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Background: Hadal trenches (>6000 m) are the deepest oceanic regions on Earth and depocenters for organic materials. However, how these enigmatic microbial ecosystems are fueled is largely unknown, particularly the proportional importance of complex polysaccharides introduced through deposition from the photic surface waters above. In surface waters, Bacteroidetes are keystone taxa for the cycling of various algal-derived polysaccharides and the flux of carbon through the photic zone. However, their role in the hadal microbial loop is almost unknown. Results: Here, culture-dependent and culture-independent methods were used to study the potential of Bacteroidetes to catabolize diverse polysaccharides in Mariana Trench waters. Compared to surface waters, the bathypelagic (1000–4000 m) and hadal (6000–10,500 m) waters harbored distinct Bacteroidetes communities, with Mesoflavibacter being enriched at ≥ 4000 m and Bacteroides and Provotella being enriched at 10,400–10,500 m. Moreover, these deep-sea communities possessed distinct gene pools encoding for carbohydrate active enzymes (CAZymes), suggesting different polysaccharide sources are utilised in these two zones. Compared to surface counterparts, deep-sea Bacteroidetes showed significant enrichment of CAZyme genes frequently organized into polysaccharide utilization loci (PULs) targeting algal/plant cell wall polysaccharides (i.e., hemicellulose and pectin), that were previously considered an ecological trait associated with terrestrial Bacteroidetes only. Using a hadal Mesoflavibacter isolate (MTRN7), functional validation of this unique genetic potential was demonstrated. MTRN7 could utilize pectic arabinans, typically associated with land plants and phototrophic algae, as the carbon source under simulated deep-sea conditions. Interestingly, a PUL we demonstrate is likely horizontally acquired from coastal/land Bacteroidetes was activated during growth on arabinan and experimentally shown to encode enzymes that hydrolyze arabinan at depth. Conclusions: Our study implies that hadal Bacteroidetes exploit polysaccharides poorly utilized by surface populations via an expanded CAZyme gene pool. We propose that sinking cell wall debris produced in the photic zone can serve as an important carbon source for hadal heterotrophs and play a role in shaping their communities and metabolism. 4KfyME3i8Y2_7Ng17-RaMr Video Abstract [ABSTRACT FROM AUTHOR]

Glycans facilitate critical biological functions and control the mammalian gut microbiota composition by supplying differentially accessible nutrients to distinct microbial subsets. Therefore, identifying unique glycan substrates that support defined microbial populations could inform therapeutic avenues to treat diseases via modulation of the gut microbiota composition and metabolism. However, examining heterogeneous glycan mixtures for individual microbial substrates is hindered by glycan structural complexity and diversity, which presents substantial challenges to glycomics approaches. Fortuitously, gut microbes encode specialized sensor proteins that recognize unique glycan structures and in-turn activate predictable, specific, and dynamic transcriptional responses. Here, we harness this microbial machinery to indicate the presence and abundance of compositionally similar, yet structurally distinct glycans, using a transcriptional reporter we develop. We implement these tools to examine glycan mixtures, isolate target molecules for downstream characterization, and quantify the recovered products. We assert that this toolkit could dramatically enhance our understanding of the mammalian intestinal environment and identify host-microbial interactions critical for human health. Detecting distinct glycans within heterogeneous mixtures is hindered by glycan structural complexity and diversity. Here the authors exploit the ability of gut microbes to sense different glycan structures in order to develop quantitative glycan biosensors by coupling bacterial detection machinery to an optimised luciferase reporter. [ABSTRACT FROM AUTHOR]

