In order to determine the inserted sequences, chromosomes of the eight mutant were isolated and completely digested by restriction enzyme PstI. As shown in Fig. 3, there is a PstI site outside the transposon kanamycin-resistant gene and hence the nucleotides of K. pneumoniae next to the kanamycin-resistant gene could be included with PstI digestion. The M4 strain grew poorly on the kanamycin/
chloramphinicol- containing medium and hence was not included for the following study. The PstI-digested fragments were cloned into vector pUC19, and the transformants selected on Knanmycin containing plates.
As shown in Fig11B, except M3, six recombinant clones namely pM1, pM2, pM5, pM6, pM7, and pM8 were obtained and further confirmed by PstI digestion. After subjected to more insertion enzymes digestion, the restriction pattern of pM1 and pM6 appeared to be identical (Fig. 11C).
Finally, the clones were sent to commercial service for sequence determination using the primers M13R and M13F.
The Blast tool (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) was then applied to analyze the determined sequences and the inserted genes by the transposon include a putative glycosidase encoding gene (Fig.
12A), lacI (Fig. 12B), gene encodes RelE/ParE family or carbon
20
starvation protein A (Fig. 12C), and yehZ gene (Fig. 12D). YehZ is homologous to OsmF, an osmotically inducible protein (Checroun and Gutierrez 2004).
21 unexpected protein size of D27-3 (Fig. 6A, lane 7) and D27-4 (Fig. 6A, lane 8) were found. Analysis of pD27-3 revealed that two DEN2EDIII the one still carrying the signal peptide as a cytosolic form. As shown in the lane 2 and lane 8 in Fig 6A, one major band was observed indicating that most of the MrkA had been transported out to the periplasmic space.
The differential transported level is probably due to different exposure of the signal peptide on the recombinant MrkA to be recognized by the signal peptidase.
Although all recombinant fimbriae could be detected by anti-MrkA
22
antibody, the polymer pattern analysis revealed only the recombinant fimbriae D27-4 could be comparable with that of the wild type carrying pmrkABCD. This is probably due to the smallest size of the monomeric form of D27-4 could be better assembly into polymeric form. As shown in Fig.8, the better detection of D27-3 and D27-5 by IFM analysis indicated the two recombinant epitopes are properly exposed to be detected by anti-DEN2EDIII antibody.
The previous study has shown that the recombinant type 3 fimbriae inserted PERV (Porcine endogenous retrovirus) envelope amino acid sequence can inducePERV envelope antibody in mouse (陳欣瑜 2008).
We’ve expected that the purified recombinant fimbriae can induce immune reaction in animals as well. However, the purified fimbriae, even from D27-4 (lane 8 in Fig.9A) with the largest amount, were not enough for the following immunization study. Since the purified fimbriae appeared to be good, alternative way such as improving the cultured condition for stable fimbrial expression might be helpful to obtain enough amounts of the fimbriae for immunization.
Part II
Identification of the regulator(s) involved in controlling the expression of type 3 fimbriae
Fimbriae are an important pathogenic factor for bacteria to attach to the host cell. Regulation to control the expression of the fimbriae is conceivably important. Herein, the random mutagenesis employed had identified several genes which may have a role for the expression of type
23
3 fimbriae. The analysis of transposon insertion site of M2 mutant revealed a putative glycosidase. Glycosidase (also called Glycoside hydrolase), catalyzes the hydrolysis of the glycosidic linkage to generate two smaller sugars, has been found in essentially all domains of life. In bacteria and prokaryotes, glycosidase are found both as intracellular and extracellular enzymes that are largely involved in nutrient acquisition (Dwek 1996). The enzyme beta-galactosidase (LacZ) is one of the important occurrences of glycoside hydrolases in bacteria. The white colony of M2 mutant could be resulted from the disruption of the glycosidase gene and hence affected the bacterial digestion of X-gal. The insertion site of M5 was mapped to lacI gene. This could be easily explained by the inability of LacI to repress the LacZ activity and hence a deep blue colony was observed.
