A conserved inhibitory interdomain interaction regulates DNA-binding activities of hybrid two-component systems in Bacteroides.

Hybrid two-component systems (HTCSs) comprise a major class of transcription regulators of polysaccharide utilization genes in Bacteroides. Distinct from classical two-component systems in which signal transduction is carried out by intermolecular phosphotransfer between a histidine kinase (HK) and a cognate response regulator (RR), HTCSs contain the membrane sensor HK and the RR transcriptional regulator within a single polypeptide chain. Tethering the DNA-binding domain (DBD) of the RR with the dimeric HK domain in an HTCS could potentially promote dimerization of the DBDs and would thus require a mechanism to suppress DNA-binding activity in the absence of stimulus. Analysis of phosphorylation and DNA-binding activities of several HTCSs from Bacteroides thetaiotaomicron revealed a DBD suppression mechanism in which an inhibitory interaction between the DBD and the phosphoryl group-accepting receiver domain (REC) decreases autophosphorylation rates of HTCS-RECs and represses DNA-binding activities in the absence of phosphorylation. Sequence analyses and structure predictions identified a highly conserved sequence motif correlated with a conserved inhibitory domain arrangement of REC and DBD. The presence of the motif, as in most HTCSs, or its absence, in a small subset of HTCSs, is likely predictive of two distinct regulatory mechanisms evolved for different glycans. Substitutions within the conserved motif relieve the inhibitory interaction and result in elevated DNA-binding activities in the absence of phosphorylation. Our data suggest a fundamental regulatory mechanism shared by most HTCSs to suppress DBD activities using a conserved inhibitory interdomain arrangement to overcome the challenge of the fused HK and RR components. IMPORTANCE: Different dietary and host-derived complex carbohydrates shape the gut microbial community and impact human health. In Bacteroides, the prevalent gut bacteria genus, utilization of these diverse carbohydrates relies on different gene clusters that are under sophisticated control by various signaling systems, including the hybrid two-component systems (HTCSs). We have uncovered a highly conserved regulatory mechanism in which the output DNA-binding activity of HTCSs is suppressed by interdomain interactions in the absence of stimulating phosphorylation. A consensus amino acid motif is found to correlate with the inhibitory interaction surface while deviations from the consensus can lead to constitutive activation. Understanding of such conserved HTCS features will be important to make regulatory predictions for individual systems as well as to engineer novel systems with substitutions in the consensus to explore the glycan regulation landscape in Bacteroides.

Human gut microbes express functionally distinct endoglycosidases to metabolize the same N-glycan substrate.

Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-ß-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract.

Genome sequence and annotation of Bacteroides sp aff. Thetaiotaomicron strain isolated from blood.

This study is focused on genome sequence and annotation of the Bacteroides strain isolated from the blood of a patient with descending colon cancer. According to a comparison of the 16S ribosomal RNA sequence with the National Center for Biotechnology Information database, this strain was identified as Bacteroides sp. aff. Thetaiotaomicron. The next-generation sequencing of the strain was performed in a GENEWIZ laboratory (Jiangsu, China) on Illumina HiSeq device. According to CAZy classification, metabolic pathways related to carbohydrate metabolism of this strain engage the following enzymes: 427 glycosylhydrolases, 277 glycosyltransferases, 137 carbohydrate-binding modules, 48 carbohydrate esterases, and 24 polysaccharide lyases. According to the KEGG pathway database, Bacteroides sp. aff thetaiotaomicron strain is reported to incorporate 199 pathway associated genes. Bacteroides sp. aff. Thetaiotaomicron exposes the capacity of metabolizing a variety of polysaccharides. Its genome is enriched with an expanded repertoire of enzymes for the hydrolysis of glycosidic bonds and, thus, likely to hydrolyze most of glycosidic bonds in biological polysaccharides. The advanced capabilities of the studied strain to recognize and respond to environmental signals are expressed in the rich representation of one- and two-component signal transduction systems.

A Metabolic Pathway for Activation of Dietary Glucosinolates by a Human Gut Symbiont.

Consumption of glucosinolates, pro-drug-like metabolites abundant in Brassica vegetables, has been associated with decreased risk of certain cancers. Gut microbiota have the ability to metabolize glucosinolates, generating chemopreventive isothiocyanates. Here, we identify a genetic and biochemical basis for activation of glucosinolates to isothiocyanates by Bacteroides thetaiotaomicron, a prominent gut commensal species. Using a genome-wide transposon insertion screen, we identified an operon required for glucosinolate metabolism in B. thetaiotaomicron. Expression of BT2159-BT2156 in a non-metabolizing relative, Bacteroides fragilis, resulted in gain of glucosinolate metabolism. We show that isothiocyanate formation requires the action of BT2158 and either BT2156 or BT2157 in vitro. Monocolonization of mice with mutant BtΔ2157 showed reduced isothiocyanate production in the gastrointestinal tract. These data provide insight into the mechanisms by which a common gut bacterium processes an important dietary nutrient.

Gut microbiome and serum metabolome alterations in obesity and after weight-loss intervention.

Emerging evidence has linked the gut microbiome to human obesity. We performed a metagenome-wide association study and serum metabolomics profiling in a cohort of lean and obese, young, Chinese individuals. We identified obesity-associated gut microbial species linked to changes in circulating metabolites. The abundance of Bacteroides thetaiotaomicron, a glutamate-fermenting commensal, was markedly decreased in obese individuals and was inversely correlated with serum glutamate concentration. Consistently, gavage with B. thetaiotaomicron reduced plasma glutamate concentration and alleviated diet-induced body-weight gain and adiposity in mice. Furthermore, weight-loss intervention by bariatric surgery partially reversed obesity-associated microbial and metabolic alterations in obese individuals, including the decreased abundance of B. thetaiotaomicron and the elevated serum glutamate concentration. Our findings identify previously unknown links between intestinal microbiota alterations, circulating amino acids and obesity, suggesting that it may be possible to intervene in obesity by targeting the gut microbiota.

Prioritization of polysaccharide utilization and control of regulator activation in Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron is a human gut symbiotic bacterium that utilizes a myriad of host dietary and mucosal polysaccharides. The proteins responsible for the uptake and breakdown of many of these polysaccharides are transcriptionally regulated by hybrid two-component systems (HTCSs). These systems consist of a single polypeptide harboring the domains of sensor kinases and response regulators, and thus, are thought to autophosphorylate in response to specific signals. We now report that the HTCS BT0366 is phosphorylated in vivo when B. thetaiotaomicron experiences the BT0366 inducer arabinan but not when grown in the presence of glucose. BT0366 phosphorylation and transcription of BT0366-activated genes requires the conserved predicted sites of phosphorylation in BT0366. When chondroitin sulfate is added to arabinan-containing cultures, BT0366 phosphorylation and transcription of BT0366-activated genes are inhibited and the bacterium exhibits diauxic growth. Whereas 20 additional combinations of polysaccharides also give rise to diauxic growth, other combinations result in synergistic or unaltered growth relative to bacteria experiencing a single polysaccharide. The different strategies employed by B. thetaiotaomicron when faced with multiple polysaccharides may aid its competitiveness in the mammalian gut.