X-gal plate are shown. (B) Analysis of the recombinant clone (in pUC19) by restriction enzyme PstI. Lanes 1: pM1; 2: pM2; 3: pM5; 4: pM6; 5: pM8; 6: pM7. (C) Analysis of the recombinant clone of pM1 (lanes 1, 3, 5) and pM6 (lanes 2, 4, 6) by restriction enzymes BamHI (lanes 1, 2), EcoRI (lanes 3, 4) and HindIII (lanes 5, 6).

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50 (A) M2 (B) M5 (C) M7 (D) M8

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Fig. 12. The tansposon insertion site and the genes beside the insertion site. The arrows mark the transposon insertion