The l -Arabinan Utilization System of Geobacillus stearothermophilus

Abstract

Geobacillus stearothermophilus T-6 is a thermophilic soil bacterium that has a 38-kb gene cluster for the utilization of arabinan, a branched polysaccharide that is part of the plant cell wall. The bacterium encodes a unique three-component regulatory system (araPST) that includes a sugar-binding lipoprotein (AraP), a histidine sensor kinase (AraS), and a response regulator (AraT) and lies adjacent to an ATP-binding cassette (ABC) arabinose transport system (araEGH). The lipoprotein (AraP) specifically bound arabinose, and gel mobility shift experiments showed that the response regulator, AraT, binds to a 139-bp fragment corresponding to the araE promoter region. Taken together, the results showed that the araPST system appeared to sense extracellular arabinose and to activate a specific ABC transporter for arabinose (AraEGH). The promoter regions of the arabinan utilization genes contain a 14-bp inverted repeat motif resembling an operator site for the arabinose repressor, AraR. AraR was found to bind specifically to these sequences, and binding was efficiently prevented in the presence of arabinose, suggesting that arabinose is the molecular inducer of the arabinan utilization system. The expression of the arabinan utilization genes was reduced in the presence of glucose, indicating that regulation is also mediated via a catabolic repression mechanism. The cluster also encodes a second putative ABC sugar transporter (AbnEFJ) whose sugar-binding lipoprotein (AbnE) was shown to interact specifically with linear and branched arabino-oligosaccharides. The final degradation of the arabino-oligosaccharides is likely carried out by intracellular enzymes, including two ␣-L-arabinofuranosidases (AbfA and AbfB), a ␤-L-arabinopyranosidase (Abp), and an arabinanase (AbnB), all of which are encoded in the 38-kb cluster.The natural degradation of biomass from plants is a key step in the carbon cycle (53,69,79). This process is carried out mainly by microorganisms that can be found either free or as part of the digestive system in higher animals (76). The three main polysaccharides in the plant cell wall are cellulose, hemicellulose, and pectin, which are rigidified by lignin, a heterogeneous aromatic polymer (28, 60). Pectin is a complex polysaccharide and may account for up to 30% of the dry weight of the plant cell wall (46). Arabinan is a pectic polysaccharide consisting of a backbone of ␣-1,5-linked L-arabinofuranosyl units, which are further decorated mainly with ␣-1,2-and ␣-1,3-linked arabinofuranosides (46).Three general strategies are taken by the microbial world for plant cell wall degradation and can be described as follows. Anaerobic bacteria, such as Clostridium spp., have evolved unique multienzyme complexes, named cellulosomes, that integrate many cellulolytic and hemicellulolytic enzymes and mediate both the attachment of the cell to the crystalline polymer and its controlled hydrolysis (9,16,23,65). Aerobic fungi, such as Trichoderma and Aspergillus, secrete a large variety of free cellulases, hemicellulases...

The Geobacillus paradox: why is a thermophilic bacterial genus so prevalent on a mesophilic planet?

“…The genome of strain T-6 has a hemicellulose utilization island of 76 kb, divided equally between two subclusters providing functionality for utilizing xylan/xylose and arabinan/arabinose (Shulami et al, 1999(Shulami et al, , 2011. The region also contains several mobile elements, suggesting that the island could have been assembled by horizontal gene transfer.…”

The abp gene in Geobacillus stearothermophilus T‐6 encodes a GH27 β‐l‐arabinopyranosidase

“…Arabinan, a branched polysaccharide, is part of rhamnogalacturonan I (RG-I) and is also one of the polysaccharides that assemble hemicellulose [3,4]. The arabinan backbone consists of a-1,5-linked L-arabinofuranosyl units, and is decorated mainly with a-1,2-and a-1,3-linked arabinofuranosides [5]. The L-arabinose residues are hence very abundant in plants, and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides, such as arabinoxylans and pectic arabinogalactans.…”

“…Geobacillus stearothermophilus T-6 is a thermophilic soil bacterium that possesses an extensive system for the utilization of xylan, galactan and arabinan [5][6][7]. This bacterium produces a limited number of extracellular enzymes (e.g., extracellular xylanase, arabinanase, and galactanase) [8] that cleave the high molecular weight polysaccharides backbone to short decorated oligosaccharides.…”

Biochemical and Structural Insights into Xylan Utilization by the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

“…Thus, ␣-glucuronidases cleave the ␣-1,2-glycosidic bond between 4-O-methyl ␣-glucuronic acid and the xylopyranosyl unit of xylo-oligosaccharides (10). To date, most of the cloned microbial ␤-xylosidases fall within glycoside hydrolase (GH) 4 families 3, 30, 39, 43, 51, 52, and 54 (2,(11)(12)(13)(14), whereas characterized ␣-glucuronidases are assigned to either GH family 67 (6,15,16) or GH family 115 (17). With a number of advantages over mesophilic enzymes, thermostable enzymes are especially thought to improve hydrolytic performance and overall economy of the process of biofuel production from the plant cell wall.…”

“…The complete degradation of xylan requires the synergistic activity of several hemicellulolytic enzymes, such as ␤-1,4-endoxylanase, ␤-xylosidase, ␣-glucuronidase, ␣-L-arabinosidase, and acetylxylan esterase (1). To facilitate a concerted action of these enzymes for hemicellulose degradation, several microorganisms have evolved gene clusters encoding the different hemicellulolytic enzymes (1,(3)(4)(5)(6). The transport mechanism for xylan degradation products has been fairly well described in bacteria such as Streptomyces lividans and Geobacillus stearothermophilus (7,8).…”