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Bacterial strains and plasmids. Pactamycin producing S. pactum ATCC 27456 was purchased from American Type Culture Collection (ATCC). Escherichia coli DH10B was used as a host strain for the construction of recombinant plasmids. E. coli ET12567 was used as a donor strain in conjugation experiments. pJET1.2 (Fermentas) and pGEM-T (Promega) were used as cloning vectors. pTMN002 is a pJTU1278+ derivative containing an OriT transfer element required for conjugation. 1,2 All bacterial strains and plasmids used in this study are listed in Table 1.General DNA manipulations. Genomic DNA of S. pactum ATCC 27456 was prepared by standard protocol 3 or using the DNeasy Tissue Kit (QIAGEN). DNA fragments were recovered from an agarose gel by using the QIAquick Gel Extraction Kit (QIAGEN). Restriction endonucleases were purchased from Invitrogen or Promega. Preparation of plasmid DNA was done by using a QIAprep Spin Miniprep Kit (QIAGEN). All other DNA manipulations were performed according to standard protocols. 3,4 PCR was performed in 30 cycles using a Mastercycler gradient thermocycler (Eppendorf) and Platinum Taq DNA polymerase (Invitrogen) or Platinum Pfx DNA polymerase (Invitrogen). Oligodeoxyribonucleotides for PCR primers were synthesized by Sigma-Genosys, and are shown in Table 2. The nucleotide sequences of the gene fragments were determined at the Center for Genome Research and Biocomputing (CGRB) Core Laboratories, Oregon State University.Construction of ptmA and ptmT knock-out plasmids. For the construction of ptmA knock-out plasmid, two 1 kb PCR fragments upstream and downstream of the ptmA gene were generated with primers ptmA-F1/ptmA-R1 and ptmA-F2/ptmA-R2 (see Table 2), respectively, and separately cloned into pJET1.2 cloning vector for DNA sequencing. HindIII and EcoRI digested upstream DNA fragment, EcoRI and XbaI digested downstream DNA fragment, and HindIII and XbaI digested pTMN002 were ligated to create pTMW034, which was introduced into S. pactum by conjugation with the E. coli donor strain ET12567 (pUZ8002). Apramycin resistant strains representing single crossover mutants were obtained and subsequently grown on nonselective mannitolsoy flour agar containing MgCl 2 (10 mM, MS-Mg) to allow the formation of double crossover recombinants. Apramycin sensitive colonies were counter-selected by replica plating onto MS-Mg agar with and without apramycin (50 µg mL −1 ). The resulting double-crossover candidate strains were confirmed by PCR amplification with external primers (ptmA-P1 and ptmA-P2, Table 2) flanking the targeted ptmA gene. The resulting PCR fragment from putative double crossover mutants was subcloned into pGEM-T Easy vector and sequenced to confirm that part of ptmA has been removed from the genomic DNA. A similar approach was used for the construction of ptmT knock-out plasmid pTMW037, except that the PCR fragments upstream and downstream of the ptmT gene were generated with primers ptmT-F1/ptmT-R1 and ptmT-F2/ptmT-R2, respectively. pTMW037 was introduced into S. pactum wild type strain or Δp...