Supplemental Table and Figures
Functional characterization of a novel Mn2+ dependent protein serine/threonine kinase
KpnK, produced by Klebsiella pneumoniae strain MGH78578
Vijaya Bharathi Srinivasan, Vasanth Vaidyanathan, Amitabha Mondal, Manjunath
Venkataramaiah and Govindan Rajamohan*
Council of Scientific Industrial Research (CSIR)-Institute of Microbial Technology, Bacterial
signaling and Drug resistance laboratory, Sector 39 A, Chandigarh-160036, India
Running title: Characterization of Ser/Thr kinase from K. pneumoniae MGH78578
* Corresponding Author
Telephone: 91-172-26665258
Fax: 91-172-2690585
E-mail: [email protected]
Table S1
Primers used in this study
Primer name
Primer sequences
pkKP-F
5’-TCATCATATGCACGATAAGGCTTTTACTTTTCAGACG-3’
pkKP-R
5’-TACGGGATCCTTAATACATTGGCGTTAACTGAAGCGG-3’
pkKPint-F
5’-TATGTGGTAAAGTTCTATCGTCCTGAGCGC-3’
pkKPint-R
5’-GGGCTGAATTCCTCATAGGCCTCGACAATA-3’
KPN_cpxR-F
5’-GCGTCATATGAATAAAATCCTGTTAGTTGATGATG-3’
KPN_cpxR-R
5’-CTCAGGATCCTCATGAAGCGGAAACCATCAGATAC-3’
pkKPprom-F
5’-CAGAAGGATTATGAAGAGGATTTTAAA-3’
pkKPprom-R
5’-ATCGATAATGGTATCCGGGCGAAGCGT-3’
kmrARTnt
5’-TGCTGGAGCATTTCTACTGGGGGTCGGTGT-3’
kmrARTct
5’-CTCCTGGGCCATCAGCAGCTCAAAA-3’
eefBRTnt
5’-TTCTCGGTAACCATTATCTCGGCGATGATG-3’
eefBRTct
5’-TGATCTCCCCCTGGTCCTCCACCGG-3’
acrBRTnt
5’-AAGAGCACGCACCATTACACCGACAG-3’
acrBRTct
5’-TTCCTCACCCGGACGCTGGCTCCAGTC-3’
rpoBRTnt
5’-GCGGTTGGTCGTATGAAGTT-3’
rpoBRTct
5’-TGGCGTTGATCATATCCTGA-3’
Figures
Figure S1
Figure S1: In silico analysis of KpnK
A) Structure based sequence alignment of KpnK with protein kinase homologues. The
accession numbers of protein kinases from different bacteria ; Escherichia coli:
NC_002655, K. pneumoniae MGH78578: YP_001337815.1, K. pneumoniae NTUHK2044: NC_012731, Salmonella enterica subsp. enterica serovar Newport: EDX48829.1,
Shigella flexneri: YP_690976.1, Enterobacter cloacae: ADO50623.1, Yersinia pestis:
NP_671107.1, Citrobacter koseri: YP_001454680.1, Vibrio cholerae O1: NC_002505.1,
Multiple sequence alignments were made in CLUSTAL W, and formatting was from the
ESPript
server
(http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).
The
secondary
structural elements of E. coli YihE are shown on the lines above the sequence alignment
using the PDB file 1ZYL. The arrows indicate β-sheet, the coils indicate α-helices, TT
indicates β turns and η indicates 310 helices. Residues strictly conserved have a black
background and indicated by bold letters; residues conserved between groups are boxed.
The phosphotransferase Brenner's motif [197-RLHGDCHAGN-206] and crucial residues
(Asp201, Asp217, Asp219, Asp220, Arg270, Arg273, Tyr277, Trp280) known for
substrate binding are highly conserved in KpnK.
B) Similarity and Identity index was generated using sequences of K. pneumoniae
MGH78578: YP_001337815.1, K. pneumoniae NTUH-K2044: NC_012731, Escherichia
coli: NC_002655, Shigella flexneri: YP_690976.1, Shigella dysenteriae: EGI90318.1,
Citrobacter koseri: YP_001454680.1, Salmonella enterica subsp. enterica serovar
Newport:
EDX48829.1,
Enterobacter
cloacae:
ADO50623.1,
Yersinia
pestis:
NP_671107.1, Vibrio cholerae O1: NC_002505.1, Erwinia billingiae : NC_014306,
Pseudomonas carotovorum Pf0-1 (P. carotovorum) : CP000094.2.
