Structural insights into xyloglucan recognition by an ABC transporter from a Gram-positive, thermophilic bacterium.

Cell-Surface and Periplasmic Deconstruction of Xylan by Oligotrophic Bacterium Caulobacter vibrioides.

Bacterial species that inhabit diverse nutritional niches, such as plants, the human gut, and aquatic environments, exhibit common and unique molecular mechanisms for acquiring nutrients from plant polysaccharides like xylan. The oligotrophic bacterium Caulobacter vibrioides (Syn. Caulobacter crescentus) shows unique adaptations for growth in low-nutrient conditions and codes for a repertoire of genes that facilitate xylan utilisation as a carbon source. Investigation of xylan and xylan-derivative utilisation by C. vibrioides shows membrane-bound xylanase-mediated xylan deconstruction on the cell surface and further deconstruction of the xylo-oligosaccharides in the periplasm by xylosidases and other accessory enzymes. Proton motive force disruption studies suggest involvement of TonB-dependent transporters, Major Facilitator Superfamily transporters, or ATP-Binding Cassette transporters in the transport of xylo-oligosaccharides and other xylan-derivatives across the membranes of C. vibrioides. Results from our studies using enzyme assays and chromatographic analyses suggest that bacteria deconstruct xylan in a surface-bound manner leading to efficient uptake of xylo-oligosaccharides across the outer membrane, without loss of nutrients to the environment. This proposed model for C. vibrioides xylan utilisation highlights both unique and similar mechanistic features found in gut and plant pathogenic bacteria, advancing the molecular understanding of nutrient acquisition in oligotrophic Gram-negative bacteria.

Comparative genomic analysis of hemicellulose-degrading potential in bacterial isolates from the anterior intestine of Eisenia andrei (Bouché, 1972)

In Silico Insights into Carbohydrate-Active Enzymes (CAZymes) of Bacillus subtilis T7: Lignin and Polysaccharide Degradation Mechanisms

In silico structural characterization of carbohydrate-active enzymes (CAZymes) in Bacillus subtilis T7 reveals mechanistic insight into the strain’s capacity for consolidated bioprocessing of untreated lignocellulosic biomass. Homology models for 13 CAZymes were constructed using SWISS-MODEL, with Cu2+ and FAD cofactors incorporated into the AA10 lytic polysaccharide monooxygenase and AA3 oxidoreductase models, respectively. Blind molecular docking across the full protein surface identified energetically favorable binding pockets on GH9 endoglucanase and Abhydrolase_1. Among 12 enzyme–ligand pairs screened, Abhydrolase_1 exhibited the highest affinity for xylotetraose (−7.7 kcal/mol) and GH9 showed the strongest preference for cellotetraose (−7.0 kcal/mol). Site-specific docking confirmed six hydrogen bonds with Gly32, Phe33, Thr34, Ser36, Arg179, and His256, supplemented by two carbon–hydrogen bonds with Ile180 and Ser39, anchoring xylotetraose within the Abhydrolase_1 binding cavity, and seven hydrogen bonds stabilizing cellotetraose in the GH9 catalytic groove, with key contacts at Tyr141, Trp145, Asp194, Trp193, Arg254, Tyr255, and Tyr354. One-hundred nanosecond all-atom molecular dynamics simulations (GROMACS 2023.2, CHARMM36 force field, triplicate runs) confirmed overall structural integrity for both proteins: Abhydrolase_1 maintained a compact conformation (Rg = 18.11 ± 0.09 Å; backbone RMSD 2–3 Å), while GH9 was similarly stable (Rg = 30.36 ± 0.33 Å; RMSD 2–5 Å). Ligand dynamics were more variable—xylotetraose remained bound within the Abhydrolase_1 active site for approximately 75 ns before partial displacement, whereas cellotetraose exhibited dynamic association along the GH9 catalytic channel, consistent with processive substrate translocation in endoglucanases. These computational findings line up with the strain’s experimentally observed hydrolytic clearance zones (cellulase 24.5 mm; xylanase 11.6 mm), 63.4% alkali lignin decolorization, transient accumulation of ferulic acid and vanillin, and a hydrogen yield of 1.41 mol H2/mol substrate from untreated food waste. Together they give a molecular-level picture of substrate-specific CAZyme recognition in B. subtilis T7 and support its potential as a pretreatment-free platform for lignocellulosic biohydrogen production.