Protein synthesis is crucial for cell growth and survival yet one of the most energy‐consuming cellular processes. How, then, do cells sustain protein synthesis under starvation conditions when energy is limited? To accelerate the translocation of mRNA–tRNAs through the ribosome, bacterial elongation factor G (EF‐G) hydrolyzes energy‐rich guanosine triphosphate (GTP) for every amino acid incorporated into a protein. Here, we identify an EF‐G paralog—EF‐G2—that supports translocation without hydrolyzing GTP in the gut commensal bacterium Bacteroides thetaiotaomicron. EF‐G2's singular ability to sustain protein synthesis, albeit at slow rates, is crucial for bacterial gut colonization. EF‐G2 is ~10‐fold more abundant than canonical EF‐G1 in bacteria harvested from murine ceca and, unlike EF‐G1, specifically accumulates during carbon starvation. Moreover, we uncover a 26‐residue region unique to EF‐G2 that is essential for protein synthesis, EF‐G2 dissociation from the ribosome, and responsible for the absence of GTPase activity. Our findings reveal how cells curb energy consumption while maintaining protein synthesis to advance fitness in nutrient‐fluctuating environments. Synopsis: Gut‐commensal Bacteroides thetaiotaomicron harbors a second paralog of translation elongation factor EF‐G, EF‐G2. Here, EF‐G2 is found to increase bacterial fitness in the nutrient‐fluctuating gut environment by mediating slow translation without GTP hydrolysis. EF‐G2 is abundant specifically during carbon starvation when energy is limited.EF‐G2 promotes ribosome translocation and protein synthesis without consuming GTP.A unique insert conserved in all Bacteroides dictates EF‐G2's energy‐saving activity.Protein synthesis mediated by EF‐G2 is essential for gut colonization. [ABSTRACT FROM AUTHOR]

The gut microbiome plays an integral role in health and disease, and diet is a major driver of its composition, diversity, and functional capacity. Given the dynamic development of the gut microbiome in infants and children, it is critical to address two major questions: (a) Can diet modify the composition, diversity, or function of the gut microbiome, and (b) will such modification affect functional/clinical outcomes including immune function, cognitive development, and overall health? We synthesize the evidence on the effect of nutritional interventions on the gut microbiome in infants and children across 26 studies. Findings indicate the need to study older children, assess the whole intestinal tract, and harmonize methods and interpretation of findings, which are critical for informing meaningful clinical and public health practice. These findings are relevant for precision health, may help identify windows of opportunity for intervention, and may inform the design and delivery of such interventions. [ABSTRACT FROM AUTHOR]

Two-component systems (TCSs) are ubiquitous signaling pathways, typically comprising a sensory histidine kinase (HK) and a response regulator, which communicate via intermolecular kinase-to-receiver domain phosphotransfer. Hybrid HKs constitute non-canonical TCS signaling pathways, with transmitter and receiver domains within a single protein communicating via intramolecular phosphotransfer. Here, we report how evolutionary relationships between hybrid HKs can be used as predictors of potential intermolecular and intramolecular interactions ('phylogenetic promiscuity'). We used domain-swap genes chimeras to investigate the specificity of phosphotransfer within hybrid HKs of the GacS–GacA multikinase network of Pseudomonas brassicacearum. The receiver domain of GacS was replaced with those from nine donor hybrid HKs. Three chimeras with receivers from other hybrid HKs demonstrated correct functioning through complementation of a gacS mutant, which was dependent on strains having a functional gacA. Formation of functional chimeras was predictable on the basis of evolutionary heritage, and raises the possibility that HKs sharing a common ancestor with GacS might remain components of the contemporary GacS network. The results also demonstrate that understanding the evolutionary heritage of signaling domains in sophisticated networks allows their rational rewiring by simple domain transplantation, with implications for the creation of designer networks and inference of functional interactions. [ABSTRACT FROM AUTHOR]

The Bacteroidetes are numerically abundant Gram-negative organisms of the distal human gut with a greatly expanded capacity to degrade complex glycans. A subset of these are adept at scavenging host glycans within this environment, including mucin O -linked glycans, N -linked glycoproteins and highly sulfated glycosaminoglycans (GAGs) such as heparin (Hep) and chondroitin sulfate (CS). Several recent biochemical studies have revealed the specific polysaccharide utilization loci (PULs) within the model symbiont Bacteroides thetaiotaomicron for the deconstruction of these host glycans. Here we discuss the Sus-like paradigm that defines glycan uptake by the Bacteroidetes and the salient details of the PULs that target heparin/heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (DS)/hyaluronic acid (HA), respectively, in B. thetaiotaomicron. The ability of the Bacteroidetes to target highly sulfated host glycans is key to their success in the gut environment but can lead to inflammation in susceptible hosts. Therefore, our continued understanding of the molecular strategies employed by these bacteria to scavenge carbohydrate nutrition is likely to lead to novel ways to alter their metabolism to promote host health. [ABSTRACT FROM AUTHOR]

