FIG. 7.

Construction of transcriptional fusions linking the epoA promoter region to the bar reporter gene. Plasmid pFOM5 was prepared from pFOM4 (Fig. 4) by ligating a 2.3-kb HindIII-XbaI fragment including epoA into HindIII-XbaI sites of pUC118. Plasmid pFOM6 was obtained from pFOM5 by introducing a ClaI site upstream of the ATG start codon of epoA by site-specific mutation (12) with a primer (5′-AAATCCCCCATCGATGAAGCCTC-3′). The Streptomyces hygroscopicus bar gene was PCR amplified from pARK22 (19) with two primers, barU (5′-AAGGATCGATATGAGCCCAGAACGACGCCC-3′) and barR (5′-GCTTGGATCCTCAGATCTCGGTGACGGGC-3′), which included ClaI and BamHI sites. The termini were cut with ClaI and BamHI and replaced with hph from pDH25 (3) (pDHBAR). Next, a ClaI-XbaI fragment including bar and the trpC terminator (TtrpC) was removed from pDHBAR and inserted into pFOM6 cleaved with ClaI/XbaI so as to replace the epoA structural gene and put the bar reporter gene under the control of the epoA promoter (pFOM7). pFOM8 was prepared by inserting a cassette containing the hph gene into the XbaI site of pFOM7. pFOM15 was prepared by first deleting the putative epoA promoter region from pFOM7 by digestion with HindIII and ClaI and then by using the same procedure to insert the hph cassette. Abbreviations: PepoA, the P. decumbens epoA promoter region; bar, the S. hygroscopicus bialaphos resistance gene.