Assessment of human microbiota stability across longitudinal samples using iteratively growing-partitioned clustering.

Microbiome research is advancing rapidly, and every new study should definitively be based on updated methods, trends and milestones in this field to avoid the wrong interpretation of results. Most human microbiota surveys rely on data captured from snapshots-single data points from subjects-and have permitted uncovering the recognized interindividual variability and major covariates of such microbial communities. Currently, changes in individualized microbiota profiles are under the spotlight to serve as robust predictors of clinical outcomes (e.g. weight loss via dietary interventions) and disease anticipation. Therefore, novel methods are needed to provide robust evaluation of longitudinal series of microbiota data with the aim of assessing intrapersonally short-term to long-term microbiota changes likely linked to health and disease states. Consequently, we developed microbiota STability ASsessment via Iterative cluStering (µSTASIS)-a multifunction R package to evaluate individual-centered microbiota stability. µSTASIS targets the recognized interindividual variability inherent to microbiota data to stress the tight relationships observed among and characteristic of longitudinal samples derived from a single individual via iteratively growing-partitioned clustering. The algorithms and functions implemented in this framework deal properly with the sparse and compositional nature of microbiota data. Moreover, the resulting metric is intuitive and independent of beta diversity distance methods and correlation coefficients, thus estimating stability for each microbiota sample rather than giving nonconsensus magnitudes that are difficult to interpret within and between datasets. Our method is freely available under GPL-3 licensing. We demonstrate its utility by assessing gut microbiota stability from three independent studies published previously with multiple longitudinal series of multivariate data and respective metadata.

Comparative Binding Study of Gliptins to Bacterial DPP4-like Enzymes for the Treatment of Type 2 Diabetes Mellitus (T2DM).

The role of the gut microbiota and its interplay with host metabolic health, particularly in the context of type 2 diabetes mellitus (T2DM) management, is garnering increasing attention. Dipeptidyl peptidase 4 (DPP4) inhibitors, commonly known as gliptins, constitute a class of drugs extensively used in T2DM treatment. However, their potential interactions with gut microbiota remain poorly understood. In this study, we employed computational methodologies to investigate the binding affinities of various gliptins to DPP4-like homologs produced by intestinal bacteria. The 3D structures of DPP4 homologs from gut microbiota species, including Segatella copri, Phocaeicola vulgatus, Bacteroides uniformis, Parabacteroides merdae, and Alistipes sp., were predicted using computational modeling techniques. Subsequently, molecular dynamics simulations were conducted for 200 ns to ensure the stability of the predicted structures. Stable structures were then utilized to predict the binding interactions with known gliptins through molecular docking algorithms. Our results revealed binding similarities of gliptins toward bacterial DPP4 homologs compared to human DPP4. Specifically, certain gliptins exhibited similar binding scores to bacterial DPP4 homologs as they did with human DPP4, suggesting a potential interaction of these drugs with gut microbiota. These findings could help in understanding the interplay between gliptins and gut microbiota DPP4 homologs, considering the intricate relationship between the host metabolism and microbial communities in the gut.

Holdemanella biformis improves glucose tolerance and regulates GLP-1 signaling in obese mice.

Impaired glucose homeostasis in obesity is mitigated by enhancing the glucoregulatory actions of glucagon-like peptide 1 (GLP-1), and thus, strategies that improve GLP-1 sensitivity and secretion have therapeutic potential for the treatment of type 2 diabetes. This study shows that Holdemanella biformis, isolated from the feces of a metabolically healthy volunteer, ameliorates hyperglycemia, improves oral glucose tolerance and restores gluconeogenesis and insulin signaling in the liver of obese mice. These effects were associated with the ability of H. biformis to restore GLP-1 levels, enhancing GLP-1 neural signaling in the proximal and distal small intestine and GLP-1 sensitivity of vagal sensory neurons, and to modify the cecal abundance of unsaturated fatty acids and the bacterial species associated with metabolic health. Our findings overall suggest the potential use of H biformis in the management of type 2 diabetes in obesity to optimize the sensitivity and function of the GLP-1 system, through direct and indirect mechanisms.

The effect of inulin and resistant maltodextrin on weight loss during energy restriction: a randomised, placebo-controlled, double-blinded intervention.

