• 沒有找到結果。

克雷白氏肺炎桿菌CG43中KvhAS雙分子調控系統的功能分析

N/A
N/A
Protected

Academic year: 2021

Share "克雷白氏肺炎桿菌CG43中KvhAS雙分子調控系統的功能分析"

Copied!
43
0
0

加載中.... (立即查看全文)

全文

(1)

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 中 KvhAS 雙分子

調控系統的功能分析

Functional analysis of the

Two-component System KvhAS in

Klebsiella pneumoniae CG43

研究生:梁婉君

學號:9128511

(2)

CONTENTS

CONTENTS...1

ABSTRACT...2

INTRODUCTION...5

MATERIALS AND METHODS...11

RESULTS...19

DISCUSSION ...27

REFERENCES...32

TABLES ...43

(3)

摘要

根據本實驗室過去的EMSA(Electrophoretic mobility shift assay)研究,我們發

現KvhAS 及其同源雙分子調控系統 KvgAS 和 KvhR 可能具有互相調控關係。為

了更進一步研究彼此的交互作用,因此構築了一系列的突變株,包括kvhA-kvgA-

kvhA-kvhR-kvgA-kvhR-kvhA-kvgA-kvhR-。經過比較這些突變株的生長及菌體低

速離心狀態後,將這些突變株分成兩群:第一群菌帶有kvgA 或 kvhR 突變,呈現 快速生長及低黏度的表現型;而以老鼠腹膜炎模式分析,刪除kvgA 或 kvhR 會導 致老鼠半致死率增加。第二群菌包含 kvhA- 和 kvhA-kvhR- 突變株,這些突變株 呈現和母株 LacZ16 相同的生長速率及菌體黏度。將含有 kvhA 的質體轉型入 kvhA-kvhR-雙基因突變株,我們發現 kvhA-kvhR-突變株會由第二群轉成第一群; 且同時含有kvhA 及 kvhR 基因突變時,細菌對人類血清的抵抗能力下降。這些結 果顯示KvhA 和 KvhR 之間可能有交互作用,而這些突變株在菌體黏度及致病力 表現不同程度的差異,以莢膜多糖體(capsular polysaccharide)為最可能引起變化 的因子。因此,我們利用LacZ 為報導蛋白,分析莢膜多糖體基因的啟動子在這 些突變株的表現差異。結果顯示:莢膜多糖體基因組開放骨架 1~2 或 16~17 的啟 動子活性在第一群突變株中表現降低,而在kvhR-降低的活性會因為kvhA 基因同 時突變而回復;然而,開放骨架 3~15 的啟動子活性在任一突變株中並沒有顯著 差異。另外,當壓力調節的主控子RpoS 基因突變時,kvhAS 的啟動子活性明顯 上升,進一步分析發現在 kvhAS 基因的上游-379 至-476 片段可能是 RpoS 調控 的區域,而此區也是 KvhA 的自我調控區域。我們還發現 kvhAS 的啟動子活性 會隨鎂離子濃度升高而上升,這個結果暗示KvhAS 和 PhoPQ 有交互作用。除此 之外,大量表現KvhA 蛋白質會改變菌體對不同抗生素的感受性,這結果顯示著 KvhA 可能調控細菌對藥物的感受性。

(4)

ABSTRACT

Previous EMSA (Electrophoretic mobility shift assay) study in Klebsiella

pneumoniae CG43 genome has shown a possibility of the presence of a regulatory

circuit in-between the homologous two components (2CS) KvhAS, KvgAS and KvhR. In order to investigate further the regulatory interaction, a series of mutants including kvhA-kvgA-, kvhA-kvhR-, kvgA-kvhR- and kvhA-kvgA-kvhR- derived from K.

pneumoniae LacZ16 were constructed. Comparative analysis of the growth and

phenotype of the mutants allowed us to classify the mutants into two groups: the Group I carrying either kvgA or kvhR mutation displayed a less mucoid and a faster growth rate by comparing to the parental strain LacZ16. In a mouse peritonitis model, the deletion of either kvgA or kvhR resulted in an increase of LD50. Group II

includes kvhA- and kvhA-kvhR- mutants, which exert a similar phenotype and growth rate with that of the parental strain. Complementation of the kvhA-kvhR- with a kvhA containing plasmid converts the mutant into Group I. Only with both kvhA- and

kvhR- mutations, the bacteria revealed a decreasing activity of resistance to human serum. The results suggested that a cross-talk is present in-between KvhA and KvhR. Mutations of these genes affect both bacterial mucoidity and virulence at different levels indicating that change of capsular polysaccharide (CPS) is most likely the causing factor. We therefore attempt further to analyze promoters of the CPS

(5)

encoding genes in the mutants using LacZ as a reporter. The promoter activity of either Porf1-2 or Porf16-17 in Group I mutants appeared to be reduced, which suggests

that KvgA and KvhR are the regulators for cps gene expression. The decreasing activity in kvhR- was found to be reversed in kvhA-kvhR-, indicating a direct interaction between KvhA and KvhR. In addition, we have shown that mutation of the global stress regulator RpoS increased the promoter activity of kvhAS. The putative regulatory region of kvhAS by RpoS was localized between -379 and -476 bp upstream of kvhAS, which appeared to contain also the auto-regulating region of KvhA. The promoter activity of kvhAS was found to increase by the addition of MgSO4, suggesting an interaction with PhoPQ regulon. Moreover, changes of

resistance activity to antibiotics were noted by overexpression of KvhA in the bacterial, implying its involvement in drug resistance.

(6)

INTRODUCTION

Klebsiella pneumoniae is a commonly reported nosocomial Gram-negative

bacteria. As an opportunistic pathogen, it often causes pneumonia, septicemia, bacteremia, suppurative lesion, wound infection, burn infection, and urinary or respiratory tract infections in chronic alcoholics or immunocompromised patients. There are five major virulence factors identified to participate in K. pneumoniae infections, which include capsular polysaccharide, lipopolysaccharide, adhesin, iron-acquisition system, and serum resistance factors (Podschun and Ullmann, 1998). Besides these virulence factors, very little is known about other genes that may participate in the pathogenesis of K. pneumoniae.

To persist in nature, bacteria must be able to compete and survive under various growth conditions. To accomplish this task, they possess regulatory systems that permit them to recognize and adapt to a changing environment. These adaptations are brought about largely through changes in gene expression that are coordinated by global regulatory networks. Pathogenic bacteria often use two-component systems (2CSs) to control expression of the genes encoding bacterial toxins, adhesins, and other virulence-associated molecules that promote their survival in the host (Locht, 1999). The 2CSs composed of a membrane-bound sensor protein and a cytoplasmic

(7)

response regulator are commonly used to regulate bacterial gene expression in response to environmental changes. On stimulation by an extracellular signal, a histidine kinase domain of the sensor protein is autophosphorylated and the signal transduction occurs with transfer of the phosphoryl group from the phosphorylated kinase to an aspartic acid residue in the receiver domain of the cognate response regulator. Phosphorylation of the regulator protein, which is often a transcription factor containing a C-terminal DNA-binding domain, increases its binding affinity to target promoters and thereby influences the expression of the target genes (Foussard

et al., 2001; Chang et al., 1998; Mizuno, 1998) .

