Co‑cultivation of the anaerobic fungus Caecomyces churrovis with Methanobacterium bryantii enhances transcription of carbohydrate binding modules, dockerins, and pyruvate formate lyases on specific substrates

Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus. C. churrovis possess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal–methanogen physical associations and fungal cell wall development and remodeling.

Sugar beet cold-induced PMT5a and STP13 carriers are poised for taproot proton-driven plasma membrane sucrose and glucose import

SummaryAs the major sugar-producing crop in the northern hemisphere, sugar beet taproots store sucrose at a concentration of about 20 %. While the vacuolar sucrose loader TST has already been identified in the taproot, sugar transporters mediating sucrose uptake across the plasma membrane of taproot parenchyma cells remained unknown.We electrophysiologically examined taproots for proton-coupled sugar uptake and identified potentially involved transporters by transcriptomic profiling. After cloning, the transporter features were studied in the heterologous Xenopus laevis oocyte expression system using the two-electrode voltage clamp technique. Insights into the structure were gained by 3D homology modeling.As with glucose, sucrose stimulation of taproot parenchyma cells caused inward H+-fluxes and plasma membrane depolarization, indicating a sugar/proton symport mechanism. As one potential candidate for sugar uploading, the BvPMT5a was characterized as a H+-driven low-affinity glucose transporter, which does not transport sucrose. BvSTP13 operated as a high-affinity H+/sugar symporter, transporting glucose and to some extent sucrose due to a binding cleft plasticity. Both transporter genes were upregulated upon cold exposure, with the transport capacity of BvSTP13 being more cold-resistant than BvPMT5a.Identification of BvPMT5a and BvSTP13 as taproot sugar transporters could improve breeding of cold-tolerant sugar beet to provide a sustainable energy crop.

Effect of Short-Term Exposure to Titanium Dioxide Nanoparticles on Intestinal Absorption of Glucose by Ex Vivo Everted Rat Gut Sac Model

Titanium dioxide nanoparticles (TiO2 NPs) as food additives were widely found in various foodrelated products, especially in high-sugar foods. The daily intake of TiO2 NPs in the diet may therefore expose the small intestine to TiO2 NPs and affect its physiological functions, including the absorption of nutrients. It is speculated that TiO2 may cause serious health hazards by increasing sugar uptake. To explore this possibility, transport of glucose from small intestine was studied using an everted gut sac model prepared from small intestine of young healthy male SD rats. The translocation of TiO2 NPs and the morphological changes of small intestine were also observed after exposure of intestinal lumen to TiO2 NPs for 2 h. The results showed that TiO2 NPs can enter into enterocyte but hardly cross the intestinal epithelium. No change on microstructure of gut epithelia and expression of glucose transporter was found, and there is no obvious impact on intestinal absorption and metabolism of glucose. These results suggest that short-term exposure to TiO2 NPs has little influence on intestinal absorption of glucose. More attention should be paid to the chronic effect of dietary consumption of TiO2 NPs on nutrient absorption.

Sucrose-dependence of sugar uptake, quorum sensing and virulence of the rice blight pathogen Xanthomonas oryzae pv. oryzae

Virulence of Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial leaf blight of rice, depends on induction of host SWEET sucrose efflux transporters. It remained unknown whether secreted sucrose serves bacterial nutrition or host defense. Here we identified the sux sucrose uptake/utilization locus of Xoo and demonstrate that it is necessary and sufficient for sucrose acquisition. Induction of sux genes during infection closely tracked induction of rice SWEET11a. sux mutants were defective in swimming, swarming, extracellular polysaccharide (EPS) production and biofilm formation. EPS synthesis in mutants was restored by the quorum-sensing factor DSF. Notably, transcripts for rate limiting steps in DSF production were unaffected by sucrose, transcripts of the DSF receptor were sucrose-inducible and increased during infection, indicating sensitization to DSF in response to sucrose supply. Sucrose induced the sigma factors transcripts for RpoN1 and RpoN2 that regulate swimming, EPS and virulence. Furthermore, in contrast to Xanthomonas axonopodis pv. manihotis, virulence of Xoo depended critically on sux gene function. Together, pathogen-induced sucrose efflux from host cells likely induces bacterial sigma factors and sensitizes quorum signaling necessary for biofilm formation and colonization of the xylem, serves as energy source for swimming against the xylem stream, and as nutrient for growth.

