Investigating the Function of an Arabinan Utilization Locus Isolated from a Termite Gut Community

Arnal, Grégory; Bastien, Géraldine; Monties, Nelly; Abot, Anne; Leberre, Véronique Anton; Bozonnet, Sophie; O’Donohue, Michael; Dumon, Claire

doi:10.1128/aem.02257-14

Cited by 19 publications

(15 citation statements)

References 36 publications

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“…This observation is manifested in the large number of microbes reported to harbor enzymes for its hydrolysis (29)(30)(31)(32)(33)(34)(35), including Bacteroides spp., Bacillus subtilis, and Bacillus stearothermophilus. In addition, arabinan is an important product for the food and biotechnology industries (4,5).…”

Section: Discussionmentioning

confidence: 99%

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

Wefers

Dong

Abdelhamid

et al. 2017

Appl Environ Microbiol

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The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 ␣-L-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 ␤-L-arabinopyranosidase (CpAbp27A), and two GH127 ␤-Larabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para-nitrophenyl (pNP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are ␣-L-arabinofuranosidases that exhibit diverse substrate specificities, cleaving ␣-1,2, ␣-1,3, and ␣-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved ␤-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and L-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticus. IMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus. The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as ␤-L-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.

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Section: Discussionmentioning

confidence: 99%

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

Wefers

Dong

Abdelhamid

et al. 2017

Appl Environ Microbiol

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“…3B). While previously identified Bacteroidetes PULs were predicted to target polysaccharides like starch and pectin (2,8,(54)(55)(56), recent metagenomic analyses of mammalian rumen microbiota have yielded a Bacteroidaleslinked PUL that putatively targets cellulose (45,57). Unique PULs putatively targeting at least one of these polysaccharides were detected in P. americana Bacteroidetes isolate genomes ( Fig.…”

Section: P Americana Harbors New Cultivablementioning

confidence: 94%

Cultivable, Host-Specific Bacteroidetes Symbionts Exhibit Diverse Polysaccharolytic Strategies

León

Jahnes

Duan

et al. 2020

Appl Environ Microbiol

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Beneficial gut microbes can facilitate insect growth on diverse diets. The omnivorous American cockroach, Periplaneta americana (Insecta: Blattodea), thrives on a diet rich in plant polysaccharides and harbors a species-rich gut microbiota responsive to host diet. Bacteroidetes are among the most abundant taxa in P. americana and other cockroaches, based on cultivation-independent gut community profiling, and these potentially polysaccharolytic bacteria may contribute to host diet processing. Eleven Bacteroidetes isolates were cultivated from P. americana digestive tracts, and phylogenomic analyses suggest that they were new Bacteroides, Dysgonomonas, Paludibacter, and Parabacteroides species distinct from those previously isolated from other insects, humans, and environmental sources. In addition, complete genomes were generated for each isolate, and polysaccharide utilization loci (PULs) and several non-PUL-associated carbohydrate-active enzyme (CAZyme)-coding genes that putatively target starch, pectin, and/or cellulose were annotated in each of the isolate genomes. Type IX secretion system (T9SS)- and CAZyme-coding genes tagged with the corresponding T9SS recognition and export C-terminal domain were observed in some isolates, suggesting that these CAZymes were deployed via non-PUL outer membrane translocons. Additionally, single-substrate growth and enzymatic assays confirmed genomic predictions that a subset of the Bacteroides and Dysgonomonas isolates could degrade starch, pectin, and/or cellulose and grow in the presence of these substrates as a single sugar source. Plant polysaccharides enrich P. americana diets, and many of these gut isolates are well equipped to exploit host dietary inputs and potentially contribute to gut community and host nutrient accessibility. IMPORTANCE Gut microbes are increasingly being recognized as critical contributors to nutrient accessibility in animals. The globally distributed omnivorous American cockroach (Periplaneta americana) harbors many bacterial phyla (e.g., Bacteroidetes) that are abundant in vertebrates. P. americana thrives on a highly diverse plant-enriched diet, making this insect a rich potential source of uncharacterized polysaccharolytic bacteria. We have cultivated, completely sequenced, and functionally characterized several novel Bacteroidetes species that are endemic to the P. americana gut, and many of these isolates can degrade simple and complex polysaccharides. Cultivation and genomic characterization of these Bacteroidetes isolates further enable deeper insight into how these taxa participate in polysaccharide metabolism and, more broadly, how they affect animal health and development.

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“…1) through (meta)genomic, transcriptomic, and proteomic approaches (93)(94)(95)(96). PULs have been identified in species outside the Bacteroides genus in diverse environments, including the ruminal Prevotella bryantii (97), the freshwater dweller Flavobacterium johnsoniae (98), and the gut microbiota of the termite Pseudacanthotermes militaris (99).…”

Section: Pulomicsmentioning

confidence: 99%

Polysaccharide Utilization Loci: Fueling Microbial Communities

et al. 2017

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The complex carbohydrates of terrestrial and marine biomass represent a rich nutrient source for free-living and mutualistic microbes alike. The enzymatic saccharification of these diverse substrates is of critical importance for fueling a variety of complex microbial communities, including marine, soil, ruminant, and monogastric microbiota. Consequently, highly specific carbohydrate-active enzymes, recognition proteins, and transporters are enriched in the genomes of certain species and are of critical importance in competitive environments. In Bacteroidetes bacteria, these systems are organized as polysaccharide utilization loci (PULs), which are strictly regulated, colocalized gene clusters that encode enzyme and protein ensembles required for the saccharification of complex carbohydrates. This review provides historical perspectives and summarizes key findings in the study of these systems, highlighting a critical shift from sequence-based PUL discovery to systems-based analyses combining reverse genetics, biochemistry, enzymology, and structural biology to precisely illuminate the molecular mechanisms underpinning PUL function. The ecological implications of dynamic PUL deployment by key species in the human gastrointestinal tract are explored, as well as the wider distribution of these systems in other gut, terrestrial, and marine environments.

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Investigating the Function of an Arabinan Utilization Locus Isolated from a Termite Gut Community

Cited by 19 publications

References 36 publications

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

Cultivable, Host-Specific Bacteroidetes Symbionts Exhibit Diverse Polysaccharolytic Strategies

Polysaccharide Utilization Loci: Fueling Microbial Communities

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