A novel two-component system kvgASXR (Klebsiella virulence gene) has been isolated by PCR-supported genomic subtractive hybridization from a highly virulent strain K. pneumoniae CG43 (楊淑理, 民國八十九年), which is not found in the genome of K. pneumoniae strain MGH78578 (http://genome.wustl.edu/gsc/). A 2CS exhibiting high sequence identity with that of the kvgAS was isolated later in our laboratory also from K. pneumoniae CG43 and designated kvhAS (kvg homolog), which is present in the genome of K. pneumoniae MGH78578 (賴旻初, 民國八十九). In contrast to kvgAS, which is present in approximately 15% of the laboratory-collected clinical isolates (Lai et al., 2000), kvhAS prevails in almost all the strains. As well as the KvgAS, KvhAS displayed a moderate similarity to BvgAS

(8)

(Bordetella virulence gene) of B. pertussis and EvgAS (Escherichia virulence gene), the E. coli ortholog of BvgAS (Konig et al., 2002; Bock et al., 2001; Utsumi et al., 1992). These sensors are classified as unorthodox type sensor, each of which is composed of an N-terminal periplasmic region and a C-terminal cytoplasmic region containing four functional domains of a linker, transmitter, receiver and an output, the Hpt module. A multistep phosphorelay occurs among the alternating histidine and aspartate residues (His-Asp-His-Asp) before the response regulator is activated (Stock, 2000). Although EvgAS is highly homologous to BvgAS, domain swapping of the liberated signaling modules had shown that the C-terminal Hpt domain of the sensor protein endows a specificity for the corresponding regulator, and the receiver domain of the response regulator determines the specific interaction (Perraud et al., 1998, 2000).

Lipopolysaccharide (LPS) and capsular polysaccharide (CPS) are two of the virulence factors of K. pneumoniae. The production of a hydrophilic CPS (K antigen) is characteristic of the genus Klebsiella, and is involved mainly in the resistance, by acting as a physical barrier, to phagocytosis by polymorphonuclear cells (Kabha et al., 1995 ; Held et al., 2000). Klebsiella K2 CPS biosynthetic pathway is similar to that of the group I CPS in E. coli, and the regulatory strategy of colanic acid biosynthesis employed by E. coli may therefore serve as a model (Whitfield et al., 1999, 2003).

(9)

Colanic acid is produced by enzymes encoded by the cps genes, which are regulated by rcsC/yojN/rcsB and also by lon protease for degradation of RcsA. RcsC is a sensor protein, RcsB is its cognate response regulator, and YojN is a histidine-containing phosphotransfer protein that transfers the phosphoryl group from RcsC to RcsB. The activated RcsB together with the co-activator RcsA subsequently coordinate the activation of the cps operon transcription (Chen et al., 2001). In addition to Rcs system, the rmpA2 gene was isolated from a 200-kb virulence plasmid of K.

pneumoniae CG43 also encodes an activator for CPS synthesis. Deletion of rmpA2

was shown to reduce the mucoidy and virulence of the bacteria (Lai et al., 2003).

In bacteria, rpoS gene encodes the alternative sigma factor σs (RpoS) subunit of RNA polymerase to control the expression of genes involved in cell survival during the cessation of growth (stationary phase) and provide cross-protection to various stresses (Hengge-Aronis, 2002). Expression of σs increases dramatically at the onset of stationary phase, which is regulated at the transcriptional, post-transcriptional and post-translational levels (Loewen et al., 1998). A most complex regulatory system is also essential for Salmonella virulence in mice, which includes the GacA/GacS two component system and quorum sensing control to affect rpoS expression (Ibanez-Ruiz

et al., 2000). The recent study of DNA microarray analysis suggested that rpoS

(10)

al., 2003). In E. coli, CpxR-P in conjunction with RpoS activates expression of the cpxRA two-component system, suggesting an involvement of the Cpx system in

stationary-phase survival (De Wulf, 1999).

While the physically linked sequences of kvhAS compared with that of the evgAS, several homologous genes were identified (黃騰逸, 民國九十一年). The putative acid-resistance genes yfdXL which show a high homology with the evgAS-regulated

yfdX were identified downstream and upstream of kvhAS respectively. The emrKY and yhiUV which are located nearby the EvgAS genes were also identified, however,

elsewhere in K. pneumoniae MGH78578 genome. Overexpression of the evgA response regulator up-regulates the drug transporter genes emrKY and yhiUV in E.coli, which led to resistance to a wide range of toxic compounds, such as erythromycin, doxorubicin, novobiocin, crystal violet, rhodamine 6G, TPP, benzalkonium, SDS, and deoxycholate (Nishino et al., 2001, 2002; Eguchi et al., 2003). The 2CS BvgAS positively controlled transcription of several virulence genes, which include the genes for toxins and adhesins, in B. pertussis and B. bronchiseptica. On the other hand, the BvgAS system negatively controlled the expression of a set of genes, the majority of these genes encode putative metabolic functions involved in redox reactions and amino acid transport (Schneider et al., 2002). It has also been demonstrated that the expression of bvgAS genes is regulated by the addition of MgSO4, nicotinic acid or

(11)

elevated growth temperatures (Scarlato et al., 1991). As an ortholog of EvgAS and BvgAS, KvhAS is likely playing a similar function.

The emergence and spread of multiple antibiotic-resistant K. pneumoniae, especially the extended-spectrum β-lactamase-producing strains, urge a need for new

antibiotics with an innovative mode of action (Gupta et al., 2003). It has been proven that two-component systems play an important role in sensing cell disruption by drugs and thereby activating expression of some resistance factors (Ohki et al., 2003). Higher eukaryotes including mammals often use a distinct signal transduction system that incorporates serine, threonine, and tyrosine phosphorylation. As for the selectivity of antimicrobial agents, histidine kinases appear to be potent targets (Matsushita et al., 2002). Previous studies have shown the possibility of the presence of a regulatory circuit connected by kvhAS, kvgAS and kvhR, a response regulator located at the downstream of kvgAS. In this study, we attempt to elucidate the relationship between these 2CS paralogs (Fig. 1). A series of mutant strains including

kvhA, kvgA, kvhR were constructed in order to assign functional role of the 2CSs in

the bacteria. The environmental signal(s) sensed by KvhS and the target genes under control by kvhAS were also investigated.

