The human gut microbiota (HGM) is profoundly influenced by dietary carbohydrates, impacting its composition and function. Manipulating the HGM through dietary interventions using indigestible carbohydrates offers a promising strategy to enhance gut health by selectively increasing beneficial microorganisms. Understanding the glycan metabolic abilities of specific HGM species is crucial for developing systematic methods for microbiota manipulation. The HGM possesses a diverse array of carbohydrate-active enzymes (CAZymes) to break down complex carbohydrates, highlighting the importance of carbohydrate availability in microbiota adaptation. While the influence of specific carbohydrates on niche acquisition in animal models and infants is known, the relationship between carbohydrate availability and bacterial growth in the adult human gut remains largely unclear due to its complex and highly variable composition. Bifidobacteria, a core component of the HGM, are characterized by their diverse carbohydrate metabolism genes. Glycan metabolism is essential for their adaptation to the intestinal environment. While the glycan utilization of several bifidobacterial species has been studied (e.g., infant-dominant species utilize human milk oligosaccharides (HMOs), while adult-dominant species utilize plant-derived polysaccharides), the carbohydrate utilization of B. pseudocatenulatum, a prevalent species in the adult gut, remains under-investigated. Long-chain xylans (LCXs), abundant in plant cell walls and the human diet, are complex polysaccharides. In the HGM, a limited number of Bacteroidetes and Firmicutes species can hydrolyze LCXs, with endo-1,4-β-xylanases playing a key role in cleaving the xylose backbone. These enzymes release oligosaccharides which can then be utilized by other bacteria. Bifidobacteria were previously considered secondary LCX consumers, lacking the ability to cleave the xylose backbone. This study focuses on investigating the LCX utilization potential of B. pseudocatenulatum and its ecological implications.

The literature extensively documents the intricate interplay between the human gut microbiota and host health. Dietary glycans serve as major nutrients for the gut microbiota, significantly influencing its composition. Dietary interventions using indigestible carbohydrates offer a strategy for selectively increasing beneficial microorganisms. Studies have explored the diverse array of CAZymes within the HGM, emphasizing the role of carbohydrate availability in microbiota adaptation. While research has shown carbohydrate availability's impact on niche acquisition in animal models and infants, the relationship between specific carbohydrate availability and bacterial growth in the complex adult human gut requires further investigation. Bifidobacteria, a crucial part of the HGM, possess a rich repertoire of carbohydrate metabolism genes, suggesting that glycan metabolism is essential for their adaptation to the intestinal environment. Previous studies have investigated the glycan utilization patterns of various bifidobacterial species, highlighting the differences between infant-dominant and adult-dominant taxa. Infant-dominant species often utilize HMOs, while adult-dominant species utilize plant-derived polysaccharides, including the degradation products of LCXs. LCXs are major components of plant cell walls and are commonly found in human diets, yet relatively little is known about the bifidobacteria’s ability to utilize them directly. Previous studies have shown that some, but not all strains of Bifidobacteria can use LCX derived oligosaccharides.

This study employed a multi-faceted approach combining genomic analysis, phenotypic characterization, in vitro co-culture experiments, and a human dietary intervention trial. **Genome Sequencing and Analysis:** The genomes of 35 B. pseudocatenulatum strains were sequenced using various platforms (Illumina MiSeq, Oxford Nanopore Technologies MinION, and Pacific Biosciences PacBio RS2). Genome assembly, gene prediction, and pan-genome analysis were performed to identify carbohydrate-active enzymes (CAZymes). The average gene copy number of CAZyme families was calculated for each genome. **Carbohydrate Utilization Analysis:** The ability of B. pseudocatenulatum strains to utilize various carbohydrates (XOS, arabinoxylan, xylan) was assessed by measuring growth and organic acid production. **Cloning, Expression, and Purification of Recombinant BpXyn10A:** The GH10 domain of the BpXyn10A gene was cloned, expressed in E. coli, and purified. **Endo-Xylanase Activity Assay:** Endo-xylanase activity was measured in cell supernatants and cell fractions using a Xylanase Assay Kit. **Purified BpXyn10A-Added Culture:** The effect of adding purified recombinant BpXyn10A to cultures of LCX-utilizing and non-utilizing B. pseudocatenulatum strains was investigated. **RNA-seq Analysis:** RNA-seq was performed on B. pseudocatenulatum YIT 11952 cultured with various carbohydrates to assess the expression levels of genes in the LCX utilization pathway. **Co-culture Experiments:** Co-culture experiments were conducted with B. pseudocatenulatum strains and other HGM species (Bacteroides ovatus and B. longum subsp. longum) to evaluate the impact of BpXyn10A on interspecies interactions. **Cereal Intervention Study:** A dietary intervention study involved 27 adult participants who consumed LCX-rich cereal for 14 days. Faecal samples were collected to assess changes in B. pseudocatenulatum abundance and the overall composition of the HGM using qPCR and 16S rRNA gene amplicon sequencing. Statistical analysis was performed using appropriate tests (Mann–Whitney U tests, Wilcoxon signed-rank tests, Kruskal-Wallis test, PERMANOVA).