Since the available sequences of the M7 clone contained only K.
pneumonia genes, the transposon insertion site was speculated. As shown in Fig. 12C, the disrupted genes could be the gene encoding plasmid stabilization system protein of RelE/ParE family or carbon starvation protein A, CstA. RelE/ParE is a family of TA systems, which typically consist of pairs of genes: one for a stable toxin that can cause cell death by disrupting an essential cellular process and the other for a labile antitoxin that can bind the toxin and block activity of the toxin (Anantharaman and Aravind 2003; Engelberg-Kulka, Amitai et al. 2006).
The deletion of TA systems has been shown to decrease biofilm formation initially (8 h) on three different surfaces and then increased biofilm formation (24 h) by decreasing biofilm dispersal (Kim, Wang et al. 2009).
24
CstA is involved in peptide transport that would assist the cell in escaping carbon starvation (Schultz and Matin 1991). If disruption of the relE/parE gene or cstA could influence the MrkA expression remains to be investigated.
The disrupted gene of M8 is yehZ gene which is located within yehZYXW operon and coding for a putative glycine/betaine/choline transport protein (ABC superfamily). The yehZYXW encoded ABC transporter as an additional element of the global stress response controlled by sigma(s) (Checroun and Gutierrez 2004). The analysis of a yehZ-lacZ transcriptional fusion demonstrated that yehZ is inducible not only by osmolarity, but also upon entry into stationary phase. CstA and YehZ both are related with membrane transporters. Whether they have influences on MrkA expression awaits to be clarified.
While sequencing the other mutant clones, transposase sequence has been identified indicating some unexpected recombination events had occurred. How to improve the transposon delivery system has to be taken into consideration.
25
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30
Table 1. Bacteria strains used in this study
Strains Genotypes or relevant properties Reference or source
E. coli
JM109 RecA1 supE44 endA1 hsdR17 gyrA96 RelA1
thiΔ(lac-proAB)
Laboratory stock
S17-1 λ pir Tpr Smr recA, thi, pro,
hsdR-M+[RP4-2-Tc::Mu:KmrTn7](λ pir)
Laboratory stock
K. pneumoniae
CG43 K2 serotype Laboratory stock
CG43S3 ΔrspL, Str Laboratory stock
CG43S3-Z01 CG43S3 ΔlacZ Smr Laboratory stock
CG43S3-Z01ΔrcsB Z01ΔrcsB Smr Laboratory stock
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Table 2. Plasmids used and constructed in this study
Plasmids Relevant characteristic Reference or source
yT&A Cloning vector;Apr Yeastern Biotech
pGEMT Cloning vector;Apr Promega
pMrkABC mrkABC gene cluster cloned into pGEMT vector,Apr Laboratory stock pMrkABCD 1 kb fragment amplified using primer pairs, phw03 and MZ006,
and cloned into pMrkABC by AscI and ApaI site, Apr
Laboratory stock
pA-2 insert DENIIEDIII part2 in the pA25 PmeI site This study pD-1 insert DENIIEDIII part1 in the pD27 PmeI site This study pD-2 insert DENIIEDIII part2 in the pD27 PmeI site This study pD-3 insert DENIIEDIII part3 in the pD27 PmeI site This study pD-4 insert DENIIEDIII part4 in the pD27 PmeI site This study pD-5 insert DENIIEDIII part5 in the pD27 PmeI site This study
pLacZ15
A derivative of pYC016, containing a promoterless lacZ from K. pneumoniae CG43S3 as the reporter, Cmr
Laboratory stock
32
pMrkA-lacZ primers pmrkA4 and pmrkA5 were used to get 545 bp sequence, BamHI and BglII were used to cut into pLacZ15
