TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Relevant characteristic(s) and/or construction | Source or reference |
|---|---|---|
| Strains | ||
| A. hydrophila SSU | CDC, Atlanta, GA | |
| SSU-R | Rifr strain of A. hydrophila SSU | Laboratory stock |
| WT/pBReno | WT A. hydrophila SSU-R transformed with pBReno plasmid; Rifr Tcr | This study |
| Enolase mutant | The chromosomal enolase gene was deleted from WT A. hydrophila SSU-R that was transformed with the plasmid pBReno; Sm/Spr Rifr Tcr | This study |
| E. coli | ||
| SM10 | Kmr λpir | 24 |
| JM109 | endA1 recA1 gyrA96 thi-1 hsdR17(rK− mK+) relA1 supE44 Δ(lac-proAB) [F′ traD36 proAB lacIqZΔM15] | Promega |
| ES1301 mutS | lacZ53 mutS201::Tn5 thyA36 rha-5 metB1 deoC | Promega |
| HMS174 (DE3) | RecA-mutated K-12 background E. coli strain that carries a chromosomal copy of the T7 RNA polymerase gene under the control of the lacUV5 promoter; used as the host strain for the pET expression system | Novagen |
| Plasmids | ||
| pHPΩ45 | Contains a 2.0-kb Sm/Spr gene cassette | 33 |
| pET30a | pBR322-derived expression vector with T7 lac promoter and up- and down-stream His tags; Kmr | Novagen |
| pDMS197 | A suicide vector; R6K ori sacB Tcr | 10 |
| pALTER-1 | Vector for the site mutagenesis; Tcr Aps | Promega |
| pBReno | The coding region of A. hydrophila enolase gene was cloned in pBR322 at the ScaI/PstI sites and under the control of the Ap promoter of the vector; Tcr | This study |
| pETUD | pET30a vector containing up- and downstream DNA flanking sequences to the enolase gene; Kmr | This study |
| pETUDSm/Sp | The Sm/Sp cassette was ligated with the up- and downstream DNA flanking sequences to the enolase gene in pET30a vector; Sm/Spr Kmr | This study |
| pBUDSm/Sp | The Sm/Sp cassette was ligated with the up- and downstream DNA flanking sequences to the enolase gene in pBluescript vector; Sm/Spr Apr | This study |
| pDMSUDSm/Sp | Suicide vector pDMS197 containing the Sm/Sp cassette ligated with up- and downstream DNA flanking sequences to the enolase gene; used to generate the enolase gene deletion mutant of A. hydrophila; Kmr Tcr | This study |
| pALTER-1/eno | The native enolase gene of A. hydrophila was cloned into pALTER-1 vector at the BamHI/HindIII sites for the site mutagenesis; Tcr Aps | This study |
| pALTER/eno343K/Q | pALTER-1 vector containing the mutated A. hydrophila enolase gene eno(K343Q); Tcs Apr | This study |
| pALTER/eno394K/M | pALTER-1 vector containing the mutated A. hydrophila enolase gene eno(K394M); Tcs Apr | This study |
| pALTER/eno420K/L | pALTER-1 vector containing the mutated A. hydrophila enolase gene eno(K420L); Tcs Apr | This study |
| pALTER/eno427K/N | pALTER-1 vector containing the mutated A. hydrophila enolase gene eno(K427N); Tcs Apr | This study |
| pALTER/eno430K/R | pALTER-1 vector containing the mutated A. hydrophila enolase gene eno(K430R); Tcs Apr | This study |
| pET30aeno | The native enolase gene of A. hydrophila was cloned in pET30a and fused with the downstream His tags of the vector for hyperexpression; Kmr | This study |
| pET30aeno343K/Q | The mutated enolase gene of A. hydrophila eno(K343Q) was cloned in pET30a and fused with the downstream His tags of the vector for hyperexpression; Kmr | This study |
| pET30aeno394K/M | The mutated enolase gene of A. hydrophila eno(K394M) was cloned in pET30a and fused with the downstream His tags of the vector for hyperexpression; Kmr | This study |
| pET30aeno420K/L | The mutated enolase gene of A. hydrophila eno(K420L) was cloned in pET30a and fused with the downstream His tags of the vector for hyperexpression; Kmr | This study |
| pET30aeno427K/N | The mutated enolase gene of A. hydrophila eno(K427N) was cloned in pET30a and fused with the downstream His tags of the vector for hyperexpression; Kmr | This study |
| pET30aeno430K/R | The mutated enolase gene of A. hydrophila eno(K430R) was cloned in pET30a and fused with the downstream His tags of the vector for hyperexpression; Kmr | This study |