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Dietary intake of phytate has various reported health benefits. Previous work showed that the gut microbiota can convert phytate to short-chain fatty acids (SCFAs), but the microbial species and metabolic pathway are unclear. Here we identified Mitsuokella jalaludinii as an efficient phytate degrader, which works synergistically with Anaerostipes rhamnosivorans to produce the SCFA propionate. Analysis of published human gut taxonomic profiles revealed that Mitsuokella spp., in particular M. jalaludinii, are prevalent in human gut microbiomes. NMR spectroscopy using 13 C-isotope labelling, metabolomic and transcriptomic analyses identified a complete phytate degradation pathway in M. jalaludinii, including production of the intermediate Ins(2)P/myo-inositol. The major end product, 3-hydroxypropionate, was converted into propionate via a synergistic interaction with Anaerostipes rhamnosivorans both in vitro and in mice. Upon [ 13 C 6 ]phytate administration, various 13 C-labelled components were detected in mouse caecum in contrast with the absence of [ 13 C 6 ] InsPs or [ 13 C 6 ]myo-inositol in plasma. Caco-2 cells incubated with co-culture supernatants exhibited improved intestinal barrier integrity. These results suggest that the microbiome plays a major role in the metabolism of this phytochemical and that its fermentation to propionate by M. jalaludinii and A. rhamnosivorans may contribute to phytate-driven health benefits., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Mucin-degrading microbes are known to harbor glycosyl hydrolases (GHs) which cleave specific glycan linkages. Although several microbial species have been identified as mucin degraders, there are likely many other members of the healthy gut community with the capacity to degrade mucins. The aim of the present study was to systematically examine the CAZyme mucin-degrading profiles of the human gut microbiota. Within the Verrucomicrobia phylum, all Akkermansia glycaniphila and muciniphila genomes harbored multiple gene copies of mucin-degrading GHs. The only representative of the Lentisphaerae phylum, Victivallales, harbored a GH profile that closely mirrored Akkermansia. In the Actinobacteria phylum, we found several Actinomadura, Actinomyces, Bifidobacterium, Streptacidiphilus and Streptomyces species with mucin-degrading GHs. Within the Bacteroidetes phylum, Alistipes, Alloprevotella, Bacteroides, Fermenitomonas Parabacteroides, Prevotella and Phocaeicola species had mucin degrading GHs. Firmicutes contained Abiotrophia, Blautia, Enterococcus, Paenibacillus, Ruminococcus, Streptococcus, and Viridibacillus species with mucin-degrading GHs. Interestingly, far fewer mucin-degrading GHs were observed in the Proteobacteria phylum and were found in Klebsiella, Mixta, Serratia and Enterobacter species. We confirmed the mucin-degrading capability of 23 representative gut microbes using a chemically defined media lacking glucose supplemented with porcine intestinal mucus. These data greatly expand our knowledge of microbial-mediated mucin degradation within the human gut microbiota., (© 2022. The Author(s).)

Alzheimer's disease (AD) represents the most common form of dementia in the elderly with no available disease modifying treatments. Altered gut microbial composition has been widely acknowledged as a common feature of AD, which potentially contributes to progression or onset of AD. To assess the hypothesis that Candida rugosa lipase (CRL), which has been shown to enhance gut microbiome and metabolite composition, can rebalance the gut microbiome composition and reduce AD pathology, the treatment effects in APPswe/PS1de9 (APP/PS1) mice were investigated. The analysis revealed an increased abundance of Acetatifactor and Clostridiales vadin BB60 genera in the gut; increased lipid hydrolysis in the gut lumen, normalization of peripheral unsaturated fatty acids, and reduction of neuroinflammation and memory deficits post treatment. Finally, we demonstrated that the evoked benefits on memory could be transferred via fecal matter transplant (FMT) into antibiotic-induced microbiome-depleted (AIMD) wildtype mice, ameliorating their memory deficits. The findings herein contributed to improve our understanding of the role of the gut microbiome in AD's complex networks and suggested that targeted modification of the gut could contribute to amelioration of AD neuropathology., (© 2022. The Author(s).)

