The human gut microbiota, including Bacteroides, is required for the degradation of otherwise undigestible polysaccharides. The gut microbiota uses polysaccharides as an energy source, and fermentation products such as short-chain fatty acids are beneficial to the human host. This use of polysaccharides is dependent on the proper pairing of a TonB protein with polysaccharide-specific TonB-dependent transporters; however, the formation of these protein complexes is poorly understood. In this study, we examine the role of 11 predicted TonB homologs in polysaccharide uptake. We show that two proteins, TonB4 and TonB6, may be functionally redundant. This may allow for the development of drugs targeting Bacteroides species containing only a TonB4 homolog with limited impact on species encoding the redundant TonB6.

Understanding how members of the human gut microbiota prioritize nutrient resources is one component of a larger effort to decipher the mechanisms defining microbial community robustness and resiliency in health and disease. This knowledge is foundational for development of microbiota-directed therapeutics. To model how bacteria prioritize glycans in the gut, germfree mice were colonized with 13 human gut bacterial strains, including seven saccharolytic Bacteroidaceae species. Animals were fed a Western diet supplemented with pea fiber. After community assembly, an inducible CRISPR-based system was used to selectively and temporarily reduce the absolute abundance of Bacteroides thetaiotaomicron or B. cellulosilyticus by 10- to 60-fold. Each knockdown resulted in specific, reproducible increases in the abundances of other Bacteroidaceae and dynamic alterations in their expression of genes involved in glycan utilization. Emergence of these \"alternate consumers\" was associated with preservation of community saccharolytic activity. Using an inducible system for CRISPR base editing in vitro, we disrupted translation of transporters critical for utilizing dietary polysaccharides in Phocaeicola vulgatus, a B. cellulosilyticus knockdown-responsive taxon. In vitro and in vivo tests of the resulting P. vulgatus mutants allowed us to further characterize mechanisms associated with its increased fitness after knockdown. In principle, the approach described can be applied to study utilization of a range of nutrients and to preclinical efforts designed to develop therapeutic strategies for precision manipulation of microbial communities.

Glycoside hydrolases (GHs) are carbohydrate-active enzymes essential for many environmental (e.g., carbon cycling) and biotechnological (e.g., biofuels) processes. The complete processing of carbohydrates by bacteria requires many enzymes acting synergistically. Here, I investigated the clustered or scattered distribution of 406,337 GH-genes and their association with transporter genes identified in 15,640 completely sequenced bacterial genomes. Different bacterial lineages displayed conserved levels of clustered or scattered GH-genes, but overall, the GH-genes clustering was generally higher than in randomized genomes. In lineages with highly clustered GH-genes (e.g., Bacteroides, Paenibacillus), clustered genes shared the same orientation. These codirectional gene clusters potentially facilitate the genes\' co-expression by allowing transcriptional read-through and, at least in some cases, forming operons. In several taxa, the GH-genes clustered with distinct types of transporter genes. The type of transporter genes and the distribution of the so-called GH:TR-genes clusters were conserved in selected lineages. Globally, the phylogenetically conserved clustering of the GH-genes with transporter genes highlights the central function of carbohydrate processing across bacterial lineages. In addition, in bacteria with the most identified GH-genes, the genomic adaptations for carbohydrate processing also mirrored the broad environmental origin of the sequenced strains (e.g., soil and mammal gut) suggesting that a combination of evolutionary history and the environment selects for the specific supragenic organization of the GH-genes supporting the carbohydrate processing in bacterial genomes.

Commercially-supplied potato galactan (PG) is widely used as a model polysaccharide in various bioactivity studies. However, results using this galactan are not always consistent with the stated composition. Here, we assessed its composition by fractionating this commercial PG and purified its primary components: PG-A, PG-B and PG-Cp with weight-averaged molecular weights of 430, 93, and 11.3 kDa, respectively. PG-Cp consists of free β-1,4-galactan chains, whereas PG-A and PG-B are type I rhamnogalacturonans with long β-1,4-galactan side chains of up to 80 Gal residues and short β-1,4-galactan side chains of 0 to 3 Gal residues that display a \"trees in lawn\" pattern. Structures of these polysaccharides correlate well with their activities in terms of galectin-3 binding and gut bacterial growth assays. Our study clarifies the confusion related to commercial PG, with purified fractions serving as better model polysaccharides in bioactivity investigations.

Marine herbivorous fish that feed primarily on macroalgae, such as those from the genus Kyphosus, are essential for maintaining coral health and abundance on tropical reefs. Here, deep metagenomic sequencing and assembly of gut compartment-specific samples from three sympatric, macroalgivorous Hawaiian kyphosid species have been used to connect host gut microbial taxa with predicted protein functional capacities likely to contribute to efficient macroalgal digestion. Bacterial community compositions, algal dietary sources, and predicted enzyme functionalities were analyzed in parallel for 16 metagenomes spanning the mid- and hindgut digestive regions of wild-caught fishes. Gene colocalization patterns of expanded carbohydrate (CAZy) and sulfatase (SulfAtlas) digestive enzyme families on assembled contigs were used to identify likely polysaccharide utilization locus associations and to visualize potential cooperative networks of extracellularly exported proteins targeting complex sulfated polysaccharides. These insights into the gut microbiota of herbivorous marine fish and their functional capabilities improve our understanding of the enzymes and microorganisms involved in digesting complex macroalgal sulfated polysaccharides. IMPORTANCE This work connects specific uncultured bacterial taxa with distinct polysaccharide digestion capabilities lacking in their marine vertebrate hosts, providing fresh insights into poorly understood processes for deconstructing complex sulfated polysaccharides and potential evolutionary mechanisms for microbial acquisition of expanded macroalgal utilization gene functions. Several thousand new marine-specific candidate enzyme sequences for polysaccharide utilization have been identified. These data provide foundational resources for future investigations into suppression of coral reef macroalgal overgrowth, fish host physiology, the use of macroalgal feedstocks in terrestrial and aquaculture animal feeds, and the bioconversion of macroalgae biomass into value-added commercial fuel and chemical products.

