國立交通大學
生物科技研究所
碩士論文
克雷白氏肺炎桿菌中纖毛表現之分析
Study on the fimbrial expression in
Klebsiella pneumoniae
研究生:廖朝陽
指導教授:彭慧玲 博士
致謝
兩年的碩士班生涯就此告一段落,回憶過去的種種,無論在生活中或是研究 工作上都讓我學習到許多;感謝身邊的人一直以來的照顧及支持,讓我有所成長。 特別是我的指導老師彭慧玲博士,在研究上給我很大的發揮空間,也提供許 多寶貴的想法與意見。一直以來,給您增添了不少的麻煩;像是過去的許多報告、 對於一些觀念的謬思,或是這次的論文,老師都不辭辛勞、費心費力地給予教導, 在此致上我最深的感激。此外,這篇論文的完成,亦要感謝中興大學的黃秀珍老 師以及交通大學的林志生老師細心地指正與建議,讓此篇論文能夠更加完整。 感謝實驗室的夥伴們,豐富了我的碩士班生活。特別是盈蓉學姊耐心的指 導,讓我學習到許多實驗上的技巧;而健誠學長亦提供許多研究上的資訊以及充 分的討論,讓我收穫不少。此外,也要感謝格維和登魁這兩位實驗室的同學一路 上共同分享喜憂,伴隨著我一路成長;祝福你們都有美好的前程。 還有靜柔學姊、育聖學長、智凱學長、靖婷學姊、秉熹、雲龍、嘉怡、佳瑩、 振宇、純珊,謝謝這段時間的幫忙與包容,讓此求學期間增添了許多美好的回憶。 最後要感謝爸爸、媽媽、大姐、二姐以及我的好室友仲翔,謝謝您們給我鼓舞與 照顧,僅次論文獻給我的家人以及各位,謝謝您們。Contents
Page Contents………..………... Ⅰ Table content……….………..……… Ⅲ Figure content………..……….………….. Ⅳ Abbreviation……….………... Ⅵ Abstract in Chinese………..……….…... 1 Abstract……….……….……... 3 Introduction………..……….………... 5Materials and Methods……….……….……… 15
Results Part Ι: Expressional analyses of the putative fimbriae in Klebsiella pneumoniae... 23
Part ΙΙ: Characterization of the putative regulators encoding by the genes respectively located in the vicinity to kpb, kpd, or kpf gene cluster……... 28
Discussion………..………….. 33
Reference.……….….……….…... 36
Table
Page
Table 1. Bacterial strains and plasmids used in this study……….….. 46
Table 2. Primers used in this study………..…. 48
Table 3. Measurements of the fimbrial promoter activity on different
culture conditions………...… 49
Table 4. Measurements of the fimbrial promoter activity on different carbon
sources and bovine serum additions………... 50
Figure
Page
Fig. 1. Organization of the putative fimbrial gene clusters in K.pneumoniae
NTUH-K2044………...………..………...…...… 52
Fig. 2. Promoter activity measurement in K. pneumoniae CG43S3Z01 and CG43S3Z01phgS-………...………....… 53
Fig. 3. Sequence alignment of PhgS with other representative members of MarR family………...….………....………... 54
Fig. 4. Nucleotide sequence of PkpgA……… 55
Fig. 5. Nucleotide sequence of PmrkA………...………. 56
Fig. 6. Activity measurements of PkpgA, PkpgA1, PkpgA2, PmrkA, and Pmrkt…...…. 57
Fig. 7. Schematic representation of the entericidin loci....……….…...…. 58
Fig. 8. Organization of kpbRABCD and the BLAST result of KpbR... 59
Fig. 9. Over-expression of KpbR in E. coli JM109….………...……….. 60
Fig. 10. Over-expression of KpdR in K. pneumoniae and the organization of kpdRABCD………. 63
Fig. 11. PCR detection of blaTEM-116 in K. pneumoniae NTUH-K2044 and CG43S3………..……….... 64
Fig. 12. Ampicillin susceptibility assay……….………... 65
Fig. 14. Biofilm formation capability... 68
Fig. 15. Effect of KpdR over-expression on the capsular synthesis………….…… 69
Fig. 16. Organization of kpfABCD and kpfR genes, and the BLAST result of
Abbreviation
BLAST basic local alignment search tool
BCIP 5-bromo-4-chloro-3-indolyl phosphate
ESBL extended-spectrum beta-lactamase
2CS two component system
kDa kiloDalton
AFA a-fimbrial adhesins
NFA non-fimbrial adhesins
CPS capsular polysaccharide
PG peptidoglycan
IPTG isopropyl-1-thio-β-D-galactopyranoside
LB Luria-Bertani
mRNA messenger RNA
NBT nitro blue tetrazolium chloride
ONPG ο-nitrophenyl-β-D-galactopyranoside PCR polymerase chain reaction
PAGE polyacrylamide gel electrophoresis
Lrp leucine-responsive regulatory protein
IHF integration host factor
CAP catabolite gene activator protein
H-NS histone-like protein
Dam deoxyadenosine methylase
TFA trifluoroacetic acid
中文摘要
克雷白氏肺炎桿菌是伺機性感染的革蘭氏陰性菌。利用生物資訊分析方法 HMMER,我們在一株從台灣大學附設醫院的病患身上所分離出具有高致病性的 NTUH-K0244 菌株的基因體中找到九套線毛基因組。除了已被廣泛研究的第一型 (fim)和第三型(mrk)線毛之外,其餘七套的功能以及特性都未曾被報導。我 們分別將它們命名為kpa、kpb、kpc、kpd、kpe、kpf 和 kpg。為了了解各套線毛 在特定條件下的表現情形,我們建構啟動子的活性分析系統。在各種測試條件 下,第一型以及第三型線毛的啟動子活性都比其他七套要高。此外,這兩套線毛 的活性在添加葡萄糖或甘油的培養環境下皆有顯著上升的情形。相對地,在高滲 透壓力、過氧化物的存在,或偏鹼環境的刺激下,都造成活性的降低。 另一方面,我們在 kpd 基因組上游找到一個未知功能的基因,經由胺基酸 序列比對(BLAST),我們認為其轉譯蛋白可能以雙分子訊息傳遞系統中調控子 的角色來調控kpd 基因組表現,因而命名為 kpdR。有趣的是,在克雷白氏肺炎 桿菌中大量表現 KpdR 蛋白時,我們發現 β−lactamase TEM-116 蛋白明顯增加因 而大大提高細菌對ampicillin 的抵抗力;相對的,其生物膜的形成受到抑制。雖 然,我們分別在CG43S3 和 NTUH-K2044 兩株菌中可利用核酸增殖反應(PCR)得到 bla TEM-116,但經由比對現有的NTUH-K2044 基因資料庫卻找不到該基因。
今後,將確認 bla TEM-116的位置,並建構 kpdR 及 bla SHV-1a的突變菌株,進一步 了解 KpdR 與細菌對抗β-內酰胺類抗生素能力的關係。同時,我們在 kpb 及
kpf 基因組上游的位置分別找到類似的調控子基因,並分別命名為 kpbR 和 kpfR。而在克雷白氏肺炎桿菌中分別大量表現 KpbR 和 KpfR 蛋白時,我們發現 細菌的生長都會受到抑制。 最後,我們發現移除座落於 mrk 及 fim 基因組之間的調控基因 phgS 與 phgM,不僅會顯著降低mrk 啟動子的活性,kpg 線毛的啟動子活性也明顯下降; 而進一步,分別在這兩個啟動子Pkpg和Pmrk片段中移除過去報導我們預測 PhgS 的辨認序列,原先受PhgS 影響的現象已不復見,這些結果顯示 PhgS 和 PhgM 在 調控kpg 與 mrk 線毛的表現上扮演特定的角色。
Abstract
There are nine fimbiral operons identified in the genome of Klebsiella
pneumoniae NTUH-K2044, a highly invasive strain isolated from Taiwan university hospital, by the HMMER search. The fim and mrk fimbriae had been described
previously, but the others are novel fimbrial operons and are respectively named kpa,
kpb, kpc, kpd, kpe, kpf, and kpg. The putative promoters of the nine operons were isolated, cloned in a LacZ reporter plasmid and the activities measured. In K.
