Simulated digestions of free oligosaccharides and mucin-type O-glycans reveal a potential role for Clostridium perfringens.

The development of a stable human gut microbiota occurs within the first year of life. Many open questions remain about how microfloral species are influenced by the composition of milk, in particular its content of human milk oligosaccharides (HMOs). The objective is to investigate the effect of the human HMO glycome on bacterial symbiosis and competition, based on the glycoside hydrolase (GH) enzyme activities known to be present in microbial species. We extracted from UniProt a list of all bacterial species catalysing glycoside hydrolase activities (EC 3.2.1.-), cross-referencing with the BRENDA database, and obtained a set of taxonomic lineages and CAZy family data. A set of 13 documented enzyme activities was selected and modelled within an enzyme simulator according to a method described previously in the context of biosynthesis. A diverse population of experimentally observed HMOs was fed to the simulator, and the enzymes matching specific bacterial species were recorded, based on their appearance of individual enzymes in the UniProt dataset. Pairs of bacterial species were identified that possessed complementary enzyme profiles enabling the digestion of the HMO glycome, from which potential symbioses could be inferred. Conversely, bacterial species having similar GH enzyme profiles were considered likely to be in competition for the same set of dietary HMOs within the gut of the newborn. We generated a set of putative biodegradative networks from the simulator output, which provides a visualisation of the ability of organisms to digest HMO and mucin-type O-glycans. B. bifidum, B. longum and C. perfringens species were predicted to have the most diverse GH activity and therefore to excel in their ability to digest these substrates. The expected cooperative role of Bifidobacteriales contrasts with the surprising capacities of the pathogen. These findings indicate that potential pathogens may associate in human gut based on their shared glycoside hydrolase digestive apparatus, and which, in the event of colonisation, might result in dysbiosis. The methods described can readily be adapted to other enzyme categories and species as well as being easily fine-tuneable if new degrading enzymes are identified and require inclusion in the model.

Characterization of an antifungal ß-1,3-glucanase from Ficus microcarpa latex and comparison of plant and bacterial ß-1,3-glucanases for fungal cell wall ß-glucan degradation.

MAIN CONCLUSION: Each ß-1,3-glucanase with antifungal activity or yeast lytic activity hydrolyzes different structures of ß-1,3-glucans in the fungal cell wall, respectively. Plants express several glycoside hydrolases that target chitin and ß-glucan in fungal cell walls and inhibit pathogenic fungal infection. An antifungal ß-1,3-glucanase was purified from gazyumaru (Ficus microcarpa) latex, designated as GlxGluA, and the corresponding gene was cloned and expressed in Escherichia coli. The sequence shows that GlxGluA belongs to glycoside hydrolase family 17 (GH17). To investigate how GlxGluA acts to degrade fungal cell wall ß-glucan, it was compared with ß-1,3-glucanase with different substrate specificities. We obtained recombinant ß-1,3-glucanase (designated as CcGluA), which belongs to GH64, from the bacterium Cellulosimicrobium cellulans. GlxGluA inhibited the growth of the filamentous fungus Trichoderma viride but was unable to lyse the yeast Saccharomyces cerevisiae. In contrast, CcGluA lysed yeast cells but had a negligible inhibitory effect on the growth of filamentous fungi. GlxGluA degraded the cell wall of T. viride better than CcGluA, whereas CcGluA degraded the cell wall of S. cerevisiae more efficiently than GlxGluA. These results suggest that the target substrates in fungal cell walls differ between GlxGluA (GH17 class I ß-1,3-glucanase) and CcGluA (GH64 ß-1,3-glucanase).

Exploring the Palynological, Chemical, and Bioactive Properties of Non-Studied Bee Pollen and Honey from Morocco.

Bee products are known for their beneficial properties widely used in complementary medicine. This study aims to unveil the physicochemical, nutritional value, and phenolic profile of bee pollen and honey collected from Boulemane-Morocco, and to evaluate their antioxidant and antihyperglycemic activity. The results indicate that Citrus aurantium pollen grains were the majority pollen in both samples. Bee pollen was richer in proteins than honey while the inverse was observed for carbohydrate content. Potassium and calcium were the predominant minerals in the studied samples. Seven similar phenolic compounds were found in honey and bee pollen. Three phenolic compounds were identified only in honey (catechin, caffeic acid, vanillic acid) and six phenolic compounds were identified only in bee pollen (hesperidin, cinnamic acid, apigenin, rutin, chlorogenic acid, kaempferol). Naringin is the predominant phenolic in honey while hesperidin is predominant in bee pollen. The results of bioactivities revealed that bee pollen exhibited stronger antioxidant activity and effective α-amylase and α-glycosidase inhibitory action. These bee products show interesting nutritional and bioactive capabilities due to their chemical constituents. These features may allow these bee products to be used in food formulation, as functional and bioactive ingredients, as well as the potential for the nutraceutical sector.

