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.

Cloning, functional analysis, and transcriptional regulation of the Bacillus subtilis araE gene involved in L-arabinose utilization.

The Bacillus subtilis araR locus (mapped at about 294 degrees on the genetic map) comprises two open reading frames with divergently arranged promoters, the regulatory gene, araR, encoding a repressor, and a partially cloned gene, termed araE by analogy to the Escherichia coli L-arabinose permease gene. Here, we report the cloning and sequencing of the entire araE gene encoding a 50.4-kDa polypeptide. The araE gene is monocistronic (as determined by Northern blot analysis), and its putative product is very similar to a number of prokaryotic proton-linked monosaccharide transporters (the group I family of membrane transport proteins). Insertional inactivation of the araE gene leads to a conditional Ara- phenotype dependent on the concentration of L-arabinose in the medium. Therefore, we assume that araE encodes a permease involved in L-arabinose transport into the cell. The araE promoter region contains -10 and -35 regions (as determined by primer extension analysis) very similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigmaA, and the -35 region of the transcription start point for araE is located 2 bp from the -35 region of the araR gene. Transcriptional studies demonstrated that the expression from the araE promoter is induced by L-arabinose, repressed by glucose, and negatively regulated by AraR. These observations are consistent with a model according to which in the absence of L-arabinose, AraR binds to a site(s) within the araE/araR promoter, preventing transcription from the araE promoter and simultaneously limiting the frequency of initiation from its own promoter; the addition of L-arabinose will allow transcription from the araE promoter and increase the frequency of initiation from the araR promoter.

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.

Assessment of phenotypic and genetic diversity in the yeast genus Metschnikowia.

The taxonomy of the yeast genus Metschnikowia has undergone profound changes over the past century. Major developments, from the capacity to obtain pure cultures of parasitic species to progress associated with the extensive use of molecular biology tools in yeast systematics, are briefly reviewed. Results from past work and new data are combined to evaluate evolutionary relationships and clarify the classification of both terrestrial and aquatic species. Recent physiological studies, including the utilization of non-conventional carbon and nitrogen sources, and characteristics like lipolytic activity and maximum temperatures for growth, are presented. The assessment of the genetic diversity within the genus by restriction analysis of the mitochondrial DNA and by the production of specific DNA probes has been explored. The results indicate the potential application of the latter in rapid identification procedures.

Ribosomal DNA spacer probes for yeast identification: studies in the genus Metschnikowia.

To test whether DNA probes derived from ribosomal DNA spacer sequences are suitable for rapid and species-specific yeast identification, a pilot study was undertaken. A 7.7 kb entire ribosomal DNA unit of the type strain of Metschnikowia reukaufii was isolated, cloned and mapped. A 0.65 kb BamHI-HpaI fragment containing non-transcribed spacer sequences was amplified and selected for testing as a 32P hybridization probe with total DNA from the type strains of M. reukaufii, M. pulcherrima, M. lunata, M. bicuspidata, M. australis, M. zobellii, M. krissii, five other strains identified as M. reukaufii and strains of Schizosaccharomyces pombe, Hansenula canadensis, Saccharomyces cerevisiae and Yarrowia lipolytica. The probe hybridized exclusively with DNA from the type strain and four other strains of M. reukaufii. DNA from one strain labelled M. reukaufii did not hybridize with the probe. Subsequent % G + C comparison and DNA-DNA reassociation with the type strain revealed that the non-hybridizing strain does not belong to the species M. reukaufii.

Cloning and characterization of araA, araB, and araD, the structural genes for L-arabinose utilization in Bacillus subtilis.

Two recombinant plasmids, pSNL1 and pSNL2, carrying structural genes for L-arabinose utilization were isolated from a Bacillus subtilis gene library. Both plasmids complemented araD mutations in a Rec- B. subtilis strain and in Escherichia coli. Moreover, pSNL1 also complemented araB mutations in both species and efficiently transformed araA Rec+ B. subtilis strains to Ara+. Detailed physical mapping of both plasmids in addition to transformation experiments involving defined restriction fragments from the pSNL1 insert unambiguously determined the gene order to be araD, araB, and araA, an order different from that found in E. coli.

Isolation of constitutive mutants for L-arabinose utilization in Bacillus subtilis.

Constitutive mutants for L-arabinose utilization were isolated from Bacillus subtilis 168T+ and showed resistance to D-fucose, a nonmetabolizable analog of L-arabinose. The mutations that conferred the constitutive phenotype (Arac) were mapped between cysB and hisA. All the mutants showed an isomerase activity which was reduced to 50 to 70% in the presence of L-arabinose and to 10% in the presence of glucose.