pneumoniae CG43S3Z01, the promoters of type 1 fimbriae (PfimA and PfimB) and type 3
fimbriae (PmrkA) had higher level of activity than those of the other fimbrial promoters.
The activity of PfimA, PfimB, and PmrkA were enhanced under static cultures and the
cultures addition with glucose or glycerol. On the other hand, the expression of fimB
and mrkA were suppressed while the bacteria subject to higher osmotic pressure (>
200 mM NaCl), oxidative stress (60 μM H2O2), or the pH switch from 5.5 to 8.
Interestingly, the overexpression of KpdR, a putative response regulator located
upstream of the kpd gene cluster, appeared to decrease the biofilm formation and
increase expression of β-lactamase TEM-116 in both K. pneumoniae NTUH-K 2044
and CG43S3Z01. This implied a regulatory role of KpdR in the biofilm formation and
also the resistance to the β-lactam drug. Searching for the gene of blaTEM-116 and
over-expressions of two other putative regulators, KpbR and KpfR, in K. pneumoniae
appeared to interfere growth of the bacteria.
Finally, the deletion of phgS or phgM was found to reduce not only the
expression of type 3 fimbriae but also the activity of PkpgA. Truncation of the predicted
consensus sequences for PhgS binding from PkpgA or PmrkA abolished the PhgS
dependent expression. It was suggested that a regulatory role of PhgS for the
Introduction
The emergence of Klebsiella pneumoniae is a striking topic worldwide
Klebsiella pneumoniae, a gram-negative opportunistic pathogen, is a common cause of community-acquired and nosocomial infections (1). Although the incidence
of community-acquired K. pneumoniae has decreased, the mortality rate due to
Klebsiella pneumonia remains high (2). In western countries, most K. pneumoniae infections occur in lungs and urinary tract. While K. pneumoniae has been the leading
cause of liver abscess in Taiwan (3). It has been shown that patients with diabetic
mellitus in Taiwan are more susceptible to K. pneumoniae infection than those
without diabetes (3). In addition to liver abscess that involves destructive clinical
syndromes, the commonly emerging infections include metastatic meningitis and
endophthalmitis, osteomyelitis, and brain abscess (3, 4). Although several virulence
factors, such as polysaccharide capsule, different adhesins, lipopolysaccharide, and
iron-scavenging proteins have been identified (1), the pathogenicity of K. pneumoniae
has not been completely elucidated.
Individuals most at risk for K. pneumoniae infections are infants, the elderly, and
those with compromised defense mechanisms. Patients that suffer from Klebsiella
gastrointestinal tract by antibiotic-resistant K. pneumoniae isolates (5, 6). These
resistant strains produce extended-spectrum beta-lactamases and are of particular
concern as the infections lead to relatively high mortality rates due to treatment failure
and subsequent septicemia (7).
β-lactamases genes are prevalent in Klebsiella pneumoniae
Many Gram-negative bacteria produce β-lactamases for the resistance to
β-lactam antibiotics. The enzymes evolved from bacterial penicillin-binding proteins
which involved in peptidoglycan synthesis. The encoding genes are found on
chromosome or on transmissible plasmids (8). Plasmid-encoded β-lactamases are
often expressed in large amounts (9), while chromosomally encoded β-lactamases are
typically expressed at low levels until induced by the presence of the substrates. In
Gram-negative bacteria, β-lactamases are exported to the periplasm, whereas in
Gram-positive bacteria they are secreted from the cell (10). β-lactamases enzymes
exhibit diversity in both structure and function, and are divided into four classes (A-D)
based on their primary sequence and catalytic mechanism. Class A is the most
numerous and is a serine hydrolase as the Class C and D enzymes. The class B are
zinc-dependent β-lactamases. Historically, these enzymes were described as penicillinases because they were able to catalyze penicillin hydrolysis. The ubiquitous
belongs to class A, and was the first β-lactamase for which the crystal structure was
solved (11). Class A β-lactamases have a molecular mass of approximately 29 kDa
and are comprised of 260-280 residues, of which nine appear to be highly conserved:
four residues, Ser-70, Lys-73, Ser-130 and Glu-166 are critical for catalysis whereas
the five remaining residues Gly-45, Pro-107, Asp-131, Ala-134 and Gly-236 likely
play a role in structural integrity (12). Furthermore, three additional residues: Asn (in
most sequences)-132, Lys/Arg-234 and Ser/Thr-235 also play important roles in
enzyme activity. Sequence analysis revealed similarity between class A β-lactamases
and the low molecular weight PBPs such as PBP4 of Escherichia coli (13). In
addition, the crystal structure of class A β-lactamases revealed homology with the
catalytic domain of PBP5 from E. coli (14). However, even though the PBPs and
β-lactamases share strong structural homologies and have a common ancestry,
β-lactamases are thought to have lost the ability to interact with PG while function
efficiently as resistance enzymes (15).
The first plasmid-mediated β-lactamase in gram-negative bacteria, tem-1, was
described in the early 1960s. Another common plasmid-mediated β-lactamase found
in K. pneumoniae and E. coli is shv-1. Although SHV-1 was originally characterized
as a plasmid-mediated β-lactamase (16), recent data showing that many clinical K.
suggest its production may be intrinsic to K. pneumoniae,. Many new β-lactam
antibiotics have been designed to resist to the hydrolytic action. Resistance to these
new beta-lactam antibiotics due to extended-spectrum beta-lactamases (ESBLs) also
have emerged subsequently. ESBLs were commonly derived from tem-1 and shv-1
β-lactamases by mutations to alter the hydrolytic abilities and spectrums (17). Over
100 tem and shv types of β-lactamases have been characterized
(http://www.lahey.org/studies/webt.asp).
