TABLE 1.
Bacterial strains and primers used in this study
| Strain, plasmid, or primer | Characteristic(s)a | Source and/or reference |
|---|---|---|
| Strains | ||
| E. coli DH5α F′ | F′ endA1hsdR17(rκ− mκ−) supE44thi-1recA1gyrA(Nalr) relA1 Δ(lacZYA-argF)U169deoR [φ80dlacΔ(lacZ)M15] | 44 |
| E. coli β2155 | thrB1004 pro thi strA hsdS lacZΔM15 (F′ lacZΔM15 laqIqtraD36proA+proB+)Δdap::erm(Ermr) recA::RPA-2-tet(Tcr)::Mu-Km (Kmr) λ pir | 11 |
| E. coli TOP10 | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1deoRaraD139 Δ(araleu)7697 galU galK rpsL(Strr) endA1 nupG | TOPO TA cloning |
| A. pleuropneumoniae wt | A. pleuropneumoniae serotype 7 isolate 76 | 1 |
| A. pleuropneumoniae ΔarcA | Unmarked arcA deletion mutant of A. pleuropneumoniae wt | 8 |
| A. pleuropneumoniae Δfrd | Unmarked frd deletion mutant of A. pleuropneumoniae wt | This work |
| Plasmids | ||
| pCR2.1-TOPO | E. coli cloning vector for fast and efficient cloning of Taq polymerase-amplified PCR products | TOPO TA cloning; Invitrogen, Groningen, The Netherlands (52) |
| pFRD811 | pCR2.1-TOPO-based plasmid containing a 1,300-bp PCR product obtained with primers ofrd_3 and ofrd_4, starting at position 1534 downstream of the frdA start codon and ending 251 bp downstream of the frdC start codon | This work |
| pFRD820 | PCR product obtained with primers ofrd_1 and ofrd_2, digested with BsmBI and PspOMI and ligation of the 1,231-bp fragment into pFRD811 digested with PspOMI and BsmBI, resulting in pFRD820 | This work |
| pEMOC2 | Transconjugation vector based on pBluescript SK with mobRP4, a polycloning site, Cmr, and transcriptional fusion of the omlA promoter with the sacB gene | Accession no. AJ868288 (3) |
| pFRD710 | pEMOC2-based plasmid carrying the truncated frdABC fragment excised from pFRD820 using NotI and PspOMI and ligated into pEMOC2 restricted with NotI and PspOMI | This work |
| pLS88 | Broad host range shuttle vector from Haemophilus ducreyi; Strr Smr Kmr | 58 |
| pFRD1300 | pLS88-based complementation plasmid carrying the PCR product of primers ofrd_5 and ofrd_6 encoding the entire frdABCD operon and 477-bp upstream sequence cloned into the EcoRI restriction site | This work |
| Primers | ||
| ofrd_1 | 5′-ATGCGGGCCCCGGGGTTTCCACTTCTAATA-3′; forward primer containing an internal PspOMI site (underlined) comprising positions 477-458 upstream of the frdA gene start codon | This work |
| ofrd_2 | 5′-ATGCCGTCTCATTCACCGCGACAACCTTCAG-3′; reverse primer containing an internal BsmBI site (underlined) comprising positions 734-753 of the frdA gene | This work |
| ofrd_3 | 5′-ATGCCGTCTCATGAATTCATTTTAGATGTGGCGCA-3′; forward primer containing an internal BsmBI site (underlined) comprising positions 1534-1553 of the frdA gene | This work |
| ofrd_4 | 5′-ATGCGCGGCCGCGCATGGTAAAGTAATGCGGC-3′; reverse primer containing an internal NotI site (underlined) comprising positions 232-251 of the frdC gene | This work |
| ofrd_5 | 5′-ACGTACCAATTGGATTACAGTGCGATTACTGC-3′; forward primer containing an internal MfeI site (underlined) comprising positions 477-458 upstream of the frdA gene start codon | This work |
| ofrd_6 | 5′-ACGTACCAATTGCGGGGTTTCCACTTCTAATA-3′; reverse primer containing an internal MfeI site (underlined) comprising position 18 upstream to position 2 downstream of the frdD stop codon | This work |
| ofrd_7 | 5′-CAATGCGAATGCGGTGGTTA-3′; forward primer comprising positions 522-541 of the frdA gene | This work |
| ofrd_8 | 5′-CGTTTCGCCGGTTGTGATTT-3′; reverse primer comprising positions 1714-1733 of the frdA gene | This work |
a
Nalr, nalidixic acid resistance; Ermr, erythromycin resistance; Tcr, tetracycline resistance; Kmr, kanamycin resistance; Strr, streptomycin resistance.