2 folds serial dilution

CG43

yor5

-yco6

-U9451

Fig. 5. Comparison of CPS production of CG43-S3, CG43-S3-yor5^-, CG43-S3-yco6^-, and U9451. The CPS were extracted with hot-phenol and treated with protease K, appeared to be measured and probed with K2 antiserum. The frames represent equal amount of CPS analyzed with ScanAlyze (Michael Eisen , Stanford University).

0 2 4 6 8 10 12 14

S3 yor5- yco6-

galU-Bacterial Strains Glucuronic acid content (μg/109 CFU)

Fig. 6. Glucuronic acid contents of CG43, CG43-yor5^- , CG43-yco6^- and U9451.

CPS of the bacteria from 500 µl of overnight culture was extracted and then hydrolyzed by H2SO4, and then 3-hydroxydiphenol was added to measure the absorbance at 520 nm. Glucuronic acid was used as standard for the quantification of uronic acid.

A

Fig. 7. Both Yor5 and Yco6 affected the polymeric CPS production. The CPS was extracted with hot-phenol, treated with protease K and analyzed with 5% DOC PAGE. Panel A shows immune blot analysis probed with polyclonal K2 antiserum.

Panel B shows the alcian blue-silver stained DOC PAGE. CPS-P represents high molecular weight polymeric CPS. CPS-O represents low molecular weight

CG43 yor5^- yco6^- U9451

CPS-P

CPS-O

B

CPS-P

CPS-O

CG43 yco5^- yor6^- U9415

Fig. 8. Neither yor5 nor yco6 mutation affected the oligomeric CPS synthesis. The CPS were extracted with hot-phenol and treated with protease K and the CPS analyzed with alcian blue-silver stained 10% DOC PAGE. Alcian blue is a cationic dye and capable of binding to negative CPS to increase the sensitivity in CPS detection.

kDa M 1 2 3 4 5 6 7 8

200.0 116.3 66.3 55.4

36.5 31.0

Fig. 9 Purification of the His₆-Yor5 recombinant protein. The His6-Yor5 was overexpressed in E. coli NovaBlue (DE3) and purified through Ni⁺² affinity column.

The purified proteins were resolved by 12.5% SDS PAGE and then stained with Coomassie blue. Lane 1, protein molecular weight marker; lane 2, the whole cell lysate; lane 3, soluble proteins; lane 4, binding buffer elution； lane 5:washing buffer elution; lane 6 to lane 8, the purified fractions.

21.5

14.4 6.0

Yor5

1 2 3 4 5 6

A

(min) 30 0 1 5 15 30

B

1.6 1.4 1.2

Yor5 1

0.8

0.6 Control

0.4 0.2 0

2.5 10

0 5 15 30 45 60 (min)

Fig. 10. Assessment of the phosphatase activity of the recombinant Yor5. The phosphatase activity of Yor5 was determinated by measurement of the decomposition of Para-nitrophenyl phosphate (pNPP) at 37 ℃ (A). Tube 1 to 5 each contained 4 µg/ml Yor5 in PNPP reaction buffer. Tube 6 is the blank containing only the PNPP reaction buffer. The phosphatase activity of Yor5 with PNPP as the substrate is 3.2×10^-7 moles/min/mg (B).

ig. 11. Purification of the His₆-Yco6E23 recombinant protein. Panel A: Yco6E23

TM helix

is a truncated form of Yco6 Panel B His6-Yco6E23 was overexpressed in E. coli and purified by Ni⁺² affinity column and resolved on 12.5% SDS PAGE using Coomassie Blue-staining. M, protein marker; lane 1, total protein; lane 2, soluble proteins; lane 3, binding buffer elution; lane 4: washing buffer elution; lanes 5 to 7, the eluted fractions.

Panel C: Purification of the His6-Yco6E23 recombinant protein by raising the imidazole concentration of wash buffer to 125 mM, M: protein maker; Lane 1: is the purified recombinant protein Yco6E23.

Yco6E23 + + + +

0 5 15 30 (min)

Yco6E23 + + + + Yor5 + + + +

Yor5 - - - -

High MW

Yco6E23

Yco6E23 complex

Fig. 12. In vitro dephosphorylation assay. The reaction mixes were separated with 12.5 % SDS PAGE, and the radioactive proteins were visualiz by InstantImager^TM (Packard Instrument Company).

ig. 13. Identification of Yco6E23 complex. The protein samples were separated Yco6E23

