Table S1

Bacterial strains and plasmids used in this study

Strain or plasmid / Relevant characteristics a / Source or reference
Bacterial strains
E. coli
DH5a / supE44 DlacU169 (f80 lacZ DM15) hsdR17 recA1 endA1 gyrA96 thi-1 relAI / Laboratory collection
S17-1lpir / λ-pir lysogen; thi pro hsdR hsdM+recA RP4 2-Tc::Mu-Km::Tn7(Tpr Smr) / (6)
BL21 StarTM (DE3) / F- ompT hsd SB(rB- mB-) gal dcm rne131 (DE3) / Novagen
DHP1 / F- glnV44(AS) recA1 endA1 gyrA96(Nalr) thi1 hsdR17 spoT1 rfbD1 cya- / (1)
V. vulnificus
CMCP6 / Clinical isolate / (2)
MQ4 / CMCP6 ∆lacZ ∆hlyU / (4)
Plasmid
pGEM-T Easy / A vector for the cloning of PCR products with blue/white screening, Apr / Promega
pGEM-hlyU / pGEM-T easy derivative containing the hlyU gene and its SB site, Apr / This study
pET47 / Protein expression vector with N-terminal His6 tag, Kmr / Novagen
pET-hlyU / pET47 derivative containing the hlyU gene, Kmr / This study
pET-hlyU(K18A) / pET with hlyU K18A, Kmr / This study
pET-hlyU(N22A) / pET with hlyU N22A, Kmr / This study
pET-hlyU(R25A) / pET with hlyU R25A, Kmr / This study
pET-hlyU(S54A) / pET with hlyU S54A, Kmr / This study
pET-hlyU(Q55A) / pET with hlyU Q55A, Kmr / This study
pET-hlyU(H56A) / pET with hlyU H56A, Kmr / This study
pET-hlyU(L57A) / pET with hlyU L57A, Kmr / This study
pET-hlyU(W59A) / pET with hlyU W59A, Kmr / This study
pET-hlyU(R61A) / pET with hlyU R61A, Kmr / This study
pET-hlyU(K70A) / pET with hlyU K70A, Kmr / This study
pET-hlyU(Y77A) / pET with hlyU Y77A, Kmr / This study
pET-hlyU(L17A / L91A) / pET with hlyU L17A & L91A, Kmr / This study
pMMB208 / A broad-host-range expression vector, Cmr IncQ lacIq Ptac, polylinker from M13mp19, Cmr / (5)
pMMB-hlyU / pMMB208 derivative containing the hlyU gene and its SB site, Cmr / (4)
pMMB-hlyU(K18A) / pMMB208 with hlyU , K18A, Cmr / This study
pMMB-hlyU(N22A) / pMMB208 with hlyU N22A, Cmr / This study
pMMB-hlyU(E23A) / pMMB208 with hlyU E23A, Cmr / This study
pMMB-hlyU(R24A) / pMMB208 with hlyU R24A, Cmr / This study
pMMB-hlyU(R25A) / pMMB208 with hlyU R25A, Cmr / This study
pMMB-hlyU(S38A) / pMMB208 with hlyU S38A, Cmr / This study
pMMB-hlyU(G40A) / pMMB208 with hlyU G40A, Cmr / This study
pMMB-hlyU(S49A) / pMMB208 with hlyU S49A, Cmr / This study
pMMB-hlyU(Q50A) / pMMB208 with hlyU Q50A, Cmr / This study
pMMB-hlyU(S51A) / pMMB208 with hlyU S51A, Cmr / This study
pMMB-hlyU(S54A) / pMMB208 with hlyU S54A, Cmr / This study
pMMB-hlyU(Q55A) / pMMB208 with hlyU Q55A, Cmr / This study
pMMB-hlyU(H56A) / pMMB208 with hlyU H56A, Cmr / This study
pMMB-hlyU(L57A) / pMMB208 with hlyU L57A, Cmr / This study
pMMB-hlyU(W59A) / pMMB208 with hlyU W59A, Cmr / This study
pMMB-hlyU(R61A) / pMMB208 with hlyU R61A, Cmr / This study
pMMB-hlyU(R62A) / pMMB208 with hlyU R62A, Cmr / This study
pMMB-hlyU(T68A) / pMMB208 with hlyU T68A, Cmr / This study
pMMB-hlyU(K70A) / pMMB208 with hlyU K70A, Cmr / This study
pMMB-hlyU(Q73A) / pMMB208 with hlyU Q73A, Cmr / This study
pMMB-hlyU(T74A) / pMMB208 with hlyU T74A, Cmr / This study
pMMB-hlyU(V75A) / pMMB208 with hlyU V75A, Cmr / This study
pMMB-hlyU(L17A) / pMMB208 with hlyU L17A, Cmr / This study
pMMB-hlyU(L91A) / pMMB208 with hlyU L91A, Cmr / This study
pMMB-hlyU(L17A / L91A) / pMMB208 with hlyU L17A & L91A, Cmr / This study
pMMB-hlyU(Y77A) / pMMB208 with hlyU K18A, Cmr / This study
pTL61T / A vector containing promoterless lacZ gene used for transcriptional gene fusion studies, Apr / (3)
pTL-PrtxA / pTL61T derivative containing a 803-bp of rtxA1 promoter region, Apr / (4)
