GENE REPLACEMENT ANALYSIS OF THE STREPTOMYCES-VIRGINIAE BARA GENE ENCODING THE BUTYROLACTONE AUTOREGULATOR RECEPTOR REVEALS THAT BARA ACTS AS A REPRESSOR IN VIRGINIAMYCIN BIOSYNTHESIS

Virginiae butanolides (VBs), which are among the butyrolactone autoreg ulators of Streptomyces species, act as a primary signal in Streptomyc es virginiae to trigger virginiamycin biosynthesis and possess a speci fic binding protein, BarA. To clarify the in vivo function of BarA in the VB-mediated signal pathway that leads to virginiamycin biosynthesi s, two barA mutant strains (strains NH1 and NH2) were created by homol ogous recombination. In strain NH1, an internal 99-bp EcoT14I fragment of barA was deleted, resulting in an in-frame deletion of 33 amino ac id residues, including the second helix of the probable helix-turn-hel ix DNA-binding motif, With the same growth rate as wild-type S. virgin iae on both solid and liquid media, strain NH1 showed no apparent chan ges in its morphological behavior, indicating that the VB-BarA pathway does not participate in morphological control in S. Virginiae. In con trast, virginiamycin production started 6 h earlier in strain NHL than in the wild-type strain, demonstrating for the first time that BarA i s actively engaged in the control of virginiamycin production and impl ying that BarA acts as a repressor in virginiamycin biosynthesis. In s train NH2, an internal EcoNI-SmaI fragment of barA was replaced with a divergently oriented neomycin resistance gene cassette, resulting in the C-terminally truncated BarA retaining the intact helix-turn-helix motif, In strain NH2 and in a plasmid-integrated strain containing bot h intact and mutated barA genes, virginiamycin production was abolishe d irrespective of the presence of VB, suggesting that the mutated BarA retaining the intact DNA-binding motif was dominant over the wild-typ e BarA, These results further support the hypothesis that BarA works a s a repressor in virginiamycin production and suggests that the helix- turn-helix motif is essential to its function. In strain NH1, VB produ ction was also abolished, thus indicating that BarA is a pleiotropic r egulatory protein controlling not only virginiamycin production but al so autoregulator biosynthesis.