In the process of yielding biofuels from cellulose degradation, traditional enzymatic hydrolysis, such as β-glucosidase catalyzing cellobiose, can barely resolve the contradiction between cellulose degradation and bioenergy conservation. However, it has been shown that cellobiose phosphorylase provides energetic advantages for cellobiose degradation through a phosphorolytic pathway, which has attracted wide attention. Here, the cellobiose phosphorylase gene from Caldicellulosiruptor bescii (CbCBP) was cloned, expressed, and purified. Analysis of the enzymatic properties and kinetic mechanisms indicated that CbCBP catalyzed reversible phosphorolysis and had good thermal stability and broad substrate selectivity. In addition, the phosphorolytic reaction of cellobiose by CbCBP proceeded via an ordered Bi Bi mechanism, while the synthetic reaction proceeded via a ping pong Bi Bi mechanism. The present study lays the foundation for optimizing the degradation of cellulose and the synthesis of functional oligosaccharides., (© 2022. The Author(s).)

The heightened cardiovascular disease (CVD) risk observed among omnivores is thought to be linked, in part, to gut microbiota-dependent generation of trimethylamine-N-oxide (TMAO) from L-carnitine, a nutrient abundant in red meat. Gut microbial transformation of L-carnitine into trimethylamine (TMA), the precursor of TMAO, occurs via the intermediate γ-butyrobetaine (γBB). However, the interrelationship of γBB, red meat ingestion and CVD risks, as well as the gut microbial genes responsible for the transformation of γBB to TMA, are unclear. In the present study, we show that plasma γBB levels in individuals from a clinical cohort (n = 2,918) are strongly associated with incident CVD event risks. Culture of human faecal samples and microbial transplantation studies in gnotobiotic mice with defined synthetic communities showed that the introduction of Emergencia timonensis, a human gut microbe that can metabolize γBB into TMA, is sufficient to complete the carnitine → γBB → TMA transformation, elevate TMAO levels and enhance thrombosis potential in recipients after arterial injury. RNA-sequencing analyses of E. timonensis identified a six-gene cluster, herein named the γBB utilization (gbu) gene cluster, which is upregulated in response to γBB. Combinatorial cloning and functional studies identified four genes (gbuA, gbuB, gbuC and gbuE) that are necessary and sufficient to recapitulate the conversion of γBB to TMA when coexpressed in Escherichia coli. Finally, reanalysis of samples (n = 113) from a clinical, randomized diet, intervention study showed that the abundance of faecal gbuA correlates with plasma TMAO and a red meat-rich diet. Our findings reveal a microbial gene cluster that is critical to dietary carnitine → γBB → TMA → TMAO transformation in hosts and contributes to CVD risk., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Butyrate is the primary energy source for colonocytes and is essential for mucosal integrity and repair. Butyrate deficiency as a result of colonic dysbiosis is a putative factor in ulcerative colitis (UC). Commensal microbes are butyrogenic, while others may inhibit butyrate, through hydrogenotropic activity. The aim of this study was to quantify butyrogenic and hydrogenotropic species and determine their relationship with inflammation within the colonic mucus gel layer (MGL). Mucosal brushings were obtained from 20 healthy controls (HC), 20 patients with active colitis (AC) and 14 with quiescent colitis (QUC). Abundance of each species was determined by RT-PCR. Inflammatory scores were available for each patient. Statistical analyses were performed using Mann-Whitney-U and Kruskall-Wallis tests. Butyrogenic R. hominis was more abundant in health than UC (p < 0.005), prior to normalisation against total bacteria. Hydrogenotropic B. wadsworthia was reduced in AC compared to HC and QUC (p < 0.005). An inverse correlation existed between inflammation and R. hominis (ρ - 0.460, p < 0.005) and B. wadsworthia (ρ - 0.646, p < 0.005). Other hydrogenotropic species did not widely colonise the MGL. These data support a role for butyrogenic bacteria in UC. Butyrate deficiency in UC may be related to reduced microbial production, rather than inhibition by microbial by-products.

