Mode of action of AraR, the key regulator of <scp>l</scp>‐arabinose metabolism in <i>Bacillus subtilis</i>

Mota, Luı́s Jaime; Tavares, Paulo; Sá‐Nogueira, Isabel

doi:10.1046/j.1365-2958.1999.01484.x

Cited by 56 publications

(102 citation statements)

References 33 publications

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“…At the level of gene expression abfA and abf2 are induced by arabinose and repressed by glucose (Raposo et al, 2004). AraR, the key regulator of arabinose utilization in B. subtilis, in the absence of the inducer (arabinose) represses and tightly controls the transcription of both abfA and abf2 by binding to two in-phase operators in both the promoter region of the araABDLMNPQ-abfA operon and the promoter region of abf2 (Mota et al, 1999;Raposo et al, 2004). Glucose repression of the expression of the two AF genes is achieved by CcpA, the master regulator of carbon catabolite repression, associated mainly with coeffector HPr(Ser-P) (Inácio & de Sá-Nogueira, 2007).…”

Section: Enzymic Characterization Of Purified Abfa and Abf2mentioning

confidence: 99%

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

2008

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Bacillus subtilis produces a-L-arabinofuranosidases (EC 3.2.1.55; AFs) capable of releasing arabinosyl oligomers and L-arabinose from plant cell walls. Here, we show by insertion-deletion mutational analysis that genes abfA and xsa(asd), herein renamed abf2, encode AFs responsible for the majority of the intracellular AF activity in B. subtilis. Both enzyme activities were shown to be cytosolic and functional studies indicated that arabino-oligomers are natural substrates for the AFs. The products of the two genes were overproduced in Escherichia coli, purified and characterized. The molecular mass of the purified AbfA and Abf2 was about 58 kDa and 57 kDa, respectively. However, native PAGE gradient gel analysis and cross-linking assays detected higher-order structures (.250 kDa), suggesting a multimeric organization of both enzymes. Kinetic experiments at 37 6C, with p-nitrophenyl-a-L-arabinofuranoside as substrate, gave an apparent K m of 0.498 mM and 0.421 mM, and V max of 317 U mg "1 and 311 U mg "1 for AbfA and Abf2, respectively. The two enzymes displayed maximum activity at 50 6C and 60 6C, respectively, and both proteins were most active at pH 8.0. AbfA and Abf2 both belong to family 51 of the glycoside hydrolases but have different substrate specificity. AbfA acts preferentially on (1A5) linkages of linear a-1,5-L-arabinan and a-1,5-linked arabino-oligomers, and is much less effective on branched sugar beet arabinan and arabinoxylan and arabinogalactan. In contrast, Abf2 is most active on (1A2) and (1A3) linkages of branched arabinan and arabinoxylan, suggesting a concerted contribution of these enzymes to optimal utilization of arabinosecontaining polysaccharides by B. subtilis.

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Section: Enzymic Characterization Of Purified Abfa and Abf2mentioning

confidence: 99%

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

2008

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“…For this method we selected the gene that encodes the repressor of the arabinose metabolism genes, namely araR (Mota et al, 1999(Mota et al, , 2001Sa-Nogueira & Mota, 1997), for use in the selective marker cassette, and the promoter of the araABDLMNPQ-abfA operon for use in the counter-selective marker cassette. This new mutation system will be convenient for the introduction of multiple mutations, and we discuss its use in large-scale deletion mutations of the B. subtilis genome.…”

Section: Introductionmentioning

confidence: 99%

Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter

et al. 2008

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We have developed a system for the induction of marker-free mutation of Bacillus subtilis. The system features both the advantages of the use of antibiotic-resistance markers for mutant selection, and the ability to efficiently remove the markers, leaving unmarked mutations in the genome. It utilizes both a selective marker cassette and a counter-selective marker cassette. The selective marker cassette contains a chloramphenicol-resistance gene and the araR gene, which encodes the repressor for the arabinose operon (ara) of B. subtilis. The counter-selective marker cassette consists of a promoterless neomycin (Nm)-resistance gene (neo) fused to the ara promoter. First, the chromosomal araR locus is replaced with the counter-selective marker cassette by double-crossover homologous recombination and positive selection for Nm resistance. The selective marker cassette is connected with upstream and downstream sequences from the target locus, and is integrated into the upstream region of the target locus by a double-crossover event. This integration is also positively selected for, using chloramphenicol resistance. In the resultant strain, AraR, encoded by araR on the selective marker cassette, represses the expression of neo in the absence of L-arabinose. Finally, the eviction of the selective marker cassette together with the target locus is achieved by an intra-genomic single-crossover event between the two downstream regions of the target locus, and can be selected for based on Nm resistance, because of the excision of araR. The counter-selective marker cassette remaining in the genome, whose expression is switched on or off based on the excision or introduction of the selective marker cassette, is used again for the next round of deletion. Using this system, the 3.8 kb iolS-csbC region and the 41.8 kb hutM-csbC region have been efficiently and successfully deleted, without leaving markers in the target loci. The positive selection and simple procedure will make it a useful tool for the construction of multiple mutations.