(12)

MATERIALS AND METHODS

Plasmids, bacterial strains, and growth conditions. The bacterial strains and

plasmids used in this study are listed in Table 1 and 2. Tryptone, yeast extract, and agar were purchased from Difco Laboratories (Detroit, Mich). E. coli, K. pneumoniae CG43 and its derivatives were propagated at 37°C in Luria-Bertani (LB) broth supplemented with the appropriate antibiotics unless otherwise indicated. The density of the bacterial culture was determined by measuring the absorbance at 600 nm (OD600).

Recombinant DNA technique. The recombinant DNA experiment was carried out by

standard procedures as described (Joseph et al., 2001). Plasmid DNA was prepared using VIOGENE Miniprep kit. Restriction endonucleases and DNA modifying enzymes were purchased from New England Biolab (Beverly, MA), MBI (Hanover, MD), or Roche Molecular Biochemicals (Mannheim, Germany), and were used according to the recommendation of the suppliers.

Construction of the gene-deletion mutants. K. pneumoniae CG43LacZ16 derived

mutants with deletion specific at either of kvhA, kvgA, and kvhR genes were constructed by allelic exchange strategy (Skorupski et al., 1996). The pKAS46 derivative containing a deletion in kvgA constructed previously was delivered from E.

(13)

coli S17-1 λ-pir into kvhA- and kvhR- respectively by conjugation. The transconjugants were selected by plating with a 105-fold dilution of the culture on a minimal medium containing 25 µg/ml kanamycin and 100 µg/ml ampicillin for the integration of the plasmid. Subsequently, the resulting strains were cultured without selection to late logarithmic phase followed by selection on the plates containing 500 µg/ml streptomycin for loss of the vector sequence. In addition, a plasmid containing a deletion in kvhR was transferred from E. coli S17-1 λ-pir into kvhA- and kvhA-kvgA

-respectively. The plasmid containing a tetracycline gene insertion in rpos was transferred also from E. coli S17-1λ-pir into kvhA-. The transconjugants were selected on appropriate medium and the mutations were all confirmed by PCR and Southern analysis.

Expression and purification of the histidine-tagged KvhA. The recombinant

His6-tagged response regulator, His6-KvhA, was constructed (pHP4005) and the

plasmid was transformed into E. coli BL21-RIL (DE3). The transformants carrying pHP4005 were cultured in LB broth to log phase, and expression of the His6-KvhA

recombinant protein was induced with 0.5 mM IPTG for 4 h. The IPTG (isopropyl-β-D-thiogalactopyranoside)-induced bacterial cells were harvested by

centrifugation at 4°C, 6000 rpm for 15 min, resuspended in 1 X binding buffer (5 mM imidazole, 0.5 M NaCl and 20 mM Tris-HCl pH 7.9), and then disrupted by

(14)

sonication. To purify His6-KvhA, the cell lysate was centrifuged at 13,500 rpm for 20

min at 4°C, and the supernatant was then applied to a column containing His-Bind Resin (Novagen, Madison, WI). The column was washed with 1 X binding buffer, and then washed with 1 X wash buffer (60 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl pH7.9). Finally, the His6-KvhA protein was eluted with 1 X elution buffer (1

M imidazole, 0.5 M NaCl, and 20 mM Tris-HCl pH 7.9), dialyzed with electrophoretic mobility shift assay buffer (20 mM Tris-HCl pH 8.0, 4 mM MgCl2, 50

mM KCl, 1 mM CaCl2, 1 mM dithiothreitol), and then concentrated by PEG-20,000 at

4°C.

Electrophoretic mobility shift assays. The probes used in the electrophoretic

mobility shift assay were the restriction fragments of the upstream region of kvhR. The restricted fragments were separated on a 0.8% agarose gel and recovered, and 5’ end-labelled with [γ-32P]-ATP (25 µCi/ml) using T4 polynucleotide kinase. The reaction mixture was incubated for 60 min at 37°C. The His6-KvhA was incubated in

1 X gel shift binding buffer (20 mM Tris-HCl, pH 8.0, 0.4 mM MgCl2, 50 mM KCl, 1

mM CaCl2, 1 mM DTT, and 10% glycerol) for 30 min at 25°C (Karimova et al. 1996).

Bovine serum albumin (1 µg) and the radioactively-labelled DNA fragments were then added and incubated at 37°C for 20 min. The samples were applied to 5% non-denaturing polyacrylamide (acrylamide: bis-acrylamide, 19: 1) gels that were

(15)

pre-electrophoresed at a constant voltage of 100 V for 1 h at 4°C. Electrophoresis was performed at 100 V for 6 h at 4°C and the gels were read by InstantImagerTM (Packard Instrument Company).

Mouse lethality assay. Female BALB/c mice with an average age of four weeks were

acclimatized in an animal house for 7 days. The tested bacterial strains were cultured in LB medium at 37°C overnight. Four mice of a group were injected intraperitoneally with bacteria resuspended in 0.2 ml of saline in 10-fold graded doses. The LD50, based on the number of survivors after 10 days, was calculated and

expressed as CFU (colony forming units) as described (Reed et al., 1938).

Resistance to serum killing. Fresh blood collected from healthy volunteers was

centrifuged to obtain nonimmune human sera (NHS). NHS was incubated at 56°C for 30 min to obtain heat-inactivated human sera (HI-NHS). The serum-resistant activity of bacteria was determined essentially as described (Lai et al., 2003).One hundred microliters of overnight grown bacteria re-suspended in saline was mixed with 100 µl of NHS or HI-NHS, and the mixture was incubated at 37°C for 30 min. The number of viable bacteria in the mixture was then determined by plating onto LB agar.

Extraction and quantification of CPS. CPS was extracted by the method as

(16)

cultured in LB broth overnight were mixed with 100 µl of 1% Zwittergent 3-14 detergent in 100 mM citric acid (pH 2.0), and then the mixture was incubated at 50°C for 20 min. After centrifugation 13,500 rpm for 10 min, 250 µl of the supernatant was transferred to a new tube, and CPS was precipitated with 1 ml of absolute ethanol at 4°C for 20 min and then centrifugated 13,500 rpm for 25 min. The pellet was dried at 37°C and dissolved in 200 µl of distilled water, and a 1,200-µl volume of 12.5 mM borax in H2SO4 was added. The mixture was vigorously vortexed, boiled for 5 min,

and cooled, and then 20 µl of 0.15% 3-hydroxydiphenol was added and the absorbance at 520 nm was measured. The uronic acid content was determined from a standard curve of glucuronic acid and expressed as micrograms per 109 CFU (Blumenkrantz et al., 1973).

Capsular polysaccharides purification. 1.5 ml bacteria cultured in LB broth

overnight at 37°C were harvested by centrifugation at 13,200 rpm for 10 min. The pellet was resuspended with 500 µl phosphate-buffered saline (PBS) and mixed with 500 µl phenol, subsequently extracted at 65°C for 5 min then put on ice for 5 min and repeated for three times. The sample was centrifuged at 10,000 g for 5 min and transferred aqueous phase to dialyze against water overnight. Subsequently, the sample was treated with DNase and RNase at 37°C for 2 h and proteinase K at 50°C overnight then dialyzed against water overnight again as described (Kalka-Moll et al.,

(17)

2001).