Biofilm formation is linked to virulence and colonization
In addition to the prevalence of ESBLs, recent studies suggest that biofilm
formation may also be an important virulence factor for K. pneumoniae. Biofilms are
organized communities of bacteria living on surface environments. Growth as a
biofilm is linked to virulence and colonization for a variety of bacterial pathogens,
including Pseudomonas aeruginosa, which causes chronic, life threatening respiratory
infections in cystic fibrosis patients (18, 19). It has been demonstrated in vitro that
bacteria growing within biofilms are more resistant to antibiotic treatment than
bacteria growing planktonically (20). While expression of β-lactamases have been
shown to inhibit bacterial biofilm formation and this anti-biofilm effect was specific
to class A and D β-lactamases (21). A model was proposed in which β-lactamases
macromolecular complexes participating in surface attachment. The interference leads
to affect the subsequent biofilm development (21).
Microbial adhesion to surfaces is the onset of the development of a biofilm.
Adhesion to host tissue receptors is the first step in successful colonization by many
Gram-negative pathogens. Adhesion fimbriae are specialized surface structures
responsible for the successful recognition and binding of these bacteria to their host
receptors. In addition, these fimbriae are responsible for maintaining this contact
during the first stages of bacterial colonization. The resulting specific adherence may
enhance bacterial persistence at the site of infections , but adherence also has been
identified as a virulence factor that facilitates tissue attack, invasion, and mucosal
inflammation.
Fimbriae are long filamentous polymeric protein structures located at the surface
of bacterial cells. The word “fimbriae” comes from the Latin word for “thread” or
“fibre” (22). These adhesive organelles are also referred to as “pili”, which comes
from the Latin word for “hair” or “hairlike strucutre” (23). Practically all
Gram-negative bacterial species that have been examined so far were found to
produce one or more types of fimbriae. While a few Gram-positive bacteria were also
proteins, and genes encoding a major subunit, minor subunit, a periplasmic chaperone,
a relatively large outer membrane usher and an adhesin protein (25). Despite their
functional differences, the conserved genetic organization implicates that these
different fimbriae evolved from a common ancestor (25).
Besides fimbriae, amorphous adhesins also exist, called a-fimbrial adhesins
(AFA) or non-fimbrial adhesins (NFA). These adhesins are present at the outer
membrane of the bacterial cell as single proteins or large multi-unit aggregates (25).
Another distinct group of adhesins is type 4 pili, which is present in a wide variety of
Gram-negative bacterial pathogens on one pole of the bacterial cell. They are, besides
adherence, associated with twitching motility (26, 27).
Polysaccharide capsule is a major pathogenicity factor
K. pneumoniae is enveloped by a prominent polysaccharide capsule that produce large, sticky colonies when plated on an agar plate with nutrient media. The strains
with the hypermucoviscosity phenotype demonstrate extremely high viscosity
determined by a string test (28). In Taiwan, the hypermucoviscosity-positive strains
were more prevalent for cases of K. pneumoniae liver abscess than from other sites of
K. pneumoniae infection (29).
with certain infection sites (30). In animal models, K1 and K2 strains are the most
virulent (1). The seroepidemiologic surveys indicated that Klebsiella K1 was rare
among North American and European clinical isolates (30, 31, 32). However, the K1
serotype was found the most frequently in Taiwan (33).
Genome-wide study of the putative chaperone-usher assembly fimbriae in K.
pneumoniae NTUH-K2044
In this study, two invasive strains of K. pneumoniae are studied and the
properties compared. K. pneumoniae NTUH-K2044 of K1 antigen, isolated from the
blood of a previously healthy 40 years-old man suffering from community-acquired
primary liver abscess and metastatic meningitis, is a hypermucoviscous strain (28). K.
pneumoniae CG43, a K2 serotype, is also a liver abscess isolate (34).
The sequence analysis revealed nine fimbrial operons identified by the HMMER
search in the genome of K. pneumoniae NTUH-K2044. Except mrk (type 3 fimbriae)
and fim (type 1 fimbriae), the other novel fimbrial operons were respectively named
kpa, kpb, kpc, kpd, kpe, kpf, and kpg. Upstream of kpb, kpd and kpf gene clusters, respectively, a gene encoding putative response regulator that contains both receiver
domain and DNA-binding domain was identified and named kpbR, kpdR, and kpfR.
namely phgS and phgM.
PecS/M plays a critical role in virulence regulation in Erwinia chrysanthemi
PecS was originally discovered to negatively regulate the expression of cellulose
and pectate lyase in E. chrysanthemi (35). The pecS mutant showed an increased rate
of infection, which is likely resulted of the overproduction of extracellular macerating
enzymes and resistance to oxidative stress (36). In addition to pectate lyase, PecS has
also been shown to negatively affect the expression of cellulase, indigoidine, a type
Ⅱsecretion pathway and flagella biosynthesis (35, 37, 38). Pectate lyases and
cellulases are extracellular enzymes that attack components of the plant cell wall and
are crucial in the pathogenesis of E. chrysanthemi (39). Indigoidine is a blue pigment
upregulated in pecS mutants and is involved in the resistance to oxidative stress (35,
36). Moreover, PecS could positively regulate the expression of polygalacturonase (40,
41).
PecS was classified into the MarR/SlyA family of transcriptional regulators.
MarR was the first member crystallized in the family (42). Although members of the
MarR/SlyA family show little sequence homology, their structures share significant
similarities. They include a common triangular shape with winged helix-turn-helix
regulatory molecules (39).
Cross-talk regulation between the expression of kpg and mrk genes
The factors mediating transcriptional regulation of fimbriae expression in
response to environmental signals either belong to the family of “local” regulators or
to the family of “global” regulators (43). The local regulators are encoded by the
respective fimbrial operons and their function is restricted to the operon by which
they are encoded. Global regulators control the expression of a variety of operons.
The regulatory activity of global regulators is superimposed over the local control of
each individual operon (44).
Global regulators which are known to be involved in fimbriae expression are,
e.g., leucine-responsive regulatory protein (Lrp), integration host factor (IHF),
catabolite gene activator protein (CAP), the histone-like protein (H-NS), and
deoxyadenosine methylase (Dam) (43). Lrp, IHF and CAP are DNA bending proteins.
The H-NS protein changes the degree of supercoiling of the DNA. Dam is an enzyme
involved in the methylation of GATC sites (43). Many fimbiral operons were found to
encode one or two local regulators. These genes probably act as transcriptional
activators of the respective operons, but their precise mode of action is not well
Specific aims
This study could be classified into three parts. In part Ι, the optimal condition for
the expression of the fimbriae is investigated using LacZ reporter system. Except for
fim and mrk gene clusters that have been well characterized in K. pneumoniae, the conditions for the optimal expression of the fimbrial operons are mysterious.
As reported by Clegg, et al. in 2006 (45), the transposon insertion in the 5’
non-coding sequence of kpg gene cluster lowered the expression level of type 3
fimbriae indicating a possible interacting regulation. It has been shown in our
laboratory that the transcription of mrk is PhgS/M dependent (46). Thus, the part ΙΙ is
to investigate the role of PhgS in regulation of kpg and mrk expression.
