GENE REPLACEMENT ANALYSIS OF THE STREPTOMYCES-VIRGINIAE BARA GENE ENCODING THE BUTYROLACTONE AUTOREGULATOR RECEPTOR REVEALS THAT BARA ACTS AS A REPRESSOR IN VIRGINIAMYCIN BIOSYNTHESIS
H. Nakano et al., GENE REPLACEMENT ANALYSIS OF THE STREPTOMYCES-VIRGINIAE BARA GENE ENCODING THE BUTYROLACTONE AUTOREGULATOR RECEPTOR REVEALS THAT BARA ACTS AS A REPRESSOR IN VIRGINIAMYCIN BIOSYNTHESIS, Journal of bacteriology, 180(13), 1998, pp. 3317-3322
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.