Bryantii B14 Research Articles

CRISPR-based screening of small RNA modulators of bile susceptibility in Bacteroides thetaiotaomicron

Microbiota-centric interventions are limited by our incomplete understanding of the gene functions of many of its constituent species. This applies in particular to small RNAs (sRNAs), which are emerging as important regulators in microbiota species yet tend to be missed by traditional functional genomics approaches. Here, we establish CRISPR interference (CRISPRi) in the abundant microbiota member Bacteroides thetaiotaomicron for genome-wide sRNA screens. By assessing the abundance of different protospacer-adjacent motifs, we identify the Prevotella bryantii B14 Cas12a as a suitable nuclease for CRISPR screens in these bacteria and generate an inducible Cas12a expression system. Using a luciferase reporter strain, we infer guide design rules and use this knowledge to assemble a computational pipeline for automated gRNA design. By subjecting the resulting guide library to a phenotypic screen, we uncover the sRNA BatR to increase susceptibility to bile salts through the regulation of genes involved in Bacteroides cell surface structure. Our study lays the groundwork for unlocking the genetic potential of these major human gut mutualists and, more generally, for identifying hidden functions of bacterial sRNAs.

Impact of nicotine and cotinine on macrophage inflammatory plasticity via vesicular modifications in gastrointestinal bacteria

ObjectivesThis study aimed to elucidate mechanistic explanation(s) for compositional changes to enteric microbiota by determining the impacts of continuous nicotine/cotinine exposure on representative gastrointestinal bacteria and how these alterations impact innate immune cell plasticity. MethodsIn vitro cultures of the gastrointestinal bacteria (Bacteroides fragilis 25285, Prevotella bryantii B14, and Acetoanaerobium sticklandii SR) were continuously exposed to nicotine or cotinine. Supernatant samples were collected for fermentation acid analysis. Vesicles were collected and analyzed for physiological changes in number, size, and total protein cargo. Cultured macrophages were stimulated to a tolerogenic phenotype, exposed to control or altered (nicotine or cotinine – exposed) vesicles, and inflammatory plasticity assessed via inflammatory cytokine production. ResultsNicotine/cotinine exposure differentially affected metabolism of all bacteria tested in a Gram (nicotine) and concentration-dependent (cotinine) manner. Physiological studies demonstrated changes in vesiculation number and protein cargo following nicotine/cotinine exposures. Continuous exposure to 1 μM nicotine and 10 μM cotinine concentrations reduced total protein cargo of Gram (-) – 25285 and B14 vesicles, while cotinine generally increased total protein in Gram (+) - SR vesicles. We found that theses physiological changes to the vesicles of 25285 and SR formed under nicotine and cotinine, respectively, challenged the plasticity of tolerogenic macrophages. Tolerogenic macrophages exposed to vesicles from 1 μM nicotine, and 5 or 10 μΜ cotinine cultures produced significantly less IL-12p70, TNFα, or KC/GRO, regardless of macrophage exposure to nicotine/cotinine. ConclusionsNicotine/cotinine exposure differentially alters bacterial metabolism and vesicle physiology, ultimately impacting the inflammatory response of tolerogenic macrophages.

Na+-Coupled Respiration and Reshaping of Extracellular Polysaccharide Layer Counteract Monensin-Induced Cation Permeability in Prevotella bryantii B14.

Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P. bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P. bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P. bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.

Short-Chain Fatty Acids Modulate Metabolic Pathways and Membrane Lipids in Prevotella bryantii B14.

Short-chain fatty acids (SCFAs) are bacterial products that are known to be used as energy sources in eukaryotic hosts, whereas their role in the metabolism of intestinal microbes is rarely explored. In the present study, acetic, propionic, butyric, isobutyric, valeric, and isovaleric acid, respectively, were added to a newly defined medium containing Prevotella bryantii B14 cells. After 8 h and 24 h, optical density, pH and SCFA concentrations were measured. Long-chain fatty acid (LCFA) profiles of the bacterial cells were analyzed via gas chromatography-time of flight-mass spectrometry (GC-ToF MS) and proteins were quantified using a mass spectrometry-based, label-free approach. Cultures supplemented with single SCFAs revealed different growth behavior. Structural features of the respective SCFAs were identified in the LCFA profiles, which suggests incorporation into the bacterial membranes. The proteomes of cultures supplemented with acetic and valeric acid differed by an increased abundance of outer membrane proteins. The proteome of the isovaleric acid supplementation showed an increase of proteins in the amino acid metabolism. Our findings indicate a possible interaction between SCFAs, the lipid membrane composition, the abundance of outer membrane proteins, and a modulation of branched chain amino acid biosynthesis by isovaleric acid.

Inhibition of Bacteroidetes and Firmicutes by select phytochemicals

Current research indicates that changes in gut microbiota can impact the host, but it is not always clear how dietary and environmental factors alter gut microbiota. One potential factor is antimicrobial activity of compounds ingested by the host. The goal of this study was to determine the antimicrobial activity of common plant secondary metabolites against pure cultures of paired, structurally and phylogenetically distinct gastrointestinal bacteria of human or bovine origin: Prevotella bryantii B14, Bacteroides fragilis 25285, Acetoanaerobium (Clostridium) sticklandii SR and Clostridioides difficile 9689. When growth media were amended with individual phytochemicals (the alkaloids: berberine, capsaicin, nicotine, piperine and quinine and the phenolic: curcumin), growth of each species was inhibited to varying degrees at the three greatest concentrations tested (0.10–10.00 mg mL−1). The viable cell numbers of all the cultures were reduced, ≥4-logs, by berberine at concentrations ≥1.00 mg mL−1. Quinine performed similarly to berberine for B14, 25285, and SR at the same concentrations. The other phytochemicals were inhibitory, but not as much as quinine or berberine. Nicotine had activity against all four species (≥2-log reduction in viable cell number at 10.00 mg mL−1), but had stronger activity against the Gram-positive bacteria, SR and 9689, (≥4-log reductions at 10.00 mg mL−1). In conclusion, the phytochemicals had varying spectra of antimicrobial activity. These results are consistent with the hypothesis that ingested phytochemicals have the ability to differentially impact gut microbiota through antimicrobial activity.

