The Bacteroidetes Aequorivita sp. and Kaistella jeonii Produce Promiscuous Esterases With PET-Hydrolyzing Activity

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.

A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein O-Glycosylation in the Bacteroidetes

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species—Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked β1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

512 Late-Breaking: A Postbiotic from Lactobacillus Acidophilus Fermentation (LAF) Accelerates Progression of Compositional and Functional Gut Microbiome Clusters and Promotes Body Weight in Nursery Pigs

This study investigated the effects of a postbiotic from Lactobacillus acidophilus fermentation (LAF) on pig gut microbiome during nursery. Piglets (n = 32) at weaning (day 21 ± 2) were randomized based on BW (7.4 ± 1.7 kg) and received basal diets that met NRC nutrient requirements for phase 1 (d 0–14) and phase 2 (d 14–32) post-wean. Treatments included i) NC, negative control; ii) PC, antibiotic positive control, iii) LAFa (Dia-V™ Nursery, Diamond V, IA) supplemented at 1000 ppm in phase 1 and 1500 ppm in phase 2, and iv) LAFb (Dia-V™ Nursery) supplemented at 2000 and 1000 ppm during phase 1 and 2, respectively. Fecal samples were collected on d 0, 7, 14, and 32 post-wean and subjected to DNA extraction and long read Nanopore shotgun metagenomics to assess composition, function and progression of gut microbiome and their correlations with BW. On d7, PC and LAF supplemented pigs numerically were closer to microbiome of d 14 and 32 compared to NC pigs. From d 0 to 32, pigs progressed through five compositional clusters (P < 0.05). By d 32, more pigs from PC and LAF groups were exhibiting a compositional cluster that was characterized by dominance of members of Bacteroidetes phylum including several Prevotella species. From d 0 to d 32, four microbiome functional clusters were observed (P < 0.05) with varying abundances of carbohydrate-active-enzymes (CAZy). Pigs in LAF groups compared to other treatments exhibited clusters with greater abundances of CAZy that was correlated with greater BW on d 32 (P = 0.004). The 2000 ppm supplementation of LAF in phase 1 and 1500 ppm in phase 2 numerically increased all measured of diversity compared to 1000 ppm. Overall, while PC and LAF groups promoted more similar microbiome compositional clusters compared to NC, LAF pigs exhibited superior functional clusters.

PSXV-7 The mixture of herbal extracts that change the microbiome of the cattle rumen

Today, the search for new antibacterial natural agents based on plant raw materials is relevant. These raw materials have promising secondary metabolites that reduce the formation of methane in the ruminant scar. The aim of this study was to evaluate the effect of the following extracts: oak bark+birch leaves (I group) (250 ml/head per day), oak bark + St. John’s wort grass (250 ml / head per day; II group) and birch leaves + St. John’s wort grass (III group; 250 ml/head per day) on the rumen microbiota of large cattle, against the background of control (C). Extracts added to the water of calves with rumen fistula (dairy breed, age 12 months, diet -70 % of hay, 30 % of grain feed, duration is 14 days). The microflora analyzed using high efficiency 16S rRNA (Illumina) gene sequencing. Plant extracts prepared by purification, grinding (1–2 mm) and extraction in a water bath (for 30 min, 70 ° C) and filtration. According to the study results, the inclusion of extracts led to increase of the Bacteroidetes phylum for 1.11–3.69 % in the practical groups in comparison with C. A decrease was obtained for Firmicutes for 2.06–3.55 % (p 0.05) and Proteobacteria. In the I group bacteria of the class Bacteroides increased by 0.9–5.64% and Candidatus Saccharibacteria increased by 0.25% (P = 0.05). In the III group the value increased by 0.64 % (P = 0.05). Compared to C at the level of the family Prevotellaceae dominated in all experimental groups by 1.04–9.11 % (P ≤ 0.05), in group III Bacteroidaceae dominated by 0.27 % and Rikenellaceae by 0.14 %. This contributes to the regulation of opportunistic microorganisms without harmful effects on the rumen microbiota. This research was performed with financial support from the project 0761-2019-0005.

