Functional domains of the Bacillus subtilis transcription factor AraR and identification of amino acids important for nucleoprotein complex assembly and effector binding

Abstract

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.

FIG. 1.

Schematic representation of the B. subtilis arabinose regulon. Expression of all ara genes is repressed by AraR and induced by arabinose. Distinct mechanisms of AraR binding to its operator regions in the different promoters allow a tight or flexible control of the system. AbfA, AbnA, and Xsa are enzymes involved in the degradation of arabinose-containing polysaccharides. AraE is a permease, the main transporter of arabinose into the cell, also responsible for the uptake of xylose and galactose. AraNPQ are components of an ABC-type high-affinity transport system for arabinose and/or arabinose oligomers. Intracellular catabolism of arabinose into xylulose 5-P, which is further catabolized through the pentose phosphate pathway, is carried out by AraA, AraB, and AraD. The function of AraL and AraM is unknown.

FIG. 2.

Sequence alignment of AraR-like proteins and three members of the GntR family (N-terminal region) or two members of LacI/GalR family (C-terminal region) and one periplasmic binding protein. Residues that are typical of the entire GntR or LacI/GalR family are depicted with gray characters on a black background; residues characteristic of AraR homologous proteins are highlighted with white characters on a gray background (see Materials and Methods). Positions of AraR mutations obtained by random mutagenesis (circles) or site-directed mutagenesis (triangles) are shown. Black triangles or circles represent mutations leading to a constitutive phenotype, and open triangles or circles denote changes that resulted in an AraR superrepressor phenotype. Letters representing the introduced residues are shown above the symbols. The secondary structures (arrows represent beta strands; bars represent alpha-helices) of FadR (amino acid residues 1 to 73) and PurR (positions 60 to 296) are shown below the alignment according to van Aalten et al. (54) and Schumacher et al. (50), respectively. The microorganisms of source and accession numbers are as follows: B. su, B. subtilis (P96711); B. li, B. licheniformis (Q62R80 and Q62UH0); B. hd, B. halodurans (Q9KBQ0); B. st, Geobacillus stearothermophilus (Q9S470); C. ac, Clostridium acetobutylicum (Q97JE6); E. fa, Enterococcus faecium (gi48825728); P. pe, Pediococcus pentosaceus (gi48870639); L. pl, Lactobacillus plantarum (Q88S80); O. ih, Oceanobacillus iheyensis (Q8EMP1); HutC P. pu, histidine utilization repressor of Pseudomonas putida (P22773); GntR, B. su gluconate utilization repressor of B. subtilis (P10585); FadR E. co, fatty-acid metabolism regulator of E. coli (P09371); LacI E. co, lactose repressor of E. coli (P03023); RbsB E. co, ribose-binding protein of E. coli (P02925); PurR E. co, purine repressor of E. coli (P15030).

FIG. 3.

AraR modeling. A) Close-in view of the arabinose binding site on the C-terminal domain of AraR, highlighting in blue shades the residues mutated by site-directed mutagenesis using the model and in green shades the residues found by random mutagenesis. Arabinose is represented by balls and sticks, and the fold of the protein is represented by a tube. B) Colored molecular surface of the C-terminal domain of AraR highlighting the residues potentially involved in the dimer contact and the mutations performed on them. The dimer contact is made up of hydrophobic residues, surrounded by polar and charged residues. C) Close-in view of the N-terminal region of AraR, highlighting in blue shades the residues mutated by site directed mutagenesis and in green shades the residues found by random mutagenesis. The fold of the protein is represented by a tube. The structure is oriented so that the part interacting with the DNA (using the similarity with FadR) (29) is facing the viewer. D) Schematic drawing of the domain structure of LacI/GalR repressors adapted from Lewis et al. (20). The DBDs, the N-terminal subdomains (NSD) and the C-terminal subdomains (CSD), the effector binding cleft, and the hinge helix, which is formed upon operator binding and connects the DBD to the core of the repressor, are indicated. Panels A, B and C were rendered with PyMol (6).

FIG. 4.

In vivo characterization of mutant AraR proteins. β-Galactosidase activities of B. subtilis strains carrying an araAB′-lacZ fusion and an araR allele integrated at the amyE locus and grown in the absence or presence of arabinose (in gray and black bars, respectively) are shown. Amino acid substitutions (obtained by random or site-directed mutagenesis) leading to an AraR⁻ phenotype (A) or AraR^S phenotype (B) are indicated for the mutated position and substituted amino acid by use of the standard one-letter designation. Values represent the averages of three independent experiments, each assayed in duplicate. Error bars represent the standard deviation. R.F. indicates the repression factor, calculated as the ratio between values obtained in the presence and in the absence of inducer. M.U., Miller units.

FIG. 5.

AraR accumulation in the cell determined by Western immunoblot analysis. Equal amounts of the soluble fractions of cell extracts obtained from B. subtilis cultures harboring a wild-type, mutant, or no araR allele and grown in the absence (−) or presence (+) of inducer were prepared as described in Materials and Methods. Mutant proteins are designated for the mutated position and substituted amino acid by use of the standard one-letter code.