(A)
(B) (C)
(D)
61
Figure 1. Schematic representation of the feoA, feoB and feoC deletions and the verification of deletions by PCR.
(A) The mutants were confirmed using PCR with specific primer pairs (small black arrow) located upstream and downstream of the target genes; the dashed lines represent deleted regions of target genes. PCR verification for deletion strains of (B) ∆feoA and ∆feoA∆fur (B)
∆feoB, ∆feoB∆fur, ∆feoB∆sitCD (∆SB), ∆feoB∆efeUOB (∆BE) and ∆feoB∆sitCD∆efeUOB (∆ESB) (C) ∆feoC and ∆feoC∆fur; small arrows denote the size of PCR product in bp of wild-type and deletion strains
62 (A)
(B)
Figure 2. Schematic representation of the sitCD deletions and the verification of deletions by PCR.
The mutants were confirmed using PCR with specific primer pairs (small black arrow) located upstream and downstream of the target genes; the dashed lines represent deleted regions of target genes. PCR verification for deletion strains of (B) ∆sitCD, ∆sitCD∆fur,
∆sitCD∆efeUOB (∆SE) and ∆sitCD∆efeUOB∆feoB (∆ESB); small arrows denote the size of PCR product in bp of wild-type and deletion strains.
63 (A)
(B)
Figure 3. Schematic representation of the efeUOB deletions and the verification of deletions by PCR.
(A) The mutants were confirmed using PCR with specific primer pairs (small black arrow) located upstream and downstream of the target genes; the dashed lines represent deleted regions of target genes. PCR verification for deletion strains of (B) ∆efeUOB, ∆efeUB∆fur,
∆sitCD∆efeUOB (∆SE), ∆feoB∆efeUOB (∆BE), and ∆efeUOB∆sitCD∆feoB (∆ESB); small arrows denote the size of PCR product in bp of wild-type and deletion strains
64
(A) LB (B) LB + Fe
(C) LB + DIP (D) LB + DFX
Figure 4. Growth analysis of K. pneumoniae strains under iron-replete or iron-depleted media.
The optical density at 600 nm of CG43S3 and deletion mutant strains grown in (A) LB or (B) LB loaded with 50 µM ferric sulfate [Fe2(SO4)3] and 50 µM ascorbic acid or (C) LB loaded with 200 µM DIP or (D) LB loaded with 200 µM DFX was recorded every hour and plotted.
65
Figure 5. Growth analysis of K. pneumoniae ∆fur-derived strains.
The optical density at 600 nm of CG43S3, ∆fur and ∆fur-derived double deletion mutants grown in LB at 37 °C was recorded every hour and plotted.
66 (A)
(B)
(C)
67
Figure 6. Diagrammatic representation of K. pneumoniae CG43 feoABC, sitABCD and efeUOB promoter region and Pfeo::lacZ, Psit::lacZ and Pefe::lacZ fusion construction.
The upstream, non-coding sequence of feo, sitABCD and efeUOB, along with predicted transcriptional factor are shown. The open reading frame are shown in large grey arrows, the predicted position of -10 and -35 box are indicated in gray vertical line and putative transcriptional factors are indicated in black vertical line. The PCR primers (small black arrow) used to amplify DNA fragments harboring (A) Pfeo , (B) Psit and (C) Pefe regions that cloned in the lacZ fusion constructs are depicted. The dashed boxes indicate the putative RstA, Fnr, Fur, MntR, CpxR binding sequences aligned with the promoters and identical nucleotides are underlined.
68 (A) Shaking culture
(B) Static culture
Figure 7. Analysis of growth phase and static-cultured effects on activity of Pfeo, Psit and Pefe in K. pneumoniae CG43S3 ∆lacZ (Z01)
Psit
Pfeo1 P
feo2
Pefe
69
(A) The β-galactosidase actitvities of Z01 carrying placZ15-Pfeo1, Pfeo2, Psit and Pefe were determined at different growth phase: lag phase, early log phase, late log phase and stationary phase. (B) The effect of anaerobic condition on the β-galactosidase actitvities of Pfeo1, Pfeo2, Psit and Pefe were determined after statically growing until reaching approximately of OD600
0.6.
70 (A)
(B) Z01
Figure 8. Analysis of fur deletion effect and iron-depletion effect on Pfeo, Psit and Pefe
activity
(A) The β-galactosidase activities of late log-phased (OD600 0.8-0.9) cultures of Z01 and Z01∆fur carrying Pfeo1, Pfeo2, Psit and Pefe grown in LB medium agitatedly were determined and expressed as Miller unts. (B) The response of Pfeo1, Pfeo2, Psit and Pefe to iron chelators were tested by growing the cells agitatedly in LB medium, LB medium with DIP or DFX
Z01 Z01∆fur
Pfeo1
Pefe Pfeo2
Psit
71
unil late log phase and harvested. Their β-galactosidase activities were then determined as Miller units.