Capsular polysaccharides pattern analysis by Alcian Blue-silver stain. CPS was

resolved on 10% (w/v) acrylamide gel and immediately immersed in alcian blue solution (0.005% alcian blue, 40% ethanol, and 5% acidic acid in dH2O) for 30 min,

then changed to fresh solution overnight. The gel was rinsed in dH2O for 5 min and

oxidized in 0.7% (w/v) sodium metaperiodate for 10 min, then washed five times with dH2O for 5 min. The gel was stained by 10% silver solution (Bio-Rad silver

concentrate) for 10 min, and then washed with dH2O for 5 min. The color was

developed with 1.5% (w/v) developer (Bio-Rad) and agitated until dark precipitation formed, then stopped with 5% acetic acid for 10 min and rinsed in dH2O (Karlyshev

and Wren, 2001).

Anti-phagocytosis assay. THP-1 (ATCC TIB202), originally isolated from a child

with acute leukemia, are mature cells in the monocyte/macrophage lineage with a normal diploid karyotype. These nonadherent cells were maintained in continuous culture in RPMI 1640 cell culture media (GIBCO/BRL, Grand Island, N.Y.) supplemented with 10% fetal bovine serum (GIBCO/BRL) in an atmosphere of 5% CO2 at 37°C. THP-1 cells were centrifuged at 1000 rpm for 5 min and resuspended

(18)

tissue culture plates and treated with phorbol 12-myristate 13-acetate (PMA) (Calbiochem Co., La Jolla, Calif.) for 48 h to induce maturation of the monocytes and became macrophage-like; differentiated macrophages were identified by morphological features and their ability to adhere to plastic. Before experimentation, THP-1 cells were washed twice with PBS to remove nonadherent cells and resuspended in 0.5 ml RPMI without fetal bovine serum, then incubated with 50 µl of overnight cultured bacteria previously washed one time and diluted in PBS (6×108/ml) for 2 h. Subsequently, wells were washed twice with PBS and incubated for 2 h with fresh RPMI medium containing gentamycin (100 µg/ml) to kill extracellular bacteria. Then wells were washed three times with PBS and phagocytosed bacteria were quantified by plating them on LB agar plates after THP-1 lyses with 500 µl of 0.1% Triton X-100 as described (Perez-Perez et al., 1995; Cortes et al., 2002).

β-galactosidase activity assay. β-galactosidase was assayed according to the method

of Miller (Miller. 1972). Fresh M9 medium was inoculated with one-tenth volumes of the overnight M9 cultured bacteria. Samples of bacteria were taken 100 µl in the early or late logarithmic growth phase (optical density at 600 nm 0.4 or 0.7), and mixed with 900 µl Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM

MgSO4, 50 mM β-mercaptoethanol), 17 µl of 0.1% SDS and 35 µl chloroform for 10

(19)

(ONPG) was added and vortexed for 10 s, then incubated at 30°C until yellow color was apparent. Finally, reaction was stopped by adding 500 µl of stop solution (1M Na2CO3) and the OD420 of supernatant was measured. One unit of β-galactosidase is

defined as the hydrolysis of 1nmol ONPG per min per mg protein. Signals were added in the early logarithmic growth phase, and after incubated 1 h, β-galactosidase activity

was tested as described above.

Drug susceptibility assay. Antibiotic susceptibility testing was performed by disk

diffusion method. An overnight culture of bacteria was spread onto LB agar, then disks were placed onto the plates and zones of inhibition were measured after 16 h of incubation at 37°C. Standard antibiotic disks were obtained from Difco (Detroit, Mich), Becton Dickinson (Sparks, Md) and Oxoid Ltd (Basingstoke, Hampshire), and the following disc concentrations were used: erythromycin, 15 µg; novobiocin 5 µg; ceftazidime 30 µg; cefamandole 30 µg; cefotaxime 30 µg; cephalothin 30 µg; fosfomycin 50 µg; nalidixic acid 30 µg; methicillin 5 µg; penicillin 10 µg; piperacillin 100 µg; ticarcillin 75 µg; carbenicillin 100 µg; sulfamethoxazole 23.75 µg + trimethoprim 1.25 µg; polymyxin B 300 IU.

(20)

RESULTS

Characterization of the mutants derived from LacZ16. Previous EMSA study has

shown that KvgA bound to either the promoter of kvhA or kvhR, suggesting presence of a regulatory circuit among kvhAS, kvgAS and kvhR (林靖婷, 民國九十 年). The gene organizations are shown in Fig. 1. In order to investigate further the interaction, a series of mutants were constructed on K. pneumoniae LacZ16 deriving

kvhA-kvgA-, kvhA-kvhR-, kvgA-kvhR-, and kvhA-kvgA-kvhR-. Comparative analysis of the growth and sedimentation rates of the mutants allowed the classification of these mutants into two groups: the Group I carrying either kvgA or kvhR mutation that displayed a less mucoid phenotype and a faster growth rate than that of the parental strain LacZ16. Group II includes kvhA- and kvhA-kvhR- mutants, which exert similar growth rate and phenotype with that of the parental strain LacZ16 (Fig. 2A and 2B).

The reduction of mucoidy in strains of Group I was also evident as determined by the inability of the colony to form a string using a toothpick. The amount of CPS produced in these mutants were further quantified by measuring the glucuronic acid contents. As shown in Table 4, Group I strains synthesized much less K2 CPS than the parental LacZ16 strain. A slight reduction of CPS and colony mucoidy also appeared in kvhA-kvhR-, which belongs to Group II. Complementation of the kvhA-kvhR- with

(21)

pA415, a DNA fragment corresponding to the entire kvhA locus with its putative promoter amplified by A201/AS02 primer set and cloned into pRK415, converted both phenotype and growth of the mutant from Group II into Group I (Fig. 3A and 3B).

The His6-KvhA binds specifically to kvhR promoter. The response regulator KvhA, like many other response regulators of the two-component system, possesses a helix-turn-helix motif at the C-terminus. In order to demonstrate its interaction with

kvhR promoter, pHP4005 which is pET30C containing the entire kvhA was

transformed into E. coli BL21-RIL (DE3), and the IPTG-induced His6-KvhA was

purified by affinity chromatography (Fig. 4). Binding capability of the His6-KvhA to

kvhR promoter wasassessed by electrophoretic mobility shift assay. As presented in Fig. 5, specific DNA-binding activity of the His6-KvhA was demonstrated. The

mobility of His6-KvhA appeared to be retarded with the addition of 32P-labelled PkvhR.

The DNA binding appeared to be specific because the shifted band disappearedin the presence of an excess amount of unlabelled PkvhR, but not in the presence of pUC19

DNA.

