Materials and Methods - 克雷白氏肺炎桿菌CG43中二價鐵螯合系統FeoABC、SitABCD和EfeUOB之特性探討與比較

2. 1. Plasmid, primers, bacterial strains and growth conditions

The bacterial strains, plasmids and primers used and constructed in present study are describe in Table 1, Table 2 and Table 3 respectively. K. pneumoniae CG43 is clinical isolated strains from Chang Gung Memorial Hospital Linkou branch. E. coli and K. pneumoniae strains were generally cultured aerobically at 37 °C in Luria-Bertani (LB) broth or on M9 minimal medium supplemented with appropriate antibiotics. For static culture, 1: 20 dilution of overnight culture was added to fresh media and incubated for 20 hr. Otherwise indicated, the concentrations of antibiotics used include streptomycin (500 µg/ml), ampicillin (100 µg/ml), chloramphenicol (35 µg/ml), and kanamycin (25 µg/ml). The iron chelators added in media include deferoxamine mesylate salt (DFX, Sigma Aldrich), 2, 2’-dipyridy (DIP, Sigma Aldrich) and disodium salt dehydrate (EDTA, USB cooperation) with final concentration of 200 µM.

2. 2. DNA manipulation

Plasmids was purified by using High-Speed Mini kit (Geneaid). All DNA-modifying and –restriction enzymes was recommended by the manufacture (Fermentas). PCR amplifications were performed with Blend Taq DNA polymerase (TOYOBO) or Taq DNA polymerase (MDbio Inc), PCR products and DNA fragments were purified using the Gel/ PCR DNA Fragments Extraction it (Geneaid). The primers used in this study were synthesized by MDBio, Inc or Integrated DNA technology (IDT).

15 2. 3. Bioinformatics analysis

Homology search analysis and gene annotation were performed with the BLAST program provided by NCBI (http://www.ncbi.nlm.nih.gov) or vector NTI (Invitrogen Vector NTI^M advance). Promoter prediction was carried out by SofBerry provider (http://www.softberry.com).

2. 4. Construction of gene-deletion mutants

The deletion strains constructed in this study are listed in Table 1. All the strains were made by allelic exchanged. Approximately 1000-bp sequences flanking both sides of the deleted region (feoA, feoC, sitCD, and efeUOB) were PCR amplified with respective primer pairs (Table 3). The amplified DNA fragments were then cloned into plasmid pKAS46 [127], a suicide vector containing rpsL, which allows positive selection with streptomycin for loss of the vector, to generate an in frame deletion plasmid. The resulting plasmid was then mobilized to K. pneumoniae CG43S3, K. pneumoniae CG43S3 ∆lacZ [128], K. pneumoniae CG43S33 fur [19] or K.

pneumoniae CG43S3-derived strains, through conjugation from E. coli S17-1 λpir. The transconjugants, which carried constructed plasmid integrated in the chromosome via homologous recombination, were selected by ampicillin and kanamycin on minimal media (M9). Several of the colonies was grown in LB at 37 °C for 8 hours and then spread onto a LB plate containing streptomycin. The streptomycin-resistant and kanamycin-sensitive colonies were selected and verified by PCR and deletion of gene verified by PCR by designed primer sets described in Table 3.

16 2.5. Construction of a pLacZ reporter system

The putative promoter regions of feoABC (named as Pfeo1 and Pfeo2), sitABCD (named as Psit) and efeUOB (named as Pefe) were PCR amplified from CG43S3 by the designed primer pairs (Table 3) and subcloned into placZ15 [128] to fuse them with promoterless lacZ reporter system.

The promoter-reporter plasmids placZfeo1, placZfeo2, placZsit, and placZefe, were individually mobilized into K. pneumoniae strains by conjugations from E. coli S17-1 λpir.

2.6. Measurement of bacterial growth in iron depletion or repletion conditions

Cultures of the parental strains CG43S3, along with deletion mutant strains were grown overnight in LB. 1:200 of diluted overnight cultures were inoculated into LB or LB with iron-depleted or iron-replete conditions. Iron-depletion condition was created by adding DFX or DIP to a final concentration of 200 µM; iron-repletion condition was created by adding ferric sulfate [(Fe)2(SO4)3] and ascorbic acid to a final concentration of 50 µM into LB broth. The cultures were incubated at 37 °C agitatedly and the optical density was recorded every hour as the absorbance at 600 nm (OD600).