Planktonic cultures, of a rationally designed consortium, demonstrated emergent properties that exceeded the sums of monoculture properties, including a >200% increase in cellobiose catabolism, a >100% increase in glycerol catabolism, a >800% increase in ethanol production, and a >120% increase in biomass productivity. The consortium was designed to have a primary and secondary-resource specialist that used crossfeeding with a positive feedback mechanism, division of labor, and nutrient and energy transfer via necromass catabolism. The primary resource specialist was Clostridium phytofermentans (a.k.a. Lachnoclostridium phytofermentans), a cellulolytic, obligate anaerobe. The secondary-resource specialist was Escherichia coli, a versatile, facultative anaerobe, which can ferment glycerol and byproducts of cellobiose catabolism. The consortium also demonstrated emergent properties of enhanced biomass accumulation when grown as biofilms, which created high cell density communities with gradients of species along the vertical axis. Consortium biofilms were robust to oxic perturbations with E. coli consuming O 2 , creating an anoxic environment for C. phytofermentans. Anoxic/oxic cycling further enhanced biomass productivity of the biofilm consortium, increasing biomass accumulation ~250% over the sum of the monoculture biofilms. Consortium emergent properties were credited to several synergistic mechanisms. E. coli consumed inhibitory byproducts from cellobiose catabolism, driving higher C. phytofermentans growth and higher cellulolytic enzyme production, which in turn provided more substrate for E. coli. E. coli necromass enhanced C. phytofermentans growth while C. phytofermentans necromass aided E. coli growth via the release of peptides and amino acids, respectively. In aggregate, temporal cycling of necromass constituents increased flux of cellulose-derived resources through the consortium. The study establishes a consortia-based, bioprocessing strategy built on naturally occurring interactions for improved conversion of cellulose-derived sugars into bioproducts.

Fundamental restoration ecology and community ecology theories can help us better understand the underlying mechanisms of fecal microbiota transplantation (FMT) and to better design future microbial therapeutics for recurrent Clostridioides difficile infections (rCDI) and other dysbiosis-related conditions. In this study, stool samples were collected from donors and rCDI patients one week prior to FMT (pre-FMT), as well as from patients one week following FMT (post-FMT). Using metagenomic sequencing and machine learning, our results suggested that FMT outcome is not only dependent on the ecological structure of the recipients, but also the interactions between the donor and recipient microbiomes at the taxonomical and functional levels. We observed that the presence of specific bacteria in donors (Clostridioides spp., Desulfovibrio spp., Odoribacter spp. and Oscillibacter spp.) and the absence of fungi (Yarrowia spp.) and bacteria (Wigglesworthia spp.) in recipients prior to FMT could predict FMT success. Our results also suggested a series of interlocked mechanisms for FMT success, including the repair of the disturbed gut ecosystem by transient colonization of nexus species followed by secondary succession of bile acid metabolizers, sporulators, and short chain fatty acid producers.

Human milk oligosaccharides (HMOs) are a mixture of structurally diverse carbohydrates that contribute to shape a healthy gut microbiota composition. The great diversity of the HMOs structures does not allow the attribution of specific prebiotic characteristics to single milk oligosaccharides. We analyze here the utilization of four disaccharides, lacto-N-biose (LNB), galacto-N-biose (GNB), fucosyl-α1,3-GlcNAc (3FN) and fucosyl-α1,6-GlcNAc (6FN), that form part of HMOs and glycoprotein structures, by the infant fecal microbiota. LNB significantly increased the total levels of bifidobacteria and the species Bifidobacterium breve and Bifidobacterium bifidum. The Lactobacillus genus levels were increased by 3FN fermentation and B. breve by GNB and 3FN. There was a significant reduction of Blautia coccoides group with LNB and 3FN. In addition, 6FN significantly reduced the levels of Enterobacteriaceae family members. Significantly higher concentrations of lactate, formate and acetate were produced in cultures containing either LNB or GNB in comparison with control cultures. Additionally, after fermentation of the oligosaccharides by the fecal microbiota, several Bifidobacterium strains were isolated and identified. The results presented here indicated that each, LNB, GNB and 3FN disaccharide, might have a specific beneficial effect in the infant gut microbiota and they are potential prebiotics for application in infant foods.

