The length of glycine rich linker in DNA binding domain is critical for optimal functioning of quorum sensing
master regulatory protein HapR
Naorem Santa Singh1, $, Sangita Kachhap2, $, Richa Singh1, Balvinder Singh2,* and Saumya Raychaudhuri1,*
1
Molecular Biology and Microbial Physiology Division, Institute of Microbial Technology (Council of Scientific and
Industrial Research), Sector 39A Chandigarh 160036, India.
2
Bioinformatics Centre, Institute of Microbial Technology (Council of Scientific and Industrial Research), Sector 39A
Chandigarh 160036, India.
*To whom correspondence should be addressed: Tel: +91-172-6665256; Fax: +91-172-2690585;
Email: saumya@rocketmail.com, saumya@imtech.res.in
Saumya Raychaudhuri, Molecular Biology and Microbial Physiology Division, Institute of Microbial Technology, Sector
39A, Chandigarh 160036, India.
Correspondence may also be addressed to : Tel : +91-172-2690557; Fax: +91-172-2690632;
Email: bvs@imtech.res.in
Balvinder Singh, Bioinformatics Centre, Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
$: Authors have contributed equally.
Table S1 and S2
Fig. S1.
Fig. S2.
Table S1 Name of strains and plasmids
Strains/ Plasmids
Description
V. cholerae strains
V2
Non-O1, non-O139, Sergroup O37
V2S
V2S-C
V2S -RV2G
V2S-RV2G39+1
V2S-RV2G39+2
V2S-RV2G39+3
V2S-RV2G39+4
E. coli strains
Nova blue
BL21(DE3)
XL1-Blue
Non-O1, non-O139, Sergroup O37, hapR::pCD, Cmr (17
μg ml –1)
Non-O1, non-O139, Serogroup O37, hapR::pCD carrying
pKK177-3R1, Cmr (17 μg ml –1), Apr(100 μg ml –1)
Non-O1, non-O139, Serogroup O37, hapR::pCD having
pSV2G Cmr (17 μg ml –1), Apr(100 μg ml –1)
Non-O1, non-O139, Serogroup O37, hapR::pCD having
pSV2G39+1 Cmr (17 μg ml –1), Apr(100 μg ml –1)
Non-O1, non-O139, Serogroup O37, hapR::pCD having
pSV2G39+2 Cmr (17 μg ml –1), Apr(100 μg ml –1)
Non-O1, non-O139, Serogroup O37, hapR::pCD having
pSV2G39+3 Cmr (17 μg ml –1), Apr(100 μg ml –1)
Non-O1, non-O139, Serogroup O37, hapR::pCD having
pSV2G39+4 Cmr (17 μg ml –1), Apr(100 μg ml –1)
E. coli K-12, recA endA, lacIq, lacY
E. coli B, F– ompT lon, with a λ prophage carrying the T7
RNA polymerase
E. coli recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1
Source/reference
Ranjan K Nandy, National
Institute of Cholera and Enteric
Diseases (NICED), India
Dongre et al.,(2011)
Dongre et al.,(2011)
Dongre et al.,(2011)
This study
This study
This study
This study
Novagen
Novagen
Stratagene
lac [F´ proAB lacIqZΔM15 Tn10 (Tetr)]
Plasmids
pKK177-3R1
pET15b
HapR-pET15b
pSV2G
pSV2G G39+1
pSV2G G39+2
pSV2G G39+3
pSV2G G39+4
Apr
Apr, N-terminal 6His-tag expression vector
612, 615, 618, 621, 624 bp fragments of hapR (ORF)
containing G39,G39+1,G39+2 G39+3 and G39+4 respectively
were cloned into NdeI-BamHI site of pET15b
612 bp fragment of hapR (ORF) from V2 containing G39
was cloned into SmaI – HindIII site of pKK177-3RI.
pSV2G with one glycine insertion after G39
pSV2G with two glycine insertion after G39
pSV2G with three glycine insertion after G39
pSV2G with four glycine insertion after G39
Table S2 List of primers
Sequence
5’-TAACCCGGGATGGACGCATCAATC-3’
5’-CCCAAGCTTCTAGTTCTTATAGATAC-3’
5’-GGGAATTGCATATGATGGACGCATCAATCGAAAAAC-3’
5’-CGCGGATCCCTAGTTCTTATAGATACACAG-3’
5’-TGGTTTAGTCGAGAGCTACTGCCG-3’
5’-GAGTGAAGGCCAAAGTCATTG-3’
5’-GATCGGAATTCGAATGCGCAATACTGGTTAAC-3’
5’-GATCGGGATCCGATAACGTGTGGTAATGACATG
5’-GGAATTCGACTCATGGGGACTTGC-3’
5’-GGAATTCTCAGTTGCCGCTCCGGCCA-3’
5’-GGTCGAGGTGGTGGTCACGCAGATATTGCC-3’
5’-GGCAATATCTGCGTGACCACCACCTCGACC-3’
5’-GGTCGAGGTGGTGGTGGTCACGCTGATATTGCC-3’
5’-GGCAATATCAGCGTGACCACCACCACCTCGACC-3’
5’-CGAGGTGGTGGTGGTGGTCACGCTGATATTGC-3’
5’-GCAATATCAGCGTGACCACCACCACCACCTCG-3’
5’-CGAGGTGGTGGTGGTGGTGGTCACGCTGATATTGCCG-3’
5’-CGGCAATATCAGCGTGACCACCACCACCACCACCTCG-3’
Giesla Stroz, National Institute
of Health, USA.
Novagen
This study
Dongre et al.,(2011)
This study
This study
This study
This study
Primer Name
SmaI HapR (cloning and sequencing)
HindIII HapR (cloning and sequencing)
NdeI HapR (cloning and sequencing)
BamHI HapR (cloning and sequencing)
pvc0900F (promoter vc0900)
pvc0900R (promoter vc0900)
YF13(promoter apha)
YF12(promoter apha)
phapAF (promoter hapA)
phapAR (promoter hapA)
G39+1F (insertion)
G39+1R (insertion)
G39+2F(insertion)
G39+2R(insertion)
G39+3F(insertion)
G39+3R(insertion)
G39+4F(insertion)
G39+4R(insertion)
Fig. S1 MALDI-TOF spectra of wild type HapR (HapRV2G) and its glycine linker variants along with their expected
molecular weights in Daltons . BSA was used as a control.
Fig.S2 RSMD (root mean square deviation) of backbone atoms of (A) protein and (B) DNA with respect to corresponding
minimized HapR –DNA complex.