A quantitative assay for agarase activity determination using agarose-iodine complex.

Rapid and simple spectrophotometric methods are required to detect various oligosaccharides produced by agar-hydrolysing enzymes. Herein, we present a quantitative agarose-iodine assay for agarase activity determination via the detection of the extent of agarose degradation. The agarose-iodine complex becomes reddish orange upon the addition of Lugol solution, and the enzymatic activity can be detected with ultraviolet-visible spectroscopy at 600 nm. The main advantages of this modified Lugol assay are high sensitivity, simple detection, and cost effectiveness. A novel definition of the unit to measure and compare the activities of agarases is also suggested.

Metagenome-assembled genomes and gene catalog from the chicken gut microbiome aid in deciphering antibiotic resistomes.

Gut microbial reference genomes and gene catalogs are necessary for understanding the chicken gut microbiome. Here, we assembled 12,339 microbial genomes and constructed a gene catalog consisting of ~16.6 million genes by integrating 799 public chicken gut microbiome samples from ten countries. We found that 893 and 38 metagenome-assembled genomes (MAGs) in our dataset were putative novel species and genera, respectively. In the chicken gut, Lactobacillus aviarius and Lactobacillus crispatus were the most common lactic acid bacteria, and glycoside hydrolases were the most abundant carbohydrate-active enzymes (CAZymes). Antibiotic resistome profiling results indicated that Chinese chicken samples harbored a higher relative abundance but less diversity of antimicrobial resistance genes (ARGs) than European samples. We also proposed the effects of geography and host species on the gut resistome. Our study provides the largest integrated metagenomic dataset from the chicken gut to date and demonstrates its value in exploring chicken gut microbial genes.

3D-printed microfluidics integrated with optical nanostructured porous aptasensors for protein detection.

Microfluidic integration of biosensors enables improved biosensing performance and sophisticated lab-on-a-chip platform design for numerous applications. While soft lithography and polydimethylsiloxane (PDMS)-based microfluidics are still considered the gold standard, 3D-printing has emerged as a promising fabrication alternative for microfluidic systems. Herein, a 3D-printed polyacrylate-based microfluidic platform is integrated for the first time with a label-free porous silicon (PSi)-based optical aptasensor via a facile bonding method. The latter utilizes a UV-curable adhesive as an intermediate layer, while preserving the delicate nanostructure of the porous regions within the microchannels. As a proof-of-concept, a generic model aptasensor for label-free detection of his-tagged proteins is constructed, characterized, and compared to non-microfluidic and PDMS-based microfluidic setups. Detection of the target protein is carried out by real-time monitoring reflectivity changes of the PSi, induced by the target binding to the immobilized aptamers within the porous nanostructure. The microfluidic integrated aptasensor has been successfully used for detection of a model target protein, in the range 0.25 to 18 µM, with a good selectivity and an improved limit of detection, when compared to a non-microfluidic biosensing platform (0.04 µM vs. 2.7 µM, respectively). Furthermore, a superior performance of the 3D-printed microfluidic aptasensor is obtained, compared to a conventional PDMS-based microfluidic platform with similar dimensions.

A detailed in silico analysis of the amylolytic family GH126 and its possible relatedness to family GH76.