The CPS and LPS patterns of the mutants. The CPS and LPS patterns of each

(22)

without SDS supplement and 15% (w/v) SDS-PAGE respectively. The resolved LPS was visualized by silver staining, and the CPS was visualized by staining with Alcian Blue followed by silver staining. We have found that K. pneumoniae CPS was barely detectable without the use of Alcian Blue. The CPS pattern of kvhA- appeared to be the same as that of wild type, however, kvhA-kvhR- showed less high molecular weight CPS, which migrated as a broad smear at the top of the gel (Fig. 6). When the

kvhA-kvhR- mutant was complemented with a kvhA carrying plasmid, an obvious increase of the low molecular weight CPS components was noted. In addition, the Group I mutants included kvgA-, kvhR-, kvhA-kvgA-, kvgA-kvhR- and kvhA-kvgA-kvhR

-showed more high molecular weight CPS comparing with that of the parental strain LacZ16 (Fig. 6). As shown in Fig. 7, LPS was clearly visible in all the mutants, showing the characteristic O-antigen banding pattern.

Anti-phagocytosis activity of the mutants. To investigate the mutation effect on the

bacterial susceptibility to macrophage, the cell line THP-1 was used. After 2 h incubation with each of the mutant bacteria, the THP-1 cells were washed and the ingested bacteria were quantified by plating onto LB plates. As shown in Fig. 8, the Group I mutants kvgA-, kvhR-, kvhA-kvgA-, kvgA-kvhR- and kvhA-kvgA-kvhR

-significantly lost their resistant activity to phagocytosis, whereas, the kvhA- and

(23)

kvhA-kvhR- with a kvhA carrying plasmid appeared to reduce its anti-phagocytosis activity.

Effects of kvhA, kvgA and kvhR mutation on the bacterial virulence. In a mouse

peritonitis model, the virulence of the mutants were compared. As shown in Table 5, deletion of kvgA or kvhR resulted in an increase of the LD50. The kvhA mutation,

however, had no apparent effects. In the complement-mediated killing test, only

kvhA-kvhR- appeared to show a decreasing resistance to human serum. Complementation of the mutant with pA415, the kvhA carrying plasmid, slightly restored the resistance. While in the heat inactivated human sera, the sensitivity to complement-mediated killing was also reduced.

Effects of kvhA, kvgA and kvhR on CG43 cps gene expression. In E. coli K-12, the

amount of colanic acid produced has been reported to be correlated with the transcriptional level of cps genes (Arakawa et al., 1995). We reasoned that the production of CPS affected by kvhA, kvgA and kvhR might also be resulted from differential cps genes expression in K. pneumoniae CG43, which carries a K2 capsular type. Thus, five lacZ reporter fusion constructs, porf1Z15 and porf2Z15, which comprise 800 bp and 440 bp non-translated region upstream of cps gene orf1-2 (Porf1-2::lacZ) respectively, porf3Z15 and porf4Z15, which carry 950 bp and 500 bp

(24)

non-translated region upstream of the cps operon orf3-15 (Porf3-15::lacZ) respectively,

and porf162Z15 (Porf16-17::lacZ), which contains the putative promoter of cps gene orf16 and orf17 were generated (Fig. 9). The lacZ fusion plasmids were then

transformed respectively into K. pneumoniae LacZ16 derived mutants. As shown in Fig. 10, the β-galactosidase activities of porf1Z15 (A), porf2Z15 (B), porf3Z15 (C), porf4Z15 (D) and porf162Z15 (E) were measured. The promoter activities of either

Porf1-2 or Porf16-17 in Group I mutants appeared to be reduced, and the decreasing

activity in kvhR- was found to be reversed in kvhA-kvhR-. However, no change of the promoter activity of Porf3-15 in any of the mutants was noted.

Effects of the rpoS and kvhA mutations on the promoter activity of kvhAS. To

investigate the regulatory roles of RpoS and KvhA on kvhAS expression, three putative promoters of kvhAS, pA15, which contains 467 bp non-translated region upstream of kvhAS, pF15, which contains 379 bp upstream of kvhAS, and pE15, which comprises 180 bp non-translated region upstream of kvhAS, were cloned into placZ15 using lacZ as a reporter (Fig. 11). The lacZ fusion plasmids were then respectively transformed into K. pneumoniae LacZ16 and its derived mutants kvhA-, rpoS-, and

rpoS-kvhA-. As shown in Fig. 12, the promoter activity of pA15 increased about 2-fold in rpoS-. However, the pE15 and pF15 promoter activities remained to be similar in any of these strains. The results suggested that the putative regulatory region of kvhAS

(25)

by RpoS is located -379 to -476 bp upstream of kvhAS, which contains also the auto-regulating region of KvhA.

Identification of the signaling molecules for kvhAS expression. The

β-galactosidase activity of pA15 in wild-type LacZ16 grown in M9 was measured by supplying the culture with either of 25 mM MgSO4, 50 mM MgSO4, 10 mM nacin, 2

mM EDTA, 0.2 mM 2,2-dipyridyl, 0.2 mM paraquat or 0.4 mM paraquat for 1 h at 37°C. As shown in Fig. 13, the promoter activity of kvhAS was found to increase by the addition of MgSO4. However, the rest of the reagents showed no apparent effects

on kvhAS expression.

Effect of the phoP mutation on the promoter activity of kvhAS. PhoPQ is a

two-component system that governs virulence, mediates the adaptation to Mg2+-limiting environments, and regulates numerous cellular activities in several gram-negative species (Groisman, 2001). To investigate the relationship between

kvhAS and phoPQ, pA15, pE15 and pF15 were respectively transformed into K. pneumoniae LacZ16 and its derived mutant phoP-. As shown in Fig. 14, the promoter activities of pA15 and pE15 decreased in phoP-, however, the pF15 promoter activity in phoP- remained the same as that in wild type. The results suggested that the putative regulatory element on kvhAS by an unknown activator is located -379 bp to

(26)

-476 bp and -1 bp to -180 bp upstream of kvhAS. Most likely, some unknown repressor binding region is within -180 bp to -379 bp upstream of kvhAS. These regulatory factors are likely under-controlled by PhoP or even cross-talking to each other.

Effect of KvhA overexpression on bacterial resistance activity to antibiotics. In

order to investigate the effect of KvhA overexpression on the bacterial drug resistance, pHAm-c, a DNA fragment carrying the entire kvhA locus cloned into pETm-c, and pHANm-c, a DNA fragment corresponding to the kvhA locus without helix-turn-helix (HTH) DNA-binding motif, which was amplified by AS07/HA01 primer set and cloned into pETm-c, were constructed. The plasmids pETm-c, pHAm-c and pHANm-c were then transformed into K. pneumoniae CG43S3 respectively. The strains were exposed to various antimicrobial agents in a disk diffusion assay. The data were summarized in Table 6, and the photographs were shown in Fig. 15. Overexpression of KvhA conferred the bacteria resist more to several antibiotics including cephalothin, piperacillin, ticarcillin, and carbenicillin of β-Lactam group.