PURPOSE: The objective of this study was to investigate the additive effects of combining energy restriction with dietary fibres on change in body weight and gut microbiota composition. METHODS: The study was a 12-week randomised, placebo-controlled, double-blinded, parallel intervention trial. A total of 116 overweight or obese participants were assigned randomly either to 10 g inulin plus 10 g resistant maltodextrin or to 20 g of placebo supplementation through 400 mL of milk a day, while on a - 500 kcal/day energy restricted diet. RESULTS: Altogether, 86 participants completed the intervention. There were no significant differences in weight loss or body composition between the groups. The fibre supplement reduced systolic (5.35 ± 2.4 mmHg, p = 0.043) and diastolic (2.82 ± 1.3 mmHg, p = 0.047) blood pressure to a larger extent than placebo. Furthermore, a larger decrease in serum insulin was observed in the placebo group compared to the fibre group (- 26.0 ± 9.2 pmol/L, p = 0.006). The intake of fibre induced changes in the composition of gut microbiota resulting in higher abundances of Parabacteroides and Bifidobacteria, compared to placebo. The effects on blood pressure and glucose metabolism were mainly observed in women, and could be attributed to a higher gut microbiota diversity after intervention. Finally, the fibre group experienced a higher degree of gastrointestinal symptoms, which attenuated over time. CONCLUSIONS: Supplementation of inulin and resistant maltodextrin did not provide an additional weight loss during an energy-restricted diet, but reduced both systolic and diastolic blood pressure. Furthermore, the fibre supplement did stimulate the growth of potentially beneficial bacteria genera. CLINICAL TRIAL REGISTRY: The study was registered at http://www.clinicaltrials.gov , NCT03135041.

Bifidobacterium pseudocatenulatum CECT 7765 supplementation improves inflammatory status in insulin-resistant obese children.

PURPOSE: The relationships between gut microbiota and obesity-related co-morbidities have been increasingly recognized. Low-grade inflammation may be the main factor in the pathogenesis of such disorders. We investigated the effect of the potential probiotic Bifidobacterium pseudocatenulatum CECT 7765 on cardiometabolic risk factors, inflammatory cytokines and gut microbiota composition in obese children with insulin resistance. METHODS: The study included 48 obese children (10-15 years old) with insulin resistance. They received dietary advice and were assigned to take the capsules with or without probiotic (109-10 CFU) daily for 13 weeks. Clinical, biochemical and gut microbiome measurements were made at baseline and at the end of the intervention. RESULTS: There was a significant improvement in body mass index in all children after the intervention, suggesting that weight changes are related to the dietary advice. A significant decrease in circulating high-sensitive C-reactive protein (P = 0.026) and monocyte chemoattractant protein-1 (P = 0.032) and an increase in high-density lipoprotein cholesterol (P = 0.035) and omentin-1 (P = 0.023) in children receiving probiotic supplementation were observed compared to the control group. Regarding gut microbiota, probiotic administration significantly increased the proportion of the Rikenellaceae family members, particularly of the Alistipes genus. CONCLUSIONS: The beneficial effects of the intervention on inflammatory markers and lipid profile suggest that B. pseudocatenulatum CECT 7765 intake together with dietary recommendations can improve inflammatory status in children with obesity and insulin resistance. These effects are parallel to increases in bacterial groups associated with a lean phenotype. The modulation of gut microbiota with probiotic supplementation can be considered an effective tool to ameliorate some obesity-related disorders in children.

Bacillus subtilis exhibits MnmC-like tRNA modification activities.

The MnmE-MnmG complex of Escherichia coli uses either ammonium or glycine as a substrate to incorporate the 5-aminomethyl or 5-carboxymethylaminomethyl group into the wobble uridine of certain tRNAs. Both modifications can be converted into a 5-methylaminomethyl group by the independent oxidoreductase and methyltransferase activities of MnmC, which respectively reside in the MnmC(o) and MnmC(m) domains of this bifunctional enzyme. MnmE and MnmG, but not MnmC, are evolutionarily conserved. Bacillus subtilis lacks genes encoding MnmC(o) and/or MnmC(m) homologs. The glycine pathway has been considered predominant in this typical gram-positive species because only the 5-carboxymethylaminomethyl group has been detected in tRNALysUUU and bulk tRNA to date. Here, we show that the 5-methylaminomethyl modification is prevalent in B. subtilis tRNAGlnUUG and tRNAGluUUC. Our data indicate that B. subtilis has evolved MnmC(o)- and MnmC(m)-like activities that reside in non MnmC homologous protein(s), which suggests that both activities provide some sort of biological advantage.