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“…Based on the composition of these sequences and available data in the literature, it was expected that the affinity for these sequences will be different (9). Previously, the affinities of AraR to the various operators have been measured using DNA footprinting experiments and have shown apparent affinity constant (K app ) ranges between 40 nM and Ͼ250 nM (9). The full-length AraR binds DNA with higher affinity as compared with DBD.…”

Section: Arar Binds Diverse Sequence Operators With Similar Affinities-mentioning

confidence: 99%

“…The larger C terminus domain binds L-arabinose and belongs to the LacI/GalR family (16,17). AraR operators are palindromic DNA sequences, and the consensus operator is 16 bp in length (9). To understand how AraR recognizes diverse operators, we selected the ORA1, ORR3, ORE1, and ORX1 operators for structural and biochemical analysis.…”

mentioning

confidence: 99%

“…DNA footprinting experiments have shown that AraR may bind these different operators with varying affinities (9 previously determined the x-ray crystal structure of the N terminus domain of AraR bound to two natural operators, ORA1 and ORR3 (13). These structures reveal that the two AraR-DBD monomers are bound to each bipartite operator and that recognition of each monomer by DNA is through the core recognition motif TXG present in one half-site of the operator (where X is any base).…”

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confidence: 99%

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Plasticity in Repressor-DNA Interactions Neutralizes Loss of Symmetry in Bipartite Operators

Jain

Narayanan

Nair

2016

Journal of Biological Chemistry

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Transcription factor-DNA interactions are central to gene regulation. Many transcription factors regulate multiple target genes and can bind sequences that do not conform strictly to the consensus. To understand the structural mechanism utilized by the transcription regulators to bind diverse target sequences, we have employed the repressor AraR from Bacillus subtilis as a model system. AraR is known to bind to eight different operator sites in the bacterial genome. Although there are differences in the sequences of four of these operators, ORE1, ORX1, ORA1, and ORR3, the AraR-DNA binding domain (AraR-DBD) as well as full-length AraR unexpectedly binds to each of these sequences with similar affinities as measured by fluorescence anisotropy experiments. We have determined crystal structures of AraR-DBD in complex with two different natural operators ORE1 and ORX1 up to 2.07 and 1.97 Å resolution, respectively. These structures were compared with the previously reported structures of AraR-DBD bound to two other natural operators (ORA1 and ORR3). Interactions of two molecules of AraR-DBD with the symmetric operator, ORE1, are identical, but their interaction with the non-symmetric operator ORX1 results in breakdown of the symmetry in protein-DNA interactions. The novel interactions observed are accompanied by local conformational change in the DNA. ChIP-sequencing (ChIP-Seq) data on other transcription factors has shown that they can bind to diverse targets, and hence the plasticity exhibited by AraR may be a general phenomenon. The ability of transcription factors to form alternate interactions may be important for employment in new functions and evolution of novel regulatory circuits.The binding of proteins to cognate DNA sequences with high specificity is critical for a number of cellular processes. Among such proteins, the ability to detect and bind target sequences present at different locations in the whole genome is especially crucial for the function of transcription factors. This ability enables the transcriptional regulatory pathways to function with remarkable precision and sensitivity. The specificity of transcription factor for its cognate DNA sequence is largely achieved due to non-covalent interactions between the amino acid side chains and the functional groups present on the DNA bases in the major and minor groove (1). In addition, the shape of the DNA also plays a significant role in specific protein-DNA recognition (2-5). The availability of transcription factor binding data spanning entire genomes has led to identification of new targets for transcription factors. It has been observed that a single transcription factor can bind to several different sites throughout the genome. Sequence deviations have been seen in target sites that are recognized by the same transcription factor. Examples include glp repressors of Escherichia coli and NdgR from Streptomyces coelicolor, which recognize 13 and 19 different operator sequences, respectively (6, 7). The sequence differences suggest the possibility o...

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Mode of action of AraR, the key regulator of l‐arabinose metabolism in Bacillus subtilis

Cited by 56 publications

References 33 publications

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter

Plasticity in Repressor-DNA Interactions Neutralizes Loss of Symmetry in Bipartite Operators

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