Filifactor alocis, a Gram-positive anaerobic bacterium, is now a proposed diagnostic indicator of periodontal disease. Because the stress response of this bacterium to the oxidative environment of the periodontal pocket may impact its pathogenicity, an understanding of its oxidative stress resistance strategy is vital. Interrogation of the F. alocis genome identified the HMPREF0389&#95;00796 gene that encodes for a putative superoxide reductase (SOR) enzyme. SORs are non-heme, iron-containing enzymes that can catalyze the reduction of superoxide radicals to hydrogen peroxide and are important in the protection against oxidative stress. In this study, we have functionally characterized the putative SOR (FA796) from F. alocis ATCC 35896. The recombinant FA796 protein, which is predicted to be a homotetramer of the 1Fe-SOR class, can reduce superoxide radicals. F. alocis FLL141 (∆FA796::ermF) was significantly more sensitive to oxygen/air exposure compared to the parent strain. Sensitivity correlated with the level of intracellular superoxide radicals. Additionally, the FA796-defective mutant had increased sensitivity to hydrogen peroxide-induced stress, was inhibited in its ability to form biofilm and had reduced survival in epithelial cells. Collectively, these results suggest that the F. alocis SOR protein is a key enzymatic scavenger of superoxide radicals and protects the bacterium from oxidative stress conditions.

The ketone body D-β-hydroxybutyrate (DBHB) has gained attention owing to its cellular signalling function; however, its effect on the human colonic microbiota remains unclear. Here, DBHB dynamics in the human colon were investigated using an in vitro colonic microbiota model, which maintained most of the operational taxonomic units detected in the original faeces. Over 54% of 0.41% (w/v) DBHB was metabolised by microbiota models originating from seven faecal samples after 30 h of fermentation (regarded as DBHB utilisers); however, <19% of DBHB was metabolised by microbiota models from five faecal samples (regarded as non-utilisers of DBHB). In utilisers, DBHB administration increased the relative abundance of the genus Coprococcus, correlated with increased butyrogenesis. Increased butyrogenesis was not observed in DBHB non-utilisers. Based on PICRUSt analysis, the relative abundance of β-hydroxybutyrate dehydrogenase was maintained in microbiota models from DBHB utilisers following DBHB administration; however, it decreased in microbiota models from non-utilisers. After 21 h of fermentation, the intracellular glutamate concentration, which is indicative of growth, showed a positive correlation with DBHB utilisation (R 2  = 0.70). Human colonic microbiotas with high growth activity demonstrate efficient utilisation of DBHB for increased butyrate production, which affords health benefits.

Uncontrolled oxidative stress, reported in Salmonella and HIV infections, colorectal cancer or severe acute malnutrition, has been associated with anaerobic gut microbiome alteration, impaired butyrate production, mucosal immunity dysregulation and disruption of host-bacterial mutualism. However, the role of major antioxidant molecules in the human body, such as glutathione, ascorbic acid and uric acid, has been neglected in this context. Here, we performed an in vitro metabolomics study of the 3 most odorous anaerobic microbes isolated from the human gut in our laboratory (Clostridium sporogenes, Clostridium subterminale and Romboutsia lituseburensis) when grown in anaerobiosis or in aerobiosis with these 3 antioxidant molecules via gas and liquid chromatography-mass spectrometry (GC/MS and LC/MS). There was no growth or volatile organic compound production in aerobic cultures without the 3 antioxidant molecules. In anaerobiosis, the major metabolic products of the bacteria were thiols, alcohols and short-chain fatty acid esters. The production of alkanes, cycloheptatriene and, paradoxically, increased butyrate production, was observed in the cultures grown in aerobiosis with the 3 antioxidant molecules. The qualitative shift suggests specific molecular mechanisms that remain to be elucidated. The increased production of butyrate, but also isobutyrate and isovalerate in vitro suggests that these 3 antioxidant molecules contributed to the maintenance and active resilience of host-bacterial mutualism against mucosal oxygen and uncontrolled oxidative stress in vivo.

Fecal microbiota transplantation (FMT) has become an effective strategy to treat metabolic diseases, including type 2 diabetes mellitus (T2DM). We previously reported that the intestinal microbiome had significant difference between individuals with normal glucose tolerance and T2DM in Chinese Kazak ethnic group. In this study, we investigated the effects of transplanted fecal bacteria from Kazaks with normal glucose tolerance (KNGT) in db/db mice. The mice were treated with 0.2 mL of fecal bacteria solution from KNGT daily for 10 weeks. We showed that the fecal bacteria from KNGT successfully colonized in the intestinal tract of db/db mice detected on day 14. In the FMT-treated db/db mice, the levels of fasting blood glucose, postprandial glucose, total cholesterol, triglyceride, and low-density lipoprotein-cholesterol were significantly downregulated, whereas high-density lipoprotein-cholesterol levels were upregulated. In the FMT-treated db/db mice, Desulfovibrio and Clostridium coccoides levels in gut were significantly decreased, but the fecal levels of Akkermansia muciniphila and colon histone deacetylase-3 (HDAC3) protein expression were increased. At 8 weeks, both intestinal target bacteria and HDAC3 were correlated with glycolipid levels; Akkermansia muciniphila level was positively correlated with HDAC3 protein expression (r = +0.620, P = 0.037). Our results suggest that fecal bacteria from KNGT could potentially be used to treat diabetic patients.