Laboratory stock
33
Table 3. Primers used in this study
Primer Sequence 5'-->3'
SL1138-3-1 GTTTAAACggaaaggaatgtcatactcta SL1138-3-2 GTTTAAACaaacacaacatggaacaatagtt SL1138-3-3 GTTTAAACagatcccttttgagataatggatttggaa SL1138-3-4 GTTTAAACcagtcaacccaatcgtaacagaa SL1138-3-5 GTTTAAACgagacagctacatcatcataggagta SL1139-3-1 GTTTAAACgtttctgctatttccttcacaactttaaactt SL1139-3-2 GTTTAAACatcttacatggagaaccgtccccttc SL1139-3-3 GTTTAAACacgactgtaatcaggcgacctaaaacatgtct SL1139-3-4 GTTTAAACtctccgaatggaggttctgcttctat
SL1139-3-5 GTTTAAACactcctttcttaaaccagttgag
34
Table 4.DENIIEDIII inserted sequence and insertion site
Insertion site DENIIEDIII inserted sequence
A25-2 NMFKQTQHGTIVIRVQYEGDGSPCKMFKQNT
D27-1 PVFRKGMSYSMCTG KFKVVKEIAETFKQPT
D27-2 PVFKQTQHGTIVIRVQYEGDGSPCKMFKQPT
D27-3 PVFKQIPFEIMDLEKRHVLGRLITLFKQPT
D27-4 PVFKPVNPIVTEKDSPVNIEAE PPFGEFKQPT
D27-5 PVFKRDSYIIIGVEPGQLKLNWFKKGMFKQPT
underline: DENIIEDIII amino acid
bold: Tn7 inserted sequence(43)
35
Fig. 1. Organization of the fimbriae encoding gene cluster in K. pneumoniae C3091 (http://www.ncbi.nlm.nih.gov/).
The mrkABCDF is coding for type 3 fimbriae. The gene cluster from fimB to fimK encode the regulatory system, for the expression and assembly, and structural components of type 1 fimbriae. The genes named pecM and pecS are homologs of the genes coding for the regulators for the expression of virulence genes in Erwinia chrysanthem (Reverchon, Nasser et al.
1994).
Type 3 fimbriae gene cluster Type 1 fimbriae gene cluster
36
Fig. 2. Downstream genes of the type 3 fimbriae gene cluster. The genes KP4551, KP4552, and KP4554 were predicted byNCBI ( http://www.ncbi.nlm.nih.gov/ ) to encode c-di-GMP binding domain protein (PilZ domain protein), transcription factor with an HTH domain, and c-di-GMP phosphodiesterase (EAL) domain protein, respectively.
37
Fig. 3. Schematic presentation of the transposon mutagenesis. The plasmid pUTmini-Tn5Km2 was mobilized from E.
coli S17-1 λ pir to K. pneumoniae CG43S3Z01rcsB-, and the mutants selected on M9 agar plates containing kanamycin, chloramphinicol, and X-gal.
38
Fig. 4. Amino acid sequences of the major subunit of type 3 fimbriae. The arrows indicate A25 and D27 are the foreign amino acids insertion sites. The oblique line boxes mark the -helix and the arrow boxes are-sheet of the protein.
39
(A)
(B)
* : primer
Fig. 5. Amino acid sequences of DEN2EDIII. (A) Multiple-sequence alignment of domain III region (aa 295 to 395) of four DV serotypes.
Identical residues are shaded as dark gray areas, and conserved residues are shaded as light gray areas (Abd-Jamil, Cheah et al. 2008).The 5 fragmented amino acid sequences of are marked with different colors.
(B) Sequences of the five peptides, the primers used for the PCR amplification are labeled.
40
(A)
(B)
(C)
41
Fig 6. Analysis of expression of the recombinant fimbriae. E. coli JM109 carrying each of the recombinant clones were grown overnight at 37°C, and the lysates were collected and heated at 95°C for 30 mins before subjected to the analysis. (A) SDS-13.5% polyacrylamide gel stained with Coomassie blue. (B) and (C) are western blot analysis against anti-MrkA antibody and anti-DENEDIII antibody, respectively.
The arrowheads mark the recombinant MrkA. Lanes M, molecular weight marker; 1, E. coli JM109; 2, JM109[pmrkABCD]; 3,
JM109[pD27]; 4, JM109[pA25-2]; 5, JM109[pD27-1]; 6, JM109[pD27-2]; 7, JM109[pD27-3]; 8, JM109[pD27-4]; 9, JM109[pD27-5].