However, the bacteria with an ovexpression of KvhA appeared to be more susceptible to fosfomycin and sulfonamides (trimethoprim + sulfamethoxazole), which are inhibitors of cell wall synthesis and metabolism respectively. While removing the HTH domain from KvhA, the overexpression effects on the bacterial resistance

(27)
(28)

DISCUSSION

Cross-talk among the 2CSs that a sensor transfers a phosphate group to a ‘non-cognate’ response regulator within a genome has recently been noted (Verhamme et al. 2002). We have previously isolated novel 2CSs kvhAS, kvgAS and

kvhR from K. pneumoniae CG43. The deduced amino acid sequence of kvhA shares

58.5 % and 57.0 % similarity with that of kvgA and kvhR respectively, suggesting they are paralogous response regulators. Previous EMSA study has also shown a possibility of the presence of a regulatory circuit in-between these homologous two-components. We further demonstrate the relationship of these 2CS paralogs and also the functional role of KvhA in this study.

On the basis of the comparative analysis of the mutant phenotypes, the deletion of either kvgA or kvhR reduced the bacteria CPS production which leads to the phenotypes of less mucoid and a faster growth rate. We have also shown that both KvgA and KvhR are likely involved in regulating not only the amount but also the form of CPS. Although kvhA deletion showed no apparent effect on the production of CPS, the mutation however, rendered a null effect of the kvhR mutation. We believe that KvhA interacts with KvhR in some way to suppress the activity of KvhR.

(29)

phagocytosis by polymorphonuclear cells by acting as a physical barrier (Alberti et al., 1996; Alvarez et al., 2000). The assay of anti-phagocytosis activity and virulence determination in a mouse peritonitis model, deletion of either kvgA or kvhR resulted in an increase of LD50. The results indicated that kvgA and kvhR may be involved partly

in the bacterial pathogenicity. LPS consists of lipid A, core, and O-polysaccharide antigen that are essential for the microorganism to resist complement-mediated killing (Cortes et al., 2002). Only with both kvhA and kvhR mutations revealed a decrease of resistant activity to human serum. Although the mutants display LPS core and O-antigen, kvhA-kvhR- seems to product less or poor O-antigen so that susceptible to human serum.

To investigate further the kvgA-, kvhR- mutation effect on the cps gene expression, the Group I mutants kvgA-, kvhR-, kvhA-kvgA-, kvgA-kvhR- and kvhA-kvgA-kvhR

-appeared to reduce the expression of cps gene orf1-2 and orf16-17. The putative regulatory region of orf1-2 in Group I mutants is located between -1 to -400 bp upstream of orf1-2, and the putative regulatory region of orf16-17 by Group I mutants is located between -1 to -450 bp upstream of orf16-17. The decreasing cps gene expression in kvhR- was found to be reversed in kvhA-kvhR-. Analysis of the ORF showed that orf1 is a homolog of S. typhimurium LT2 galF and E. coli galU. While

(30)

mannose-1-phosphate guanylyltransferase (GDP-mannose pyrophosphorylase) and phosphomannomutase respectively. Both enzymes have been reported to be involved in the biosynthesis of mannose (Arakawa et al., 1995). KvgA and KvhR may hence play a role as regulators for mannose synthesis. Notably, the cps expression regulated by many factors, such as RcsC, YojN, RcsB, RcsA and RmpA2 has been demonstrated (Lai et al., 2003). We believe that KvgA and KvhR may very likely interact with either Rcs system or RmpA2.

The mutation of the global stress regulator RpoS appeared to enhance the promoter activity of kvhAS, suggesting a negative regulation. Although a predicted RpoS binding site is present about -270 bp upstream of kvhAS, the RpoS regulatory region determined in Fig. 11 is located between -379 and -476 bp upstream of kvhAS, which will be further investigated in vitro by EMSA. The expression of kvhAS appeared to be positively auto-regulated by KvhA, which was shown to share a common regulatory region with RpoS. Previous study has shown that kvgAS was positively regulated by RpoS. It is hence expressions of kvhAS and kvgAS are working likely differentially to adapt a diverse environment.

Whole-genome transcriptional profiles of E. coli had shown that evgA is down-regulated by paraquat (Pomposiello et al., 2001). Previous data obtained in our

(31)

laboratory had also shown that addition of paraquat or 2’, 2’ dipyridyl to K.

pneumoniae cells activates the expression of kvgAS (Lai et al., 2003). However, these

reagents conferred no apparent effects on kvhAS expression. The expression of kvhAS was found in this study to increase by addition of MgSO4. In contrast, phoPQ was

activated at low Mg2+ concentration. In E. coli, a novel interaction was demonstrated between the EvgAS and PhoPQ systems (Eguchi, 2004). The genome-wide analyses suggested the presence of a signaling network connecting the Rcs system with the PhoPQ system for a coordinate regulating of the extracellular polysaccharide synthesis in response to the external concentration of divalent cations (Hagiwara, 2003). We therefore proposed that the expression of kvhAS was affected by PhoP indirectly through an interaction with some unknown factors.

Overexpression of KvhA conferred the bacteria a significant resistance to

β-lactams, indicating that the response regulator-mediated drug resistance is of great

potential as the major determinant for the bacterial drug resistance. In E. coli, overexpression of 2CSs BaeR, EvgA, RcsB and DcuR have been shown to confer a high-level β-lactam resistance (Hirakawa, 2003). The cps genes were induced by a

subset of β-lactams but not by agents inhibiting proteins synthesis or DNA replication,

indicating that cps expression is specific and not due to stresses accompanying cell death or by a general inhibition of peptidoglycan synthesis (Sailer et al., 2003). Thus,

(32)

the presence of a signaling network built by KvhAS, PhoPQ and Rcs systems is likely responsible to regulate the cell surface structures, by modifying lipopolysaccharide (LPS) and/ or capsular polysaccharide (CPS), in response to the external states. Finally, study on searching for the target genes regulated by KvhAS using cDNA subtraction method is currently underway.

In conclusion, as shown in Fig. 16, we propose a regulatory circuit that RpoS activates the expression of kvgAS but represses that of kvhAS. Both kvhAS and kvhR are under-controlled by kvgAS and there may be a cross-talk between each other to regulate CPS synthesis and LPS modification. However, the pathway is yet to be identified. In addition, kvhAS senses a high-level concentration of Mg2+ and in response to affect the drug susceptibility.

