TABLE 1.
Bacterial strains and plasmids
| Strain/plasmid | Relevant genotype | Source or reference |
|---|---|---|
| Strains | ||
| DH5α | supE44 ΔlacU169 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 φ80dlacZΔM15 | BRL |
| Rosetta(DE3) | F−ompT hsdSB (rB− mB−) gal dcm lacY1 (DE3) | Novagen |
| BW25113 | lacIqrrnBT14 ΔlacZWJ16hsdR514 ΔaraBADAH33 ΔrhaBADLD78 | 16 |
| JW1836 | BW25113 ΔyebF::Kanr | 5 |
| JW3832 | BW25113 ΔdsbA::Kanr | 5 |
| JW2861 | BW25113 ΔdsbC::Kanr | 5 |
| Plasmids | ||
| pLysSRARE | Camr | Novagen |
| pTrc99A | Ampr | 3 |
| pTrcHis60 | pTrc99A derivative, Ampr | 36 |
| pET2160 | pET21d derivative, Ampr | 10 |
| pMLD232 | pTrc99A::cmi | 8 |
| pMLD234 | pTrcHis60::cmi | This study |
| pFFG11 | pET2160::cmi | This study |
| pFFG12 | pET2160::cmiΔ1-23 | This study |
| pMLD307 | pTrc99A::cmiΔ117 | This study |
| pMLD308 | pTrc99A::cmiΔ116-117 | This study |
| pMLD309 | pTrc99A::cmiΔ115-117 | This study |
| pMLD310 | pTrc99A::cmiΔ114-117 | This study |
| pMLD311 | pTrc99A::cmiΔ113-117 | This study |
| pMLD312 | pTrc99A::cmiΔ112-117 | This study |
| pMLD314 | pTrcHis60::yebF | This study |
| pMLD316 | pTrcHis60::yebF1-32-cmi29-117 | This study |
| pFFG11-Cys31Alaa | pET2160::cmiCys31Ala | This study |
| pMLD232-Cys31Alaa | pTrc99A::cmiCys31Ala | This study |
a
For clarity, only plasmids expressing the Cys31A mutated form of Cmi are indicated here, but all the other point mutations mentioned in the text, from Lys27Ala to Tyr114Ala, were similarly introduced in both the pFFG11 and pMLD232 plasmids. Genes encoding C-terminally truncated forms of Cmi (from Δ117 to Δ112-117) were expressed only in the pTrc99A vector. The oligonucleotides used for construction of these different expression vectors and site-directed mutagenesis experiments are shown in Table S1 in the supplemental material.