Pangenome-wide and molecular evolution analyses of the Pseudomonas aeruginosa species.

BACKGROUND: Drug treatments and vaccine designs against the opportunistic human pathogen Pseudomonas aeruginosa have multiple issues, all associated with the diverse genetic traits present in this pathogen, ranging from multi-drug resistant genes to the molecular machinery for the biosynthesis of biofilms. Several candidate vaccines against P. aeruginosa have been developed, which target the outer membrane proteins; however, major issues arise when attempting to establish complete protection against this pathogen due to its presumably genotypic variation at the strain level. To shed light on this concern, we proposed this study to assess the P. aeruginosa pangenome and its molecular evolution across multiple strains. RESULTS: The P. aeruginosa pangenome was estimated to contain more than 16,000 non-redundant genes, and approximately 15 % of these constituted the core genome. Functional analyses of the accessory genome indicated a wide presence of genetic elements directly associated with pathogenicity. An in-depth molecular evolution analysis revealed the full landscape of selection forces acting on the P. aeruginosa pangenome, in which purifying selection drives evolution in the genome of this human pathogen. We also detected distinctive positive selection in a wide variety of outer membrane proteins, with the data supporting the concept of substantial genetic variation in proteins probably recognized as antigens. Approaching the evolutionary information of genes under extremely positive selection, we designed a new Multi-Locus Sequencing Typing assay for an informative, rapid, and cost-effective genotyping of P. aeruginosa clinical isolates. CONCLUSIONS: We report the unprecedented pangenome characterization of P. aeruginosa on a large scale, which included almost 200 bacterial genomes from one single species and a molecular evolutionary analysis at the pangenome scale. Evolutionary information presented here provides a clear explanation of the issues associated with the use of protein conjugates from pili, flagella, or secretion systems as antigens for vaccine design, which exhibit high genetic variation in terms of non-synonymous substitutions in P. aeruginosa strains.

Microbiota diversity and gene expression dynamics in human oral biofilms.

BACKGROUND: Micro-organisms inhabiting teeth surfaces grow on biofilms where a specific and complex succession of bacteria has been described by co-aggregation tests and DNA-based studies. Although the composition of oral biofilms is well established, the active portion of the bacterial community and the patterns of gene expression in vivo have not been studied. RESULTS: Using RNA-sequencing technologies, we present the first metatranscriptomic study of human dental plaque, performed by two different approaches: (1) A short-reads, high-coverage approach by Illumina sequencing to characterize the gene activity repertoire of the microbial community during biofilm development; (2) A long-reads, lower-coverage approach by pyrosequencing to determine the taxonomic identity of the active microbiome before and after a meal ingestion. The high-coverage approach allowed us to analyze over 398 million reads, revealing that microbial communities are individual-specific and no bacterial species was detected as key player at any time during biofilm formation. We could identify some gene expression patterns characteristic for early and mature oral biofilms. The transcriptomic profile of several adhesion genes was confirmed through qPCR by measuring expression of fimbriae-associated genes. In addition to the specific set of gene functions overexpressed in early and mature oral biofilms, as detected through the short-reads dataset, the long-reads approach detected specific changes when comparing the metatranscriptome of the same individual before and after a meal, which can narrow down the list of organisms responsible for acid production and therefore potentially involved in dental caries. CONCLUSIONS: The bacteria changing activity during biofilm formation and after meal ingestion were person-specific. Interestingly, some individuals showed extreme homeostasis with virtually no changes in the active bacterial population after food ingestion, suggesting the presence of a microbial community which could be associated to dental health.

Regulation of expression and catalytic activity of Escherichia coli RsmG methyltransferase.