The abundance of mRNA transcripts of bacteroidetal polyethylene terephthalate (PET) esterase genes may indicate a role in marine plastic degradation

Polyethylene terephthalate (PET) is an important synthetic polymer accumulating in nature 2 and recent studies have identified microorganisms capable of degrading PET. While the majority of 3 known PET hydrolases originate from the Actinobacteria and Proteobacteria, here we describe the 4 first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific 5 Hidden-Markov-Model (HMM)-based search algorithm we identified two promiscuous and cold6 active esterases derived from Aequorivita sp. (PET27) and Chryseobacterium jeonii (PET30) acting 7 on PET foil and powder. Notably, one of the enzymes (PET30) was able to hydrolyze PET at 8 temperatures between 4° - 30°C with a similar turnover rate compared to the well-known Ideonella 9 sakaiensis enzyme (IsPETase). 10 PET27 and PET30 homologues were detected in metagenomes encompassing a wide range 11 of different global climate zones. Additional transcript abundance mapping of marine samples imply 12 that these promiscuous enzymes and source organisms may play a role in the long-term 13 degradation of microplastic particles and fibers.

Genomic and chemical decryption of the Bacteroidetes phylum for its potential to biosynthesize natural products

With progress in genome sequencing and data sharing, 1000s of bacterial genomes are publicly available. Genome mining – using bioinformatics tools in terms of biosynthetic gene cluster (BGC) identification, analysis and rating – has become a key technology to explore the capabilities for natural product (NP) biosynthesis. Comprehensively, analyzing the genetic potential of the phylum Bacteroidetes revealed Chitinophaga as the most talented genus in terms of BGC abundance and diversity. Guided by the computational predictions, we conducted a metabolomics and bioactivity driven NP discovery program on 25 Chitinophaga strains. High numbers of peerless strain-specific metabolite buckets confirmed the upfront predicted biosynthetic potential and revealed a tremendous uncharted chemical space. Sourcing this dataset, we isolated the new iron chelating nonribosomally synthesized cyclic tetradeca- and pentadecalipodepsipeptide antibiotics chitinopeptins with activity against Candida, produced by C. eiseniae DSM 22224 and C. flava KCTC 62435, respectively.

Gliding motility of a uranium tolerant Bacteroidetes bacterium Chryseobacterium sp. strain PMSZPI: Insights into the architecture of spreading colonies

Uranium tolerant soil bacterium Chryseobacterium sp. strain PMSZPI moved over solid agar surfaces by gliding motility thereby forming spreading colonies which is a hallmark of members of Bacteroidetes phylum. PMSZPI genome harbored orthologs of all the gld and spr genes considered as core bacteroidetes gliding motility genes of which gldK, gldL, gldM, and gldN were co-transcribed. Here, we present the intriguing interplay between gliding motility and cellular organization in PMSZPI spreading colonies. While nutrient deficiency enhanced colony spreading, high agar concentrations and presence of motility inhibitor like 5-hydroxyindole reduced the spreading. A detailed in situ structural analysis of spreading colonies revealed closely packed cells forming multiple layers at center of colony while the edges showed clusters of cells periodically arranged in hexagonal lattices interconnected with each other. The cell migration within the colony was visualized as branched structures wherein the cells were buried within extracellular matrix giving rise to fern like patterns. PMSZPI colonies exhibited strong iridescence that showed correlation with gliding motility. Presence of uranium reduced motility and iridescence and induced biofilm formation. This is a first report of gliding motility and iridescence in a bacterium from uranium enriched environment that could be of significant interest from an ecological perspective.