Aims/Introduction: Gut dysbiosis is generally associated with type 2 diabetes mellitus. However, the effect of habitual dietary intake on gut dysbiosis in Japanese patients with type 2 diabetes mellitus has not yet been explicated. This study investigated whether alteration of the gut microbiota was influenced by dietary intake of sucrose in Japanese patients with type 2 diabetes mellitus. Materials and Methods: In this cross‐sectional study, 97 patients with type 2 diabetes mellitus and 97 healthy individuals were matched by age and sex, and then, fecal samples were obtained. Next‐generation sequencing of the 16S ribosomal ribonucleic acid gene was carried out, and functional profiles for the gut microbiota were analyzed. We selected the top 30 gut microbial genera and top 20 functional profiles for the gut microbiota specified by the weighted average difference method. The association between gut microbial genera or functional profiles and habitual dietary intake was investigated by Spearman's rank correlation coefficient, and then, clustering analysis was carried out to clarify the impact of habitual dietary intake. Results: The Actinobacteria phylum was highly abundant in patients with type 2 diabetes mellitus, whereas the Bacteroidetes phylum was less abundant. Diabetic‐type gut microbes, specifically Bacteroides and Bifidobacterium, were altered by sucrose intake at the genus level. Furthermore, sucrose intake was associated with glycolysis/gluconeogenesis in the diabetic‐type functional profiles of the gut microbiota. Conclusions: The gut microbiota and functional profiles for the gut microbiota in patients with type 2 diabetes mellitus were significantly different from those in healthy individuals. Furthermore, we showed that sucrose intake was closely associated with these differences. [ABSTRACT FROM AUTHOR]

The human gut microbiota (HGM), which is critical to human health, utilises complex glycans as its major carbon source. Glycosaminoglycans represent an important, high priority, nutrient source for the HGM. Pathways for the metabolism of various glycosaminoglycan substrates remain ill-defined. Here we perform a biochemical, genetic and structural dissection of the genetic loci that orchestrates glycosaminoglycan metabolism in the organism Bacteroides thetaiotaomicron. Here, we report: the discovery of two previously unknown surface glycan binding proteins which facilitate glycosaminoglycan import into the periplasm; distinct kinetic and genetic specificities of various periplasmic lyases which dictate glycosaminoglycan metabolic pathways; understanding of endo sulfatase activity questioning the paradigm of how the 'sulfation problem' is handled by the HGM; and 3D crystal structures of the polysaccharide utilisation loci encoded sulfatases. Together with comparative genomic studies, our study fills major gaps in our knowledge of glycosaminoglycan metabolism by the HGM. Glycosaminoglycans (GAGs) are an important nutrient source for the gut microbiome. Here, the authors characterize the genetic loci that underpins glycosaminoglycan utilization in Bacteroides thetaiotaomicron; providing insights into the metabolism of GAGs by a predominant member of the gut microbiota. [ABSTRACT FROM AUTHOR]

Gut residential hundred trillion microbial cells are indispensable for maintaining gut homeostasis and impact on host physiology, development and immune systems. Many of them have displayed excellence in utilising dietary- and host-derived complex glycans and are producing useful postbiotics including short-chain fatty acids to primarily fuel different organs of the host. Therefore, employing individual microbiota is nowadays becoming a propitious target in biomedical for improving gut dysbiosis conditions of the host. Among other gut microbial communities, Bacteroides and Bifidobacteria are coevolved to utilise diverse ranges of diet- and host-derived glycans through harmonising distinct glycan utilisation systems. These gut symbionts frequently share digested oligosaccharides, carbohydrate-active enzymes and fermentable intermediate molecules for sustaining gut microbial symbiosis and improving fitness of own or other communities. Genomics approaches have provided unprecedented insights into these functions, but their precise mechanisms of action have poorly known. Sympathetic glycan-utilising strategy of each gut commensal will provide overview of mechanistic dynamic nature of the gut environment and will then assist in applying aptly personalised nutritional therapy. Thus, the review critically summarises cutting edge understanding of major plant- and host-derived glycan-utilising systems of Bacteroides and Bifidobacteria. Their evolutionary adaptation to gut environment and roles of postbiotics in human health are also highlighted. [ABSTRACT FROM AUTHOR]