42
(A)
(B)
Fig 7. Polymer pattern analysis of the recombinant fimbriae. E. coli JM109 carrying each of the recombinant clones were grown overnight at 37°C and the total cell lysates collected by centrifugation. (A) SDS–8%
polyacrylamide gel stained with Coomassie blue. (B) Western blot analysis by anti-MrkA antibody. Lanes M, Marker; 1, E. coli JM109; 2, JM109[pmrkABCD]; 3, JM109[pD27]; 4, JM109[pA25-2]; 5,
JM109[pD27-1]; 6, JM109[pD27-2]; 7, JM109[pD27-3]; 8, JM109[pD27-4]; 9, JM109[pD27-5].
43
(A)
(B)
Fig. 8. Immunofluorescence microscopy (IFM) analysis of the recombinant E. coli displaying with the type 3 fimbriae using the antibody (A)anti-MrkA and (B)anti-DENDIII. (a) JM109, (b) JM109 [pmrkABCD], (c) JM109[pD27], (d) JM109[pA25-2], (e)
JM109[pD27-1], (f) JM109[pD27-2], (g) JM109[pD27-3], (h) JM109[pD27-4], (i) JM109[pD27-5].
44
(A)
(B)
Fig 9. Analysis of the purified fimbriae. The E. coli JM109 carrying each of the recombinant plasmids were cultured for 24 h in 24 ml LB broth. The bacteria were collected by centrifugation for 3 min (8,000 rpm, 4°C) and were suspended in 1 ml phosphate-buffered saline (PBS).
The bacterial suspension was heated at 65°C 2 h and homogenized in blender for 20 min at ambient temperature. 30% saturate ammonium sulfate was added and stored at 4°Covernight. The pellet was collected by centrifugation (20,000 g, 90 min) and suspended in 40 l PBS. The purified fimbriae were identified by (A) SDS–8% polyacrylamide gel stained with Coomassie blue. (B) Western blot analysis using anti-MrkA
45
antibody. Lanes M, Marker; 1, E. coli JM109; 2, JM109[pMrkABCD]; 3, JM109[pD27]; 4, JM109[pA25-2]; 5, JM109[pD27-1]; 6,
JM109[pD27-2]; 7, JM109[pD27-3]; 8, JM109[pD27-4]; 9, JM109[pD27-5].
46
(A)
(B)
Fig. 10. Analysis of the expression of the type 3 fimbriae in K.
pneumoniae CG43S3Z01 and K. pneumoniae CG43S3Z01rcsB-. The bacteria (lanes 1 to 4) were cultured statically in LB for 20 h. The bacteria (lanes 6 to 9) were in shaking culture to OD 0.8. Total cell lysates were heated at 95°C for 30 min before subjected to analysis. (A) SDS–13.5% polyacrylamide gel stained with Coomassie blue;
(B)Western blot analysis using anti-MrkA antibody. The arrowheads mark the recombinant MrkA. Lanes M, Marker; 1 and 6,
47
CG43S3Z01[placZ]; 2 and 7, CG43S3Z01rcsB-(placZ);3 and 8,
CG43S3Z01(pmrkA-lacZ); 4 and 9, CG43S3Z01rcsB- (pmrkA-lacZ); 5, E. coli JM109.
48
(A)
(B)
(C)
49
Fig. 11. Analysis of the transposon insertion mutant strains. (A) The mutant strains (M1~M8) with color alteration on X-gal plate are shown.
(B) Analysis of the recombinant clone (in pUC19) by restriction enzyme PstI. Lanes 1: pM1; 2: pM2; 3: pM5; 4: pM6; 5: pM8; 6: pM7. (C) Analysis of the recombinant clone of pM1 (lanes 1, 3, 5) and pM6 (lanes 2, 4, 6) by restriction enzymes BamHI (lanes 1, 2), EcoRI (lanes 3, 4) and HindIII (lanes 5, 6).
50
(A)
M2 (B)
M5 (C)
M7 (D)
M8
51
Fig. 12. The tansposon insertion site and the genes beside the insertion site. The arrows mark the transposon insertion sites.