The complete natamycin (NTM) biosynthetic gene cluster of Streptomyces chattanoogensis was cloned and confirmed by the disruption of pathway-specific activator genes. Comparative cluster analysis with its counterpart in Streptomyces natalensis revealed different cluster architecture between these two clusters. Compared with the highly conserved coding sequences, sequence variations appear to occur frequently in the intergenic regions. The evolutionary change of nucleotide sequence in the intergenic regions has given rise to different transcriptional organizations in the two clusters and resulted in altered gene regulation. These results provide insight into the evolution of antibiotic biosynthetic gene clusters. In addition, we cloned a pleitropic regulator gene, adpA ch , in S. chattanoogensis. Using the genetic system that we developed for this strain, adpA ch was deleted from the genome of S. chattanoogensis. The DadpA ch mutant showed a conditionally sparse aerial mycelium formation phenotype and defects in sporulation; it also lost the ability to produce NTM and a diffusible yellow pigment normally produced by S. chattanoogensis. RT-PCR analysis revealed that transcription of adpA ch was constitutive in YEME liquid medium. By using rapid amplification of 59 complementary DNA ends, two transcription start sites were identified upstream of the adpA ch coding region. Quantitative transcriptional analysis showed that the expression level of the NTM regulatory gene scnRI decreased 20-fold in the DadpA ch mutant strain, while the transcription of the other activator gene scnRII was not significantly affected. Electrophoretic mobility shift assay (EMSA) showed that AdpA ch binds to its own promoter but fails to bind to the promoter region of scnRI, indicating that the control of scnRI by AdpA ch is exerted in an indirect way. This work not only provides a platform and a new potential target for increasing the titre of NTM by genetic manipulation, but also advances the understanding of the regulation of NTM biosynthesis.

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