Affiliation:
1. Department of Biotechnology, Faculty of Life Science and Biotechnology, Fukuyama University, Fukuyama, Hiroshima, Japan
Abstract
ABSTRACT
The
Bacillus subtilis
rhaEWRBMA
(formerly
yuxG-yulBCDE
) operon consists of four genes encoding enzymes for
l
-rhamnose catabolism and the
rhaR
gene encoding a DeoR-type transcriptional regulator. DNase I footprinting analysis showed that the RhaR protein specifically binds to the regulatory region upstream of the
rhaEW
gene, in which two imperfect direct repeats are included. Gel retardation analysis revealed that the direct repeat farther upstream is essential for the high-affinity binding of RhaR and that the DNA binding of RhaR was effectively inhibited by
l
-rhamnulose-1-phosphate, an intermediate of
l
-rhamnose catabolism. Moreover, it was demonstrated that the CcpA/P-Ser-HPr complex, primarily governing the carbon catabolite control in
B. subtilis
, binds to the catabolite-responsive element, which overlaps the RhaR binding site.
In vivo
analysis of the
rhaEW
promoter-
lacZ
fusion in the background of
ccpA
deletion showed that the
l
-rhamnose-responsive induction of the
rhaEW
promoter was negated by the disruption of
rhaA
or
rhaB
but not
rhaEW
or
rhaM
, whereas
rhaR
disruption resulted in constitutive
rhaEW
promoter activity. These
in vitro
and
in vivo
results clearly indicate that RhaR represses the operon by binding to the operator site, which is detached by
l
-rhamnulose-1-phosphate formed from
l
-rhamnose through a sequence of isomerization by RhaA and phosphorylation by RhaB, leading to the derepression of the operon. In addition, the
lacZ
reporter analysis using the strains with or without the
ccpA
deletion under the background of
rhaR
disruption supported the involvement of CcpA in the carbon catabolite repression of the operon.
IMPORTANCE
Since
l
-rhamnose is a component of various plant-derived compounds, it is a potential carbon source for plant-associating bacteria. Moreover, it is suggested that
l
-rhamnose catabolism plays a significant role in some bacteria-plant interactions, e.g., invasion of plant pathogens and nodulation of rhizobia. Despite the physiological importance of
l
-rhamnose catabolism for various bacterial species, the transcriptional regulation of the relevant genes has been poorly understood, except for the regulatory system of
Escherichia coli
. In this study, we show that, in
Bacillus subtilis
, one of the plant growth-promoting rhizobacteria, the
rhaEWRBMA
operon for
l
-rhamnose catabolism is controlled by RhaR and CcpA. This regulatory system can be another standard model for better understanding the regulatory mechanisms of
l
-rhamnose catabolism in other bacterial species.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
16 articles.
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