Co‑cultivation of the anaerobic fungus Caecomyces churrovis with Methanobacterium bryantii enhances transcription of carbohydrate binding modules

Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid forming anaerobic fungus. C. churrovis possess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal-methanogen physical associations and fungal cell wall development and remodeling.

Characterization of simultaneous uptake of xylose and glucose in Caldicellulosiruptor kronotskyensis for optimal hydrogen production

Background Caldicellulosiruptor kronotskyensis has gained interest for its ability to grow on various lignocellulosic biomass. The aim of this study was to investigate the growth profiles of C. kronotskyensis in the presence of mixtures of glucose–xylose. Recently, we characterized a diauxic-like pattern for C. saccharolyticus on lignocellulosic sugar mixtures. In this study, we aimed to investigate further whether C. kronotskyensis has adapted to uptake glucose in the disaccharide form (cellobiose) rather than the monosaccharide (glucose). Results Interestingly, growth of C. kronotskyensis on glucose and xylose mixtures did not display diauxic-like growth patterns. Closer investigation revealed that, in contrast to C. saccharolyticus, C. kronotskyensis does not possess a second uptake system for glucose. Both C. saccharolyticus and C. kronotskyensis share the characteristics of preferring xylose over glucose. Growth on xylose was twice as fast (μmax = 0.57 h−1) as on glucose (μmax = 0.28 h−1). A study of the sugar uptake was made with different glucose–xylose ratios to find a kinetic relationship between the two sugars for transport into the cell. High concentrations of glucose inhibited xylose uptake and vice versa. The inhibition constants were estimated to be KI,glu = 0.01 cmol L−1 and KI,xyl = 0.001 cmol L−1, hence glucose uptake was more severely inhibited by xylose uptake. Bioinformatics analysis could not exclude that C. kronotskyensis possesses more than one transporter for glucose. As a next step it was investigated whether glucose uptake by C. kronotskyensis improved in the form of cellobiose. Indeed, cellobiose is taken up faster than glucose; nevertheless, the growth rate on each sugar remained similar. Conclusions C. kronotskyensis possesses a xylose transporter that might take up glucose at an inferior rate even in the absence of xylose. Alternatively, glucose can be taken up in the form of cellobiose, but growth performance is still inferior to growth on xylose. Therefore, we propose that the catabolism of C. kronotskyensis has adapted more strongly to pentose rather than hexose, thereby having obtained a specific survival edge in thermophilic lignocellulosic degradation communities.

Pectin Induced Colony Expansion of Soil-Derived Flavobacterium Strains

The genus Flavobacterium is characterized by the capacity to metabolize complex organic compounds and a unique gliding motility mechanism. Flavobacteria are often abundant in root microbiomes of various plants, but the factors contributing to this high abundance are currently unknown. In this study, we evaluated the effect of various plant-associated poly- and mono-saccharides on colony expansion of two Flavobacterium strains. Both strains were able to spread on pectin and other polysaccharides such as microcrystalline cellulose. However, only pectin (but not pectin monomers), a component of plant cell walls, enhanced colony expansion on solid surfaces in a dose- and substrate-dependent manner. On pectin, flavobacteria exhibited bi-phasic motility, with an initial phase of rapid expansion, followed by growth within the colonized area. Proteomic and gene expression analyses revealed significant induction of carbohydrate metabolism related proteins when flavobacteria were grown on pectin, including selected SusC/D, TonB-dependent glycan transport operons. Our results show a positive correlation between colony expansion and the upregulation of proteins involved in sugar uptake, suggesting an unknown linkage between specific operons encoding for glycan uptake and metabolism and flavobacterial expansion. Furthermore, within the context of flavobacterial-plant interactions, they suggest that pectin may facilitate flavobacterial expansion on plant surfaces in addition to serving as an essential carbon source.