(33)

REFERENCES

楊淑理,國立清華大學碩士論文,肺炎克雷白氏株特異性基因的鑑定- kvgASQR 基因群的序列及表現分析,中華民國八十九年六月。 賴旻初,國立交通大學碩士論文,克雷白氏肺炎桿菌kvgAII 基因之選殖與表現 分析,中華民國八十九年八月。 林靖婷,國立交通大學碩士論文,克雷白氏肺炎桿菌kvgAS雙分子訊息傳遞系統 的功能探討,中華民國九十年六月。 黃騰逸,國立交通大學碩士論文,克雷白氏肺炎桿菌 CG43 中 KvhAS 雙分子調控 系統的特性分析,中華民國九十一年六月。 賴怡琪,國立清華大學博士論文,克雷白氏肺炎菌 CG43 致病基因相關的搜尋 (RmpA2 調控莢膜生合成的機轉),民國九十一年七月。

Alberti S, Alvarez D, Merino S, Casado MT, Vivanco F, Tomas JM, Benedi VJ. Analysis of complement C3 deposition and degradation on Klebsiella pneumoniae. Infect Immun. 1996 Nov;64(11):4726-32.

Alvarez D, Merino S, Tomas JM, Benedi VJ, Alberti S. Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient

(34)

Klebsiella pneumoniae clinical isolates. Infect Immun. 2000 Feb;68(2):953-5.

Arakawa Y, Wacharotayankun R, Nagatsuka T, Ito H, Kato N, Ohta M. Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid. J Bacteriol. 1995 Apr;177(7):1788-96.

Blumenkrantz N, Asboe-Hansen G. New method for quantitative determination of uronic acids. Anal Biochem. 1973 Aug;54(2):484-9.

Bock A, Gross R. The BvgAS two-component system of Bordetella spp.: a versatile modulator of virulence gene expression. Int J Med Microbiol. 2001 May;291(2):119-30.

Chang C, Stewart RC. The two-component system: Regulation of diverse signaling pathways in prokaryotes and eukaryotes. Plant Physiol. 1998 Jul;117(3):723–31.

Chen MH, Takeda S, Yamada H, Ishii Y, Yamashino T, Mizuno T. Characterization of the RcsC-->YojN-->RcsB phosphorelay signaling pathway involved in capsular synthesis in Escherichia coli. Biosci Biotechnol Biochem. 2001 Oct;65(10):2364-7.

(35)

of the contribution of the capsular polysaccharide and the lipopolysaccharide O side chain to the virulence of Klebsiella pneumoniae in a murine model of pneumonia. Infect Immun. 2002 May;70(5):2583-90.

De Lorenzo V, Timmis KN. Analysis and construction of stable phenotypes in Gram-negative bacteria with Tn5- and Tn10- derived minitransposons. Methods Enzymol. 1994;235:386-405.

De Wulf P, Kwon O, Lin EC. The CpxRA signal transduction system of Escherichia

coli: growth-related autoactivation and control of unanticipated target operons. J

Bacteriol. 1999 Nov;181(21):6772–8.

Domenico P, Schwartz S, Cunha BA. Reduction of capsular polysaccharide production in Klebsiella pneumoniae by sodium salicylate. Infect Immun. 1989 Dec; 57(12):3778-82.

Eguchi Y, Oshima T, Mori H, Aono R, Yamamoto K, Ishihama A, Utsumi R. Transcriptional regulation of drug efflux genes by EvgAS, a two-component system in Escherichia coli. Microbiology. 2003 Oct;149(Pt 10):2819–28.

Foussard M, Cabantous S, Pedelacq J, Guillet V, Tranier S, Mourey L, Birck C, Samama J. The molecular puzzle of two-component signaling cascades. Microbes

(36)

Infect. 2001 Apr;3(5):417-24.

Groisman EA. The pleiotropic two-component regulatory system PhoP-PhoQ. J Bacteriol. 2001 Mar;183(6):1835-42.

Gupta A, Ampofo K, Rubenstein D, Saiman L. Extended spectrum beta lactamase-producing Klebsiella pneumoniae infections: a review of the literature. J Perinatol. 2003 Sep;23(6):439-43.

Hagiwara D, Sugiura M, Oshima T, Mori H, Aiba H, Yamashino T, Mizuno T. Genome-wide analyses revealing a signaling network of the RcsC-YojN-RcsB phosphorelay system in Escherichia coli. J Bacteriol. 2003 Oct;185(19):5735-46.

Held TK, Jendrike NR, Rukavina T, Podschun R, Trautmann M. Binding to and opsonophagocytic activity of O-antigen-specific monoclonal antibodies against encapsulated and nonencapsulated Klebsiella pneumoniae serotype O1 strains. Infect Immun. 2000 May;68(5):2402-9.

Hengge-Aronis R. Signal transduction and regulatory mechanisms involved in control of the sigma(S) (RpoS) subunit of RNA polymerase. Microbiol Mol Biol Rev. 2002 Sep;66(3):373-95, table of contents.

(37)

Hirakawa H, Nishino K, Hirata T, Yamaguchi A. Comprehensive studies of drug resistance mediated by overexpression of response regulators of two-component signal transduction systems in Escherichia coli. J Bacteriol. 2003 Mar;185(6):1851-6.

Hirakawa H, Nishino K, Yamada J, Hirata T, Yamaguchi A. Beta-lactam resistance modulated by the overexpression of response regulators of two-component signal transduction systems in Escherichia coli. J Antimicrob Chemother. 2003 Oct;52(4):576-82. Epub 2003 Sep 01.

Ibanez-Ruiz M, Robbe-Saule V, Hermant D, Labrude S, Norel F. Identification of RpoS (σs)-regulated genes in Salmonella enterica Serovar typhimurium. J Bacteriol. 2000 Oct;182(20):5749-56.

Joseph S, Russell DW. Molecular Cloning: a laboratory manual—3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2001.

Kabha K, Nissimov L, Athamna A, Keisari Y, Parolis H, Parolis LA, Grue RM, Schlepper-Schafer J, Ezekowitz AR, Ohman DE, et al. Relationships among capsular structure, phagocytosis, and mouse virulence in Klebsiella pneumoniae. Infect Immun. 1995 Mar;63(3):847-52.

(38)

Immunochemical and biological characterization of three capsular polysaccharides from a single Bacteroides fragilis strain. Infect Immun. 2001 Apr;69(4):2339-44.

Karimova G., Bellalous J, Ullmann A. Phosphorylation-dependent binding of BvgA to the upstream region of the cyaA gene of Bordetella pertussis. Mol Microbiol. 1996 May;20(3):489-96.

Karlyshev AV, Wren BW. Detection and initial characterization of novel capsular polysaccharide among diverse Campylobacter jejuni strains using alcian blue dye. J Clin Microbiol. 2001 Jan;39(1):279-84.

Konig J, Bock A, Perraud AL, Fuchs TM, Beier D, Gross R. Regulatory factors of

Bordetella pertussis affecting virulence gene expression. J Mol Microbiol Biotechnol.