2.7. Measurement of promoter activity through β-galactosidase activity assay

β-galactosidase activity was determined according to the method of Miller [129]. In brief, overnight culture was diluted 1 : 20 in LB broth supplemented with appropriate antibiotic and incubated at 37 °C until it reached the lag phase (OD600 = 0.2), early (OD600 = 0.5-0.6) logarithmic, late logarithmic (OD600 = 0.8-0.9) or stationary growth phase (OD600 >1.0). 100 µl of the bacterial culture was added with 900 µl of 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, followed by

vigorous shaking and incubated for 10 min at 30 °C. Subsequently, 200 µl of 4 mg/ml o-nitrophenyl-β-galactopyranoside (ONPG) was added and mixture was mixed thoroughly with vortex for 3 s, then incubated at 30 °C until yellow color was apparent. Finally, the reaction was stopped by adding 500 µl of stop solution (1 M Na2CO3) and the absorbance the supernatant was measure at OD420.The activity was expressed as Miller units in which one unit of β-galactosidase is defined as the hydrolysis of 1 nmol ONPG per min per mg or protein. Each sample was assayed in triplicate, and at least 3 independent experiments were conducted. The data shown were calculated from one representative experiment, and are presented as the means and standard deviations from triplicate samples.

2.8. Sedimentation test

The capsular polysaccharide biosynthesis was analyzed by sedimentation test. The bacterial strains were cultured overnight in LB broth at 37ºC and subjected to centrifugation at 4,000 x g for 5 min. Pictures of bacterial cells centrifuged by low speed sedimentation of were taken.

2.9. Biofilm formation assay

Overnight grown bacteria were diluted 1: 100 in LB broth supplemented with appropriate antibiotic and then inoculated 150 µl into each well of a 96-well microtiter dish (DPP), and propagated statically at 37°C for 24 hr. After removal of the bacteria, each well was washed by de-ionized water twice before added with150 μl of 1% (w/v) crystal violet and placing on an orbital shaker for 50 min at 60 rpm. After washing each well three times with de-ionized water, 200 µl

95% ethanol was added to solubilize the dye for 1 hr. The capability of biofilm formation was quantified by the absorbance at 595 nm (ELx800, BIO-TEK).

2.10. H2O2 sensitivity test

Sensitivity of deletion strains to H2O2 stress was determined by disk diffusion method on LB plate and LB plate added with 0.2 mM of different iron chelators (DFX, DIP or EDTA).

Overnight cultures were diluted 1:20 to a fresh LB grown agitatedly to an OD600 of 0.6-0.7. The cultures were then supplemented with or without iron chelators for another 1 hr. Filter paper disks (6-mm diameter; Becton Dickinson) spotted with 5 µl hydrogen peroxide (30 %) were added onto the disks. The plates were then incubated at 37 °C for 16 hr. The diameters of inhibition zones were measured. The effect of iron chelators (deferoxamine, DIP and EDTA) was determined by its addition 1 hr prior to that H2O2.

2.11. Western blot analysis of the expression of type 3 fimbriae

Total cellular lysate from the bacteria were resolved by 12 % SDS-PAGE to determine the expression of type 3 fimbriae in CG43S3. The electrophoretic proteins were then transferred onto polyvinylidene difluoride (PVDF) membrane (Milipore, Billerica, MA, USA). After incubation with 5 % skimmed at 4 °C overnight, the membrane was washed three times with 1x Tris-buffered saline (TBS). Subsequently, the membrane was incubated at room temperature for 2 h with 25,000 fold diluted anti-MrkA serum. After washed three times with 1x TBS, a 5,000 fold diluted alkaline phosphate-conjugated immunoglobulin G (IgG) was added and the incubation continued for 1h.

The bound antibodies were detected by chromogenic reagent BCIP (5-bromo-4-chloro-3-indolyl

phosphate), NBT (Nitro blue tetrazolium) and alkaline phosphatase butter (100mM NaCl, 5 mM MgCl2, 100 mM Tris-HCl pH 9.5).

3.0 Results

3.1. Generation of the specific gene deletion mutants

The coding sequence of feoA (290-bp), feoB (2297-bp), and feoC (266-bp) were individually deleted by allelic exchange method, and the deletions were confirmed by PCR analysis. Besides ∆feoA, ∆feoB, and ∆feoC, the double mutation mutants including ∆feoA∆fur,

∆feoB∆fur, and ∆feoC∆fur, were also obtained since Fur negatively regulates the iron uptake systems. As shown in Fig. 1A, the specific primer pairs are delFeoA-check(+)/delFeoA-check(-) for the deletion of feoA, delFeoB-check(+)/delFeoB-check(-) for feoB deletion, and delFeoC-check(+)/delFeoC-check(-) for feoC deletion, respectively. The expected sizes of the PCR products are respectively 590-bp for feoA deletion (Fig.1B), 386-bp for feoB deletion (Fig.1C), and 870-bp forfeoC deletion (Fig. 1D).