Effect of ligand sensing on flagellar bundle formation in bacteria

Peritrichously flagellated E. coli swims in liquid media by rotating the flagellar bundle. The direction of rotation of each flagellum is governed by a transmembrane rotary nanomotor, which receives signals from ligand-specific receptors. Attractants bias the motor to rotate in CCW direction causing flagella to bundle and provide thrust for locomotion. Recent studies have shown that sensing not only leads to increase in CCW bias but also increases the motor rotation speed. Despite the detailed studies on bacterial motility, the effect of ligand sensing on the synchronization of flagellar filaments leading to bundle formation and changes in bundle geometry are not clear. In this work, we performed real-time imaging of the flagellar bundle of swimming cells in metabolising (glucose) and non-metabolisable (2-Deoxy-d-glucose) attractants. We characterized bundles during swimming by measuring visible distal length and the spread of filaments at poles. We show that sensing of attractant by receptor leads to the formation of tight bundles when compared to control buffer. Contrary to previous studies, the swimming speeds were proportional to the bundle tightness with the latter dependent not only on the bias but also on the torque exerted by the motor. We further show that the observed wiggles in the swimming trajectory of cells is directly proportional to the spread angles of bundle and is effected by both motor CCW bias and torque. Mutant cells, which were rendered non-motile due to the absence of the PTS (phosphotransferase system) sugar uptake mechanism, exhibited motility when exposed to the non-metabolisable attractant confirming that mere sensing can induce torque in flagellar motor. These results clarify the role of sensing and metabolism on bundle formation and its impact on the motility of cells.

Characterization of Simultaneous Uptake of Xylose and Glucose in Caldicellulosiruptor Kronotskyensis for Optimal Hydrogen Production

BackgroundCaldicellulosiruptor kronotskyensis has gained interest for its ability to grow on various lignocellulosic biomass. The aim of this study was to investigate the growth profiles of C . kronotskyensis in the presence of mixtures of glucose-xylose. Recently, we characterized a diauxic-like pattern for C. saccharolyticus on lignocellulosic sugar mixtures. In this study we aimed to investigated further whether C . kronotskyensis has adapted to uptake glucose in the disaccharide form (cellobiose) rather than the monosaccharide (glucose). ResultsInterestingly, growth of C . kronotskyensis on glucose and xylose mixtures did not display diauxic-like growth patterns. Closer investigation revealed that, in contrast to C. saccharolyticus , C . kronotskyensis does not possess a second uptake system for glucose. Both C. saccharolyticus and C . kronotskyensis share the characteristics of preferring xylose over glucose. Growth on xylose was twice as fast (μ max = 0.57 h -1 ) as on glucose (μ max = 0.28 h -1 ). It was found that C . kronotskyensis takes up glucose and xylose simultaneously with the same transporter. A study of the sugar uptake was made with different glucose-xylose ratios to find a kinetic relationship between the two sugars for transport into the cell. High concentrations of glucose inhibited xylose uptake and vice versa. The inhibition constants were estimated to be K I,glu = 0.01 cmol·L -1 and K I,xyl = 0.001 cmol·L -1 , hence glucose uptake was more severely inhibited by xylose uptake. Bioinformatic analysis indicated the lack of another sugar uptake system in C . kronotskyensis as compared to C. saccharolyticus . Therefore, it was investigated whether glucose uptake by C . kronotskyensis was in the form of cellobiose. Indeed, cellobiose is taken up faster than glucose, nevertheless, the growth rate on each sugar remained similar. ConclusionsC . kronotskyensis possesses a xylose transporter that might take up glucose at an inferior rate even in the absence of xylose. Alternatively, glucose can be taken up in the form of cellobiose, but growth performance is still inferior to growth on xylose. Therefore, we propose that the catabolism of C . kronotskyensis has adapted more strongly to pentose rather than hexose thereby having obtained a specific survival edge in thermophilic lignocellulosic degradation communities.