pT18 / pBluescript II KS with the T18 fragment, multicloning site is inserted in 5’ end of T18, Apr / (1)
pT18-hlyU / pT18 derivative in which the hlyU gene was fused into T18 by KpnI and XhoI, Apr / This study
pT25 / pACYC184 with the T25 fragment, multicloning site is inserted in 3’ end of T25, Cmr / (1)
pT25-hlyU / pT25 derivative in which the hlyU gene was fused into T25 by KpnI and XhoI, Cmr / This study
pT18-zip / pT18 with leucine zipper of GCN4, Apr / (1)
pT25-zip / pT25 with leucine zipper of GCN4, Cmr / (1)
pT18-hlyU(M6A) / pT18 with hlyU M6A, Apr / This study
pT18-hlyU(E7A) / pT18 with hlyU E7A, Apr / This study
pT18-hlyU(S10A) / pT18 with hlyU S10A, Apr / This study
pT18-hlyU(V14A) / pT18 with hlyU V14A, Apr / This study
pT18-hlyU(L16A) / pT18 with hlyU L16A, Apr / This study
pT18-hlyU(L17A) / pT18 with hlyU L17A, Apr / This study
pT18-hlyU(K18A) / pT18 with hlyU K18A, Apr / This study
pT18-hlyU(E23A) / pT18 with hlyU E23A, Apr / This study
pT18-hlyU(R24A) / pT18 with hlyU R24A, Apr / This study
pT18-hlyU(Q27A) / pT18 with hlyU Q27A, Apr / This study
pT18-hlyU(Y77A) / pT18 with hlyU Y77A, Apr / This study
pT18-hlyU(E83A) / pT18 with hlyU E83A, Apr / This study
pT18-hlyU(M87A) / pT18 with hlyU M87A, Apr / This study
pT18-hlyU(L90A) / pT18 with hlyU L90A, Apr / This study
pT18-hlyU(L91A) / pT18 with hlyU L91A, Apr / This study
pT18-hlyU(L94A) / pT18 with hlyU L94A, Apr / This study
pT18-hlyU(Da1) / pT18 with hlyU with the entire helix a1 deleted, Apr / This study
pT18-hlyU(Da5) / pT18 with hlyU with the entire helix a5 deleted, Apr / This study
pT25-hlyU(M6A) / pT25 with hlyU M6A, Cmr / This study
pT25-hlyU(E7A) / pT25 with hlyU E7A, Cmr / This study
pT25-hlyU(S10A) / pT25 with hlyU S10A, Cmr / This study
pT25-hlyU(V14A) / pT25 with hlyU V14A, Cmr / This study
pT25-hlyU(L16A) / pT25 with hlyU L16A, Cmr / This study
pT25-hlyU(L17A) / pT25 with hlyU L17A, Cmr / This study
pT25-hlyU(K18A) / pT25 with hlyU K18A, Cmr / This study
pT25-hlyU(E23A) / pT25 with hlyU E23A, Cmr / This study
pT25-hlyU(R24A) / pT25 with hlyU R24A, Cmr / This study
pT25-hlyU(Q27A) / pT25 with hlyU Q27A, Cmr / This study
pT25-hlyU(Y77A) / pT25 with hlyU Y77A, Cmr / This study
pT25-hlyU(E83A) / pT25 with hlyU E83A, Cmr / This study
pT25-hlyU(M87A) / pT25 with hlyU M87A,Cmr / This study
pT25-hlyU(L90A) / pT25 with hlyU L90A, Cmr / This study
pT25-hlyU(L91A) / pT25 with hlyU L91A, Cmr / This study
pT25-hlyU(L94A) / pT25 with hlyU L94A, Cmr / This study
pT25-hlyU(Da1) / pT25 with hlyU with the entire helix a1 deleted, Cmr / This study
pT25-hlyU(Da5) / pT25 with hlyU with the entire helix a5 deleted, Cmr / This study

a Apr, ampicillin resistant; Kmr, kanamycin resistant; Cmr, chloramphenicol resistant.

References:

1. Karimova, G., J. Pidoux, A. Ullmann, and D. Ladant. 1998. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A 95:5752-5756.

2. Kim, Y. R., et al. 2003. Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients. Infect Immun 71:5461-5471.

3. Linn, T., and R. St Pierre. 1990. Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ. J Bacteriol 172:1077-1084.

4. Liu, M., A. F. Alice, H. Naka, and J. H. Crosa. 2007. The HlyU protein is a positive regulator of rtxA1, a gene responsible for cytotoxicity and virulence in the human pathogen Vibrio vulnificus. Infect Immun 75:3282-3289.

5. Morales, V. M., A. Backman, and M. Bagdasarian. 1991. A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97:39-47.

6. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering transposon mutagenesis in gram-negative bacteria. Bio/Technology 1:787-796.