The gut microbiome exerts a powerful influence on human health and disease. Elucidating the underlying mechanisms of the microbiota\'s influence is hindered by the immense complexity of the gut microbial community and the glycans they forage. Despite a wealth of genomic and metagenomic sequencing information, there remains a lack of informative phenotypic measurements. Pudlo NA, Urs K, Crawford R, Pirani A, et al. (mSystems 7: e00947-21, 2022, https://doi.org/10.1128/msystems.00947-21) decode this complexity by introducing a scalable assay to measure specific carbohydrate utilization in the dominant microbiota phylum Bacteroidetes. The results reveal a mosaic structure of glycan utilization, both genetic and functional, underpinning niche construction in the human gastrointestinal tract. This Commentary highlights the significance of their findings in connection to the field\'s growing appreciation for competition, cooperation, and horizontal gene transfer in shaping the highly complex microbial community.

Although the strategies used by bacteria to adapt to specific environmental conditions are widely reported, fewer studies have addressed how microbes with a cosmopolitan distribution can survive in diverse ecosystems. Exiguobacterium is a versatile genus whose members are commonly found in various habitats. To better understand the mechanisms underlying the universality of Exiguobacterium, we collected 105 strains from diverse environments and performed large-scale metabolic and adaptive ability tests. We found that most Exiguobacterium members have the capacity to survive under wide ranges of temperature, salinity, and pH. According to phylogenetic and average nucleotide identity analyses, we identified 27 putative species and classified two genetic groups: groups I and II. Comparative genomic analysis revealed that the Exiguobacterium members utilize a variety of complex polysaccharides and proteins to support survival in diverse environments and also employ a number of chaperonins and transporters for this purpose. We observed that the group I species can be found in more diverse terrestrial environments and have a larger genome size than the group II species. Our analyses revealed that the expansion of transporter families drove genomic expansion in group I strains, and we identified 25 transporter families, many of which are involved in the transport of important substrates and resistance to environmental stresses and are enriched in group I strains. This study provides important insights into both the overall general genetic basis for the cosmopolitan distribution of a bacterial genus and the evolutionary and adaptive strategies of Exiguobacterium. IMPORTANCE The wide distribution characteristics of Exiguobacterium make it a valuable model for studying the adaptive strategies of bacteria that can survive in multiple habitats. In this study, we reveal that members of the Exiguobacterium genus have a cosmopolitan distribution and share an extensive adaptability that enables them to survive in various environments. The capacities shared by Exiguobacterium members, such as their diverse means of polysaccharide utilization and environmental-stress resistance, provide an important basis for their cosmopolitan distribution. Furthermore, the selective expansion of transporter families has been a main driving force for genomic evolution in Exiguobacterium. Our findings improve our understanding of the adaptive and evolutionary mechanisms of cosmopolitan bacteria and the vital genomic traits that can facilitate niche adaptation.

Harnessing the microbiota for beneficial outcomes is limited by our poor understanding of the constituent bacteria, as the functions of most of their genes are unknown. Here, we measure the growth of a barcoded transposon mutant library of the gut commensal Bacteroides thetaiotaomicron on 48 carbon sources, in the presence of 56 stress-inducing compounds, and during mono-colonization of gnotobiotic mice. We identify 516 genes with a specific phenotype under only one or a few conditions, enabling informed predictions of gene function. For example, we identify a glycoside hydrolase important for growth on type I rhamnogalacturonan, a DUF4861 protein for glycosaminoglycan utilization, a 3-keto-glucoside hydrolase for disaccharide utilization, and a tripartite multidrug resistance system specifically for bile salt tolerance. Furthermore, we show that B. thetaiotaomicron uses alternative enzymes for synthesizing nitrogen-containing metabolic precursors based on ammonium availability and that these enzymes are used differentially in vivo in a diet-dependent manner.

Evaluation of bacterial succession with cultivation-dependent strategies during a spring phytoplankton bloom in the North Sea led to the isolation of 41 strains that affiliated with the genus Winogradskyella. Fifteen of the strains were selected for a taxonomic study after discarding clonal cultures. A thorough phylogenetic, genomic and phenotypic analysis of the isolates indicated that they represented eight new species that coexisted in North Sea waters. Molecular data revealed the existence of an as yet uncultivated novel species recurrently binned from the North Sea metagenomes. The metagenome-assembled genomes (MAGs) of this new Winogradskyella were used to classify it as a new Candidatus species. This study represented a new example of the use of the tandem approach of whole cell mass spectrometry linked to 16S rRNA gene sequencing in order to facilitate the discovery of new taxa by high-throughput cultivation, which increases the probability of finding more than a single isolate for new species. In addition, we demonstrated the reasons for classifying MAGs representing recurrently retrieved heterotrophic species that evade cultivation even after an important high-throughput effort. The taxonomic study resulted in the classification of eight new species and one new Candidatus species of the genus Winogradskyella for which we propose the names W. schleiferi sp. nov., W. costae sp. nov., W. helgolandensis sp. nov., W. vidalii sp. nov., W. forsetii sp. nov., W. ludwigii sp. nov., W. ursingii sp. nov., W. wichelsiae sp. nov., and Candidatus \"W. atlantica\" sp. nov.

Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon-haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon\'s ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon-haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner\'s ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host.