Control of the arabinose regulon in Bacillus subtilis by AraR in vivo: crucial roles of operators, cooperativity, and DNA looping.

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.

Mode of action of AraR, the key regulator of L-arabinose metabolism in Bacillus subtilis.

The AraR protein is a negative regulator involved in L-arabinose-inducible expression of the Bacillus subtilis araABDLMNPQ-abfA metabolic operon and of the araE/araR genes that are organized as a divergent transcriptional unit. The two ara gene clusters are found at different positions in the bacterial chromosome. AraR was overproduced in Escherichia coli and purified to more than 95% homogeneity. AraR binds specifically to DNA fragments carrying the promoter region of the ara genes. DNase I protection assays showed that AraR binds to two sequences within the promoters of the araABDLMNPQ-abfA operon and the araE gene, and to one sequence in the araR promoter. The AraR target sequences are palindromic and share high identity, defining a 16 bp AraR consensus operator sequence showing half-symmetry, ATTTGTAC. Binding of AraR to DNA was inhibited by L-arabinose but not by other sugars. The two operator sites within the araABDLMNPQ-abfA operon and araE promoters are located on the same side of the DNA helix, and a pattern of enhanced and diminished DNase I cleavage was observed between them, but not in the araR promoter. Quantitative DNase I footprinting in DNA templates containing one, two or three AraR binding sites showed that the repressor binds cooperatively to the two operator sites within the metabolic operon and araE promoters but not to the site located in the araR promoter. These results are consistent with two modes for AraR transcriptional repression that might correlate with different physiological requirements: a high level of repression is achieved by DNA bending requiring two in-phase operator sequences (metabolic operon and araE transport gene), whereas binding to a single operator, which autoregulates araR expression, is 10-fold less effective.

Negative regulation of L-arabinose metabolism in Bacillus subtilis: characterization of the araR (araC) gene.

The Bacillus subtilis araC locus, mapped at about 294 degrees on the genetic map, was defined by mutations conferring an Ara- phenotype to strains bearing the metabolic araA, araB, and araD wild-type alleles (located at about 256 degrees on the genetic map) and by mutants showing constitutive expression of the three genes. In previous work, it has been postulated that the gene in which these mutations lie exerts its effect on the ara metabolic operon in trans, and this locus was named araC by analogy to the Escherichia coli regulatory gene. Here, we report the cloning and sequencing of the araC locus. This region comprises two open reading frames with divergently arranged promoters, the regulatory gene, araC, encoding a 41-kDa polypeptide, and a partially cloned gene, termed araE, which most probably codes for a permease involved in the transport of L-arabinose. The DNA sequence of araC revealed that its putative product is very similar to a number of bacterial negative regulators (the GalR-LacI family). However, a helix-turn-helix motif was identified in the N-terminal region by its identity to the consensus signature sequence of another group of repressors, the GntR family. The lack of similarity between the predicted primary structure of the product encoded by the B. subtilis regulatory gene and the AraC regulator from E. coli and the apparently different modes of action of these two proteins lead us to propose a new name, araR, for this gene. The araR gene is monocistronic, and the promoter region contains -10 and -35 regions (as determined by primer extension analysis) similar to those recognized by RNA polymerase containing the major vegetative cell sigma factor sigmaA. An insertion-deletion mutation in the araR gene leads to constitutive expression of the L-arabinose metabolic operon. We demonstrate that the araR gene codes for a negative regulator of the ara operon and that the expression of araR is repressed by its own product.