Affiliation:
1. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, Japan
Abstract
ABSTRACT
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
Eco
T14I 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
Eco
NI-
Sma
I 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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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