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Figure I supplemental
A. hydrophila VgrG1 716
cd00233
16
A. hydrophila VgrG1 796
cd00233
96
A. hydrophila VgrG1 873
cd00233
167
10
20
30
40
50
60
70
80
....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
GKKSAKKWATVEISKADEALRYYSAQGYTLLNNYLRDRPYKQREAIDTLLSRSYLNDEPTSASEFDQAMKAYVADVEAGL 795
GNKNYKKWLKKLSPSEKEAIREYTGSDYKKINNYLRGNGGPENSLNSELDKQIENIDSAFKKKPIPENITVYRGVDMTYL 95
90
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110
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130
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....*....|....*....|....*....|....*....|....*....|....*....|....*....|....*....|
aklpaspDLDFVYRGLALDKPELAALKDQFTGvgNIIVEPGFMSTS--PDKAW-VNDTLLRIRLPAGHGGRLLGEAAHFK 872
-------GLIFQSTDGTINKTVNKQFEAKFLG--KIYKDDGFMSTSlvSESAFgGRPIILRLTVPKGSKGAYISPISGFP 166
170
180
....*....|....*....|....
GEAEMLFPTQTRLRVDRVVSSMSG 896
GELEVLLPRGSTYKINKITVSSDG 190
Figure I: Alignment of the VIP-2 reference sequence (cd00233) with the sequence present in the COOH-terminal
fragment of A. hydrophila ATCC 7966 VgrG1. In red are conserved amino-acid residues. CD Length: 201; Bit Score:
62.27; E-value: 5e-10 (taken from BLAST-Conserved Domains report). Amino acid residues in open boxes constitute
a conserved Ser-Thr-Ser motif, which plays an important role in catalysis and structure as well as in NAD binding.
Underlined sequences denote ADP-ribosylating toxin turn-turn motif, and an orange arrow shows conformational
flexibility of ligand binding pocket.
Figure II Supplemental
A.
Phage_GPD
DUF586
VIP-2
VgrG
COOH-terminal
NH2-terminal
1aa
701aa
927aa
B.
Phage_GPD
DUF586
RtxA
VgrG
COOH-terminal
NH2-terminal
1aa
gp27-like (1 to554aa)
gp5-like
(554 to 650 aa)
701aa
Figure II: Schematic representation of the conserved domains present in VgrG1. A. A. hydrophila ATCC
7966 (gi|117619461) and B. V. cholerae N16961 (gi|15641427). The gp27- and gp5-like motifs are
represented by red and blue lines, respectively (modified from Pukatzki, et al 2007) (38). The encoding
fragments representing the NH2-terminal (gray), COOH-terminal (cyan) and the full-length (gray and cyan)
of VgrG1 from A. hydrophila ATCC 7966 were cloned into a pET-30a vector to produce recombinant
proteins, and into pBI-EGFP vector for expression in the HeLa Tet-off cells. The positions of VIP-2 and
RtxA domains in VgrG1 of A. hydrophila and V. cholerae are also shown.
1163aa
Figure III Supplemental
A.
Control
act
act/ vasH pBR322-empty
act/ vasH pBR322-vasH
Figure III Supplemental
B.
2
Δact ΔvasH
1
Δact
Δact ΔvasH
Post
Δact
Pre
3
4
VgrG 2/3 (~77 kDa)
Hcp (~20 kDa)
Figure III: A. Induction of HeLa cell-rounded phenotype in co-cultures with different strains of A. hydrophila SSU. HeLa
cells were co-cultured in direct bacterial-host cell contact for 90 min with A. hydrophila SSU Δact mutant (Top right),
Δact/ΔvasH pBR322-empty (with vector alone) (Bottom left), and Δact/ΔvasH pBR322-vasH in which the Δact/ΔvasH
mutant of A. hydrophila SSU was complemented with the vasH gene using the pBR322 vector in trans (Bottom right).
Normal morphology of HeLa cells is shown in Top left panel (control). Magnification 40X. B. Detection of Hcp2 and
VgrG 2/3 in tissue culture supernatants after co-culturing of HeLa cells with different strains of A. hydrophila SSU. Pre
(lanes 1-2): filtered tissue culture supernatants after 90 min of co-culture of HeLa cells with bacteria and before they
were used as pre-conditioned media on fresh HeLa cells. Post (lanes 3-4): same conditioned tissue culture
supernatants after 120 min on HeLa cell cultures.
VgrG1-NH2::Bla
VgrG1-Full::Bla
Empty
VgrG1-NH2::Bla
VgrG1-Full::Bla
Empty
Figure IV Supplemental
~131 kDa
Pellets
~106 kDa
~131 kDa
~106 kDa
Supernatants
1
2
act
3
4
5
6
act/vasH
Figure IV: Western blot analysis of A. hydrophila SSU act (lanes 1 to 3) and act/vasH (lanes 4 to 6) mutant bacterial
pellets (top panel) and supernatants (bottom panel) using antibodies to Bla. The production of full-length VgrG1 (VgrG1Full::Bla) (lane 2) and VgrG1-NH2::Bla (lane 3) was detected in both bacterial pellet and supernatant of A. hydrophila SSU
act parental strain. In the act/vasH mutant strain, fusion proteins were detected in the pellets (lanes 5 and 6) but not in the
supernatants. Bacterial strains transformed with the empty vector were used as a control (lanes 1 and 4).
Figure V Supplemental
pBI-EGFP empty
pBI-EGFP vgrG2
Figure V: Morphological changes of HeLa Tet-off cells induced by the expression of the vgrG2 gene of A.
hydrophila SSU. Cells were stained for actin-cytoskeleton by using Alexa fluor 568-phalloidin (red), and
expression of the gene encoding EGFP was detected in HeLa cells successfully transfected with the pBIEGFP vector alone (Left) or containing the vgrG2 gene (Right). Magnification 40X.