The first complete genome of the biotechnologically important species Sulfobacillus thermotolerans has been sequenced. Its 3 317 203-bp chromosome contains an 83 269-bp plasmid-like region, which carries heavy metal resistance determinants and the rusticyanin gene. Plasmid-mediated metal resistance is unusual for acidophilic chemolithotrophs. Moreover, most of their plasmids are cryptic and do not contribute to the phenotype of the host cells. A polyphosphate-based mechanism of metal resistance, which has been previously unknown in the genus Sulfobacillus or other Gram-positive chemolithotrophs, potentially operates in two Sulfobacillus species. The methylcitrate cycle typical for pathogens and identified in the genus Sulfobacillus for the first time can fulfill the energy and/or protective function in S. thermotolerans Kr1 and two other Sulfobacillus species, which have incomplete glyoxylate cycles. It is notable that the TCA cycle, disrupted in all Sulfobacillus isolates under optimal growth conditions, proved to be complete in the cells enduring temperature stress. An efficient antioxidant defense system gives S. thermotolerans another competitive advantage in the microbial communities inhabiting acidic metal-rich environments. The genomic comparisons revealed 80 unique genes in the strain Kr1, including those involved in lactose/galactose catabolism. The results provide new insights into metabolism and resistance mechanisms in the Sulfobacillus genus and other acidophiles.

Many cases of Clostridioides difficile infection (CDI) are poorly responsive to standard antibiotic treatment strategies, and often patients suffer from recurrent infections characterized by severe diarrhea. Our group previously reported the successful cure of two patients with recurrent CDI using a standardized stool-derived microbial ecosystem therapeutic (MET-1). Using an in vitro model of the distal gut to support bacterial communities, we characterized the metabolite profiles of two defined microbial ecosystems derived from healthy donor stool (DEC58, and a subset community, MET-1), as well as an ecosystem representative of a dysbiotic state (ciprofloxacin-treated DEC58). The growth and virulence determinants of two C. difficile strains were then assessed in response to components derived from the ecosystems. CD186 (ribotype 027) and CD973 (ribotype 078) growth was decreased upon treatment with DEC58 metabolites compared to ciprofloxacin-treated DEC58 metabolites. Furthermore, CD186 TcdA and TcdB secretion was increased following treatment with ciprofloxacin-treated DEC58 spent medium compared to DEC58 spent medium alone. The net metabolic output of C. difficile was also modulated in response to spent media from defined microbial ecosystems, although several metabolite levels were divergent across the two strains examined. Further investigation of these antagonistic properties will guide the development of microbiota-based therapeutics for CDI.

Humans with metabolic and inflammatory diseases frequently harbour lower levels of butyrate-producing bacteria in their gut. However, it is not known whether variation in the levels of these organisms is causally linked with disease development and whether diet modifies the impact of these bacteria on health. Here we show that a prominent gut-associated butyrate-producing bacterial genus (Roseburia) is inversely correlated with atherosclerotic lesion development in a genetically diverse mouse population. We use germ-free apolipoprotein E-deficient mice colonized with synthetic microbial communities that differ in their capacity to generate butyrate to demonstrate that Roseburia intestinalis interacts with dietary plant polysaccharides to: impact gene expression in the intestine, directing metabolism away from glycolysis and toward fatty acid utilization; lower systemic inflammation; and ameliorate atherosclerosis. Furthermore, intestinal administration of butyrate reduces endotoxaemia and atherosclerosis development. Together, our results illustrate how modifiable diet-by-microbiota interactions impact cardiovascular disease, and suggest that interventions aimed at increasing the representation of butyrate-producing bacteria may provide protection against atherosclerosis.

