Cloning and in vivo functional analysis by disruption of a gene encoding the γ-butyrolactone autoregulator receptor from Streptomyces natalensis

A gene encoding a gamma-butyrolactone autoregulator receptor, which has a common activity as DNA-binding transcriptional repressors controlling secondary metabolism and/or morphological differentiation in Streptomyces, was cloned from a natamycin producer, Streptomyces natalensis. PCR using the primers designed for the two highly conserved regions of Streptomyces autoregulator receptors (BarA, FarA, ScbR, and ArpA) gave a 102-bp band. The sequence of this band had a high similarity to the expected region of a receptor gene. By genomic Southern hybridization with the 102-bp insert as a probe, a 687-bp intact receptor gene (sngR) was obtained from S. natalensis. To clarify the in vivo function of sngR, a sngR-disrupted strain was constructed, and the phenotypes were compared with those of the wild-type strain. The sngR-disruptants started natamycin production 6 h earlier and showed a 4.6-fold higher production of natamycin than the wild-type strain. In addition, the sporulation began earlier and the number of spores was tenfold more abundant than that of the wild-type strain. All the phenotypes were restored back to the original phenotypes of the wild-type strain by complementation with the intact sngR, indicating that the autoregulator receptor protein of S. natalensis acts as a primary negative regulator both on the biosynthesis of natamycin and sporulation.