RsmG is an AdoMet-dependent methyltransferase responsible for the synthesis of m(7)G527 in the 530 loop of bacterial 16S rRNA. This loop is universally conserved, plays a key role in ribosomal accuracy, and is a target for streptomycin binding. Loss of the m(7)G527 modification confers low-level streptomycin resistance and may affect ribosomal functioning. Here, we explore the mechanisms controlling RsmG expression and activity, which may somehow respond to the demand set by the amount of rRNA. We confirm that rsmG is the second member in a bicistronic operon and demonstrate that rsmG also has its own promoter, which appears, in actively growing cells, as a control device to offset both the relatively low stability of RsmG and inhibition of the operon promoter. RsmG levels decrease under conditions that down-regulate rRNA synthesis. However, coordination between rRNA and RsmG expression does not seem to occur at the level of transcription initiation. Instead, it might depend on the activity of an inverted repeated region, located between the rsmG promoter and ribosome binding site, which we show to work as a weak transcriptional terminator. To gain insights into the enzymatic mechanism of RsmG, highly conserved residues were mutated and the abilities of the resulting proteins to confer streptomycin resistance, to modify rRNA, and to bind AdoMet were explored. Our data demonstrate for the first time the critical importance of some residues located in the active site of Escherichia coli RsmG for the m(7)G modification process and suggest a role for them in rRNA binding and catalysis.

The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy.

Modifying RNA enzymes are highly specific for substrate-rRNA or tRNA-and the target position. In Escherichia coli, there are very few multisite acting enzymes, and only one rRNA/tRNA dual-specificity enzyme, pseudouridine synthase RluA, has been identified to date. Among the tRNA-modifying enzymes, the methyltransferase responsible for the m(2)A synthesis at purine 37 in a tRNA set still remains unknown. m(2)A is also present at position 2503 in the peptidyl transferase center of 23S RNA, where it is introduced by RlmN, a radical S-adenosyl-L-methionine (SAM) enzyme. Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. The ΔrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. Moreover, RlmN performs m(2)A(37) synthesis in vitro using a tRNA chimera as a substrate. This chimera has also proved useful to characterize some tRNA identity determinants for RlmN and other tRNA modification enzymes. Our data suggest that RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA. RlmN inactivation increases the misreading of a UAG stop codon. Since loss of m(2)A(37) from tRNA is expected to produce a hyperaccurate phenotype, we believe that the error-prone phenotype exhibited by the ΔrlmN mutant is due to loss of m(2)A from 23S rRNA and, accordingly, that the m(2)A2503 modification plays a crucial role in the proofreading step occurring at the peptidyl transferase center.

A practical guide for the computational selection of residues to be experimentally characterized in protein families.

In recent years, numerous biocomputational tools have been designed to extract functional and evolutionary information from multiple sequence alignments (MSAs) of proteins and genes. Most biologists working actively on the characterization of proteins from a single or family perspective use the MSA analysis to retrieve valuable information about amino acid conservation and the functional role of residues in query protein(s). In MSAs, adjustment of alignment parameters is a key point to improve the quality of MSA output. However, this issue is frequently underestimated and/or misunderstood by scientists and there is no in-depth knowledge available in this field. This brief review focuses on biocomputational approaches complementary to MSA to help distinguish functional residues in protein families. These additional analyses involve issues ranging from phylogenetic to statistical, which address the detection of amino acids pivotal for protein function at any level. In recent years, a large number of tools has been designed for this very purpose. Using some of these relevant, useful tools, we have designed a practical pipeline to perform in silico studies with a view to improving the characterization of family proteins and their functional residues. This review-guide aims to present biologists a set of specially designed tools to study proteins. These tools are user-friendly as they use web servers or easy-to-handle applications. Such criteria are essential for this review as most of the biologists (experimentalists) working in this field are unfamiliar with these biocomputational analysis approaches.

Streptococcus dentisani sp. nov., a novel member of the mitis group.

Genomic, taxonomic and biochemical studies were performed on two strains of α-haemolytic streptococci that showed them to be clustered with major members of the Streptococcus mitis group. These Gram-stain-positive strains were isolated from tooth surfaces of caries-free humans and showed the classical spherical shape of streptococcal species growing in chains. Sequence analysis from concatenated 16S and 23S rRNA gene and sodA genes showed that these strains belonged to the mitis group, but both of them clustered into a new phylogenetic branch. The genomes of these two isolates were sequenced, and whole-genome average nucleotide identity (ANI) demonstrated that these strains significantly differed from any streptococcal species, showing ANI values under 91 % even when compared with the phylogenetically closest species such as Streptococcus oralis and S. mitis. Biochemically, the two isolates also showed distinct metabolic features relative to closely related species, like α-galactosidase activity. From the results of the present study, the name Streptococcus dentisani sp. nov. is proposed to accommodate these novel strains, which have been deposited in open collections at the Spanish type Culture Collection (CECT) and Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ), being respectively identified as Streptococcus dentisani Str. 7746 ( = CECT 8313 = DSM 27089) and Streptococcus dentisani Str. 7747(T) ( = CECT 8312(T) = DSM 27088(T)).