Cyanochelin uptake reveals an exclusively cyanobacterial class of AMIN-domain TonB-dependent transporters

Siderophore transport is central to microbial competition, because it determines access to iron, frequently a limiting nutrient. While siderophore-mediated iron uptake via TonB-dependent transporters (TBDTs) has been extensively studied in heterotrophic bacteria, little is known about the functionality and specificity of TBDTs in cyanobacteria. In the present study we functionally characterise the import system of cyanochelin B, a photolytic β-hydroxy aspartate siderophore produced by several filamentous cyanobacteria, including Leptolyngbya sp. NIES-3755. We have identified a cyanochelin B putative transport cassete localized in the vicinity of the cyanochelin biosynthetic gene cluster in Leptolyngbya genome. By expressing the import genes heterologously in a model unicellular cyanobacterium Synechocystis sp. PCC 6803, we established that the transport cassette reconstitutes cyanochelin B-dependent growth, consistent with cyanochelin-mediated iron acquisition. Systematic gene dissection showed that the TBDT (CctA) and the substrate-binding protein (CctB), responsible for binding the siderophore in the periplasm, are alone sufficient for cyanochelin import into Synechocystis cells, with the permeases, ATPase and a cassette-associated ferredoxin supplied in trans by the host. CctA carries an N-terminal AMIN domain, a fusion found only in cyanobacterial TBDTs. The cassette accepts the structurally similar cyanochelin A but not cyanochelin C, enterobactin or pyoverdine, indicating limited promiscuity. The phylogenetic placement of cyanochelin receptors within a broader clade containing citrate-hydroxamate-type siderophore receptors suggests an evolutionary link between transport systems for chemically distinct cyanobacterial siderophores. Our study reports the first functional heterologous expression of a cyanobacterial TonB-dependent transporter and establishes Synechocystis as a promising platform for cyanobacterial xenosiderophore-uptake studies.

Multiomics Identification and Structural Dynamics of PeXyn1: A Novel GH11 Xylanase from Pleurotus eryngii for Substrate Utilization.

The widely cultivated mushroom Pleurotus eryngii utilizes lignocellulosic biomass as a growth substrate, yet its xylanases remain poorly understood. Here, we identified PeXyn1, a novel cellulose-upregulated GH11 xylanase featuring a C-terminal carbohydrate-binding module (CBM1) domain, using integrated multiomics. Recombinant PeXyn1 exhibited an optimal pH of 4.5 and temperature of 50 °C, with remarkable stability across pH 4.5-7.5. Kinetic characterization yielded a Km of 27.51 mg mL-1 and a kcat of 20.95 s-1. Purified PeXyn1 efficiently saccharified corncob, releasing 4.258 mg mL-1 reducing sugars after 2 h. Notably, CBM1 truncation increased specific activity from 10.04 ± 0.29 to 28.79 ± 2.23 U mg-1, confirming the core's hydrolytic function while CBM1 mediates substrate anchoring. Simulations revealed an induced-fit mechanism with a high xylohexaose binding affinity (-11.7 kJ mol-1). Multivalent interactions at the CBM1-catalytic interface restrict structural fluctuations, promoting a stable conformation. This work offers the first molecular insight into P. eryngii xylanases, highlighting PeXyn1 for agro-waste valorization.

AlphaFold-driven structural proteomics reveals extensive cellulosome machinery in human ruminococcal symbionts

ABSTRACT Cellulosomes are large, surface-displayed enzyme complexes that enable anaerobic bacteria to degrade recalcitrant plant polysaccharides, yet cellulosome-expressing bacteria are thought to be rare in the human gut. Here, we show that extensive sequence divergence obscures the detection of many ruminococcal cellulosomes by conventional sequence homology-based methods. Using proteome-scale AlphaFold2 structural predictions, we uncovered a substantially expanded set of putative cellulosome-producing Ruminococcus species, including six previously unrecognized human symbionts. Structure-based clustering identifies several novel cohesin families that retain conserved folds despite extreme sequence divergence and define distinct, phylogenetically conserved cellulosome architectures. The analysis reveals R. callidus and related human symbionts encode elaborate cellulosomes that are invisible to sequence-based annotation. Similarly, R. difficilis, a human gut symbiont, has been found to possess genes for an atypical cohesin-based assembly enriched in amylases and related starch-binding proteins, which may enable this microbe to degrade resistant starches that evade digestion in the upper gastrointestinal tract. Together, these findings reveal that ruminococcal cellulosomes are far more prevalent and diverse than previously appreciated and demonstrate the power of structural proteomics to uncover deeply divergent functional systems in the gut microbiome. IMPORTANCE Plant cell wall polysaccharides are a major dietary carbon source, yet their degradation relies on rare, highly specialized microbial enzyme assemblies known as cellulosomes, which have long been considered uncommon in the human gut. Using proteome-scale structure prediction combined with experimental validation, we show that cellulosomes are far more widespread and structurally diverse in human-associated Ruminococcus species than previously appreciated. We identify multiple new cohesin families and reveal distinct cellulosome architectures likely adapted to degrade different dietary substrates. Together, these findings redefine the distribution and evolution of cellulosomes in gut microbes and demonstrate the power of structural proteomics to uncover deeply diverged biological systems. Plant cell wall polysaccharides are a major dietary carbon source, yet their degradation relies on rare, highly specialized microbial enzyme assemblies known as cellulosomes, which have long been considered uncommon in the human gut. Using proteome-scale structure prediction combined with experimental validation, we show that cellulosomes are far more widespread and structurally diverse in human-associated Ruminococcus species than previously appreciated. We identify multiple new cohesin families and reveal distinct cellulosome architectures likely adapted to degrade different dietary substrates. Together, these findings redefine the distribution and evolution of cellulosomes in gut microbes and demonstrate the power of structural proteomics to uncover deeply diverged biological systems.