Yco6E23

Yor5 complex

F

with 12.5% SDS PAGE. The Western blot probed with anti-phosphotyrosine monoclonal antibody (Sigma P3300) was shown in panel A. Panel B represents the blot probed with anti-His^.tag monoclonal antibody (Novagen). M, protein maker;

lane 1, Yco6E23: lane 2, Yco6E23 (100 ℃); lane 3, Yor5; lane 4, Yco6E23+Yor5;

lane 5 (100 ℃)

ig. 14. Comparsion of the phosphotyrosine proteins between wild type and 114.0

CG43-S3-yco6^-. Panel A shows Coomassie Blue-stained SDS-PAGE; Panel B shows the corresponding Western blot probed with monoclonal anti-phosphotyrosine antibody (Sigma P3300). M, protein maker; lane 1, CG43-S3 whole cell lysate; lane 2, CG43-S3-yco6^- whole cell lysate; The arrows ( ) represent probable targets of Yco6 and the arrow ( ) represents Yco6.

ig. 15. The Yco6-interacting proteins identified using immunoprecipitation.

M 1 2 M 1 2

Immunoprecipitation was used with monoclonal anti-phosphotyrosine antibody (Sigma PT66) from the whole cell protein. Then the protein-antibody complex was cached by proteinA/G. Panel A shows Coomassie Blue-stained SDS PAGE; Panel B shows corresponding Western blot probed with monoclonal anti-phosphotyrosine antibody (Sigma P3300). M, protein maker; lane 1, CG43-S3; lane 2, CG43-S3-yco6^-. The arrows ( ) represent probable targets of Yco6 and the arrow ( ) represents Yco6.

kD

A B

ig. 16. Overexpression of ugd, orf1, orf3 and orf15 in E. coli NovaBlue (DE3).

116.0

66.2

28.5 37.6

18.4

kDa M 1 2 3 4 5 6 7 8

97.4

F

The SDS PAGE of whole cell lysate was resolved by Coomassie Blue-staining. M, protein marker; lane 1, the total proteins of E. coli NovaBlue (DE3) [pET-UGD]; lane 2, the soluble fraction of sample 1; lane 3, the total proteins of E. coli NovaBlue (DE3) [pET-ORF1]; lane 4, the soluble fraction of sample3; lane 5, the total proteins of E.

coli NovaBlue (DE3) [pET100-ORF3]; lane 6, the soluble fraction of sample5; lane 7, the total proteins of E. coli NovaBlue (DE3) [pET-ORF15]; lane 8, the soluble fraction of sample 7. Arrows represent the recombinant proteins.

ig. 17. In vitro phosphorylation assay. Yco6E23 was incubated with Ugd, Orf3 Yco6 Control 1 2 3

Yco6E23 Yco6E23 Yco6E

E23

F

and Orf15 respectively in reaction buffer containing [γ-P³²] ATP as substrates at 37 ℃ for 30 min. The reaction mixture was then separated by 12.5% SDS-PAGE. Control, Yco6E23；lane 1, Ugd and Yco6E23；lane 2, Orf3 and Yco6E23；lane 3, Orf15 and Yco6E23.

Apendix 1

J Bacteriol. 1995 Apr;177(7):1788-96

Organization of the K. pneumoniae K2 cps gene cluster. Physical map of the

ORF18

chromosomal 20-kb region carrying the K. pneumoniae cps gene cluster is shown.

The horizontal arrows below represent the putative transcriptional units. The white arrow represents the gene (ORF18) of K. pneumoniae NTUH-K2044.

Apendix 2

Sequence alignment and structural features of several PTKs. The PTK cytoplasmic domain of Yco6 from K. pneumoniae K2 is presented in the middle section. The Walker A and B ATP-binding motifs are indicated as light gray shaded boxes. The Lys⁵⁴¹ residue of Walker A, the intraphosphorylated Tyr⁵⁷⁰ residue, and the tyrosine residues of the tyrosine cluster (Tyr⁷⁰⁷, Tyr⁷⁰⁹, Tyr⁷¹¹, Tyr⁷¹³, Tyr⁷¹⁴ and Tyr⁷¹⁶) are indicated with one-letter code (Y707, Y709, Y711, Y713, Y714 and Y716 respectively).The two predicted transmembrane -helices of the PTKs termed TM1 and TM2, are indicated by dark shaded boxes. The C-terminal cytosolic PTK domain is enlarged, and sequence alignment of PTKs from Gram-negative bacteria (Klebsiella pneumoniae Yco6, E. coli K30 Wzc, E. coli E2348/69 Etk, Erwinia amylovora AmsA, Acinetobacter johnsonii Ptk, and Rastonia solanacearum EpsB) by using the Vector NTI program. Dashes indicate gaps introduced in the alignment process.

Tyrosine residues of the tyrosine cluster and the Walker A and Walker B ATP-binding motifs are in gray boxes. Catalytic lysine of the Walker A