Extracellular vesicles from mesenchymal stem cells alter gut microbiota and improve neuroinflammation and motor impairment in rats with mild liver damage.

Gut microbiota perturbation and motor dysfunction have been reported in steatosis patients. Rats with mild liver damage (MLD) show motor dysfunction mediated by neuroinflammation and altered GABAergic neurotransmission in the cerebellum. The extracellular vesicles (EV) from mesenchymal stem cells (MSC) have emerged as a promising therapeutic proxy whose molecular basis relies partly upon TGFß action. This study aimed to assess if MSC-EVs improve motor dysfunction in rats with mild liver damage and analyze underlying mechanisms, including the role of TGFß, cerebellar neuroinflammation and gut microbiota. MLD in rats was induced by carbon tetrachloride administration and EVs from normal (C-EVs) or TGFß-siRNA treated MSCs (T-EV) were injected. Motor coordination, locomotor gait, neuroinflammation and TNF-α-activated pathways modulating GABAergic neurotransmission in the cerebellum, microbiota composition in feces and microbial-derived metabolites in plasma were analyzed. C-EVs reduced glial and TNFα-P2X4-BDNF-TrkB pathway activation restoring GABAergic neurotransmission in the cerebellum and improving motor coordination and all the altered gait parameters. T-EVs also improved motor coordination and some gait parameters, but the mechanisms involved differed from those of C-EVs. MLD rats showed increased content of some Bacteroides species in feces, correlating with decreased kynurenine aside from motor alterations. These alterations were all normalized by C-EVs, whereas T-EVs only restored kynurenine levels. Our results support the value of MSC-EVs on improving motor dysfunction in MLD and unveil a possible mechanism by which altered microbiota may contribute to neuroinflammation and motor impairment. Some of the underlying mechanisms are TGFß-dependent.

Bacteroides uniformis CECT 7771 requires adaptive immunity to improve glucose tolerance but not to prevent body weight gain in diet-induced obese mice.

BACKGROUND: The metabolic disturbances of obesity can be mitigated by strategies modulating the gut microbiota. In this study, we sought to identify whether innate or adaptive immunity mediates the beneficial metabolic effects of the human intestinal bacterium Bacteroides uniformis CECT 7771 in obesity. METHODS: We evaluated the effects of orally administered B. uniformis on energy homeostasis, intestinal immunity, hormone levels, and gut microbiota in wild-type and Rag1-deficient mice with diet-induced obesity. We also assessed whether B. uniformis needed to be viable to exert its beneficial effects in obesity and to directly induce immunoregulatory effects. RESULTS: The administration of B. uniformis to obese mice improved glucose tolerance and insulin secretion, restored the caloric intake suppression after an oral glucose challenge, and reduced hyperglycemia. The pre- and post-prandial glucose-related benefits were associated with restoration of the anti-inflammatory tone mediated by type 2 macrophages and regulatory T cells (Tregs) in the lamina propria of the small intestine. Contrastingly, B. uniformis administration failed to improve glucose tolerance in obese Rag1-/- mice, but prevented the increased body weight gain and adiposity. Overall, the beneficial effects seemed to be independent of enteroendocrine effects and of major changes in gut microbiota composition. B. uniformis directly induced Tregs generation from naïve CD4+ T cells in vitro and was not required to be viable to improve glucose homeostasis but its viability was necessary to prevent body weight gain in diet-induced obese wild-type mice. CONCLUSIONS: Here we demonstrate that B. uniformis modulates the energy homeostasis in diet-induced obese mice through different mechanisms. The bacterium improves oral glucose tolerance by adaptive immunity-dependent mechanisms that do not require cell viability and prevents body weight gain by adaptive immunity-independent mechanisms which require cell viability. Video Abstract.

Gut microbiota DPP4-like enzymes are increased in type-2 diabetes and contribute to incretin inactivation.

