A binding site for activation by theBacillus subtilis AhrC protein, a repressor/activator of arginine metabolism

Abstract

InBacillus subtilis, the AhrC protein represses genes encoding enzymes of arginine biosynthesis and activates those mediating its catabolism. To determine how this repressor also functions as an activator, we attempted to clone catabolic genes by searching for insertions of the Tn917-lacZ transposon that express AhrC-dependent, arginine-inducibleβ-galactosidase activity. One such isolate was obtained. The region upstream oflacZ was subcloned inEscherichia coli in such a way that it could be replaced in theB. subtilis chromosome after appropriate manipulation. Analysis of exonuclease III-derived deletions located an AhrC-dependent, arginine-inducible promoter to within a ca. 1.9 kb fragment. The sequence revealed: the 3′ end of an ORF homologous togdh genes encoding glutamate dehydrogenase, with highest homology to the homologue fromClostridium difficile; the 5′ end of an ORF homologous to aSaccharomyces cerevisiae gene encoding Δ¹-pyrroline 5-carboxylate dehydrogenase (P5CDH), an enzyme of arginine catabolism ; and just upstream of the latter, a sequence with homology to known AhrC binding sites in the upstream part of the biosyntheticargCJBD-cpa-F cluster. The same region has also been sequenced by others as part of theB. subtilis genome sequencing project, revealing that the P5CDH gene is the first in a cluster termedrocABC. Restriction fragments containing the putative AhrC-binding sequence, but not those lacking it, showed retarded electrophoretic mobility in the presence of purified AhrC. A 277 by AhrC-binding fragment also showed anomalous mobility in the absence of AhrC, consistent with its being intrinsically bent. DNAse I footprinting localized AhrC binding to by − 16/ − 22 to + 1 (the transcription startpoint). Such a location for an activator binding site, i.e. overlapping the transcription start, is unusual.