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

Sá‐Nogueira, Isabel; Paveia, H.; Lencastre, Hermı́nia de

doi:10.1128/jb.170.6.2855-2857.1988

Cited by 14 publications

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References 12 publications

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“…In a previous work a collection of B. subtilis strains carrying mutations that mapped on the araR region was characterized (33,44). Chromosomal DNA from these strains was amplified by PCR, using oligonucleotides ARA13 and ARA18, and sequenced.…”

Section: Vol 188 2006 Characterization Of the B Subtilis Regulatormentioning

confidence: 99%

“…In previous studies, B. subtilis mutants constitutive for arabinose utilization and mutants showing growth deficiencies in minimal medium with arabinose as the sole carbon source were isolated after mutagenesis with NЈ-methyl-NЈ-nitro-N-nitrosoguanidine and mapped in the araR locus (33,44). This region of the chromosome was sequenced, and three novel single missense mutations conferring an AraR s phenotype (E-1423K, G-2153V, and G-2153D) and one responsible for a constitutive phenotype AraR Ϫ (G-1383E) were detected.…”

Section: Mutagenesis Of the Arar Allele And Isolation Of Arar Defectimentioning

confidence: 99%

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Functional Domains of theBacillus subtilisTranscription Factor AraR and Identification of Amino Acids Important for Nucleoprotein Complex Assembly and Effector Binding

et al. 2006

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The Bacillus subtilis AraR transcription factor represses at least 13 genes required for the extracellular degradation of arabinose-containing polysaccharides, transport of arabinose, arabinose oligomers, xylose, and galactose, intracellular degradation of arabinose oligomers, and further catabolism of this sugar. AraR exhibits a chimeric organization comprising a small N-terminal DNA-binding domain that contains a winged helix-turn-helix motif similar to that seen with the GntR family and a larger C-terminal domain homologous to that of the LacI/GalR family. Here, a model for AraR was derived based on the known crystal structures of the FadR and PurR regulators from Escherichia coli. We have used random mutagenesis, deletion, and construction of chimeric LexA-AraR fusion proteins to map the functional domains of AraR required for DNA binding, dimerization, and effector binding. Moreover, predictions for the functional role of specific residues were tested by site-directed mutagenesis. In vivo analysis identified particular amino acids required for dimer assembly, formation of the nucleoprotein complex, and composition of the sugar-binding cleft. This work presents a structural framework for the function of AraR and provides insight into the mechanistic mode of action of this modular repressor.The transcription factor AraR controls the expression of several Bacillus subtilis genes encoding enzymes and permeases involved in the degradation of arabinose-containing polysaccharides, uptake of L-arabinose (and possibly arabinose oligomers), xylose, and galactose, and further intracellular catabolism of arabinose and arabinose oligomers. The arabinose (ara) regulon comprises at least 13 genes (Fig. 1), the araAB DLMNPQ-abfA operon (43), the divergently arranged araE/ araR genes (42, 45) located in distinct regions of the B. subtilis chromosome, and the genes abnA and xsa positioned immediately upstream and 23 kb downstream from the operon, respectively (57). The first three genes of the 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 (41). The function of araL and araM is unknown, and genes araNPQ encode components of an ABC-type transporter most likely involved in the uptake of arabinose oligomers (43, 45). The product of the araE gene is a permease, the main transporter of arabinose into the cell (45), that is also responsible for the uptake of xylose and galactose (14). The araR gene encodes the regulatory protein of the system (27). The last gene of the metabolic operon, abfA, and the xsa gene most probably encode arabinofuranosidases involved in the intracellular degradation of arabinose oligomers (36). The abnA gene encodes an extracellular endo-arabinanase that degrades the arabinose homoglycan arabinan (16).The pathways of L-arabinose utilization in B. subtilis and Escherichia coli are identical, and the catabolic enzymes are functionally ...

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Section: Vol 188 2006 Characterization Of the B Subtilis Regulatormentioning

confidence: 99%

Section: Mutagenesis Of the Arar Allele And Isolation Of Arar Defectimentioning

confidence: 99%

Functional Domains of theBacillus subtilisTranscription Factor AraR and Identification of Amino Acids Important for Nucleoprotein Complex Assembly and Effector Binding

et al. 2006

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“…Transcriptional studies demonstrated that the expression of the araABDLMNPQabfA operon is directed by a strong A -like promoter identified upstream from the translation start site of the araA gene and that expression from the araABDLMNPQ-abfA promoter is induced by L-arabinose and repressed by glucose (24). The araR locus defined by three additional classes of mutations affecting L-arabinose utilization (17,18,25), at about 294°on the B. subtilis genetic map, was recently cloned (23). This region comprises two open reading frames with divergently arranged promoters, the regulatory gene, araR, and a partially cloned gene, termed araE.…”

Section: Insertional Inactivation Of the Arae Gene Leads To A Conditimentioning

confidence: 99%

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

Sá‐Nogueira

Ramos

1997

J Bacteriol

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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...

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“…Three additional classes of mutations affecting L-arabinose utilization were identified: (i) mutations conferring an Ara Ϫ phenotype to strains bearing the araA, araB, and araD wildtype alleles (39,40), (ii) mutations leading to constitutive expression of the three genes (47), and (iii) mutations conferring a conditional Ara Ϫ phenotype which was dependent on the concentration of sugar (39,40). These mutations were mapped at about 294Њ on the B. subtilis genetic map and define another ara locus.…”

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

Sá‐Nogueira

Mota

1997

J Bacteriol

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The Bacillus subtilis araC locus, mapped at about 294؇ 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؇ 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 A . 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.Bacillus subtilis is able to grow on L-arabinose as the sole carbon and energy source. The pathway of L-arabinose utilization in B. subtilis was described by Lepesant and Dedonder (30) and is identical to the one found in Escherichia coli (13). After entering the cell, L-arabinose is sequentially converted to L-ribulose, L-ribulose-5-phosphate, and D-xylulose-5-phosphate by the action of L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase, respectively. D-Xylulose-5-phosphate is further catabolized through the pentose phosphate pathway. The synthesis of these enzymes was shown to be inducible by L-arabinose, and the isomerase activity was shown to be subjected to catabolite repression by glucose and glycerol (30).The three metabolic genes, araA, araB, and araD, coding for L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase, respectively, have been cloned, and complementation experiments have shown that the products are functionally homologous to their E. coli counterparts (45). These genes are adjacent, with the order A-B-D (45), unlike the B-A...

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

Cited by 14 publications

References 12 publications

Functional Domains of theBacillus subtilisTranscription Factor AraR and Identification of Amino Acids Important for Nucleoprotein Complex Assembly and Effector Binding

Functional Domains of theBacillus subtilisTranscription Factor AraR and Identification of Amino Acids Important for Nucleoprotein Complex Assembly and Effector Binding

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

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

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