BACKGROUND: The gut microbiota controls broad aspects of human metabolism and feeding behavior, but the basis for this control remains largely unclear. Given the key role of human dipeptidyl peptidase 4 (DPP4) in host metabolism, we investigate whether microbiota DPP4-like counterparts perform the same function. RESULTS: We identify novel functional homologs of human DPP4 in several bacterial species inhabiting the human gut, and specific associations between Parabacteroides and Porphyromonas DPP4-like genes and type 2 diabetes (T2D). We also find that the DPP4-like enzyme from the gut symbiont Parabacteroides merdae mimics the proteolytic activity of the human enzyme on peptide YY, neuropeptide Y, gastric inhibitory polypeptide (GIP), and glucagon-like peptide 1 (GLP-1) hormones in vitro. Importantly, administration of E. coli overexpressing the P. merdae DPP4-like enzyme to lipopolysaccharide-treated mice with impaired gut barrier function reduces active GIP and GLP-1 levels, which is attributed to increased DPP4 activity in the portal circulation and the cecal content. Finally, we observe that linagliptin, saxagliptin, sitagliptin, and vildagliptin, antidiabetic drugs with DPP4 inhibitory activity, differentially inhibit the activity of the DPP4-like enzyme from P. merdae. CONCLUSIONS: Our findings confirm that proteolytic enzymes produced by the gut microbiota are likely to contribute to the glucose metabolic dysfunction that underlies T2D by inactivating incretins, which might inspire the development of improved antidiabetic therapies.

Dietary patterns drive loss of fiber-foraging species in the celiac disease patients gut microbiota compared to first-degree relatives.

BACKGROUND: Celiac disease is an autoimmune disorder triggered by dietary gluten in genetically predisposed individuals that primarily affects the small intestine. Studies have reported differentially abundant bacterial taxa in the gut microbiota of celiac patients compared with non-celiac controls. However, findings across studies have inconsistencies and no microbial signature of celiac disease has been defined so far. RESULTS: Here, we showed, by comparing celiac patients with their non-celiac 1st-degree relatives, that bacterial communities of related individuals have similar species occurrence and abundance compared with non-relatives, regardless the disease status. We also found in celiac patients a loss of bacterial species associated with fiber degradation, and host metabolic and immune modulation, as ruminiclostridia, ruminococci, Prevotella, and Akkermansia muciniphila species. We demonstrated that the differential abundance of bacterial species correlates to different dietary patterns observed between the two groups. For instance, Ruminiclostridium siraeum, Ruminococcus bicirculans, and Bacteroides plebeious, recognized as fiber-degraders, appear more abundant in non-celiac 1st-degree relatives, which have a vegetable consumption pattern higher than celiac patients. Pattern of servings per day also suggests a possible link between these species' abundance and daily calorie intake. CONCLUSIONS: Overall, we evidenced that a kinship approach could be valuable in unveiling potential celiac disease microbial traits, as well as the significance of dietary factors in shaping microbial profiles and their influence on disease development and progression. Our results pave the way for designing and adopting novel dietary strategies based on gluten-free fiber-enriched ingredients to improve disease management and patients' quality of life.

YibK is the 2'-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNA(Leu) isoacceptors.

Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few notable exceptions include the 2'-O-methyltransferase(s) that methylate the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNA(Leu)(CmAA) and tRNA(Leu)(cmnm5UmAA). Here, we have used a comparative genomics approach to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 2'-O-methylation at position 34 in both tRNA(Leu)(CmAA) and tRNA(Leu)(cmnm5UmAA). Loss of YibK methylation reduces the efficiency of codon-wobble base interaction, as demonstrated in an amber suppressor supP system. Inactivation of yibK had no detectable effect on steady-state growth rate, although a distinct disadvantage was noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase was impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK. Despite being one of the smallest characterized α/ß knot proteins, YibK independently catalyzes the methyl transfer from S-adenosyl-L-methionine to the 2'-OH of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N6-(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL.

The microbiota composition drives personalized nutrition: Gut microbes as predictive biomarkers for the success of weight loss diets.

