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J. Biol. Chem., Vol. 276, Issue 2, 1479-1485, January 12, 2001
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,
From the Biozentrum, University of Basel, Department of
Microbiology, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
Pip is a
pristinamycin-induced transcriptional regulator
protein detected in many Streptomyces species
by its ability to specifically bind sequence motifs within the promoter
of a Streptomyces pristinaespiralis multidrug resistance
gene (ptr). To investigate the possible role of Pip in
regulating multidrug resistance, it was purified from a genetically
characterized species, Streptomyces coelicolor, utilizing
an affinity matrix of the ptr promoter conjugated to magnetic beads. Reverse genetics identified the corresponding locus and
confirmed that it encoded Pip, a protein belonging to the TetR family
of procaryotic transcriptional repressors. Pip binding motifs were
located upstream of the adjacent gene pep, encoding a major
facilitator antiporter homologous to ptr. In vivo analysis
of antibiotic susceptibility profiles demonstrated that pep conferred
elevated levels of resistance only to pristinamycin I (PI), a
streptogramin B antibiotic having clinical importance. Purified
recombinant Pip was a dimer (in the presence or absence of PI) and
displayed a high affinity for its palindromic binding motifs within the
ptr promoter and the upstream region of pep. The Pip/ptr promoter complex was dissociated by PI but not
by any of the other nonstreptogramin antibiotics that were described previously as transcriptional inducers. These procaryotic
regulatory elements served as the basis for the development of systems
allowing repression or induction of cloned genes in mammalian and plant cells in response to streptogramin antibiotics (including
pristinamycin, virginiamycin, and Synercid®).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF193856.
Present address: Morphochem, Schwarzwaldallee 215, Basel, BS 4058, Switzerland.
§
Present address: Infectious Disease Group, Aventis-Hoechst Marion
Roussel, 102 route de Noisy F-93235 Romainville Cedex, France.
¶
Present address: Dept. of Developmental Biology, 279 Campus
Dr., Beckman Center B-343, Stanford University School of Medicine, Stanford, CA 94305-5329.
To whom correspondence should be addressed. Tel.:
41-61-267-2116; Fax: 41-61-267-2118;
Charles-J.Thompson{at}unibas.ch.
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