In the vicinity to kpb, kpd and kpf fimbrial gene clusters, three two component
system (2CS) response regulator encoding genes namely kpbR, kpdR, and kpfR were
identified. Whether the response regulator(s) play a role on the expression of the
Materials and Methods
Bacterial strains, plasmids and growth conditions
Bacterial strains and plasmids used in this study are listed in Table 1. E. coli
JM109 was used for cloning and Pseudomonas aeruginosa PAO1 was a positive
control strain in biofilm formation assay. Bacteria were grown at 37 ℃ in
Luria-Bertani (LB) broth or the medium supplemented with appropriate antibiotics.
Antibiotics were used where indicated at the following concentrations: ampicillin
(100 μg/ml), kanamycin (25 μg/ml), and chloramphenicol (35 μg/ml). For
β-galactosidase activity assay, the bacteria in different culture conditions were
statically grown to early logarithmic phase (optical density at 600 nm 0.4 to 0.5).
DNA manipulation
Plasmids were purified by the High-Speed Plasmid Mini kit (Geneaid, Taipei,
Taiwan). All restriction and DNA-modifying enzymes were used as recommended by
the manufacturer (Fermentas, Hanover, MD, USA). PCR amplifications were
performed with Taq DNA polymerase (MDBio, Inc, Taiwan) or Blend TaqTM-Plus-
DNA polymerase (Cosmo Bio Co., LTD.). PCR products and DNA fragments were
purified using the Gel/PCR DNA Fragments Extraction kit (Geneaid). The primers
cells was performed by following the method of Dower (47).
Construction of plasmid-mediated LacZ reporter strains in K. pneumoniae CG43S3Z01
The putative promoter containing about 600 nucleotides upstream of the
translation initiation codon of each of the fimbrial gene clusters (Fig. 1) were PCR
amplified with respective primer pairs (Table 2) from the genomic DNA of K.
pneumoniae NTUH-K0244, and the PCR product cloned into BamHI site in front of the promolerless lacZ in the low-copied plasmid, pLacZ15 (48). To identifiy the
Phg-binding site in PkpgA and PmrkA, the specific primers ZY105/pCC046,
ZY106/pCC046 and ZY107/pMrkA5 were used to generate the truncated forms of the
promoters.
Measurement of β-galactosidase activity
Miller assay was carried out as described (48). In summary, a culture was grown
under the tested condition to early logarithmic phase (optical density at 600 nm 0.4 to
0.5). A 100 μl was removed and added to the reaction mixture which containing 900
μl Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM
β-mercaptoethanol), 17 μl 0.1% SDS and 35 μl chloroform. The reaction mixture was
ONPG was added to the reaction and incubated at 30℃ until yellow color was
apparent or for 1 h if there was no obvious color change. Finally, the reaction was
stopped by adding 500 μl of stop solution (1 M Na2CO3) and the absorbance of the
supernatant was measured at OD420 (ELx800, BIO-TEK). One unit of β-galactosidase
is defined as the hydrolysis of 1 nmol ONPG per min per mg protein. Strains were
tested under different conditions of temperature, pH, oxygen levels, carbon source,
serums, osmolarity stress, and others.
Over-expression of KpbR, KpdR and KpfR
The kpbR, kpdR and kpfR were respectively amplified with specific primers
(Table 2) and cloned into a TA vector, pCR®
2.1-TOPO®
(Table 1), and then subcloned
into pETQ33, which carrying an IPTG-inducible tac-promoter preceding the multiple
cloning site, with EcoRI digestion. The resulted recombinant plasmids were then
transformed into E. coli JM109, K. pneumoniae NTUH-K2044 and K. pneumoniae
CG43S3 strains. Analysis of the overexpression was carried out while these
transformants were induced with 0.5 mM IPTG and cultured at 37℃ for 4 h.
In-gel digestion
Stained protein bands were excised from gels and were rinsed with distilled
bicarbonate and acetonitrile (1:1) for 15 min, and then the gels were transferred into
another solution containing 50 mM ammonium bicarbonate and acetonitrile (3:2) for
15 min. After repeating the destain procedure, the protein was incubated in 500 μl
acetonitrile to remove the water and dried using a speed vaccum concentrator. After
immersed in 50 μl of 25 mM ammonium bicarbonate, the protein was digested with 3
to 5 μl trypsin (20 μg/ml) at 37℃ for 12 to 16 h. The digested peptides were then
recovered using a solution containing 0.1% trifluoroacetic acid (TFA) in acetonitrile.
The resulting peptide extracts were processed to MS or MS/MS analysis by core
facilities for proteomics and structural biology research in Academia Sinica
(http://proteome.sinica.edu.tw/).
Ampicillin susceptibility assay
Ampicillin susceptibility test was performed by the broth method (49). An
overnight culture of bacteria was refreshed in LB broth with different concentrations
of ampicillin. The transformants of K. pneumoniae NTUH-K2044 and K. pneumoniae
CG43S3 carrying pETQ33 or kpdR overexpression plasmid were cultured with
addition of 0.5 mM IPTG and 25μg/ml kanamycin. The optical density at 600 nm
were measured after 16 h of incubation at 37℃.
Biofilm formation was assessed by the ability of the cells to adhere to the glass
tubes. The indicator medium contained an aliquot of 1:20 diluted overnight bacteria
culture in LB and was incubated at 37 ℃ for 48 h for biofilm formation.
Quantification of biofilm formation was carried out as the reported protocol (50) with
some modification. Essentially, the indicator medium (200 μl/well) in 96-well
microtire dishes made of PVC (TPP 96 flat) contained an aliquot of 1:10 diluted
overnight grown bacteria in LB and the plate was incubated at 37℃ for 12 h, 24 h or
48 h. The unadherent bacteria was washed with 200 μl H20 and then 200 μl of 1%
crystal violet was added to each well. After the plate was placed at room temperature
for 30 min, ddH2O was used to wash the plate three times. Finally, the crystal
violet-stained biofilm was solubilized in 200 μl of 0.1% SDS and the absorbance
determined at OD595 using spectrophotometer.
Yeast agglutination assay
The capacity of bacteria to express a D-mannose-binding phenotype was
determined by their activity to agglutinate yeastcells (Saccharomyces cerevisiae) on
glass slide. Bacterial cells were adjusted to about 1X109 CFU and mixed with yeast
cells (0.01%, in PBS) until the time agglutination occurred was observed in different
Qualitative analysis of the capsule polysaccharide (CPS)
Qualitative analysis of the CPS was performed by sedimentation test. An
overnight culture of bacteria was refreshed in LB broth with 0.5 mM IPTG and
25μg/ml kanamycin. The sedimentation test was carried after 8 h of incubation at
37℃ and centrifuged at 3000 rpm for 10 min.
