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.
Sá-Nogueira
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Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal. sanoguei@itqb.unl.pt
Ramos
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D000595:Amino Acid Sequence; D051777:AraC Transcription Factor; D001089:Arabinose / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D014157:Transcription Factors; D014158:Transcription, Genetic
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; C109577:AraE protein, bacteria; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C491627:araE protein, E coli; D001089:Arabinose