This study revealed several key findings: 1. **LCX Utilization by B. pseudocatenulatum:** All 36 B. pseudocatenulatum strains tested could utilize short-chain xylo-oligosaccharides (XOS). However, only 12 strains, all possessing the BpXyn10A gene (an endo-1,4-β-xylanase), could utilize arabinoxylan and xylan, indicating direct LCX utilization. The BpXyn10A gene showed high sequence similarity among these strains. 2. **Genetic Background of LCX Utilization:** The BpXyn10A gene was found within an arabinoxylan-hydrolyzate (AXH) utilization gene cluster. RNA-seq analysis showed that expression of BpXyn10A and surrounding genes was upregulated by xylose, XOS, and arabinoxylan. This indicates that the BpXyn10A gene was likely acquired through horizontal gene transfer and integrated into a pre-existing carbohydrate utilization pathway. 3. **Impact on Interspecies Interactions:** Co-culture experiments with Bacteroides ovatus showed that LCX-utilizing B. pseudocatenulatum strains were more competitive, even when supplied with pre-digested LCX-derived oligosaccharides. Conversely, the growth of B. ovatus was inhibited in the presence of the BpxYn10A producing strains, suggesting a complex interplay between primary and secondary consumers of LCX. 4. **In Vivo Confirmation:** A dietary intervention study using LCX-rich cereal showed that the abundance of B. pseudocatenulatum, especially strains possessing BpXyn10A, significantly increased in participants during the intervention period. The presence or absence of the BpXyn10A gene also influenced the overall composition of the gut microbiota, suggesting a substantial impact of LCX utilization on the overall gut ecosystem. These observations were corroborated by 16S rRNA amplicon sequencing analyses which showed significant shifts in alpha and beta diversity between groups.

This study demonstrates that LCX availability plays a significant role in promoting the adaptation of B. pseudocatenulatum in the adult human gut. The acquisition of the BpXyn10A gene via horizontal gene transfer has enabled certain strains to directly utilize LCX as a primary degrader, rather than relying solely on pre-digested oligosaccharides. The observed competitive advantage of LCX-utilizing strains, even in the presence of other xylanolytic species, highlights the ecological significance of this newly identified trait. The dietary intervention study convincingly demonstrated the in vivo relevance of these findings, showing a direct link between LCX consumption and the increase in B. pseudocatenulatum abundance, particularly in individuals harboring strains with BpXyn10A. The study provides crucial insight into the complex interactions within the gut microbial ecosystem, revealing the impact of a single enzyme on the overall microbiota composition.

This study provides compelling evidence for the importance of LCX utilization in the adaptation of B. pseudocatenulatum to the human gut environment. The acquisition of the BpXyn10A gene, likely through horizontal gene transfer, confers a significant competitive advantage, enabling strains to thrive in LCX-rich environments. The findings highlight the potential for manipulating the gut microbiota through targeted dietary interventions. Future research should focus on expanding our understanding of the genetic diversity within B. pseudocatenulatum and other bifidobacterial species to identify additional enzymes and pathways involved in polysaccharide utilization, potentially leading to the development of personalized dietary interventions to optimize gut health.

The study's findings are based on a limited number of participants in the dietary intervention study, and future studies with larger and more diverse populations are needed to confirm the generalizability of the results. The study focused on the impact of LCX on B. pseudocatenulatum, and further investigation is needed to explore the effects of other dietary fibers and polysaccharides on various members of the gut microbiome. While the study provided evidence for the competitive advantage of B. pseudocatenulatum strains possessing BpXyn10A, further research could investigate the specific mechanisms underlying this competitive advantage, including the production of inhibitory compounds or alterations in metabolic pathways.