The investigation of the human gut microbiome during recent years has permitted us to understand its relevance for human health at a systemic level, making it possible to establish different functional axes (e.g., the gut-brain, gut-liver, and gut-lung axes), which support the organ-like status conferred to this microecological component of our body. The human gut microbiota is extremely variable but modifiable via diet, a fact that allows targeting of microbes through defined dietary strategies to uncover cost-effective therapies to minimize the burden of non-communicable diseases such as pandemic obesity and overweight and its metabolic comorbidities. Nevertheless, randomly controlled dietary interventions regularly exhibit low to moderate degrees of success in weight control, making their implementation difficult in clinical practice. Here, we review the predictive value of the baseline gut microbiota configurations to anticipate the success of dietary interventions aimed at weight loss, mostly based on caloric restriction regimes and oral fiber supplementation. This emergent research concept fits into precision medicine by considering different diet patterns and adopting the best one, based on the individual microbiota composition, to reach significant adiposity reduction and improve metabolic status. We review the results from this fresh perspective of investigation, taking into account studies released very recently. We also discuss some future outlooks in the field and potential pitfalls to overcome with the aim of gaining knowledge in the field and achieving breakthroughs in personalized nutrition.

Species- and strain-level assessment using rrn long-amplicons suggests donor's influence on gut microbial transference via fecal transplants in metabolic syndrome subjects.

Fecal microbiota transplantation (FMT) is currently used for treating Clostridium difficile infection and explored for other clinical applications in experimental trials. However, the effectiveness of this therapy could vary, and partly depend on the donor's bacterial species engraftment, whose evaluation is challenging because there are no cost-effective strategies for accurately tracking the microbe transference. In this regard, the precise identification of bacterial species inhabiting the human gut is essential to define their role in human health unambiguously. We used Nanopore-based device to sequence bacterial rrn operons (16S-ITS-23S) and to reveal species-level abundance changes in the human gut microbiota of a FMT trial. By assessing the donor and recipient microbiota before and after FMT, we further evaluated whether this molecular approach reveals strain-level genetic variation to demonstrate microbe transfer and engraftment. Strict control over sequencing data quality and major microbiota covariates was critical for accurately estimating the changes in gut microbial species abundance in the recipients after FMT. We detected strain-level variation via single-nucleotide variants (SNVs) at rrn regions in a species-specific manner. We showed that it was possible to explore successfully the donor-bacterial strain (e.g., Parabacteroides merdae) engraftment in recipients of the FMT by assessing the nucleotide frequencies at rrn-associated SNVs. Our findings indicate that the engraftment of donors' microbiota is to some extent correlated with the improvement of metabolic health in recipients and that parameters such as the baseline gut microbiota configuration, sex, and age of donors should be considered to ensure the success of FMT in humans. The study was prospectively registered at the Dutch Trial registry - NTR4488 (https://www.trialregister.nl/trial/4488).

Intestinal microbiota is modified in pediatric food protein-induced enterocolitis syndrome.

Background: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food hypersensitivity that affects the gastrointestinal system, especially in children, who often present with more severe clinical manifestations than adults do. Although its pathogenesis is poorly understood and biomarkers are still lacking, scientific evidence suggests that gut microbiota may play an important role in the development of FPIES. Objective: We aimed to compare the composition of gut microbiota in children with FPIES with that in age- and sex-matched healthy controls. Methods: We analyzed the gut microbiota profiles in fecal samples of 17 patients with FPIES (case patients) and 12 age-matched healthy children (controls) by tag sequencing of the 16S ribosomal RNA gene hypervariable V4-V5 regions. Subjects' sociodemographic, clinical, and food diary variables were described and compared between groups by using inferential statistical tests. Nonparametric linear discriminant analysis was performed for intestinal microbiota data. Results: Patients with confirmed cases FPIES (n = 17; average patient age, 7.5 ± 3.2 years) and controls without FPIES or any atopy (n = 12, average patient age, 6.9 ± 2.7 years) were included. Fish was the main FPIES-inducing allergen in 65% of the cases. The patients with FPIES showed higher proportions of Lachnospiraceae spp (P < .0286) and a lower proportion of Ruminococcaceae spp (P < .0066), Lactobacillaceae spp (P < .0075), and Leuconostocaceae spp (P < .0173) than the controls. Conclusions: Our data clearly show a different gut microbial signature in patients with FPIES, suggesting a new potential avenue for aiding the diagnosis and clinical management of FPIES. Larger studies are needed to confirm these results.