Preparation of anti-KpdA, anti-KpfA and anti-KpgA polyclonal antibodies
Five-week-old female BALB/c mice, purchased from Laboratory Animal Center
in National Taiwan University, received injection in peritoneal cavity with about 50
μg/150 μl gel-extracted KpdA, KpfA or KpgA proteins respectively on day 1, 11 and
15. For the first injection, the same volume of Freund’s complete adjuvant was used,
and Freund’s incomplete adjuvant was used for the following injections. The KpdA,
KpfA and KpgA antisera were finally obtained by intracardic puncture. Indirect
ELISA was performed to test the titer of serum from the tail on day 15 and the serum
was harvested from the blood of the mice’s heart.
Western blot analysis
As the method described (51), total cell lysates were resolved by 12.5% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins
(ImmobilonTM-P, Millipore). Subsequently, the membranes were blocked with 5%
skim milk in PBS, the membranes were incubated with a 10,000-fold diluted KpdR,
KpfR, KpgR or MrkA anti-serum at room temperature for 2 h. Followed by
incubation with a 10,000-fold diluted alkaline phosphatase-conjugated anti-mouse or
anti-rabbit (to MrkA) immunoglobulin G at room temperature for 1 h, additional
washes were applied triply and the bound antibodies were detected by using the
chromogenic reagents BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (Nitro
blue tetrazolium).
Localization of the blaTEM-116 in K. pneumoniae NTUH-K2044
Extracted genomic DNA was treated with NotI, BamHI, and EcoRV digestions
and the fragmented DNA cloned to NotI, NotI/BamHI, BamHI/EcoRV, NotI/EcoRV,
EcoRV, and BamHI sites of pET30a individually. Then these clones in E. coli JM109 were selected with ampicillin and kanamycin.
Extraction of mRNAs from K. pneumoniae
Total RNA was isolated from mid-exponential phase of K. pneumoniae CG43S3
or NTUH-K2044 cells (OD600=0.6~0.8) by extraction with the TRI reagent
(Molecular Research Center, Cincinnati, OH). Contaminating DNA was eliminated
Total RNA (1 μg) was reverse-transcribed using SMART PCR cDNA synthesis kit
Results
Part Ⅰ
Comparative analysis of the fimbrial gene clusters present in K. pneumoniae CG43S3
In the sequenced genome of K. pneumoniae CG43S3 (http://genome.nhri.org.tw
/kp/), we could find seven other fimbrial gene clusters, except kpb and kpc. An
incomplete kpb gene cluster was found in K. pneumoniae CG43S3. The KpbD
encoding gene, which encodes a putative adhsin of kpb fimbriae, could not be
identified by PCR analysis in K. pneumoniae CG43S3. However, kpbB gene encoding
a putative chaperone could be found.
In vitro expression of the fimbrial genes under different conditions
Bacteria only fully express virulence factors when the conditions are appropriate
within a host. Therefore, bacteria must have a way of recognizing the environmental
circumstances and be able to adapt their protein expression according to these
conditions. Regulation of fimbiral gene expression triggered by environmental signals
in enteric pathogens is well documented. The environmental conditions include
temperature, carbon source, osmolarity, pH, and so on (43).
The promoter regions of each of the putative fimbrial gene clusters (except kpc
and kpe) were cloned to the lacZ reporter plasmid pLacZ15 and the β-galactosidase
majority of the fusion promoters exerted extremely low level of activity. Only the
putative promoter of kpaA, fimA, fimB and mrkA showed a certain level of detectable
activity. A slightly higher expression was noted when the strains were grown in LB
than in M9 medium (data not shown). As shown in Table 3, with exception of the PfimE
activity, all the others appeared to be higher in standing culture than in the aerated
condition. As the temperature decreased from 37℃ to 30℃, the expression of kpaA,
kpfA, kpgA, fimA, fimB and mrkA in standing culture increased. The promoter activity of fimB and mrkA at 30℃ were found to be about three times higher than those at 37
℃. While the growth at mild acidic condition (pH 5.5), apparent increase of the
expression of fimB and mrkA were also observed. Moreover, addition of 0.2%
glycerol or 10% bovine albumin serums exerted a positive effect on the promoter
activity of fimB and mrkA. Notably, the increased expression of fimA expression was
significant with the addition of 10% bovine albumin serums (Table 4). However, as
the growth at the condition adding with different concentrations of sodium chloride,
H2O2 or bile salts treatments had negative effect on the expression of fimB and mrkA
(Table 5).
The phgS deletion effect on the fimbrial expression
on each of the promoter activity was hence investigated. As shown in Fig. 2, the
activity of PmrkA was drastically decreased. Interestingly, PkpgA was also found to be
reduced greatly in the phgS deletion mutant K. pneumoniae CG43S3Z01phgS-.
The possible interacting regulation for the expression of kpg and mrk in K.
pneumoniae 43816 has been previously implicated by Clegg, et al (45). They reported an insertion in the upstream non-coding sequence of kpg fimbrial gene cluster
decreased the expression of type 3 fimbriae (mrk) (45). We speculate that PhgS is
likely the regulator for the interacting regulation between kpg and mrk.
Since phgS/M are homologous genes of pecS/M of E. chrysanthemi. PecS,
belonging to the MarR family controls the synthesis of various virulence factors (39).
The crystal structure of MarR had been recently elucidated (42). Sequence alignment
with the representative members of the MarR family indicated that PhgS shares high
similarity in the DNA-binding domain (Fig. 3). A consensus binding site for MarR
family was therefore predicted. The analysis of 11 PecS target genes revealed a
consensus binding site which further demonstrated by mutation analysis (38). The
binding element consists of a 23-bp palindrome-like sequence
(C-11G-10A-9N-8W-7T-6C-5G-4T-3A-2)T-1A0T1(T2A3C4G5A6N7N8N9C10G11). The very
conserved part -6 to 6 allows a specific interaction with PecS and the relatively
four bases G-4, A-2, T2 and C4 are required for efficient binding of PecS and the
presence of several binding sites on a promoter increases its affinity to PecS (38).
Comparative analysis with the conserved sequence, a putative palindrome with
the conserved nucleotides were found to be present on PkpcI, PkpgA, and PmrkA. Two
putative PhgS-binding sites were found on PkpgA (Fig. 4). As shown in Fig. 5, a
putative PhgS binding site is located within PmrkA which is about 380 bp upstream of
the mrkA start codon. In order to verify if the consensus sequence is required for the
PhgS dependent expression, truncation forms of Pkpg and PmrkA including PkpgA1 (420
bp), PkpgA2 (488 bp), and PmrkAt (386 bp) were generated. As shown in Fig. 6, the
deletion of phgS appeared to be no effect on the promoter activity of either PkpgA1
PkpgA2, or PmrkAt implying that the truncated sequence is a PhgS binding element.