The glycoside hydrolase (GH) family 126 was established based on the X-ray structure determination of the amylolytic enzyme CPF_2247 from Clostridium perfringens genome. Its original identification as a putative carbohydrate-active enzyme was based on its low, yet significant sequence identity to members of the family GH8, which are inverting endo-ß-1,4-glucanases. As the family GH8 forms the clan GH-M with GH48, the CPF_2247 protein also exhibits similarities with members of the family GH48. The original screening of the CPF_2247 on carbohydrate substrates demonstrated its activity on glycogen and amylose, thus classifying this protein as an "α-amylase". It should be pointed out, however, there are apparent inconsistencies concerning the exact enzyme specificity of the "amylase" CPF_2247, since it exhibits both the endo- and exo-fashion of action. The family GH126 currently counts ~1000 amino acid sequences solely from Bacteria; all belonging to the phylum Firmicutes. The present study delivers the first detailed bioinformatics study of 117 selected amino acid sequences from the family GH126, featuring the insightful sequence-structure comparison with the aim to define seven conserved sequence regions and elucidate the evolutionary relationships within the family. In addition, a comparative structural analysis of the GH126 members with representatives of other GH families adopting the same (α/α)6-barrel catalytic domain fold indicates the possible sharing a catalytic residue between the families GH126 and GH76.

A Colorimetric Assay to Enable High-Throughput Identification of Biofilm Exopolysaccharide-Hydrolyzing Enzymes.

Glycosidase enzymes that hydrolyze the biofilm exopolysaccharide poly-ß-(1→6)-N-acetylglucosamine (PNAG) are critical tools to study biofilm and potential therapeutic biofilm dispersal agents. Function-driven metagenomic screening is a powerful approach for the discovery of new glycosidase but requires sensitive assays capable of distinguishing between the desired enzyme and functionally related enzymes. Herein, we report the synthesis of a colorimetric PNAG disaccharide analogue whose hydrolysis by PNAG glycosidases results in production of para-nitroaniline that can be continuously monitored at 410 nm. The assay is specific for enzymes capable of hydrolyzing PNAG and not related ß-hexosaminidase enzymes with alternative glycosidic linkage specificities. This analogue enabled development of a continuous colorimetric assay for detection of PNAG hydrolyzing enzyme activity in crude E. coli cell lysates and suggests that this disaccharide probe will be critical for establishing the functional screening of metagenomic DNA libraries.

Enhanced production of Aspergillus niger inulinase from sugar beet molasses and its kinetic modeling.

OBJECTIVE: The fermentation medium contains many complex components (vitamins, minerals, etc.) for better growth of the microorganisms. The increasing purity and number of these components used in the medium seriously affect the cost of the microbial process. This study aimed to further optimize the concentration of the components used in the medium (yeast extract and peptone) for inulinase fabrication by Aspergillus niger from sugar-beet molasses in shake flask fermentation by using Central Composite Design (CCD) and to kinetically identify the fermentation. RESULTS: The results indicated that the optimal medium composition consisted of only 4.2% (w/v) yeast extract. By using the fermentation environment, the inulinase generation, inulinase/sucrase ratio, maximum inulinase generation rate, maximum sugar depletion rate, and substrate utilization yield were determined as 1294.5 U/mL, 1.2, 159.6 U/mL/day, 7.4 g/L/day, and 98.1%, respectively. The kinetic analysis of the fungal development (logistic model) indicated that a specific development rate and initial biomass concentration were 0.89/day and 1.79 g/L, respectively. Inulinase and sucrase productions are mixed-development associated since the α value ≠ 0 (8.46 and 4.31 U/mgX) and the ß value ≠ 0 (5.15 and 4.83 U/mgX day), respectively (Luedeking-Piret model). Besides, the maintenance value (Z) (0.009 gS/gX day) was lower than γ value (1.044 gS/gX), showing that A. niger commonly uses the substrates for enzyme fabrication and fungal development (modified Luedeking-Piret model). CONCLUSIONS: The enzyme activity was increased by optimizing the concentration of the components used. It was demonstrated that the proposed kinetic models can victoriously define fungal development, enzyme fabrication, and sugar depletion.

Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases.

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases.

Analysis of polysaccharide hydrolases secreted by Aspergillus flavipes FP-500 on corn cobs and wheat bran as complex carbon sources.