Fig. 2 and 3 respectively show the position of the designed primer pairs delSitCD-check(+)/delSitCD-check(-) and delEfeUOB-check (+)/delEfeUOB-check (-), and PCR analysis employed to confirm the sitCD and efeUOB deletion. A total of 1437-bp sitCD- and 1640-bp efeUOB-containing DNA was deleted. The derived mutants are ∆sitCD∆fur, ∆sitCD∆fur,

∆sitCD∆feoB (∆SB), ∆efeUOB∆sitCD (∆SE), ∆efeUOB∆fur, ∆efeUOB∆feoB (∆BE), and

∆efeUOB∆sitCD∆feoB (∆ESB). As shown in Fig. 2(B) and Fig. 3(B), the amplicon of 546-bp for sitCD deletion and 759-bp for efeUOB deletion were obtained.

3.2. Effect of iron availability on the growth of the specific gene deletion strains

To investigate the effect of iron availability on these iron-acquisition systems, the growth of CG43S3 along with ∆feoA, ∆feoB, ∆feoC, ∆sitCD, ∆efeUOB, ∆sitCD∆feoB (∆SB),

∆sitCD∆efeUOB (∆SE), ∆efeUOB∆feoB (∆BE) and ∆efeUOB∆sitCD∆feoB (∆ESB) mutant strains

in LB, iron-supplemented or iron-depleted medium was investigated. LB medium adding with 50 µM Fe2(SO4)3 and 50 µM ascorbic acid is the iron-supplemented condition, while iron-depleted condition was created by adding 200 µM ferrous iron chelator DIP or 200 µM ferric iron chelator DFX. All the strains grown in LB medium reached OD600 of 1.2 in 6 h (Fig. 4A) and all appeared to grow faster when supplemented with iron (Fig. 4B). As shown in Fig. 4(C) and 4(D), the bacteria grown in medium containing DIP or DFX grew slower compared to the growth in LB or LB loaded with iron. Interestingly, the deletion mutants grew slightly slower than the parental strain CG43S3 only in the LB medium added with DIP. Otherwise, they exhibited similar growth pattern.

As shown in Fig. 5, the growth of double deletion strains ∆feoA∆fur, ∆feoB∆fur, ∆feoC∆fur,

∆sitCD∆fur, ∆efeUOB∆fur and ∆feoB∆sitCD∆efeUOB∆fur (∆ESB∆fur) had similar growth pattern as ∆fur but grew slower than CG43S3 in LB medium. The ∆fur-derived strains reached OD600 of 0.25 while CG43S3 of 0.5 after 2 h growth.

3.3. Analysis of feoABC, sitABCD and efeUOB promoter region

As shown in Fig. 6(A), 6(B), and 6(C), a Fur box-like sequence could be identified in the putative promoter regions of feoABC, sitCD and efeUOB. Besides, other regulatory sequences predicted using Softberry are respectively RstA- and Fnr-box within Pfeo, ArcA- and MntR-box in Psit, and CpxR box in Pefe. The presence of Fnr-box in feoABC and ArcA-box in sitABCD promoter region suggests oxygen availability affects the expression of feoABC and sitABCD. However, whether Fnr regulates expression of feoABC remains unknown since the Fnr binding sequence overlaps with the Fur box in Pfeo. The manganese repressor MntR binding box (AAACATAGCN4GCTATGTTT) is located 81-bp upstream of the start codon of SitA. The

predicted CpxR box (GTAAAN4-8GTAAA) identified in efeUOB promoter region implies a negative control by CpxR as reported in E. coli [46].

3.4. Effect of growth phase and oxygen availability on the expression of feo, sit and efe To examine the promoter activity, the DNA fragments encompassing the putative promoter regions of feoABC, sitABCD and efeUOB were individually cloned in front of the promoterless lacZ to generate pPfeo1-lacZ, pPfeo2-lacZ, pPsit-lacZ and pPefe-lacZ.

As shown in Fig. 7A, the activities of Pfeo, Psit and Pefe gradually increased following their growth. The activity of Pfeo, Psit and Pefe in stationary phase showed approximately 1.5-fold higher than in late log phase. Among these three Fe(II) uptake systems, sit exhibits the highest expression, following by efe and feo. It is interesting to note that Pfeo1 which contains two predicted 10 and -35 promoters had a much lower activity than Pfeo2 implying the presence of repressor binding element in the region only contained by Pfeo1.