Metabolism of dietary glycans is pivotal in shaping the human gut microbiota. However, the mechanisms that promote competition for glycans among gut commensals remain unclear. Roseburia intestinalis, an abundant butyrate-producing Firmicute, is a key degrader of the major dietary fibre xylan. Despite the association of this taxon to a healthy microbiota, insight is lacking into its glycan utilization machinery. Here, we investigate the apparatus that confers R. intestinalis growth on different xylans. R. intestinalis displays a large cell-attached modular xylanase that promotes multivalent and dynamic association to xylan via four xylan-binding modules. This xylanase operates in concert with an ATP-binding cassette transporter to mediate breakdown and selective internalization of xylan fragments. The transport protein of R. intestinalis prefers oligomers of 4-5 xylosyl units, whereas the counterpart from a model xylan-degrading Bacteroides commensal targets larger ligands. Although R. intestinalis and the Bacteroides competitor co-grew in a mixed culture on xylan, R. intestinalis dominated on the preferred transport substrate xylotetraose. These findings highlight the differentiation of capture and transport preferences as a possible strategy to facilitate co-growth on abundant dietary fibres and may offer a unique route to manipulate the microbiota based on glycan transport preferences in therapeutic interventions to boost distinct taxa.

Previous studies suggested a close relationship between ruminant gut microbes and the mammary gland. In this study, shotgun metagenomic sequencing was used to reveal the differences in the intestinal microbiome potentially related to milk components in Murrah buffaloes and Chinese Holstein cattle. A PCoA based on the weighted Unifrac distances showed an apparent clustering pattern in the structure of intestinal microbiota between buffalo and cattle. We could attribute the structural difference to the genera of Sutterella, Coprococcus and Dorea. A further analysis of microbial functional features revealed that the biosynthesis of amino acids (including lysine, valine, leucine and isoleucine), lipopolysaccharide biosynthesis and cofactor/vitamin biosynthesis were enriched in the buffalo. In contrast, dairy cattle had higher levels of pyruvate metabolism and carbon fixation in photosynthetic organisms. A further correlation analysis based on different milk components and the typical microbiome uncovered a significant positive correlation between milk protein and the microbial biosynthesis of amino acids, which was also positively correlated in the genera of Parabacteroides, Dorea and Sutterella. This study will expand our understanding of the intestinal microbiome of buffalo and cattle as representative ruminants, as well as provide new views about how to improve the production and nutritional qualities of animal milk., Competing Interests: The authors declare no competing financial interests.

The digestion of dietary fibers is a major function of the human intestinal microbiota. So far this function has been attributed to the microorganisms inhabiting the colon, and many studies have focused on this distal part of the gastrointestinal tract using easily accessible fecal material. However, microbial fermentations, supported by the presence of short-chain fatty acids, are suspected to occur in the upper small intestine, particularly in the ileum. Using a fosmid library from the human ileal mucosa, we screened 20,000 clones for their activities against carboxymethylcellulose and xylans chosen as models of the major plant cell wall (PCW) polysaccharides from dietary fibres. Eleven positive clones revealed a broad range of CAZyme encoding genes from Bacteroides and Clostridiales species, as well as Polysaccharide Utilization Loci (PULs). The functional glycoside hydrolase genes were identified, and oligosaccharide break-down products examined from different polysaccharides including mixed-linkage β-glucans. CAZymes and PULs were also examined for their prevalence in human gut microbiome. Several clusters of genes of low prevalence in fecal microbiome suggested they belong to unidentified strains rather specifically established upstream the colon, in the ileum. Thus, the ileal mucosa-associated microbiota encompasses the enzymatic potential for PCW polysaccharide degradation in the small intestine.

During the course of evolution, the cellulosome, one of Nature's most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.

Porphyromonas gingivalis and Filifactor alocis are fastidious anaerobic bacteria strongly associated with chronic forms of periodontitis. Our understanding of the growth activities of these microorganisms in situ is very limited. Previous studies have shown that copy numbers of ribosomal-RNA precursor (pre-rRNA) of specific pathogen species relative to genomic-DNA (gDNA) of the same species (P:G ratios) are greater in actively growing bacterial cells than in resting cells. The method, so-called steady-state pre-rRNA-analysis, represents a novel culture-independent approach to study bacteria. This study employed this technique to examine the in situ growth activities of oral bacteria in periodontitis before and after non-surgical periodontal therapy. Sub-gingival paper-point samples were taken at initial and re-evaluation appointments. Pre-rRNA and gDNA levels of P. gingivalis and F. alocis were quantified and compared using reverse-transcriptase qPCR. The results indicate significantly reduced growth activity of P. gingivalis, but not F. alocis, after therapy. The P:G ratios of P. gingivalis and F. alocis were compared and a low-strength, but statistically significant inter-species correlation was detected. Our study demonstrates that steady-state pre-rRNA-analysis can be a valuable culture-independent approach to studying opportunistic bacteria in periodontitis.