72 (A)
(B)
Figure 9. Analysis rstA, low pH and feoC deletion effect on Pfeo activity in different culture conditions.
(A) The left panel showed the determined β-galactosidase activities of late log-phased (OD6000.8-0.9) cultures of Z01 and Z01∆rstA (The left panel) carrying Pfeo1 and Pfeo2 grown in LB medium agitatedly. The right panel showed the β-galactosidase activities of Z01 carrying Pfeo1 and Pfeo2 growing in pH 7 and pH 5.5 LB (B) The deletion effect of feoC on acitivity of Pfeo1 and Pfeo2 coupled with O2 availability (Left panel – shaking; right panel –
Z01 Z01∆rstA
Pfeo1 Pfeo2 Pfeo1 Pfeo2
73
static) and iron-depleted effect were determined by β-galaoctosidase activity and expressed as Miller unit.
74
Figure 10. Analysis of effect of manganese on activity of Pfeo, Psit and Pefe.
Z01 carrying Pfeo1, Pfeo2, Psit and Pefe grown agitatedly in LB medium suplemented with or without 0.1mM MnCl2 until reaching OD600 of 0.8 to 0.9. Then, β-galactosidase activities were determined as Miller units.
LB LB + 0.1mM MnCl2
75
Figure 11. Analysis of weak acid and cpxAR effects on activity of Pefe
The β-galactosidase activities of late log-phased (OD6000.8-0.9) cultures of Z01 and Z01∆cpxAR carrying Pefe grown in pH 7 or pH 5 LB media agitatedly were determined and expressed as Miller unts
76
(A) LB pH 5 (B) LB pH 5 + Fe
(C) LB pH 5 + DIP (D) LB pH 5 + DFX
Figure 12. Effect of iron availability on growth of K. pneumoniae strains in acidic media.
The optical density at 600 nm was recorded and plotted for CG43S3 and deletion mutant strains that grown in pH 5 LB broth (A) alone or (B) loaded with 50 µM ferric sulfate [Fe2(SO4)3] and 50 µM ascorbic acid or (C) loaded with 200 µM DIP or (D) loaded with 200 µM DFX was every hour.
77 (A) LB
(B) LB + DFX
(C) LB + DIP
78
Figure 13. Analysis of capsular polysaccharide (CPS) biosynthesis in of K. pneumoniae strains in different media.
Comparison of CPS production between CG43S3 and deletion mutant strains was done by sedimentation test. The bacterial strains were grown 16 hr in LB (A), LB with 0.2 mM DFX (B) or LB with 0.2 mM DIP (C) at 37°C and subjected to centrifugation at 4,000 rpm for 5 min. Pellet formation after centrifugation was observed and photographed.
79 (A) LB
(B) LB + DIP
(C) LB + DFX
(D) LB + EDTA
1.6 ± 0.003 1.63 ± 0.02 1.73 ± 0.03 1.56 ± 0.03 1.56 ± 0.03 1.75 ± 0.003 1.82 ± 0.03 1.7 ± 0.07
1.21 ± 0.03 1.21 ± 0.03 1.33 ± 0.03 1.21 ± 0.03 2.2 ± 0.06
1.71 ± 0.03 1.65 ± 0.03 1.7 ± 0.03 1.9 ± 0.03 1. 8 ± 0.02 1.8 ± 0.05 1.8 ± 0.05 1.8 ± 0.05 1.76 ± 0.05 1.86 ± 0.02 1.8 ± 0.02 1. 93 ± 0.02 1. 9 ± 0.02
1. 78 ± 0.03 1. 83 ± 0.05 1. 75 ± 0.05 1. 88 ± 0.02 1. 7 ± 0.05 1. 82 ± 0.02 1. 7 ± 0.05 1. 78 ± 0.05 2.23 ± 0.05 1.13 ± 0.03 1.12 ± 0.03 1. 13 ± 0.03 1. 12 ± 0.03
1. 91 ± 0.03 1. 91 ± 0.07 1.91 ± 0.15 2.08 ± 0.07 1.78 ± 0.07 1.82 ± 0.02 2.15 ± 0.05 2.16 ± 0.02 2.06 ± 0.02 1.22 ± 0.003 1.2 ± 0.03 1.22 ± 0.03 1.2 ± 0.03
80
Figure 14. Effect of deletion mutants and iron chelators on the oxidative stress response
Overnight bacterial culture was inoculated into fresh LB broth and incubated at 37°C agitatedly until reaching OD600 of 0.6-0.7.
Following that, 1 ml of each culture was suspended in pre-warmed liquefied agar and puored over the LB plates supplementd with or without iron chelators before placing filter paper disks spotted with 30% of H2O2 onto the surface culture. After 16 hr incubation at 37 °C, the inhibition zones of each plate were measured in cm (indicated as the average of triplicate samples with stanard deviations).
