Article Figures & Data
Figures
- FIG 1
CT production profiles of single-SNP strains and double-, triple-, and quadruple-SNP combination strains. SNPs and SNP combinations were introduced into the WT strain N16961, and average fold changes in CT production levels relative to the CT production in WT N16961 were calculated. Other controls included MQ1795 (hypervirulent clinical isolate) and O395ΔtoxT (negative control). Strains containing the different SNPs and SNP combinations introduced into WT N16961 are indicated by the numbers beginning with “MS” found in Table 2. The presence of SNPs identified in genes in the indicated clinical isolates is denoted by a “+,” whereas the WT N16961 version of a SNP is denoted by a “−.” A two-tailed Student t test yielded P values of ≤0.05 (*), <0.005 (**), and <0.0005 (***).
- FIG 2
TcpA production profiles of single-SNP strains and double-, triple-, and quadruple-SNP combination strains. WCE were prepared, 3 µg of total protein was loaded onto a 16% Tris-glycine polyacrylamide gel, and proteins in immunoblots were quantified using densitometry for V. cholerae virulence factor TcpA. Immunoblots (inset) are from different blots (indicated by borders), all with WT N16961 and other controls run simultaneously. Nonspecific bands serve as a loading control (L.C.) and are labeled accordingly. Average fold changes were calculated relative to the TcpA level in N16961. Other controls included MQ1795 (hypervirulent clinical isolate), classical O395, El Tor C6706, O395ΔtoxT, and O395ΔtcpA. Strains containing the different SNPs and SNP combinations introduced into WT N16961 are indicated by the numbers beginning with “MS” found in Table 2. The presence of SNPs identified in genes in the indicated clinical isolates is denoted by a “+,” whereas the WT N16961 version of a SNP is denoted by a “−.” A two-tailed Student t test yielded P values of ≤0.05 (*), <0.005 (**), and <0.0005 (***).
- FIG 3
ToxT production profiles of single-SNP strains and double-, triple-, and quadruple-SNP combination strains. WCE were prepared, 12 µg of total protein was loaded onto a 14% Tris-glycine polyacrylamide gel, and proteins in immunoblots were quantified using densitometry for V. cholerae virulence factor ToxT. Immunoblots (inset) are from different blots (indicated by borders), all with WT N16961 and other controls run simultaneously. Nonspecific bands serve as a loading control (L.C.) and are labeled accordingly. Average fold changes were calculated relative to the ToxT level in N16961. Other controls included MQ1795 (hypervirulent clinical isolate), classical O395, El Tor C6706, O395ΔtoxT, and O395ΔtcpA. Strains containing the different SNPs and SNP combinations introduced into WT N16961 are indicated by the numbers beginning with “MS” found in Table 2. The presence of SNPs identified in genes in the indicated clinical isolates is denoted by a “+,” whereas the WT N16961 version of a SNP is denoted by a “−.” A two-tailed Student t test yielded P values of ≤0.05 (*), <0.005 (**), and <0.0005 (***).
- FIG 4
Schematic representation of the involvement of each of the four genes on virulence factor expression. HapR binds to the aphA promoter, resulting in a decrease in virulence factor production, and H-NS decreases virulence factor production through a mechanism of transcriptional silencing. LuxO works in a manner contrary to that of HapR, and VieA degrades c-di-GMP, both of which result in indirect upregulation of virulence factor production. qrr, quorum regulatory RNA; sRNA, small RNA.
Tables
- TABLE 1
SNPs previously identified in MQ1795 by next-generation deep sequencinga
Gene Gene identifier Base no. N16961 MQ1795 Base Residue Base Residue hapR VC0583 219 None (T deletion) Nonfunctioning T (insertion) Functioning hns VC1130 319 G G A S luxO VC1021 656 C A T V vieA VC1652 235 C L T F ↵a Single nucleotide polymorphisms (SNPs) of interest found in the hypervirulent MQ1795 strain compared to the El Tor reference strain N16961. MQ1795 has a gain of function in the hapR gene with a mutation that results in the insertion of a thymine. The remaining SNPs (hns, luxO, and vieA) result in neither a gain of function nor a loss of function but rather result in residue changes.