Aspergillus flavipes FP-500 is a Mexican native strain that has been reported as a good producer of xylanases and pectinases; therefore, it promises a strong impact on biotechnology. To provide an overview of protein secretion by A. flavipes, we carried out a comparative proteome analysis of extracellular proteins in liquid cultures with two heterogeneous agro-industrial residues; corn cob (CC) and wheat bran (WB), as carbon sources. Extracellular proteins obtained from both cultures were identified using MS/MS spectrometry. We identified 134 proteins, which were classified into four groups: glycosyl hydrolases (GH), esterases/proteases, miscellaneous proteins, and unidentified proteins. Around 50% of the total proteins identified were GH such as xylanases, ß-xylosidases, ß-galactosidases, cellulolytic enzymes like ß-glucosidase, endoglucanases, and cellobiohydrolases. From this family, a core of 22 (16%) of the proteins identified were found in both substrates, CC and WB, whereas 30% and 54% were unique for CC and WB, respectively. In the esterases/proteases group, proteases, lipases and esterases like feruloylesterases, and acetyl-xylanesterase were identified. Proteins with diverse functions such as monophosphate dehydrogenase or N-acetylglucosaminidase were present. Here, we present strong evidences indicating that the composition and heterogeneity of the used carbon source determine the specific set of protein secreted by the fungus.

Small Molecule as Fluorescent Probes for Monitoring Intracellular Enzymatic Transformations.

All cellular processes are the results of synchronized actions of several intracellular biochemical pathways. Recent emphasis is to visualize such pathways using appropriate small molecular reagents, dye-labeled proteins, and genetically encoded fluorescent biosensors that produce a luminescence ON response either on selective binding or on reacting with an analyte that is produced through a specific biochemical/enzymatic transformation. Studying such enzymatic processes by probing the fluorescence response as the read-out signal is expected to provide important insights into crucial biochemical transformations induced by an enzyme in its native form. Many of such studies are extended for monitoring enzymatic transformations under in vitro or in vivo condition. A few of the recent reports reveal that such molecular probes are even capable of quantifying abnormal levels of enzymes in real-time and is linked to the key area of clinical diagnostics and chemical biology. A synchronized analysis of all such reports helps in developing a rationale for designing purpose-built molecular probes or chemodosimeters as well as newer reagents for studying crucial enzymatic process or quantification of the respective enzyme. In this review, an attempt will be there to highlight several recent bioimaging reagents and studies that have provided insights into crucial biochemical or enzymatic transformations.

Capillary electrophoresis with dual detection UV/C4D for monitoring myrosinase-mediated hydrolysis of thiol glucosinolate designed for gold nanoparticle conjugation.

Capillary electrophoresis (CE) with dual UV and conductivity detection was used for the first time to monitor the functionalization of gold nanoparticles (AuNPs), a process catalyzed by an enzyme, myrosinase (Myr). A thiol glucosinolate (GL-SH) designed by our group was used as substrate. Hydrolysis of free and immobilized GL-SH was characterized using off-line and on-line CE-based enzymatic assays. The developed approaches were validated using sinigrin, a well-referenced substrate of Myr. Michaelis-Menten constant of the synthetized GL-SH was comparable to sinigrin, showing that they both have similar affinity towards Myr. It was demonstrated that transverse diffusion of laminar flow profiles was well adapted for in-capillary Mixing of nanoparticles (AuNPs) with proteins (Myr) provided that the incubation time is inferior to 20 min. Only low reaction volume (nL to few µL) and short analysis time (<5 min) were required. The electrophoretic conditions were optimized in order to evaluate and to confirm the AuNPs stability before and after functionalization by CE/UV based on surface plasmon resonance band red-shifting. The hydrolysis of the functionalized AuNPs was subsequently evaluated using the developed CE-C4D/UV approach. Repeatabilities of enzymatic assays, of electrophoretic analyses and of batch-to-batch functionalized AuNPs were excellent.

Dissipation of chlorantraniliprole in contrasting soils and its effect on soil microbes and enzymes.

An experiment was set up to determine the rate of dissipation of chlorantraniliprole (CTP) from two soils with contrasting properties. The other objective of the study was to find out the effect of CTP on soil microorganisms (population, microbial biomass carbon and soil enzymes) under controlled environment. CTP residues when applied at recommended dose ((RD) (at 40 g a.i./ha)) could not be recovered either from alluvial soil or red soil at 60 days post application of CTP in a microcosm study. Higher clay content led to higher half-life in alluvial soil compared to red soil. CTP could not be recovered from RD treatment at 30 days after pesticide application under controlled environment. Faster dissipation of CTP was observed in rice rhizosphere soil with 23.89 and 34.65 days dissipation half-lives for RD and double the recommended dose (DRD) treatments, respectively. Different doses of chlorantraniliprole did not have considerable negative effect on actinomycetes, fungi, biological nitrogen fixers and phospahte solubilising bacteria except the bacteria population. Among the treatments, DRD recorded the lowest activity of dehyrodeganse, fluoresein diacetate hydrolase, acid and alkaline phosphatases followed by RD treatment. Microbial biomass carbon, ß -glycosidase and urease did not vary significantly among the different doses of CTP. In general, RD did not have negative effcts on soil microbes. Hence, CTP can be recommeded in rice pest managment maintaining existing soil microbes and soil enzymes activity.