Perceiving environmental and internal information and reacting in adaptive ways are essential attributes of living organisms. Two-component systems are relevant protein machineries from prokaryotes and lower eukaryotes that enable cells to sense and process signals. Implicating sensory histidine kinases and response regulator proteins, both components take advantage of protein phosphorylation and flexibility to switch conformations in a signal-dependent way. Dozens of two-component systems act simultaneously in any given cell, challenging our understanding about the means that ensure proper connectivity. This review dives into the molecular level, attempting to summarize an emerging picture of how histidine kinases and cognate response regulators achieve required efficiency, specificity, and directionality of signaling pathways, properties that rely on protein:protein interactions. α helices that carry information through long distances, the fine combination of loose and specific kinase/regulator interactions, and malleable reaction centers built when the two components meet emerge as relevant universal principles. [ABSTRACT FROM AUTHOR]

The human microbiota, which plays an important role in health and disease, uses complex carbohydrates as a major source of nutrients. Utilization hierarchy indicates that the host glycosaminoglycans heparin (Hep) and heparan sulfate (HS) are high-priority carbohydrates for Bacteroides thetaiotaomicron, a prominent member of the human microbiota. The sulfation patterns of these glycosaminoglycans are highly variable, which presents a significant enzymatic challenge to the polysaccharide lyases and sulfatases that mediate degradation. It is possible that the bacterium recruits lyases with highly plastic specificities and expresses a repertoire of enzymes that target substructures of the glycosaminoglycans with variable sulfation or that the glycans are desulfated before cleavage by the lyases. To distinguish between these mechanisms, the components of the B. thetaiotaomicron Hep/HS degrading apparatus were analyzed. The data showed that the bacterium expressed a single-surface endo-acting lyase that cleaved HS, reflecting its higher molecular weight compared with Hep. Both Hep and HS oligosaccharides imported into the periplasm were degraded by a repertoire of lyases, with each enzyme displaying specificity for substructures within these glycosaminoglycans that display a different degree of sulfation. Furthermore, the crystal structures of a key surface glycan binding protein, which is able to bind both Hep and HS and periplasmic sulfatases reveal the major specificity determinants for these proteins. The locus described here is highly conserved within the human gut Bacteroides, indicating that the model developed is of generic relevance to this important microbial community. [ABSTRACT FROM AUTHOR]

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls diverse functions in bacteria, including transitions from planktonic to biofilm lifestyles, virulence, motility, and cell cycle. Here we describe TolR, a hybrid two-component system (HTCS), from the β-proteobacterium Azoarcus sp. strain CIB that degrades c-di-GMP in response to aromatic hydrocarbons, including toluene. This response protects cells from toluene toxicity during anaerobic growth. Whereas wild-type cells tolerated a sudden exposure to a toxic concentration of toluene, a tolR mutant strain or a strain overexpressing a diguanylate cyclase gene lost viability upon toluene shock. TolR comprises an N-terminal aromatic hydrocarbon-sensing Per-Arnt-Sim (PAS) domain, followed by an autokinase domain, a response regulator domain, and a C-terminal c-di-GMP phosphodiesterase (PDE) domain. Autophosphorylation of TolR in response to toluene exposure initiated an intramolecular phosphotransfer to the response regulator domain that resulted in c-di-GMP degradation. The TolR protein was engineered as a functional sensor histidine kinase (TolRSK) and an independent response regulator (TolRRR). This classic two-component system (CTCS) operated less efficiently than TolR, suggesting that TolR was evolved as a HTCS to optimize signal transduction. Our results suggest that TolR enables Azoarcus sp. CIB to adapt to toxic aromatic hydrocarbons under anaerobic conditions by modulating cellular levels of c-di-GMP. This is an additional role for c-di-GMP in bacterial physiology. [ABSTRACT FROM AUTHOR]

Two-component systems are a dominant form of bacterial signal transduction. The prototypical two-component system consists of a sensor that responds to a specific input(s) by modifying the output of a cognate regulator. Because the output of a two-component system is the amount of phosphorylated regulator, feedback mechanisms may alter the amount of regulator, and/or modify the ability of a sensor or other proteins to alter the phosphorylation state of the regulator. Two-component systems may display intrinsic feedback whereby the amount of phosphorylated regulator changes under constant inducing conditions and without the participation of additional proteins. Feedback control allows a two-component system to achieve particular steady-state levels, to reach a given steady state with distinct dynamics, to express coregulated genes in a given order, and to activate a regulator to different extents, depending on the signal acting on the sensor. [ABSTRACT FROM AUTHOR]