Part Ⅱ
Characterization of the putative regulators encoding by the genes
respectively located in the vicinity to kpb, kpd, or kpf gene cluster
Characterization of the putative response regulator KpbR
The locus kpbR is located 451 bp upstream of the kpb gene cluster in a divergent
transcription orientation. As shown in Fig. 7, next to kpbR, there is a putative
entericidin locus in both KP strains. E. coli entericidin has been implicated to be
required for the bacterial programmed cell death (52). Downstream of the entericidin
loci ecnA and ecnB, a 500 bp DNA encoding elongation factor P was found (52). In
Citrobacter freundii. ecnR encoding a member of the OmpR response regulator family was found upstream of ecnB. Next to ecnB, sugE gene which has been reported to
encode a member of the SMR family of small multidrug resistance efflux pump (53)
could be identified in the three bacteria (Fig. 7). Sequence analysis revealed that
KpbR encodes a 2CS response regulator with an OmpR domain. The response
regulator of approximately 25.1 kDa contains a carboxy-terminal DNA-binding
helix-turn-helix motif and a conserved aspartate residue near the amino terminus (Fig.
8).
While overexpress KpbR in E. coli JM109, an apparent synthesis of a protein
with the predicted molecular weight of KpbR could be observed (Fig. 9). However,
transformation of pKpbR into NTUH-K2044 or CG43S3 appeared to inhibit growth
Characterization of the putative response regulator KpdR
As shown in Fig. 10A, kpdR is located upstream of kpd gene cluster in a
divergent transcription orientation with an intergenic sequence of 414 bp. The Blast
analysis revealed that KpdR is a putative response regulator with a carboxyl
DNA-binding motif and a conserved aspartate at the N-terminal CheY-like receiver
domain. Notably, the over-expression of the KpdR in both K. pneumoniae strains
enhanced the synthesis of a protein of about 28.5 kDa (Fig. 10B). The protein band
was then isolated, subjected to in-gel digestion by trypsin and MS/MS analysis for
protein identity. As shown in Fig. 10C, the specific peptide
QQLIDWMEADKVAGPLLR matched to the sequence of β-lactamase TEM-116.
Nevertheless, we found no sequence homolog of TEM-116 gene in either genome of
K. pneumoniae NTUH-K2044 or CG43 (http://genome.nhri.org.tw/kp/). In order to confirm the protein identity, two pairs of TEM116 specific primers (56) were
synthesized for amplification of L1 and L2 products as shown in Fig. 11A. As shown
in Fig. 11B, the TEM-116 specific PCR products could be detected in K. pneumoniae
NTUH-K2044 and CG43 strains indicating the TEM116 gene is present in both
strains.
To investigate if the increased synthesis of TEM-116 affects the bacterial
shown in Fig. 12, the KpdR over-expression appeared to drastically increase the
bacterial resistance to ampicillin. This further supported that over-expression of KpdR
enhances the expression of TEM-116 β-lactamase.
While overexpression of FimB in K. pneumoniae CG43 (Fig. 13A), apparently
diminished expression of MrkA, the major pilin of type 3 fimbriae was observed (Fig.
13B). However, the overexpression of KpdR had no apparent effect on the pilin
synthesis as determined by western blot hybridization in Fig. 13. In K. pneumoniae,
MrkA pilin of type 3 fimbriae has been shown as the major determinant for the
bacterial activity to form biofilm (45). In case that the effect of the KpdR
overexpression on type 3 fimbrial expression is too subtle to be detected, the effect on
the biofilm formation was analyzed. The overexpression of KpdR reduced the biofilm
formation when compared to that of wild-type bacteria carrying vector plasmid (Fig.
14). However, no apparent changes could be observed when the biofilm formation
capability was quantitatively determined using 96-well PVC-microtire dish (data not
shown). Moreover, the over-expression of KpdR slightly decreased the synthesis of
the capsular polysaccharide (Fig. 15).
Characterization of the putative response regulator KpfR
and a CheY-like receiver domain containing the conserved aspartate residue at the
amino terminus (Fig. 16). The entire coding region of kpfR has been isolated and
cloned into expression vector pETQ30a. However, the optimal condition for the
Discussion
Fimbriae are a focus for research as they often define the initial interaction with
the host and are a key target for development of interventions such as vaccines. The
investigation into the expression of these fimbriae is contributive to image of the
disease dynamics during infection. We have found in this work that transcription of
the fimbrial operons, except kpd, were enhanced by growing at 30℃. Temperature has
been shown to be an important regulator of virulence gene expression in several
genera of bacteria. Cytoplasmic membranes, nucleic acids, and ribosomes are
suggested to be the cellular thermosensors (54). The major effects of lower
temperature are a decrease in membrane fluidity and the stabilization of secondary
structures of RNA and DNA, which may affect the efficiency of translation,
transcription, and DNA replication. Specific transcription factors may involve in the
expressions of these putative fimbriae at lower temperature.
As shown in Table 4, higher levels of promoter activity were observed for both
PfimB and PmrkA as the bacteria were treated with glucose, glycerol, or serum. The mild
acidic environment also appeared to enhance both PfimB and PmrkA activities. Some
pH-responsive regulatory mechanisms involved alternative σ factors that sense an
acidifying environment (54). However, whether the expression of fim or mrk involves
intestine, a natural habitat of K. pneumoniae, where they act as detergents for the
digestion of fats. In humans, 96% to 99% of the bile salts entering the small intestine
are absorbed and returned to the liver, and only 1% to 4% pass the ileocecal valve and
enter the colon (55). K. pneumoniae could have evolved adaptive strategy for dealing
with bile salts. Nevertheless, no apparent effect of bile salts on the fimbrial expression
was found.
Since there are multiple fimbriae in K. pneumoniae, investigation of the
regulatory network in fimbrial expression is ponderable. Both K. pneumoniae
NTUH-K2044 and CG43S3 appeared to express MrkA protein in LB static culture at
37℃ (Fig. 13). The activity of PmrkA has been shown to be dropped greatly as phgS or
phgM was deleted from K. pneumoniae CG43S3Z01 (46). As shown in Fig. 2, deletion of phgS or phgM was also found to lower the activity of PkpgA (Fig. 2). A
possibly interacting regulation between kpg and mrk has been discussed in K.
pneumoniae 43816 (45). We demonstrated here that PhgS/M likely play, a role on the interacting regulation.
The entericidin locus has been implicated in determining programmed cell death
(PCD) in bacteria (52). One of the best-studied PCD in bacteria is called
toxin-antitoxin system, which consists of a pair of genes that specify two components:
toxin. In 1998, a novel chromosomal bacteriolytic module called entericidin locus was
reported in E. coli and C. freundii (52). The entericidin locus contains two genes,
ecnA and ecnB, which appear to encode two small, amphipathic α-helical lipoproteins. That putative antidote/toxin pair is positively controlled by RpoS and negatively
controlled by the osmosensing EnvZ/OmpR. Under high osmolarity in stationary
phase, the locus promotes bacteriolysis with the ecnA gene product acting as an
antidote to EcnB (52). Since, we could not find ecnA next to ecnB gene in either K.
pneumoniae NTUH-K2044 or CG43S3 genome, the involvement of the locus in regulation of KpbR is elusive.