Characterization of membrane vesicles secreted by seaweed associated bacterium Alteromonas macleodii KS62.

Recent studies have reported abundant presence of bacterial extracellular membrane vesicles in the marine environment. However, the ecological significance of these bacterial vesicles in the marine environment is only beginning to be explored. In present study, for the first time we report and characterize membrane vesicles secreted by a seaweed associated bacterium, Alteromonas macleodii KS62. Proteomics studies revealed that the vesicle proteome was rich in hydrolytic enzymes (30%) like glycoside hydrolases, proteases, sulphatases, lipases, nucleases and phosphatases. Zymography experiments and enzyme assays established that the vesicles carry active κ-carrageenan degrading enzymes. κ-carrageenan is a major polysaccharide of cell walls of certain red seaweeds like Kappaphycus. Purified membrane vesicles were successfully able to degrade Kappaphycus biomass. Based on these results, we discuss how the hydrolase-rich vesicles may play a role in red seaweed cell wall degradation so that the bacteria can invade and colonise the seaweed biomass establishing a saprophytic lifestyle. We also discuss the role of these vesicles in nutrient acquisition and their ecological significance in the marine environment.

A simplified and miniaturized glucometer-based assay for the detection of ß-glucosidase activity.

ß-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of ß-glucosidase activity. Single-factor experiments showed that optimum ß-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of ß-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from ß-dextranase, snailase, ß-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for ß-glucosidase activity. The easy-to-operate method was successfully used to detect a series of ß-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of ß-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

Mutation of a Conserved Alanine Residue in Transcription Factor AraR Leads to Hyperproduction of α-l-Arabinofuranosidases in Penicillium oxalicum.

α-l-Arabinofuranosidases are important in the degradation of plant polysaccharides and are used in several industrial processes. Although the use of filamentous fungi for the production of α-l-arabinofuranosidases is widely reported, there are few reports on strain engineering for enhanced production of these enzymes by fungi. In this study, the function of transcription factor AraR in l-arabinose release and catabolism by the fungus Penicillium oxalicum (P. oxalicum) is investigated. Also, a mutant of AraR, AraRA731V , is constructed to improve the production of α-l-arabinofuranosidases on the basis of the sequence homology between AraR and the xylanolytic gene activator XlnR. The AraRA731V -overexpressing strain can synthesize α-l-arabinofuranosidase in the absence of an inducer and shows a 54.1-fold increase in α-l-arabinofuranosidase production and a 7.4-fold increase in α-galactosidase production in the medium containing wheat bran. Determination of the transcript abundances of lignocellulolytic enzyme genes reveals significant upregulation of multiple α-l-arabinofuranosidase genes and downregulation of some cellulolytic and xylanolytic enzyme genes in the engineered strain relative to its parent. Taken together, the results suggest the conserved regulatory function of AraR in the family Trichocomaceae and provide a strategy for engineering fungal strains for enhanced α- l-arabinofuranosidase production.

Inspecting the genome sequence and agarases of Microbulbifer pacificus LD25 from a saltwater hot spring.

Neoagaro-oligosaccharides prepared by agar hydrolysis have various application fields, including the pharmaceutical, cosmetic, and food industries. In this study, an agarolytic strain was isolated from a saltwater hot spring and identified as Microbulbifer pacificus LD25 by 16S rRNA. The whole genome sequence of M. pacificus LD25 was obtained. It had a size of 4.27 Mb and comprised 3062 predicted genes in 37 contigs with a G+C content of 58.0%. Six agarases were annotated and classified into three families, namely, GH16 (AgaL1), GH86 (AgaL2, AgaL3), and GH50 (AgaL4, AgaL5, AgaL6), which shared 75-96% identities with unpublished hypothetical proteins and agarases. AgaL1, AgaL4, and AgaL6 can be successfully expressed and purified in Escherichia coli. AgaL1 and AgaL4 displayed a significantly agarolytic capability, whereas AgaL6 exhibited a rarely detectable enzymatic activity. The optimal temperature and pH required for the activity of AgaL1 and AgaL4 was 50°C and 60°C, respectively, at pH 7. The specific activities of AgaL1 and AgaL4 were achieved at 16.8 and 9.6 U per mg of protein. Both agarases were significantly inhibited in the presence of EDTA, MgO, ZnCl2, and H2O2. However, AgaL1 was resistant to 0.1% SDS and AgaL4 was slightly activated by CaCl2. Substrate hydrolysis detected by LC-MS/MS analysis indicated that neoagarobiose was the main product during AgaL1 and AgaL4 catalysis. Furthermore, AgaL4 was thermostable and retained over 93% of its relative activity after pre-incubation at 70°C for 180 min. Consequently, M. pacificus LD25 has a potential for agarase production in E. coli and industrial applications.

