01% (wt/vol) β-nicotinamide adenine dinucleotide

(NAD) as required. Transconjugation medium consisted of MH broth with 20% (wt/vol) sucrose, 10% equine serum (wt/vol), and 0.01% NAD (wt/vol). E. coli strains were routinely cultured in Luria-Bertani (LB) medium, but in the case of E. coli β2155, the medium was always supplemented with 1 mM diaminopimelic acid (DAP; buy Bioactive Compound Library Sigma-Aldrich, St. Louis, MO, USA). As required, chloramphenicol was also added at the rate of either 5.0 or 2.5 μg/ml. Table 6 Bacterial strains, plasmids and primers used in the construction of the malT mutant Bacterial strains, plasmids or primers Characteristic or sequence Source or Remark E. coli DH5a F-φ80lacZΔM15Δ(lacZYA-rgF)U169 deoR recA1 endA1 hsdR17(rk -, mk +) supE44 thi-1 gyrA96 relA1 λ- Clonetech E. coli β2155 thrB1004pro thi hsdS lacZΔM15 selleck chemical (F’lacZΔM15lacI q traD36 proA + proB +) Δdap::erm(Ermr)recA::RP4-2-tet(Tcr)Mu- Lazertinib molecular weight km(Kmr)λpir Reference no. 28 E. coli DH5α-pTOPOPCR-malT DH5α harboring pCR4-TOPO containing malT of A. pleuropneumiae CM5 This work E. coli DH5α- pTopoMC DH5a harboring pCR4-TOPO containing ΔmalT::cat This work E. coli DH5α-pEMOC2M DH5a harboring pEMOC2 containing ΔmalT::cat This work A. pleuropneumoniae MalT negative mutant of A. This work CM5 3ΔmalT pleuropneumonaie

CM5   pCR4-TOPO A linearized plasmid for cloning PCR product Invitrogen pEMOC2 A conjugation vector based on pBluesript SK with mobRP4 and Cmr Reference no. 31 pTOPOPCR-malT pCR4-TOPO

containing malT of A. pleuropneumiae CM5 This work pTopoMC pCR4-TOPO containing ΔmalT::cat This work pEMOC2M harboring pEMOC2 containing ΔmalT::cat This work malT-L malT-R ATGCAAGCAACATTTTCAAGA TTAGCTATACCCCATCATTCTCAA Primers for amplification of the malT gene of A pleuropneumoniae CM5 stopupmalT-L TTAGTTAGTTACGAGCTTTTTCACACCGTTT Primers for generation of a linearized plasmid containing a deletion of 900 bp in its malT gene cloned in pTOPOPCR-malT. stopupmalT-R TAACTAACTAATGGGAATGGCATCATTTAGA   pnmalT-L TCATCTGCAGATGCAAGCAACATTTTCAAGA Primers for amplication Amine dehydrogenase of the ΔmalT::cat and the insertion of the PstI and NotI sites into the PCR product. pnmalT-R ACAATACAGCGGCCGCTTAGCTATACCCCATCATTCTCAA   cat-L CGGTGCCCTGAATGAACT Primers for the PCR cat-R AAGCTTCGACGAATTTCTGC amplification of omlA-P driven cat gene of pEMOC2 Table 7 Bacterial strains, plasmids and primers used in the construction of the lamB mutant Bacterial strains, plasmids or primers* Characteristic or sequence Source or Remark E. coli DH5α-pTOPOFL DH5α harboring pCR4-TOPO containing lamB of A. pleuropneumia e CM5 This work E. coli DH5α-TOPOΔFLcat DH5a harboring pCR4-TOPO containing ΔlamB::cat This work E. coli DH5Δ-pEMOC2-ΔlamB DH5Δharboring pEMOC2 containing ΔlamB::cat This work A. pleuropneumoniae CM5 ΔlamB LamB negative mutant of A. pleuropneumoniae CM5 This work pTOPOFL pCR4-TOPO containing lamB of A.