A high solid digester with dewatered sludge was operated for 110 days to ascertain the interactions between bacterial and archaeal communities under ammonium stress, as well as the corresponding changes in bio-degradation mechanisms. The volatile solids reduction (95% confidence intervals in mean) changed from 31.6 ± 0.9% in the stable period (day 40-55) to 21.3 ± 1.5% in the last period (day 71-110) when ammonium concentration was elevated to be within 5,000-6,000 mgN/L. Biogas yield dropped accordingly from 11.9 ± 0.3 to 10.4 ± 0.2 L/d and carbon dioxide increased simultaneously from 35.2% to 44.8%. Anaerobranca better adapted to the ammonium stress, while the initially dominant protein-degrading microbes-Tepidimicrobium and Proteiniborus were suppressed, probably responsible for the increase of protein content in digestate. Meanwhile, Methanosarcina, as the dominant Archaea, was resistant to ammonium stress with the constant relative abundance of more than 92% during the whole operation. Nonmetric Multidimensional Scaling (NMDS) analysis was thus conducted which indicated that the gradually increased TAN dictated the bacterial clusters. The dominant Methanosarcina and the increased carbon dioxide content under ammonium stress suggested that, rather than the commonly acknowledged syntrophic acetate oxidation (SAO) with hydrogenotrophic methanogenesis, only SAO pathway was enhanced during the initial 'ammonium inhibition'.

Butyrate-producing bacteria (BPB) are potential probiotic candidates for inflammatory bowel diseases as they are often depleted in the diseased gut microbiota. However, here we found that augmentation of a human-derived butyrate-producing strain, Anaerostipes hadrus BPB5, significantly aggravated colitis in dextran sulphate sodium (DSS)-treated mice while exerted no detrimental effect in healthy mice. We explored how the interaction between BPB5 and gut microbiota may contribute to this differential impact on the hosts. Butyrate production and severity of colitis were assessed in both healthy and DSS-treated mice, and gut microbiota structural changes were analysed using high-throughput sequencing. BPB5-inoculated healthy mice showed no signs of colitis, but increased butyrate content in the gut. In DSS-treated mice, BPB5 augmentation did not increase butyrate content, but induced significantly more severe disease activity index and much higher mortality. BPB5 didn't induce significant changes of gut microbiota in healthy hosts, but expedited the structural shifts 3 days earlier toward the disease phase in BPB5-augmented than DSS-treated animals. The differential response of gut microbiota in healthy and DSS-treated mice to the same potentially beneficial bacterium with drastically different health consequences suggest that animals with dysbiotic gut microbiota should also be employed for the safety assessment of probiotic candidates.

Xyloglucan, a ubiquitous highly branched plant polysaccharide, was found to be rapidly degraded and metabolized by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum. Our study shows that at least four cellulosomal enzymes displaying either endo- or exoxyloglucanase activities, achieve the extracellular degradation of xyloglucan into 4-glucosyl backbone xyloglucan oligosaccharides. The released oligosaccharides (composed of up to 9 monosaccharides) are subsequently imported by a highly specific ATP-binding cassette transporter (ABC-transporter), the expression of the corresponding genes being strongly induced by xyloglucan. This polysaccharide also triggers the synthesis of cytoplasmic β-galactosidase, α-xylosidase, and β-glucosidase that act sequentially to convert the imported oligosaccharides into galactose, xylose, glucose and unexpectedly cellobiose. Thus R. cellulolyticum has developed an energy-saving strategy to metabolize this hemicellulosic polysaccharide that relies on the action of the extracellular cellulosomes, a highly specialized ABC-transporter, and cytoplasmic enzymes acting in a specific order. This strategy appears to be widespread among cellulosome-producing mesophilic bacteria which display highly similar gene clusters encoding the cytosolic enzymes and the ABC-transporter.