Since Fnr and ArcA binding box are found respectively in promoter region of feo and sit, activity of Pfeo, Psit and Pefe was assessed under static condition (Fig. 7B). In compared to shaking condition, static condition did not apparently increase or decrease the activity of both Pfeo1 and Pfeo3. Interestingly, Psit::lacZ expression under static culture showed as high as 2000 Miller units in compared with 1000 Miller unit observed under shaking culture (stationary phase). In contrast, Pefe::lacZ expression was decreased under the microaerobic condition, showing merely 250 Miller unit of activity. These results supported the possibility that oxygen availability may affect the transcriptional level of sit and efe in adverse manner.

3.5. Effect of fur deletion and iron-depletion on expression of feo, sit, and efe

The effect of fur deletion on the expression of the feo, sit and efe was further verified by promoter activity assay. As shown in Fig. 8A, the activity of Pfeo1, Pfeo2, Psit and Pefe were apparently induced in Z01∆fur strain. In the meanwhile, Pfeo1, Pfeo2, Psit and Pefe activity were increased in response to addition of iron chelators (Fig. 8B). Interestingly, the inducible expression of feo was more effective by DFX than by DIP. On the other hand, both sit and efe promoters were more responsive to DIP than to DFX.

3.6. Effect of feoC deletion and rstA deletion and on expression of feo, sit, and efe

To investigate whether the two-component system response regulator RstA regulates feo expression, deletion effect of rstA on activity of Pfeo1 and Pfeo2 was examined. The result from left panel of Fig. 9(A) showed no apparent effect on Pfeo1 and Pfeo2 by rstA deletion. RstA has been shown to be involved in the acid stress response by regulating asr (acid shock RNA) expression in Salmonella [77], weak acid was treated to examine whether Pfeo is also acid-inducible. However, as shown in right panel of Fig. 9A, both Pfeo1 and Pfeo2 activity were not affected by acid.

Feo system is the first ferrous iron transport system identified in Enterobacteriae, and FeoB is well known as the major ferrous iron transport system in many bacteria, but the role of FeoA and FeoC are not well studied thoroughly. FeoC was proposed to regulate Fe(II) uptake of FeoB by coordinating its iron-sulfur cluster [3, 55, 62]. In order to clarify the regulatory role of FeoC in feo expression, activities of Pfeo1 and Pfeo2 were studied in Z01∆feoC strain under different environmental conditions (shaking, static, iron-depleted and iron-supplemented culture). As shown in Fig. 9B (right panel), the promoter activity of Pfeo1 and Pfeo2 was induced by feoC deletion

when bacterial strains were cultured in LB supplemented with DFX under static condition. By contrast, the increased activity was not observed under shaking condition (left panel).

3.7. Effect of manganese on expression of feo, sit and efe.

MntR is a manganese repressor using Mn²⁺ as a cofactor to regulate Mn²⁺-related uptake systems. To investigate whether Psit is negatively regulated by MntR, the culture of CG43S3∆lacZ (Z01) was treated by 0.1mM MnCl2 and the activity of Pfeo, Psit and Pefe were examined.

Interestingly, no suppressing effect was observed in Pfeo, Psit or Pefe, instead, their activities were induced by manganese (Fig.10).

3.8. Effect of weak acid and cpxR deletion effect on the efe expression

In E. coli O157:H7, expression of efeU was low-pH induced while repressed by CpxAR at high pH. The CpxR box (GTAAAnnnnnnnnGTAAA) found in promoter region of efeUOB suggests a possible role of CpxR in regulation of efeUOB in CG43. To investigate if efeUOB expression in CG43 works similar with O157:H7, Z01 and Z01∆cpxAR carrying Pefe-lacZ were grown in pH 7 or pH 5 LB media (Fig. 11). The expression of Pefe in Z01 at pH 5 was 4-fold higher than at pH 7. Deletion of cpxAR increased Pefe-lacZ expression under both pH 7 and pH 5 media, suggesting CpxR negatively regulates the expression of efeUOB under both acid and neutral pH conditions.

3.9. Analysis of cumulative effect of acid and iron-depletion or iron-supplemented on growth From the result of promoter activity, acid and iron play a regulatory role on the expression of these specific iron-acquisition genes. Growth curve analysis of all the mutants cultured in pH 5

LB with additional Fe or Fe-depleted condition was performed. As shown in Fig. 12 (A) and (B), all growth curve of the mutant strains appeared similarly as the parental strain CG43S3 in pH 5 LB or with additional Fe. However, an apparent growth defect was observed when the mutants grown in pH 5 LB supplemented with DIP. As Fig. 12(C) shows the mutant strains displayed more acute slow-growing phenomenon of which the OD600 could only reach 0.8 at stationary phase. This may be explained by no sufficient ferrous iron Fe(II) for the bacterial growth. No growth change was observed when Fe(III) iron chelator DFX was added to the cultures (Fig. 12D).