In Korea, the blaTEM-116 has been identified recently (56, 61). The prevalence
study of TEM-type β-lactamase-producing K. pneumoniae indicated that blaTEM-116 is
the predominant type in Taiwan and Guangzhou (56, 57). Why the over-expression
of KpdR enhances the β-lactamase TEM-116 expression is considerably urgent and
important. The over-expression of KpdR also affected biofilm formation and capsular
synthesis, which supporting the mechanism that was proposed the β-lactamase
interferes with peptidoglycan remodeling complexes participating in surface
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Table 1. Bacterial strains and plasmids used in this study (A)
Strain or plasmid Description Reference
Strain K. pneumoniae CG43S3 CG43S3 Smr (58) CG43S3-Z01 CG43S3 ΔlacZ (60) CG43S3mrkA- CG43S3-Z01 ΔmrkA (46) CG43S3phgS- CG43S3-Z01 ΔphgS (46) CG43S3phgM- CG43S3-Z01 ΔphgM (46) E. coli
JM109 RecA supE44 endA1 hsdR gyrA96 relA1 thi (lac proAB)F [lacIq lacZ M15 proAB traD36] Our Lab.
S17-1λpir HsdR recA pro RP4-2 [Tc::Mu; Km::Tn7] (λpir) (59)
P. aeruginosa
PAO1 Wild-type strain, laboratory strain serogroup 05 Our Lab.
Plasmid
Yt&A vector PCR cloning vector, Apr Sigma
Pcr®
2.1-TOPO®
PCR cloning vector, Kmr Invitrogen
pLacZ15 A derivative of Pyc016, containing a promoterless lacZ from K. pneumoniae CG43S3 as the reporter, Cmr (60)
Petq33 Protein expression vector, Kmr Our Lab.
PPfimA ~500-bp BamHI fragment containing the putative fimA promoter, cloned into BamHI site of placZ15 (46)
pPfimB ~500-bp BamHI fragment containing the putative fimB promoter, cloned into BamHI site of placZ15 (46)
pPfimE ~500-bp BamHI fragment containing the putative fimE promoter, cloned into BamHI site of placZ15 (46)
(B)
Plasmid Description
pPkpaA 580-bp BamHI fragment containing the putative kpaA promoter, cloned into BamHI site of placZ15
pPkpbA 639-bp BamHI fragment containing the putative kpbA promoter, cloned into BamHI site of placZ15
pPkpbR 639-bp BamHI fragment containing the putative kpbR promoter, cloned into BamHI site of placZ15
pPkpdA 602-bp BamHI fragment containing the putative kpdA promoter, cloned into BamHI site of placZ15
pPkpdR 602-bp BamHI fragment containing the putative kpdR promoter, cloned into BamHI site of placZ15
pPkpfA 746-bp BamHI fragment containing the putative kpfA promoter, cloned into BamHI site of placZ15
pPkpgA 578-bp BamHI fragment containing the putative kpgA promoter, cloned into BamHI site of placZ15
pPkpgA1 420-bp BamHI fragment containing the putative kpgA promoter, cloned into BamHI site of placZ15
pPkpgA2 388-bp BamHI fragment containing the putative kpgA promoter, cloned into BamHI site of placZ15
pPmrkAt 386-bp BamHI fragment containing the putative mrkA promoter, cloned into BamHI site of placZ15
pKpdR 867-bp EcoRI fragment containing the kpdR of K. pneumoniae NTUH-K2044, cloned into pETQ33, Kmr
pKpbR 843-bp EcoRI fragment containing the kpbR of K. pneumoniae NTUH-K2044, cloned into pETQ33, Kmr
Table 2. Primers used in this study
Primer Sequence Complementary position
ZY001 5'-CAT ATGAAAAAATACAGCAGAGGAATGG-3' -3 relative to the kpdA start codon ZY002 5'-GCGGGTGGAGCATAACTTTG-3' +371 relative to the kpdD stop codon ZY003 5'-CATATGAAAATGAAATCACTTTGCCTGG-3' -3 relative to the kpfA start codon ZY004 5'-ATGGTGACTTTCGCCCTGGAG-3' +45 relative to the kpfD stop codon ZY005 5'-CATATGAAAAAACAACCTCGCTTTATAACC-3' -3 relative to the kpgA start codon ZY006 5'-CGAGAACGCCGAAGCTGG-3' +77 relative to the kpgD stop codon ZY007 5'- GATCCGAATTCGCCAAAACC -3' +1289 relative to the kpgC start codon ZY008 5'- GGTTTTGGCGAATTCGGATC -3' -1284 relative to the kpgC stop codon ZY009 5’-CTGCGGGATCCTGTGGGCTG-3’ -102 relative to the kpbR start codon ZY010 5’-GACCAGCAAATGACTATCGCACC-3’ +52 relative to the kpbR stop codon ZY011 5’-CCAGGGGATCCTTATGTTGATCTGC-3’ -13 relative to the kpdR start codon ZY012 5’-CTGGGCGTGGCGAGTAATG-3’ +177 relative to the kpdR stop codon ZY013 5’-CATGCAACATCTTTCATTAGGATCCTTC-3’ -30 relative to the kpfR start codon ZY014 5’-CCGACGAGTGCCATTGCCAG-3’ +41 relative to the kpfA start codon
ZY101 5'-CGCTCATGAGACAATAACCC-3' -56 relative to the blaTEM-116 start codon (56)
ZY102 5'-CAGTGAGGCACCTATCTC-3' -51 relative to the blaTEM-116 stop codon (56)
ZY013 5’-CATTTTGCCTTCCTGTTTTTG-3’ +67 relative to the blaTEM-116 start codon
ZY104 5’-CTACGATACGGGAGGGCTTACC-3’ -129 relative to the blaTEM-116 stop codon
ZY105 5'-GTAGGATCCGACGAGCGCAC-3' -442 relative to the kpgA start codon ZY106 5'-GAATGGATCCGTTGTTGTTTAAAGG-3' -373 relative to the kpgA start codon ZY107 5'-CTGGATCCTGTTGCGGTC-3' -358 relative to the mrkA start codon pCC033 5’-CGTGCGCTGGATCCTGTT-3’ -532 relative to the kpaA start codon pCC034 5’-TAAAAGATCTATGGCGGGTGC-3’ +50 relative to the kpaA start codon pCC035 5’-GCATTGAGGCGGATCCACT-3’ -597 relative to the kpbA start codon pCC036 5’-GGTCGCTAGATCTGCAGTGC-3’ +46 relative to the kpbA start codon pCC039 5’-CGGATCCGCATATGCTGA-3’ -555 relative to the kpdA start codon pCC040 5’-GCCGCTGACGGGAAGATCT-3’ +46 relative to the kpdA start codon pCC043 5’-ACAGGCCGGATCCATGAC-3’ -681 relative to the kpfA start codon pCC044 5’-GCTGGCCTTAGATCTGGCTAC-3’ +59 relative to the kpfA start codon pCC045 5’-GGATCCGGTCTCTGGTTAACAT-3’ -526 relative to the kpgA start codon pCC046 5’- CCATCCTCATAGGAGCGCTGCT-3’ +47 relative to the kpgA start codon
Table 3. Effect of culture condition on the fimbrial expression
Promoter activity (Miller units)a
Fimbriae
Growth conditions kpaA kpdA kpdR kpfA kpgA fimA fimB fimE mrkA
Aerated culture (37℃) 183.2 (±10.7) 21.7 (±1.9) 26.3 (±1.7) 23.0 (±2.8) 92.8 (±3.6) 520.4 (±27.3) 32.3 (±0.7) 96.1 (±5.5) 27.8 (±4.0) Standing culture (37℃) 272.6 (±12.9) 24.8 (±1.1) 30.4 (±1.4) 46.3 (±2.1) 92.8 (±5.7) 510.8 (±25.6) 806.2 (±37.7) 24.3 (±1.1) 791.6 (±26.1) Temperature 30℃ 372.7 (±8.0) 16.8 (±0.6) 25.3 (±0.7) 90.8 (±2.8) 221.6 (±13.1) 594.4 (±32.9) 2555.9 (±53.0) 25.1 (±0.8) 2505.9 (±59.9) a, the fimbrial expression was determined using LacZ as the promoter reporter. LacZ activity measurement was as described in Materials and Methods.