Resident bacteria in the densely populated human intestinal tract must efficiently compete for carbohydrate nutrition. The Bacteroidetes, a dominant bacterial phylum in the mammalian gut, encode a plethora of discrete polysaccharide utilization loci (PULs) that are selectively activated to facilitate glycan capture at the cell surface. The most well-studied PUL-encoded glycan-uptake system is the starch utilization system (Sus) of Bacteroides thetaiotaomicron. The Sus includes the requisite proteins for binding and degrading starch at the surface of the cell preceding oligosaccharide transport across the outer membrane for further depolymerization to glucose in the periplasm. All mammalian gut Bacteroidetes possess analogous Sus-like systems that target numerous diverse glycans. In this review, we discuss what is known about the eight Sus proteins of B. thetaiotaomicron that define the Sus-like paradigm of nutrient acquisition that is exclusive to the Gram-negative Bacteroidetes. We emphasize the well-characterized outer membrane proteins SusDEF and the α-amylase SusG, each of which have unique structural features that allow them to interact with starch on the cell surface. Despite the apparent redundancy in starch-binding sites among these proteins, each has a distinct role during starch catabolism. Additionally, we consider what is known about how these proteins dynamically interact and cooperate in the membrane and propose a model for the formation of the Sus outer membrane complex. [ABSTRACT FROM AUTHOR]

Bacteria secrete enzymes into the extracellular space to hydrolyze macromolecules into constituents that can be imported for microbial nutrition. In bacterial communities, these enzymes and their resultant products can be modeled as community property. Our goal was to investigate the impact of individual community member absence on the resulting community production of exoenzymes (extracellular enzymes) involved in lipid and protein hydrolysis. Our model community contained nine bacteria isolated from the potable water system of the International Space Station. Bacteria were grown in static conditions individually, all together, or in all combinations of eight species and exoproduct production was measured by colorimetric or fluorometric reagents to assess short chain and long chain lipases, choline-specific phospholipases C, and proteases. The exoenzyme production of each species grown alone varied widely, however, the enzyme activity levels of the mixed communities were functionally robust to absence of any single species, with the exception of phospholipase C production in one community. For phospholipase C, absence of Chryseobacterium gleum led to increased choline-specific phospholipase C production, correlated with increased growth of Burkholderia cepacia and Sphingomonas sanguinis. Because each individual species produced different enzyme activity levels in isolation, we calculated an expected activity value for each bacterial mixture using input levels or known final composition. This analysis suggested that robustness of each exoenzyme activity is not solely mediated by community composition, but possibly influenced by bacterial communication, which is known to regulate such pathways in many bacteria. We conclude that in this simplified model of a drinking water bacterial community, community structure imposes constraints on production and/or secretion of exoenzymes to generate a level appropriate to exploit a given nutrient environment. [ABSTRACT FROM AUTHOR]

Genetically engineering intestinal bacteria, such as Bacteroides thetaiotaomicron (B. theta), holds potential for creating new classes of biological devices, such as diagnostics or therapeutic delivery systems. Here, we have developed a series of B. theta strains that produce functional transgenic enzymes in response to dextran and arabinogalactan, two chemically distinct glycans. Expression systems for single glycan induction, and a novel "dual-glycan" expression system, requiring the presence of both dextran and arabinogalactan, have been developed. In addition, we have created two different chromosomal integration systems and one episomal vector system, compatible with engineered recipient strains, to improve the throughput and flexibility of gene cloning, integration, and expression in B. theta. To monitor activity, we have demonstrated the functionality of two different transgenic enzymes: NanoLuc, a luciferase, and BuGH16C, an agarase from the human intestinal bacterium, Bacteroides uniforms NP1. Together this expression platform provides a new collection of glycan-responsive tools to improve the strength and fidelity of transgene expression in B. theta and provides proof-of-concept for engineering more complex multi-glycan expression systems. [ABSTRACT FROM AUTHOR]