TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Genotype or relevant characteristics | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| JM109 | e14− (McrA−) recA1 endA1 gyrA96 thi-1 hsdR17 (rK−mK+) supE44 relA1 Δ(lac-proAB) [F′ traD36+proAB+lacIqZΔM15] | Stratagene |
| DH5α | F− φ80dlacΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK− mK+) supE44 λ−thi-1 gyrA96 relA | Invitrogen |
| SM10 | thi-1 thr leu tonA lacY supE recA RP4-2-Tc::Mu Kmr | 54 |
| BL21(DE3) | F−ompT gal dcm lon hsdSB(rB− mB−) λDE3(lacI lacUV5-T7 gene 1 ind-1 sam-7 nin-5) | Novagen |
| N 00 6496 | Wild type; O145:NM | Mohamed Karmali |
| N 01 2051 | Wild type; O145:NM | Mohamed Karmali |
| N 02 5149 | Wild type; O145:NM | Mohamed Karmali |
| P. aeruginosa | ||
| PAO1 | Wild-type strain, IATSa serotype O5 (ATCC 33351) | 31 |
| O4 | Wild-type strain, IATS serotype O4 (ATCC 33351) | ATCC |
| O11 | Wild-type strain, IATS serotype O11 (ATCC 33358) | ATCC |
| O12 | Wild-type strain, IATS serotype O12 (ATCC 33359) | ATCC |
| O12 lfnA | O12 lfnA::aacC1; Gmr | This work |
| Plasmids | ||
| pEX100T | Counterselectable, mobilizable suicide vector backbone; contains oriT and sacB; Apr | 53 |
| pUCGm | Source of small, broad-host-range gentamicin cassette aacC1; Apr Gmr | 52 |
| pET28a | Protein expression vector for creating thrombin-cleavable N-terminal His6 tag gene fusions; Kmr | Novagen |
| pUCP20 | Escherichia-Pseudomonas shuttle vector derived from pUC18; Apr Cbr | 63 |
| pVLT31 | Broad-host-range vector containing the Ptac promoter for induction of downstream gene expression; Tcr | 17 |
| pKOLD2 | Suicide vector pEX100T containing the mutant lfnA allele lfnA::aacC1; Apr Gmr | This work |
| pFuc41 | pET28a-derived expression vector encoding expression of His6-Lfn; Kmr | This work |
| pUCP20-rbs-his6-lfnA | XbaI-HindIII fragment from pFuc41, containing the ribosome binding site, N-terminal His6 tag, and lfnA coding sequence cloned into pUCP20; Apr Cbr | This work |
| pVLT31-rbs-his6-lfnA | XbaI-HindIII fragment from pFuc41, containing the ribosome binding site, N-terminal His6 tag, and lfnA coding sequence cloned into pVLT31; Tcr | This work |
| pBa1ET28a-wbuX | Untagged wbuX cloned into the NcoI and BamHI sites of pET28a; Kmr | |
| pBf3ET28a-his6-wbuX | wbuX cloned into the NheI and BamHI sites of pET28a, generating an N-terminal His6 tag fusion; Kmr | This work |
| pCr1UCP20-rbs-wbuX | XbaI-HindIII fragment from pBa1ET28a-wbuX, which contains the ribosome binding site and wild-type wbuX cloned into pUCP20; Apr Cbr | This work |
| pCs2UCP20-rbs-his6-wbuX | XbaI-HindIII fragment from pBf3ET28a-his6-wbuX, which contains the ribosome binding site and His6-wbuX fusion cloned into pUCP20; Apr Cbr | This work |
a
IATS, International Antigenic Typing Scheme.