1. Primer sequences used in this study for cloning and mutagenesis
Target GBE
VvGBE
Wild type
EGBE
F/R
5’-Sequence of Oligonucleotides-3’
Restriction enzyme
F
TACGCGGTCGCATATGAGGATTGTGTTCAC
NdeI
R
F
CCTAATCGCCTGCAGTTAGACCAGCTTGTAG
CAGGAAGACACATATGTCCGATCGTATC
PstI
NdeI
R
CAATGGCGAAAGCTTTCATTCTGCCTCC
HindⅢ
F
GCCCCGTCCATATGCTTGACCCGTTG
NdeI
R
GCGGACGAACTGCAGTCACCGGTCCTCG
PstI
F
R
F
R
F
R
F
R
F
R
F
R
AGTATGACGATCATATGACGCCGAAAACCATG
CATGCCTGCTCGAGGACCAGCTTGTAGAAC
GTCACGTACCATATGGCTCGTTACCAGTGGC
CATGCCTGCTCGAGGACCAGCTTGTAGAAC
CACCATCACCATATGAGGATTGTGTTCAC
CATGGTTTTCTGCAGGTGGAGATCGTC
GCTATTATCTCTGCAGACTCACCTGCGCC
CAATGGCGAAAGCTTTCATTCTGCCTCC
CACCATCACCATATGAGGATTGTGTTCAC
GCCACTGGTACTGCAGGTGATCGTACG
CCTTATGCCTTTCTGCAGCAAATGCGCC
GATTACGCCAAGCTCGAGTTCTGCCTCCC
NdeI
XhoI
NdeI
XhoI
NdeI
PstI
PstI
HindⅢ
NdeI
PstI
PstI
XhoI
DgGBE
Truncation
mutants
VvGBE-N1
VvGBE-N
VvGBE
N1
EGBE
Swapping
mutants
N
VvGBE
EGBE
2. The characteristics of vectors and recombinant plasmids used in this study
Strains and Vectors
Strains
Characteristics
Vibrio vulnificus MO6-24/O
Escherichia coli MC1061
Deinococcus geothermalis DSM 11300
p6xHis119
Vectors
p6xHTKNd
pTKNd6xH
[1]
[2]
[3]
∙ Pblma, constitutive, Amr
∙ His-tag (N-terminal)
∙ Pblma, constitutive, Kmr
∙ His-tag (N, C-terminal)
∙ Pblma, constitutive, Kmr
∙ His-tag (C-terminal)
Recombinant plasmids
Recombinant enzyme
Wild type
Cloning vector
VvGBE
EGBE
DgGBE
∙ p6xHis119 vector, Amr
∙ p6xHis119 vector, Amr
∙ p6xHis119 vector, Amr
VvGBE-N1 (N1_t)
∙ p6xHTKNd vector, Kmr
∙ 129-residue deletion at the N-terminal of VvGBE
VvGBE-N (N_t)
∙ p6xHTKNd vector, Kmr
∙ 240-residue deletion at the N-terminal of VvGBE
N1 swapping (N1_s)
∙ p6xHis119 vector, Amr
∙ N1 of VvGBE replaced counterpart of E.coli GBE
N swapping (N_s)
∙ pTKNd6xH vector, Kmr
∙ N domain of VvGBE replaced the counterpart of E.coli GBE
Truncation
mutants
Swapping
mutants
3. Sequences used in the phylogenetic analysis
All amino acid sequences and conserved domain searches of known GBEs were obtained
from the National Center for Biotechnology Information database
(http://www.ncbi.nlm.nih.gov): Aquifex aeolicus VF5 (gi.15606119), Bacillus cereus
(674471943), Bacillus subtilis 168 (16080150), Deinococcus geothermalis DSM 11300
(94555367), Deinococcus radiodurans R1 (15806848), Escherichia coli K-12 MG1655
(16131306), Mycobacterium tuberculosis (631464201), Salmonella enterica serovar Typhi
Ty21a (485084115), Thermococcus kodakarensis KOD1 (74502442), Thermus thermophiles
(504323830), Vibrio cholerae (669407972), Vibrio parahaemolyticus (505126546), and
Vibrio vulnificus MO6-24/O (319933217).
HPAEC analysis of glycogen resulting from adding G2 by V.vulnificus MO6-24/O
4. Glycogen in Vibrio vulnificus MO6-24/O
20
G1-G7 std
after 2 h (control)
after 2 h + Isoamylase
Response (nC)
15
10
5
0
G4
G5 G7
G6
G3
G4 G5 G6
HPAEC analysis of glycogen resulting from adding G2 by V.vulnificus MO6-24/O
-5
20
10
30
40
50
G1-G7 std
Retention time (min)
after 4 h (control)
after 4 h + Isoamylase
15
Response (nC)
20
10
5
0
HPAEC analysis of glycogen resulting from adding G2 by V.vulnificus MO6-24/O
-5
Response (nC)
30
10
20
30
40
50
G1-G7 std
Retention time after
(min)6 h (control)
after 6 h + Isoamylase
20
10
0
10
20
30
40
50
Retention time (min)
Culture condition: Cells were cultured with Luria-Bertani medium at 37°C with shaking.
Glycogen extraction and side-chain distribution analysis: After cell growth reach late
exponential phase, 1.0% maltose (w/v in final) was added. Cells were harvested by
centrifugation by 2 h time intervals after c-source addition. Glycogen was extracted and
analyzed as described by Park et al. previously (J. Bacteriol. 193(10):2517-2526, 2011).
Control means that glycogen was injected directly without debranching by isoamylase.
References for supplementary materials
1. Park, J. H., Cho, Y.-J., Chun, J., Seok, Y.-J., Lee, J. K., Kim, K.-S., Lee, K.-H., Park, S.-J. & Choi,
S. H. (2011) Complete Genome Sequence of Vibrio vulnificus M06-24/O, Journal of Bacteriology.
2. Park, J.-T., Shim, J.-H., Tran, P. L., Hong, I.-H., Yong, H.-U., Oktavina, E. F., Nguyen, H. D.,
Kim, J.-W., Lee, T. S. & Park, S.-H. (2011) Role of maltose enzymes in glycogen synthesis by
Escherichia coli, Journal of bacteriology. 193, 2517-2526.
3. Makarova, K. S., Omelchenko, M. V., Gaidamakova, E. K., Matrosova, V. Y., Vasilenko, A., Zhai,
M., Lapidus, A., Copeland, A., Kim, E. & Land, M. (2007) Deinococcus geothermalis: the pool of
extreme radiation resistance genes shrinks, PLoS One. 2, e955.