TABLE 1.
Characteristics of bacterial strains and primers used in this study
| Strain, plasmid, or primers | Characteristicsa | Source and/or references |
|---|---|---|
| Strains | ||
| E. coli β2155 | thrB1004 pro thi strA hsdS lacZΔM15 (F′ lacZΔM15 laqIqtraD36 proA+proB+) ΔdapA::erm (Ermr)recA::RPA-2-tet (Tcr)::Mu-km (Kmr) λpir | 9 |
| E. coli DH5αF′ | F′ endA1 hsdR17 (rK− mK−) supE44 thi-1 recA1 gyrA (Nalr) relA1 Δ(lacZYA-argF)U169 deoR [φ80dlacΔ(lacZ)M15] | 21 |
| A. pleuropneumoniae AP76 | A. pleuropneumoniae serotype 7, field strain | 19 |
| A. pleuropneumoniae ΔhlyX | Unmarked hlyX-negative knockout mutant of A. pleuropneumoniae AP76 | This study |
| A. pleuropneumoniae ΔaspA::luxAB | Unmarked A. pleuropneumoniae mutant carrying luxAB as a transcriptional fusion in the aspA gene | 15 |
| Plasmids | ||
| pCR2.1-TOPO | Topoisomerase I enhanced E. coli cloning vector | TOPO TA Cloning, Invitrogen, Groningen, The Netherlands (25) |
| pBluescript SK(+) | 3.0-kb cloning vector; Apr | Stratagene, La Jolla, CA |
| pEMOC2 | Transconjugation vector based on pBluescript SK with mobRP4, polycloning site, Cmr, and transcriptional fusion of the omlA promoter with the sacB gene | Accession no. AJ868288 (5) |
| pHLYX810 | 2,338-bp PCR product of primers oHLYX5 and oHLYX6, containing the hlyX ORF, cloned into pCR 2.1-TOPO | This study |
| pHLYX110 | ApaI/NotI fragment carrying the hlyX ORF from pHLYX810, ligated into pBluescript SK(+) | This study |
| pHLYX111 | Deletion of an 883-bp fragment between the BgIII and XcmI restriction sites of pHLYX110 | This study |
| pHLYX701 | ApaI/NotI fragment from pHLYX111, ligated into pEMOC2 | This study |
| pLS88 | Broad-host-range shuttle vector from Haemophilus ducreyi; Strr Smr Kmr | 33 |
| pHLYX1300 | PCR product generated from primers oHLYX9 and oHLYX10 containing hlyX ORF, ligated into plasmid pLS88 after MfeI restriction in a 5′-3′ orientation with respect to the vector-derived sulII promoter | This study |
| pHLYX1301 | PCR product generated from primers oHLYX9 and oHLYX10 containing hlyX ORF ligated into plasmid pLS88 after MfeI restriction in a 3′-5′ orientation with respect to the vector-derived sulII promoter | This study |
| Primers | ||
| oHLYX5 | 5′-TGG GGC CCT CGG TAC AAC GGT ATG TCC TT-3′; forward primer containing an ApaI restriction site, situated 986 bp upstream of the hlyX start codon | This study |
| oHLYX6 | 5′-TCG CGG CCG CCA ACG TGA GAG CTT CGT TCA-3′; reverse primer containing a NotI restriction site, situated 626 bp downstream of the hlyX ORF | This study |
| oHLYX7 | 5′-TCC GAA ACC GGA TAA TTC AC-3′; forward primer situated 507 bp upstream of the hlyX start codon | This study |
| oHLYX8 | 5′-AGC GAA AGG GTT AAT CAG CA-3′; reverse primer situated 228 bp downstream of the hlyX ORF | This study |
| oHLYX9 | 5′-ATG ACA ATT GTT TTA AAA GAC GGT AGC CCT TAT G-3′; forward primer containing an MfeI restriction site, situated 20 bp upstream of the hlyX start codon | This study |
| oHLYX10 | 5′-CTT ACA ATT GCG CCT ATA CGG TCA GTT CGT-3′; | This study |
| reverse primer containing an MfeI restriction site situated 4 bp downstream of the hlyX ORF |
a
Ermr, erythromycin resistance; Tcr, tetracycline resistance; Kmr, kanamycin resistance; Strr, streptomycin resistance; Smr, sulfonamide resistance; Kmr, kanamycin resistance.