- TABLE 2
Escherichia coli and Vibrio cholerae strains used in this studya
Strain Description Reference or source Escherichia coli S17-λpir recA thi pro hsdR− M+ [RP4-2-Tc::Mu::Kmr Tn7] (λpir) Tmpr Strr de Lorenzo and Timmis (49) Vibrio cholerae O395 Classical wild type, Strr Son et al. (9) C6706 El Tor wild type, Strr Son et al. (9) N16961 El Tor reference strain, Strr Son et al. (9) MQ1795 El Tor variant, clinical isolate Nair et al. (27) MS0005 Classical O395ΔtoxT, Str Son et al. (9) MS0014 Classical O395ΔtcpA, Str Kirn et al. (50) MS0235 El Tor N16961 hapR* Str This study MS0245 El Tor N16961 hns* Str This study MS0230 El Tor N16961 luxO* Str This study MS0240 El Tor N16961 vieA* Str This study MS0256 El Tor N16961 hapR* hns* Str This study MS0254 El Tor N16961 hapR* luxO* Str This study MS0251 El Tor N16961 hapR* vieA* Str This study MS0285 El Tor N16961 hns* luxO* Str This study MS0281 El Tor N16961 hns* vieA* Str This study MS0287 El Tor N16961 luxO* vieA* Str This study MS0279 El Tor N16961 hapR* hns* luxO* Str This study MS0295 El Tor N16961 hapR* hns* vieA* Str This study MS0297 El Tor N16961 hapR* luxO* vieA* Str This study MS0299 El Tor N16961 hns* luxO* vieA* Str This study MS0282 El Tor N16961 hapR* hns* luxO* vieA* Str This study ↵a S17-λpir was used to introduce all possible SNPs and SNP combinations into the El Tor reference strain N16961, using a previously described allelic-exchange protocol (46). SNPs introduced are indicated by asterisks. Tmpr represents trimethoprim resistance, and Strr represents streptomycin resistance.
- TABLE 3
Primers used in this study to introduce SNPs and verify sequencing
Oligonucleotide no. Oligonucleotide name Sequence (5′–3′)a Purpose 0159 ETV-hapR-Forward GATCGGAATTCCCATTTCCTACTTGAAGCTGTAGCGGTGTTGGCAG Allelic exchange 0160 ETV-hapR-SapI-Reverse GATCGGCTCTTCACGTCAGTACTCCAACTTCTTGACCGATCACATCG Allelic exchange 0161 ETV-hapR-SapI-Forward GATCGGCTCTTCAACGAACCACAAAATTCAGCACATCGTCAACCAAGTCTTC Allelic exchange 0162 ETV-hapR-Reverse GATCGAGATCTCAGTATCGCTGACTTTGGTGGCGCGTATAGTACC Allelic exchange 0163 ETV-luxO-Forward GATCGGAATTCGCTGGATATTGATATCAATATCGTGGGTACCGG Allelic exchange 0164 ETV-luxO-SapI-Reverse GATCGGCTCTTCACGCCTTGACGCTCAGTCGCCGCCCCAGTAAAAGC Allelic exchange 0165 ETV-luxO-SapI-Forward GATCGGCTCTTCAGCGTGGCAGAAGCGGCTGATGGGGGAACCCTCTTTTTGG Allelic exchange 0166 ETV-luxO-Reverse GATCGAGATCTGCTTGTTCAATGGCTTGTTTTTCGGTCATCCACAGC Allelic exchange 0167 ETV-hns-Forward GATCGGAATTCGGTTTATTTATGGCGGGTTACACCGAAGATTCCG Allelic exchange 0168 ETV-hns-SapI-Reverse GATCGGCTCTTCAGTGAAGAAAAAACTTGGACAGGCCAAGGCCGTACTCC Allelic exchange 0169 ETV-hns-SapI-Forward GATCGGCTCTTCACACTGTTGGTGTCGATATACTTGTATTTTGCAGGGCGAGG Allelic exchange 0170 ETV-hns-Reverse GATCGAGATCTCAATCATTTCATTGATACTTATTTCATAAAAACACC Allelic exchange 0171 ETV-vieA-Forward GATCGGAATTCGGTCGGGGTTCCGTTGTACACCTCATGCTCCGTC Allelic exchange 0172 ETV-vieA-SapI-Reverse GATCGGCTCTTCATCAGCGCTGTGGAAGATACGATTCTTGAGTTAAC Allelic exchange 0173 ETV-vieA-SapI-Forward GATCGGCTCTTCATGAATATCACCACACCTAGCTTAGGTGCCTGTAAACTCAG Allelic exchange 0174 ETV-vieA-Reverse GATCGAGATCTCAGTCGTTATCTCACAACACAATGGGTCGCACTGCC Allelic exchange 0212 hapR-Upstream GCATTGTATAAATGGGGCTTGGAGAATTTAGGCG Sequencing 0213 hapR-Downstream GCTCAGTGATCTGTTGACCTAATTCGCGAATGCG Sequencing 0214 hns-Upstream GCATTAGCTTTAACAGGAGAAAGCGATCCGCTCGC Sequencing 0215 hns-Downstream GCAACTAGGTTCCAGTGAGAAAAACAAGTGCCACAGC Sequencing 0216 luxO-Upstream GGCTAGGCTATGCAACATAATCAATCTTTGCAG Sequencing 0217 luxO-Downstream CCAAGTTTGCAGCTTGCGATAGATGGTTGACGG Sequencing 0218 vieA-Upstream GCAGTTGAGCCAGTTGACCTAATGACGCGTAACC Sequencing 0219 vieA-Downstream GCGGAACAAGCTCTGCAAGCGGGTATGGATAAGG Sequencing ↵a Italics represent regions of enzyme restriction sites.