Surface Exposure and Packing of Lipoproteins into Outer Membrane Vesicles Are Coupled Processes in Bacteroides.

Outer membrane vesicles (OMVs) are spherical structures derived from the outer membranes (OMs) of Gram-negative bacteria. Bacteroides spp. are prominent components of the human gut microbiota, and OMVs produced by these species are proposed to play key roles in gut homeostasis. OMV biogenesis in Bacteroides is a poorly understood process. Here, we revisited the protein composition of Bacteroides thetaiotaomicron OMVs by mass spectrometry. We confirmed that OMVs produced by this organism contain large quantities of glycosidases and proteases, with most of them being lipoproteins. We found that most of these OMV-enriched lipoproteins are encoded by polysaccharide utilization loci (PULs), such as the sus operon. We examined the subcellular locations of the components of the Sus system and found a split localization; the alpha-amylase SusG is highly enriched in OMVs, while the oligosaccharide importer SusC remains mostly in the OM. We found that all OMV-enriched lipoproteins possess a lipoprotein export sequence (LES), and we show that this signal mediates translocation of SusG from the periplasmic face of the OM toward the extracellular milieu. Mutations in the LES motif caused defects in surface exposure and recruitment of SusG into OMVs. These experiments link, for the first time, surface exposure to recruitment of proteins into OMVs. We also show that surface-exposed SusG in OMVs is active and rescues the growth of bacterial cells incapable of growing on starch as the only carbon source. Our results support the role of OMVs as "public goods" that can be utilized by other organisms with different metabolic capabilities.IMPORTANCE Species from the Bacteroides genus are predominant members of the human gut microbiota. OMVs in Bacteroides have been shown to be important for the homeostasis of complex host-commensal relationships, mainly involving immune tolerance and protection from disease. OMVs carry many enzymatic activities involved in the cleavage of complex polysaccharides and have been proposed as public goods that can provide growth to other bacterial species by release of polysaccharide breakdown products into the gut lumen. This work shows that the presence of a negatively charged rich amino acid motif (LES) is required for efficient packing of the surface-exposed alpha-amylase SusG into OMVs. Our findings strongly suggest that surface exposure is coupled to packing of Bacteroides lipoproteins into OMVs. This is the first step in the generation of tailor-made probiotic interventions that can exploit LES-related sequences to generate Bacteroides strains displaying proteins of interest in OMVs.

Monitoring Poly(ADP-ribosyl)glycohydrolase Activity with a Continuous Fluorescent Substrate.

The post-translational modification (PTM) and signaling molecule poly(ADP-ribose) (PAR) has an impact on diverse biological processes. This PTM is regulated by a series of ADP-ribosyl glycohydrolases (PARG enzymes) that cleave polymers and/or liberate monomers from their protein targets. Existing methods for monitoring these hydrolases rely on detection of the natural substrate, PAR, commonly achieved via radioisotopic labeling. Here we disclose a general substrate for monitoring PARG activity, TFMU-ADPr, which directly reports on total PAR hydrolase activity via release of a fluorophore; this substrate has excellent reactivity, generality (processed by the major PARG enzymes), stability, and usability. A second substrate, TFMU-IDPr, selectively reports on PARG activity only from the enzyme ARH3. Use of these probes in whole-cell lysate experiments has revealed a mechanism by which ARH3 is inhibited by cholera toxin. TFMU-ADPr and TFMU-IDPr are versatile tools for assessing small-molecule inhibitors in vitro and probing the regulation of ADP-ribosyl catabolic enzymes.