Characterization of abn2 ( yxiA ), Encoding a Bacillus subtilis GH43 Arabinanase, Abn2, and Its Role in Arabino-Polysaccharide Degradation

Abstract

The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-␣-1,5-L-arabinanase (EC 3.2.1.99) from Bacillus subtilis, encoded by the yxiA gene (herein renamed abn2) that contributes to arabinan degradation. Functional studies by mutational analysis showed that Abn2, together with previously characterized AbnA, is responsible for the majority of the extracellular arabinan activity in B. subtilis. Abn2 was overproduced in Escherichia coli, purified from the periplasmic fraction, and characterized with respect to substrate specificity and biochemical and physical properties. With linear-␣-1,5-L-arabinan as the preferred substrate, the enzyme exhibited an apparent K m of 2.0 mg ml ؊1 and V max of 0.25 mmol min ؊1 mg ؊1 at pH 7.0 and 50°C. RNA studies revealed the monocistronic nature of abn2. Two potential transcriptional start sites were identified by primer extension analysis, and both a A -dependent and a H -dependent promoter were located. Transcriptional fusion studies revealed that the expression of abn2 is stimulated by arabinan and pectin and repressed by glucose; however, arabinose is not the natural inducer. Additionally, trans-acting factors and cis elements involved in transcription were investigated. Abn2 displayed a control mechanism at a level of gene expression different from that observed with AbnA. These distinct regulatory mechanisms exhibited by two members of extracellular glycoside hydrolase family 43 (GH43) suggest an adaptative strategy of B. subtilis for optimal degradation of arabinopolysaccharides.Microorganisms are present in all natural environments and play fundamental roles in biogeochemical cycling or in decomposition processes. A key step in the carbon cycle is the degradation of plant cell wall polysaccharides by the concerted action of several glycosyl hydrolases (see references 2 and 35 and references therein). Due to their increased value in several biotechnological processes, namely, the conversion of several hemicellulosic substrates to fermentable sugars and the successive production of fuel alcohol, the characterization of these cellulose-and/or hemicellulose-degrading enzymes has been an important goal for achieving efficient breakdown of plant cell wall polymers (24,28,29). The gram-positive bacterium Bacillus subtilis participates in the enzymatic dissolution of plant biomass in the soil. Thus, it is able to synthesize a vast variety of glycoside hydrolases capable of the depolymerization of plant cell wall polysaccharides, such as cellulose, hemicellulose, or pectin (see references 35 and 37 and references therein). L-Arabinose, the second most abundant pentose in nature, is found in significant amounts in homopolysaccharides, branched and debranched arabinans, and heteropolysaccharides, such as arabinoxylans and arabinogalactans. In fact, B. subtilis produces exo-and endoacting arabinases capable of releasing arabinosyl oligomers and L-arabinose from plant ...

The l -Arabinan Utilization System of Geobacillus stearothermophilus

“…In B. subtilis, the arabinan utilization system resembles that of G. stearothermophilus but is based on two extracellular endo-␣-1,5-L-arabinanases (36). The resulting products, arabinose and arabinose oligomers, are transported by specific transport systems, AraE, a proton symporter, and AraNPQ, an ABCtype transporter for arabino-oligosaccharides, and presumably an additional, unidentified transporter (22).…”

A Multitask ATPase Serving Different ABC-Type Sugar Importers in Bacillus subtilis

“…2). Arabinan, a homopolymer of arabinose, is extracellularly degraded by two GH43 endo-␣-1,5-arabinanases, AbnA and Abn2 (11). The resulting products, mainly arabinooligosaccharides, enter the cell through different transport systems.…”

“…L-Arabinose, the second most-abundant pentose in plant biomass, next to Dxylose, is found in homopolysaccharides (branched and debranched arabinans) and heteropolysaccharides (arabinoxylans, arabinogalactans, etc.). B. subtilis produces exo-and endo-acting arabinases capable of releasing arabinosyl oligomers and L-arabinose from plant cell walls (10,11,33). Although many polysaccharolytic glycoside hydrolases have been purified from bacteria, including several Bacillus spp., information on ABC transporters devoted to the uptake of products resulting from the degradation of hemicellulose is limited (6).…”

“…AraN is the BPD, and AraP and AraQ are the TMDs. This transporter, which lacks the NBD protein partner, was proposed to be involved in the uptake of arabinose oligomers mainly by genomic context and in silico analysis (10,11,23).…”

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

“…In the presence of L-arabinose, three promoters, ParaBAD, ParaE, and ParaFGH, are activated, and ParaC is derepressed (16). In B. subtilis, AraR, which is structurally different from E. coli AraC, controls the transcription of several genes involved in the uptake and degradation of arabinose-containing polysaccharides, in addition to the genes corresponding to the E. coli AraC regulon (17)(18)(19). AraR comprises an N-terminal DNAbinding domain homologous to the GntR family proteins and a C-terminal effector-binding domain that shows similarity to members of the LacI/GalR family (20,21).…”