2002 May;4(3):197-203.

Lai YC, Lin GT, Yang SL, Chang HY, Peng HL. Identification and characterization of KvgAS, a two-component system in Klebsiella pneumoniae CG43. FEMS Microbiol Lett. 2003 Jan 21;218(1):121-6.

Lai YC, Peng HL, Chang HY. RmpA2, an activator of capsule biosynthesis in

Klebsiella pneumoniae CG43, regulates K2 cps gene expression at the transcriptional

(39)

Lai YC, Yang SL, Peng HL, Chang HY. Identification of genes present specifically in a virulence strain of Klebsiella pneumoniae. Infect Immun. 2000 Dec;68(12): 7149-51.

Locht C. Molecular aspects of Bordetella pertussis pathogenesis. Int Microbiol. 1999 Sep;2(3):137-44.

Loewen PC, Hu B, Strutinsky J, Sparling R. Regulation in the rpoS regulon of

Escherichia coli. Can J Microbiol. 1998 Aug;44(8):707-17.

Matsushita M, Janda KD. Histidine kinases as targets for new antimicrobial agents. Bioorg Med Chem. 2002 Apr;10(4):855–67.

Miller J H. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 1972.

Mizuno T. His-Asp phosphotransfer signal transduction. J Biochem (Tokyo). 1998 Apr;123(4)555-63.

Nishino K, Yamaguchi A. EvgA of the two-component signal transduction system modulates production of the YhiUV multidrug transporter in Escherichia coli. J Bacteriol. 2002 Apr184(8):2319–23.

(40)

Nishino K, Yamaguchi A. Overexpression of the response regulator evgA of the two-component signal transduction system modulates multidrug resistance conferred by multidrug resistance transporters. J Bacteriol. 2001 Feb;183(4):1455–8.

Ohki R, Giyanto, Tateno K, Masuyama W, Moriya S, Kobayashi K, Ogasawara N. The BceRS two-component regulatory system induces expression of the bacitracin transporter, BceAB, in Bacillus subtilis. Mol Microbiol. 2003 Aug;49(4):1135-44.

Perez-Perez GI, Shepherd VL, Morrow JD, Blaser MJ. Activation of human THP-1 cells and rat bone marrow-derived macrophages by Helicobacter pylori lipopolysaccharide. Infect Immun. 1995 Apr;63(4):1183-7.

Perraud AL, Kimmel B, Weiss V, Gross R. Specificity of the BvgAS and EvgAS phosphorelay is mediated by the C-terminal HPt domains of the sensor proteins. Mol Microbiol. 1998 Mar; 27(5):875-87.

Perraud AL, Rippe K, Bantscheff M, Glocker M, Lucassen M, Jung K, Sebald W, Weiss V, Gross R. Dimerization of signalling modules of the EvgAS and BvgAS phosphorelay systems. Biochim Biophys Acta. 2000 May 23;1478(2):341-54.

Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods and pathogenicity factors. Clin Microbiol Rev. 1998 Oct;

(41)

11(4):589-603.

Pomposiello PJ, Bennik MH, Demple B. Genome-wide transcriptional profiling of the

Escherichia coli responses to superoxide stress and sodium salicylate. J Bacteriol.

2001 Jul;183(13):3890-902.

Reed LJ, Muench H. A sample method of estimating the fifty percent endpoints. Am. J. Hyg. 1938, 27:493-497.

Sailer FC, Meberg BM, Young KD. Beta-Lactam induction of colanic acid gene expression in Escherichia coli. FEMS Microbiol Lett. 2003 Sep 26;226(2):245-9.

Scarlato V, Arico B, Prugnola A, Rappuoli R. Sequential activation and environmental regulation of virulence genes in Bordetella pertussis. EMBO J. 1991 Dec;10(12):3971-5.

Schneider B, Stubs D, Gross R. Identification and genomic organization of gene loci negatively controlled by the virulence regulatory BvgAS two-component system in

Bordetella bronchiseptica. Mol Genet Genomics. 2002 Jun;267(4):526–35.

Skorupski K, Taylor RK. Positive selection vectors for allelic exchange. Gene. 1996 Feb 22;169(1):47-52.

(42)

Stock AM, Robinson VL, Goudreau PN. Two-component signal transduction. Annu Rev Biochem. 2000;69:183-215.

Sugiura M, Aiba H, Mizuno T. Identification and classification of two-component systems that affect rpos expression in Escherichia coli. Biosci Biotechnol Biochem. 2003 Jul;67(7):1612-5.

Taylor RK, Manoil C, Mekalanos JJ. Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae. J. Bacteriol. 1989 Apr;171(4):1870-8.

Utsumi R, Katayama S, Ikeda M, Igaki S, Nakagawa H, Miwa A, Taniguchi M, Noda M. Cloning and sequence analysis of the evgAS genes involved in signal transduction of Escherichia coli K-12. Nucleic Acids Symp Ser. 1992;(27):149-50.

Venturi V. Control of rpoS transcription in Escherichia coli and Pseudomonas : why so different? Mol Microbiol. 2003 Jun;49(1):1–9.

Verhamme DT, Arents JC, Postma PW, Crielaard W, Hellingwerf KJ. Investigation of in vivo cross-talk between key two-component systems of Escherichia coli. Microbiology. 2002 Jan;148(Pt 1):69-78.

(43)

Whitfield C, Paiment A. Biosynthesis and assembly of Group 1 capsular polysaccharides in Escherichia coli and related extracellular polysaccharides in other bacteria. Carbohydr Res. 2003 Nov 14;338(23):2491-502.

Whitfield C, Roberts IS. Structure, assembly and regulation of expression of capsules in Escherichia coli. Mol Microbiol. 1999 Mar;31(5):1307-19.

參考文獻

相關文件

6 《中論·觀因緣品》,《佛藏要籍選刊》第 9 冊,上海古籍出版社 1994 年版,第 1

The first row shows the eyespot with white inner ring, black middle ring, and yellow outer ring in Bicyclus anynana.. The second row provides the eyespot with black inner ring

We propose two types of estimators of m(x) that improve the multivariate local linear regression estimator b m(x) in terms of reducing the asymptotic conditional variance while

49 參看氏著“Two problems in the history of Indian Buddhism: the layman/monk distinction and the doctrines of the transference of merit.”(1985) Bones, Stones, and Buddhist Monks:

“Find sufficiently accurate starting approximate solution by using Steepest Descent method” + ”Compute convergent solution by using Newton-based methods”. The method of

化成 reduced echelon form 後由於每一個 row 除了該 row 的 pivot 外, 只剩 free variables (其他的 pivot variable 所在的 entry 皆為 0), 所以可以很快地看出解的形式..

In this paper, we extend this class of merit functions to the second-order cone complementarity problem (SOCCP) and show analogous properties as in NCP and SDCP cases.. In addition,

本書立足中華文化大背景,較為深入系統地分析研究了回族傳統法文化的形成基礎、發展歷