Table 4. Effects of glucose, glycerol, and bovine serum on the fimbrial expression
Promoter activity (Miller units)a
Fimbriae
Growth conditions kpaA kpdA kpdR kpfA kpgA fimA fimB fimE mrkA
Standing culture 272.6 (±12.9) 24.8 (±1.1) 30.4 (±1.4) 46.3 (±2.1) 92.8 (±5.7) 510.8 (±25.6) 806.2 (±37.7) 24.3 (±1.1) 791.6 (±26.1) 15 mM 244.1 (±22.2) 22.0 (±1.9) 37.8 (±0.5) 43.0 (±1.3) 93.0 (±2.6) 504.2 (±27.1) 1217.4 (±32.7) 22.6 (±1.8) 1753.2 (±38.2) Glucose 30 mM 215.1 (±11.9) 23.2 (±0.7) 33.2 (±1.1) 45.6 (±1.9) 88.3 (±2.1) 511.2 (±26.3) 1112.6 (±39.5) 20.7 (±1.1) 1563.8 (±33.3) Glycerol 0.2% 345.1 (±9.7) 23.8 (±0.7) 34.4 (±1.1) 40.7 (±1.5) 114.1 (±9.1) 512.6 (±22.4) 3266.5 (±49.2) 25.6 (±1.8) 3431.0 (±58.7) Bovine serum 10% 309.3 (±14.8) 23.0 (±1.1) 27.4 (±1.9) 51.9 (±6.0) 108.5 (±11.4) 1872.9 (±32.6) 1984.7 (±28.3) 19.9 (±1.4) 1617.2 (±27.7) a, the fimbrial expression was determined using LacZ as the promoter reporter. LacZ activity measurement was as described in Materials and Methods.
Table 5. Effects of pH, osmotic pressure, H2O2, and bile salts on the fimbrial expression
Promoter activity (Miller units)a
Fimbriae
Growth conditions kpaA kpdA kpdR kpfA kpgA fimA fimB fimE mrkA
Standing culture 272.6 (±12.9) 24.8 (±1.1) 30.4 (±1.4) 46.3 (±2.1) 92.8 (±5.7) 510.8 (±25.6) 806.2 (±37.7) 24.3 (±1.1) 791.6 (±26.1) 5.5 270.9 (±4.7) - - 26.9 (±3.2) 104.6 (±15.6) 174.0 (±17.3) 2170.2 (±88.6) 30.3 (±7.2) 2335.1 (±94.5) 8.0 195.9 (±2.8) 27.9 (±1.8) 49.5 (±6.2) 32.2 (±1.4) 82.9 (±17.5) 368.8 (±28.4) 232.8 (±10.1) 25.9 (±2.8) 292.8 (±15.6) pH 30 mM 215.1 (±11.9) 23.2 (±0.7) 33.2 (±1.1) 45.6 (±1.9) 88.3 (±2.1) 511.2 (±26.3) 1112.6 (±39.5) 20.7 (±1.1) 1563.8 (±33.3) 0 mM 129.0 (±22.4) 23.6 (±1.4) 27.0 (±1.0) 23.0 (±2.3) 56.6 (±7.3) 202.5 (±16.1) 61.8 (±3.0) 13.8 (±1.0) 57.7 (±3.8) 200 mM 120.1 (±15.6) 23.7 (±0.9) 26.8 (±1.8) 17.6 (±17.6) 59.2 (±6.0) 220.8 (±24.3) 432.5 (±22.7) 14.1 (±0.6) 381.2 (±24.3) 800 mM 106.5 (±9.2) 18.9 (±1.4) 34.2 (±1.7) 14.8 (±1.1) 51.4 (±3.3) 165.4 (±12.9) 39.3 (±2.6) 12.5 (±0.7) 47.1 (±1.3) NaCl 1.5 M 111.3 (±8.3) - - 15.2 (±2.4) 31.4 (±2.3) 154.9 (±10.1) 28.8 (±1.5) 14.3 (±2.6) 32.3 (±2.5) H2O2 60 μM 191.5 (±19.8) 22.9 (±1.5) 32.1 (±1.5) 27.4 (±2.8) 100.2 (±2.3) 247.9 (±15.1) 606.0 (±18.4) 23.3 (±1.4) 575.2 (±19.1) Bile salts 0.3% 103.0 (±10.0) 5.2 (±0.7) 8.1 (±0.2) 20.3 (±0.3) 43.1 (±3.8) 224.1 (±6.7) 553.7 (±15.0) 7.1 (±0.7) 469.7 (±17.6) a, the fimbrial expression was determined using LacZ as the promoter reporter. LacZ activity measurement was as described in Materials and
Figure 1. Gene organization of the putative fimbrial gene clusters in K. pneumoniae NTUH-K2044. The putative promoters are
respectively boxed. The KP number is according to the annotation of the genome (http://genome.nhri.org.tw/kp/). phgM phgS regulator recombinase adhesin usher chaperone pilin subunit fim [type 1] (KP4570~4579) mrk [type 3] (KP4555~4559) kpg (KP4248~4251) kpf (KP4244~4247) kpe (KP2716~2719) kpd (KP1401~1404) kpc (KP0596~0601) kpb (KP0409~0412) kpa (KP0334~0338)
Miller units
0 200 400 600 800 K. pneumoniae CG43S3Z01 K. pneumoniae CG43S3Z01 phgS deletionPkpaA PkpdA PkpfA PkpgA PfimA PfimB PfimE PmrkA
Figure 2. Measurements of the fimbriae in K. pneumoniae CG43S3Z01 and CG43S3Z01phgS-. This assay was carried out as described in Materials and Methods while the bacteria was statically grown in LB at 37℃ to exponential growth phase.