Figure S2: Studies on expression and sub cellular localization of KpnK
A) Expression of KpnK in E.coli: Lane 1: marker, Lane 2: pET28C/BL21DE3 uninduced,
Lane 3: pET28C/BL21DE3 induced, Lane 4: KpnK-pET28C/BL21DE3 uninduced, Lane
5: KpnK-pET28C/BL21DE3 induced, purified KpnK-fraction E1, E2 (lane 6, 7)
respectively. Protein samples after 5hours of induction were subjected to SDS/PAGE
(15% gel) followed by Coomassie Brilliant Blue staining.
B) Silver staining was done as described previously [1] Lanes 1-5.
C) Sub cellular fractionation: E. coli cells harboring KpnK-pET28C plasmid was grown in
LB medium at 37°C until they reached an A600 of 1.0. The cells harvested were lysed
and fractionated to cytoplasmic and crude membrane fractions. Crude membrane fraction
was separated into inner and outer membranes by following standard procedure (20).
Total protein lysate of pET28C/BL21DE3 (lane 1), inner membrane fraction of
pET28C/BL21DE3 (lane 2), pET28C/BL21DE3 outer membrane fraction (lane 3),
followed by total protein lysate of KpnK-pET28C/BL21DE3 (lane 4), inner membrane
fraction (lane 5) and outer membrane fractions (lane 6) of KpnK-pET28C/BL21DE3.
D) Localization of KpnK protein in E. coli which has the His tag was determined by Western
blot analysis with anti-Penta-His antibody (Thermo Scientific). Total protein lysate of
KpnK-pET28C/BL21DE3
(lane
1),
inner
membrane
fraction
of
KpnK-
pET28C/BL21DE3 (lane 2), KpnK-pET28C/BL21DE3 outer membrane fraction (lane 3),
followed by total protein lysate of pET28C/BL21DE3 (lane 4), inner membrane fraction
(lane 5) and outer membrane fractions (lane 6) of pET28C/BL21DE3. Analysis revealed
the presence of band in the total lysate (lane 1) and inner membrane fraction of KpnKpET28C/BL21DE3 (lane 2) which indicted that KpnK localizes in the inner membrane
region of the cell.
Figure S3: Characterization of ∆kpnK mutant
A) Growth kinetics of WT, and ∆kpnK was assessed in LB medium pH 5.0, 6.0, 7.0, and 8.0.
B) The morphology of WT (panel i) and ∆kpnK (panel ii, iii, and iv) was determined by
scanning electron microscopy.
Figure S4: Disinfectant challenge assays
A) Sensitivity towards benzalkonium chloride by WT and kpnK mutant when cells were
exposed to different concentrations of the disinfectant (3.2 µg/ml, 6.4 µg/ml, 12.8 µg/ml,
25.6 µg/ml, 51.2 µg/ml).
A) Tolerance of WT and kpnK to different concentration of chlorhexidine. The percent
survival was calculated by comparison of viable cells in control. The datas are the means
of measurements made in triplicate performed three times. *, significant difference (P <
0.05, Student t test).
Figure S5: Protein profiling of WT and kpnK mutant strain
Membrane protein profiles were compared between the wild-type strain, and kpnK mutant.
Total protein lysate of wild-type strain (lane 1), outer membrane fractions (lane 2), inner
membrane fractions (lane 3), followed by total protein lysate of kpnK mutant (lane 4), outer
membrane fractions (lane 5), inner membrane fractions (lane 6). Equal protein concentrations
were separated by SDS-PAGE with a 5% stacking gel and a 12% separating gel and stained
with coomassie brilliant blue. Lane M has molecular weight standards.
Reference :
1. Damerval, C., Le Guilloux, Martine., Blaisonneau, Joel., et al. (1987) A simplification
of Heukeshoven and Dernick’s silver staining of proteins. Electrophoresis 3, 158-159.