3.10. Analysis of ferrous iron transporter regulation in CPS biosynthesis

The ∆fur mutant strain exhibited profound CPS biosynthesis and hence the bacterial culture was precipitated much slower than CG43S3 [19]. The Fe(II)-associated Fur metalloprotein requires iron as cofactor to regulate expression of various genes. To investigate whether Feo, Sit or Efe system acts as the major source of Fe(II) for Fur, CPS production of the strains were analyzed by subjecting the overnight-grown bacteria to low-speed centrifugation. As shown in Fig.

13(A), the CPS formed by the mutant strains showed no apparent change compared to that of the parental strain CG43S3 and CG43S3∆fur, respectively. Fig. 13(B) shows that the mutant strains grown in LB with DFX displayed more viscous phenotype than those grown in LB media and hence could not be easily precipitated down. On the other hand, the addition of DIP resulted in poor growth of all the bacteria and the bacteria could be readily precipitated (Fig. 15C).

Nevertheless, no apparent of change of the phenotype in comparing with that of the parental strain CG43S3 suggesting FeoABC, SitABCD or EfeUOB transport systems does not directly influence the CPS biosynthesis.

3.11. Analysis of deletion effects on oxidative stress response

Fe(II) could be hazardous to bacteria because of the Fenton reaction leading to ROS formation. As reported in many other bacteria, iron transport systems play a role in oxidative stress responses [12, 83, 85]. To investigate if any of the three iron transport systems is also involved, disk diffusion assay was employed with the disc immersed with 30% hydrogen peroxide (H2O2).

The effect of iron chelators (DFX, DIP and EDTA) on the oxidative stress response was also tested.

By measuring the diameters of inhibition zones of the bacteria grown agitatedly in LB,

∆sitCD∆efeUOB (∆SE), ∆sitCD∆feoB (∆SB), and ∆efeUOB∆sitCD∆feoB (∆ESB) showed increased sensitivity to H2O2 compared to CG43S3 (Fig. 14A). When comparing the H2O2

inhibition zone with that of CG43S3, only ∆feoC exhibited more susceptibility when the culture added with DIP (Fig. 14B) while ∆feoB, ∆sitCD and ∆efeUOB were more sensitive by the addition of DFX (Fig. 14C) than double deletion and triple deletion mutant strains ∆feoB∆efeUOB (∆BE),

∆sitCD∆feoB (∆SB), ∆sitCD∆efeUOB (∆SE) and ∆efeUOB∆sitCD∆feoB (∆ESB) (Fig. 14C). Fig.

14(D) showed the bacteria ∆sitCD, ∆sitCD∆feoB (∆SB) and ∆efeUOB∆sitCD∆feoB (∆ESB) which carrying deletion of sitCD displayed a more sensitive phenotype in the presence of the divalent-cation chelator EDTA.

Since FeoABC, SitABCD, and EfeUOB are repressed by Fur under LB medium, the derepressing effect of Fur on these system towards oxidative stress response was also tested. As Fig. 15 that ∆efeUOB∆sitCD∆feoB∆fur (∆ESB∆fur) is more sensitive while ∆feoB∆fur,

∆sitCD∆fur, or ∆efeUOB∆fur is slightly resistant than∆fur.

3.12. Analysis of deletion effects on the biofilm formation under different culture conditions Iron plays an important role in bacterial biofilm formation. As shown in Fig. 16, all the deletion mutants except ∆sitCD∆efeUOB (∆SE) exhibited decreased biofilm forming activity compared to CG43S3. It is interesting to note that an apparent defect of biofilm formation was found by the deletion of fur.

Addition of DIP or DFX to the medium conferred different effects on the biofilm formation activity. As shown in Fig. 17, DIP negatively influenced the biofilm formation for CG43S3 as well as all the derived mutants. By contrast, addition of DFX allows identifying the deletion effect on the biofilm formation. Compared to CG43S3, only ∆feoC exhibited an increase of biofilm formation while the other mutants showed reduced biofilm formation in the presence of DFX. The feoC deletion may induce the expression of feoAB thereby promote iron-uptake and increase biofilm formation.

3.13. Analysis of the deletion effects on the expression of type 3 fimbriae

As described by Wu et. al., expression of the major pilin MrkA of type 3 fimbriae depends on the extracellular iron availability [26]. Consistent with their findings, Fig. 18(A) shows MrkA

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