Serine/Glycine Lipid Recovery in Lipid Extracts From Healthy and Diseased Dental Samples: Relationship to Chronic Periodontitis

Toll-like receptor 2 (TLR2) activation has been implicated in the pathogenesis of periodontal disease but the identity of the TLR2 agonists has been an evolving story. The serine/glycine lipids produced by Porphyromonas gingivalis are reported to engage human TLR2 and will promote the production of potent pro-inflammatory cytokines. This investigation compared the recovery of serine/glycine lipids in periodontal organisms, teeth, subgingival calculus, subgingival plaque, and gingival tissues, either from healthy sites or periodontally diseased sites. Lipids were extracted using the phospholipid extraction procedure of Bligh and Dyer and were analyzed using liquid chromatography/mass spectrometry for all serine/glycine lipid classes identified to date in P. gingivalis. Two serine/glycine lipid classes, Lipid 567 and Lipid 1256, were the dominant serine/glycine lipids recovered from oral Bacteroidetes bacteria and from subgingival calculus samples or diseased teeth. Lipid 1256 was the most abundant serine/glycine lipid class in lipid extracts from P. gingivalis, Tannerella forsythia, and Prevotella intermedia whereas Lipid 567 was the most abundant serine/glycine lipid class recovered in Capnocytophaga species and Porphyromonas endodontalis. Serine/glycine lipids were not detected in lipid extracts from Treponema denticola, Aggregatibacter actinomycetemcomitans, or Fusobacterium nucleatum. Lipid 1256 was detected more frequently and at a significantly higher mean level in periodontitis tissue samples compared with healthy/gingivitis tissue samples. By contrast, Lipid 567 levels were essentially identical. This report shows that members of the Bacteroidetes phylum common to periodontal disease sites produce Lipid 567 and Lipid 1256, and these lipids are prevalent in lipid extracts from subgingival calculus and from periodontally diseased teeth and diseased gingival tissues.

Bacteroidetal cold-active and promiscuous esterases play a significant role in global polyethylene terephthalate (PET) degradation

Polyethylene terephthalate (PET) is an important synthetic polymer accumulating in nature and recent studies have identified microorganisms capable of degrading PET. While the majority of known PET hydrolases originate from the Actinobacteria and Proteobacteria, here we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model (HMM)-based search algorithm we identified two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Chryseobacterium jeonii (PET30) acting on PET foil and powder. Notably, one of the enzymes (PET30) was able to hydrolyze PET at temperatures between 4° - 30°C with a similar turnover rate compared to the well-known Ideonella sakaiensis enzyme (IsPETase). PET27 and PET30 homologues were detected in metagenomes encompassing a wide range of different global climate zones. Additional transcript abundance mapping of marine samples imply that these enzymes and source organisms play a significant role in the long-term degradation of microplastic particles and fibers.

High-Throughput Absolute Quantification Sequencing Revealed Osteoporosis-Related Gut Microbiota Alterations in Han Chinese Elderly

ObjectiveAccumulative evidence suggests that gut microbiota play an important role in bone remodeling and hence bone health maintenance. This study aimed to explore the association of gut microbiota with the risk of osteoporosis and to identify potential disease-related taxa, which may be promising targets in osteoporosis prevention and treatment in the future.MethodsAbsolute quantification 16S ribosomal RNA gene sequencing was used to detect absolute and relative abundances of gut microbiota in 44 patients with osteoporosis and 64 controls. In combination with one of our previous studies, a total of 175 samples were involved in the relative abundance analysis.ResultsCompared with the controls, the patients with osteoporosis had higher absolute and relative abundances of Bacteroidetes phylum, and Bacteroides and Eisenbergiella genera. The absolute abundances of Clostridium_XlVa, Coprococcus, Lactobacillus, and Eggerthella genera increased, and that of the Veillonella genus decreased in the osteoporosis group. As for relative abundance, that of the Parabacteroides and Flavonifractor genera increased, whereas that of the Raoultella genus decreased in the osteoporosis group. Controlling for potential confounders, the associations of Clostridium_XlVa, Coprococcus, and Veillonella genera with the risk of osteoporosis did not maintain significance. Ridge regression analysis suggested that Bacteroides is associated with reduced bone mineral density (BMD) and T-score at lumbar spines, and Anaerovorax is associated with increased BMD at the femoral neck. Functional predictions revealed that 10 Kyoto Encyclopedia of Genes and Genomes pathways were enriched in the osteoporosis group.ConclusionsGut microbiota compositions may contribute to the risk of osteoporosis. Several specific taxa and functional pathways are identified to associate with reduced bone density, thus providing epidemiologic evidence for the potential role of aberrant gut microbiota in osteoporosis pathogenesis.