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

Abstract

The Bacillus subtilis araR locus (mapped at about 294°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 A , 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.Bacillus subtilis can use L-arabinose as its sole carbon and energy source. The ability to utilize L-arabinose is dependent on three intracellular enzymes, L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate-4-epimerase, which sequentially convert L-arabinose to L-ribulose, L-ribulose-5-phosphate, and D-xylulose-5-phosphate, respectively (8). D-Xylulose-5-phosphate is further catabolized through the pentose-phosphate pathway. This metabolic pathway is identical to the one found in Escherichia coli, and its enzymes were previously described as inducible by L-arabinose (8).The three metabolic genes, araA, araB, and araD, coding for L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate-4-epimerase, respectively, are adjacent and constitute the first three open reading frames of a nine-cistron transcriptional unit with a total length of 11 kb (22,24). This operon, called araABDLMNPQ-abfA, is located at about 256°on the B. subtilis genetic map (18) and includes six other genes named araL, araM, araN, araP, araQ, and abfA. Analysis of the sequence of the ara operon showed that the putative products of araN, araP, and araQ are homologous to bacterial components of binding-protein-dependent transport systems and that the abfA gene most probably codes for...

Transcriptional Regulation of Genes Encoding Arabinan-Degrading Enzymes in Bacillus subtilis

“…The resulting products, arabinose, arabinobiose, arabinotriose, and arabinooligosaccharides, are transported by different systems. Arabinose enters the cell mainly through the AraE permease (33), and the uptake of arabinose oligomers most likely occurs via AraNPQ, an ABC-type transporter (32). These latter products might be further digested intracellularly by AbfA and Xsa.…”

“…Previous work by our group characterized the genes involved in the utilization of L-arabinose that belong to the araABDLMNPQabfA operon (32) and the divergently arranged araE and araR genes (31,33), located in distinct regions of the B. subtilis chromosome. The first three genes of the L-arabinose metabolic operon, araA, araB, and araD, encode the enzymes required for the intracellular conversion of L-arabinose into D-xylulose 5-phosphate, which is further catabolized through the pentose phosphate pathway (30).…”

“…The first three genes of the L-arabinose metabolic operon, araA, araB, and araD, encode the enzymes required for the intracellular conversion of L-arabinose into D-xylulose 5-phosphate, which is further catabolized through the pentose phosphate pathway (30). The product of the araE gene is a permease, the main transporter of L-arabinose into the cell (33). The araR gene encodes the regulatory protein of L-arabinose metabolism in B. subtilis, negatively controlling the expression from the L-arabinose-inducible promoters of the ara genes (22,23).…”

The LacI-Type Transcriptional Regulator AraR Acts as an l -Arabinose-Responsive Repressor of l -Arabinose Utilization Genes in Corynebacterium glutamicum ATCC 31831

“…As in C. glutamicum ATCC 31831, L-arabinose plays a key role in the regulation of L-arabinose utilization genes as a primary inducer in E. coli and B. subtilis (36)(37)(38)(39). However, their L-arabinose-responsive transcriptional regulators belong to distinct families.…”

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…The operons involved in the catabolism of secondary carbon sources are as follows (the carbon sources in parentheses): gntRKPZ (gluconate), 16,35,36) xylAB (xylose), 9) iolABCDEFGHIJ (myo-inositol), 40,41,102) trePAR (trehalose), 25,47,103) galKT (galactose), 25) glpFK (glycerol), 33) glvARC (6-P--glucoside), 34) bglPH ( -glucoside), 26,27) yjlBCD-uxaC-yjmBCD-uxuA-yjmFexuTR-uxaBA (hexuronate), 25,48) xynPB ( -xyloside), 65) yxjC-scoAE-bdh ( -hydroxybutyrate), 25,49) ara-ABDLMNPQ-abfA (arabinose) 24,52,53) and abnA xsa (arabinose), 50) kdgRKAT (hexuronate), 25,42) and kduID (galacturonate). 43) The yxkF-msmX operon probably involved in the transport of unknown sugars has been found to be under CcpA-mediated CCR.…”