The effect of iron chelators on oxidative stress response was tested by adding (B) 0.2 mM DIP or (C) 0.2 mM DFX or (D) 0.2 mM EDTA 1 h prior pouring onto agar. The small black triangles marked the larger inhibition zone in compared with CG43S3.
81
Figure 15. Effect of ∆fur-derived deletion mutants on the oxidative stress response Overnight bacterial culture was inoculated into fresh LB broth and incubated at 37°C agitatedly until reaching OD600 of 0.6-0.7. Following that, 1 ml of each culture was suspended in pre-warmed liquefied agar and puored over the plates supplementd with iron chelators before placing filter paper disks spotted with 30% of H2O2 onto the surface culture. After 16 hr incubation at 37 °C, the inhibition zones of each plate were measured (indicated as the average of triplicate samples with stanard deviations). The small black triangles marked the larger inhibition zone in compared with CG43S3∆fur.
1.97 ± 0.03 2.01 ± 0.03 2.02 ± 0.03 1.96 ± 0.03 2.06 ± 0.03 2.02 ± 0.03 2.01 ± 0.03
82
Figure 16. Biofilm formation analysis of deletion mutant strains.
The abilities of deletion mutants and ∆fur-derived mutants form biofilm were tested by growing cultures statically in plastic 96-well supplemented with LB medium at 37°C for 24 hr. The biofilm was visualized by staining with 1% crystal violet and quantified by absorbance at 595 nm.
83 (B)
Figure 17. Biofilm formation analysis of deletion strains under iron-depletion
Biofilm formation assay was tested for bacterial culture that grown in LB medium supplemented with DIP (upper panel) or DFX (lower panel) with static incubation on plastic 96-well at 37°C for 24 hr. The biofilm were visualized by staining with 1% crystal violet and quantified by absorbance at 595 nm.
84 Shaking culture
(A) LB
(B) LB
(C) LB + DFX
Figure 18. Iron-depleted effect on the iron-dependent MrkA expression under shaking condition
Total protein of bacterial cultures, approximately 10 µg per lane, were separated by SDS-PAGE, transferred to PVDF membrane and subjected to hybridization against the antibodies of anti-MrkA and anti-GAPDH (as an internal control). Proteins were collected from
85
bacterial culture treated with different growing conditions. (A) CG43S3 (WT) treated with or without iron chelator (DIP or DFX); WT and deletion mutant strains grown agitatedly at 37 °C for 16 hr in (B) LB or (C) LB added with DFX
86
Figure 19. MrkA expression of the ∆fur-derived deletion mutants under shaking condition
Total protein of bacterial cultures, approximately 10 µg per lane, were separated by SDS-PAGE, transferred to PVDF membrane and subjected to hybridization against the antibodies of anti-MrkA and anti-GAPDH (as an internal control). Proteins were collected from CG43S3 and ∆fur-derived deletion mutants after growing agitatedly at 37 °C for 16 hr.
87 Static culture
(A) LB
(B) LB + DFX
Figure 20. Iron- and oxygen-depleted effect on MrkA expression for the deletion mutants
Total protein of bacterial cultures, approximately 10 µg per lane, were separated by SDS-PAGE, transferred to PVDF membrane and subjected to hybridization against the antibodies of anti-MrkA and anti-GAPDH (as an internal control). Proteins were collected from CG43S3 and deletion mutants that grown statically at 37 °C for 24 h in (A) LB or (B) LB supplemented with DFX
88
Figure 21. Schematic representation (not to scale) of the proposed regulation of feoABC, sitABCD and efeUOB in CG43S3.
All three systems are induced by iron-depletion (low Fe) but negatively regulated by Fur-Fe2+. FeoC possibly represses expression of feo when both iron and oxygen are depleted (low Fe and low O2). The repression of CpxAR on efe expression when CG43S3 in a rather a non-acidic environment (OH-). The increased level of Mn2+ might enable repression of Fur by PerR. [ represents positively regulates;
represents negatively regulates]
89
Figure 22. Schematic representation of the proposed model of functional role and location of FeoABC, SitABCD and EfeUOB in CG43S3.
The ferrous iron-acquisition is mediated by FeoB which contains a cytoplasmic GTPase domain to hydrolysis GTP to GDP; cytoplasmic FeoC senses environmental oxygen and iron to regulate feo system; the role of FeoA is unclear yet (this model is based on a model proposed by Cartron et al. [49]). Transport of ferrous iron or/ and manganese by permease SitC and SitD is activated by the periplasmic
90
SitA which in turn facilitated by SitB that hydrolyze ATP to ADP. The acid-inducible EfeU uptakes ferrous iron with the assistance of EfeO and EfeB (yet the mechanism is little known). All three systems affect the formation of biofilm in CG43S3. The SitABCD protects CG43S3 against H2O2. In a Western blot analysis, FeoB and Sit system negatively regulate the production of major pilin of type 3 fimbriae in iron-depleted (low Fe) or / and microaerobic condition (low O2). [ represents positively regulates; represents negatively regulates]
91