FIG. 4.

TM1040 tda genes reside on a plasmid that undergoes a low frequency spontaneous loss. (A) Pigment synthesis. TM1040 (wt) produces a yellow-brown extracellular pigment that is correlated with TDA synthesis. In contrast, a tdaE:Tn mutant (strain HG1265) and a spontaneous mutant (sm; TM1040SM) are nonpigmented and have lost the ability to produce both TDA and pigment. (B) Spontaneous loss of pigment and antibiotic activity results from a loss of tda genes. PCR amplification of tdaE results in a band from wild-type (wt) and tdaE:Tn DNA, respectively, with the additional 2 kb in size of the tdaE:Tn product resulting from insertion of the transposon. No product was amplified from the spontaneous nonpigmented mutant (sm). (C) PFGE separation of total DNA obtained from TM1040 (wt), the spontaneous nonpigmented mutant (sm), and the tdaE:Tn mutant. (D) Southern blot hybridization of the PFGE gel to labeled tdaE DNA. (E) NcoI digestion of plasmid DNA isolated from TM1040 (wt), the spontaneous nonpigmented mutant (sm), and HG1265 (tdaE:Tn), respectively. The resulting patterns of DNA bands were compared to each other and to an in silico NcoI digestion of pSTM2 (see Fig. S1 in the supplemental material). (F) Southern blot hybridization of NcoI-digested plasmid DNA to tdaE.