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 autoregulators of Streptomyces species, act as a primary signal in Streptomyces virginiae to trigger virginiamycin biosynthesis and possess a specific binding protein, BarA. To clarify the in vivo function of BarA in the VB-mediated signal pathway that leads to virginiamycin biosynthesis, two barA mutant strains (strains NH1 and NH2) were created by homologous recombination. In strain NH1, an internal 99-bp EcoT14I fragment of barA was deleted, resulting in an in-frame deletion of 33 amino acid residues, including the second helix of the probable helix-turn-helix DNA-binding motif. With the same growth rate as wild-type S. virginiae on both solid and liquid media, strain NH1 showed no apparent changes in its morphological behavior, indicating that the VB-BarA pathway does not participate in morphological control in S. virginiae. In contrast, virginiamycin production started 6 h earlier in strain NH1 than in the wild-type strain, demonstrating for the first time that BarA is actively engaged in the control of virginiamycin production and implying that BarA acts as a repressor in virginiamycin biosynthesis. In strain 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 both intact and mutated barA genes, virginiamycin production was abolished irrespective of the presence of VB, suggesting that the mutated BarA retaining the intact DNA-binding motif was dominant over the wild-type BarA. These results further support the hypothesis that BarA works as a repressor in virginiamycin production and suggests that the helix-turn-helix motif is essential to its function. In strain NH1, VB production was also abolished, thus indicating that BarA is a pleiotropic regulatory protein controlling not only virginiamycin production but also autoregulator biosynthesis.
Nakano
H
H
Department of Biotechnology, Osaka University, Japan.
Takehara
E
E
Nihira
T
T
Yamada
Y
Y
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D019077:Helix-Turn-Helix Motifs; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014769:Virginiamycin / Q000096:biosynthesis*
D012097:Repressor Proteins; D014769:Virginiamycin; D015107:4-Butyrolactone