Control of the Arabinose Regulon in Bacillus subtilis by AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping

Abstract

The proteins involved in the utilization of l-arabinose by Bacillus subtilis are encoded by the araABDLMNPQ-abfA metabolic operon and by the araE/araR divergent unit. Transcription from the ara operon, araE transport gene, and araR regulatory gene is induced by l-arabinose and negatively controlled by AraR. The purified AraR protein binds cooperatively to two in-phase operators within the araABDLMNPQ-abfA (OR(A1) and OR(A2)) and araE (OR(E1) and OR(E2)) promoters and noncooperatively to a single operator in the araR (OR(R3)) promoter region. Here, we have investigated how AraR controls transcription from the ara regulon in vivo. A deletion analysis of the ara promoters region showed that the five AraR binding sites are the key cis-acting regulatory elements of their corresponding genes. Furthermore, OR(E1)-OR(E2) and OR(R3) are auxiliary operators for the autoregulation of araR and the repression of araE, respectively. Analysis of mutations designed to prevent cooperative binding of AraR showed that in vivo repression of the ara operon requires communication between repressor molecules bound to two properly spaced operators. This communication implicates the formation of a small loop by the intervening DNA. In an in vitro transcription system, AraR alone sufficed to abolish transcription from the araABDLMNPQ-abfA operon and araE promoters, strongly suggesting that it is the major protein involved in the repression mechanism of l-arabinose-inducible expression in vivo. The ara regulon is an example of how the architecture of the promoters is adapted to respond to the particular characteristics of the system, resulting in a tight and flexible control