Affiliation:
1. Mikrobielle Genetik, Universität Tübingen, D-72076 Tübingen, Germany
Abstract
ABSTRACT
A single-copy reporter system for
Staphylococcus xylosus
has been developed, that uses a promoterless version of the endogenous β-galactosidase gene
lacH
as a reporter gene and that allows integration of promoters cloned in front of
lacH
into the lactose utilization gene cluster by homologous recombination. The system was applied to analyze carbon catabolite repression of
S. xylosus
promoters by the catabolite control protein CcpA. To test if
lacH
is a suitable reporter gene, β-galactosidase activities directed by two promoters known to be subject to CcpA regulation were measured. In these experiments, repression of the
malRA
maltose utilization operon promoter and autoregulation of the
ccpA
promoters were confirmed, proving the applicability of the system. Subsequently, putative CcpA operators, termed catabolite-responsive elements (
cre
s), from promoter regions of several
S. xylosus
genes were tested for their ability to confer CcpA regulation upon a constitutive promoter, P
veg
II
. For that purpose,
cre
sequences were placed at position +3 or +4 within the transcribed region of P
veg
II
. Measurements of β-galactosidase activities in the presence or absence of glucose yielded repression ratios between two- and eightfold. Inactivation of
ccpA
completely abolished glucose-dependent regulation. Therefore, the tested
cre
s functioned as operator sites for CcpA. With promoters exclusively regulated by CcpA, signal transduction leading to CcpA activation in
S. xylosus
was examined. Glucose-dependent regulation was measured in a set of isogenic mutants showing defects in genes encoding glucose kinase GlkA, glucose uptake protein GlcU, and HPr kinase HPrK. GlkA and GlcU deficiency diminished glucose-dependent CcpA-mediated repression, but loss of HPr kinase activity abolished regulation. These results clearly show that HPr kinase provides the essential signal to activate CcpA in
S. xylosus
. Glucose uptake protein GlcU and glucose kinase GlkA participate in activation, but they are not able to trigger CcpA-mediated regulation independently from HPr kinase.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
23 articles.
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