English

This study determined the effects of aromatic amino acids (AA) and phenyl acids (phenylacetic acid and phenylpropionic acid) on fermentation of xylan by selected rumen bacteria. The ruminal bacteria were grown in a defined medium containing oat spelts xylan as the sole energy source, plus one of the following N sources: ammonia (no AA); ammonia plus a complete mixture of 20 AA commonly found in protein (CAA); ammonia plus complete AA mixture minus aromatic AA (MAR); ammonia plus phenyl acids (no AA+PA); ammonia plus complete AA mixture without aromatic AA plus phenyl acids (MAR+PA). There was varied growth response, with Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD1, and Butyrivibrio fibrisolvens JW11 not being able to grow. The supply of CAA mixture significantly (P<0.05) increased acetate production in all species, in comparison with no AA treatment, by 54, 30, 97 and 33% in Prevotella bryantii B14, Ruminococcus albus SY3, R. flavefaciens 17 and Pseudobutyrivibrio xylanivorans JK170 cultures, respectively. The addition of phenyl acids significantly (P<0.05) increased acetate yields by 34, 8 and 5% in P. bryantii B14, R. albus SY3 and P. xylanivorans JK170 cultures, respectively. Sugar utilization by all the species significantly (P<0.05) increased by 42 and 77% when CAA mixture was provided. The deletion of aromatic AA from the complete AA mixture caused a significant (P<0.05) reduction in sugar utilization in P. bryantii B14 and P. xylanivorans JK170 by 25 and 10%, respectively. Phenyl acids addition increased sugar utilization by 4.8 and 5.1% in R. albus SY3 and R. flavefaciens 17, respectively. The supply of CAA mixture significantly (P<0.05) increased microbial yield in P. bryantii B14, R. albus SY3, R. flavefaciens 17 and P. xylanivorans JK170 species. It is concluded that supplementation of complete mixture of AA is beneficial in stimulating bacterial growth in the rumen.   Key words: Aromatic amino acids, phenylacetic acid, phenylpropionic acid, rumen bacteria, xylan.

Antimicrobial activity of Brazilian propolis extracts against rumen bacteria in vitro

The antimicrobial activity of three Brazilian propolis extracts was evaluated on bacterial strains representing major rumen functional groups. The extracts were prepared using different concentrations of propolis and alcohol, resulting in different phenolic compositions. The propolis extracts inhibited the growth of Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, Prevotella albensis M384, Peptostreptococcus sp. D1, Clostridium aminophilum F and Streptococcus bovis Pearl11, while R. albus 20, Prevotella bryantii B₁4 and Ruminobacter amylophilus H18 were resistant to all the extracts. The inhibited strains showed also different sensitivity to propolis; the hyper-ammonia-producing bacteria (C. aminophilum F and Peptostreptococcus sp. D1) being the most sensitive. Inhibition of hyper-ammonia-producing bacteria by propolis would be beneficial to the animal. The extract containing the lowest amount of phenolic compounds (LLOS C3) showed the lowest antimicrobial activity against all the bacteria. The major phenolic compounds identified in the propolis extracts (naringenin, chrysin, caffeic acid, p-coumaric acid and Artepillin C) were also evaluated on four sensitive strains. Only naringenin showed inhibitory effect against all strains, suggesting that naringenin is one of the components participating to the antibacterial activity of propolis.

Search for conjugative transposon in rumen bacterium Prevotella bryantii B14

Only few plasmids and bacteriophages have been described to date in ruminal prevotella strains, therefore it appears plausible that the genetic exchange in these organisms must exploit other routes. Large conjugative transposons make possible the gene exchange process in bacteria from the genus Bacteroides, the phylogenetic relatives of ruminal prevotellas. The access to fully or partially finished genome sequences of Bacteroides and Prevotella representatives made possible the search for conserved regions within putative conjugative transposons. Multiple sequence alignment of known and putative conjugative transposon gene sequences of Bacteroides thetaiotaomicron, Prevotella intermedia, Bacteroides fragilis and Tanerella sp. was used to locate partially conserved regions within most preserved conjugative transposition genes, traG, and to construct appropriate degenerated oligonucleotide primers. These were used to amplify genome fragments from ruminal prevotella strains. Sequence analysis of the subcloned PCR products revealed the presence of a hypothetical gene in the genome of Prevotella bryantii B14, similar to the ORF BF2880 from B. fragilis YCH46, which is a part of a large conjugative transposon. Inverse PCRs were designed and performed to confirm the initial findings. A partial map of P. bryantii B14 putative conjugative transposon region was constructed, indicating an intergeneric horizontal gene transfer.

Examination of AI-2 quorum sensing System in Prevotella bryantii and Prevotella ruminicola-like strains by using bioluminiscence assay

Rumen is an example of a complex microbial ecosystem where extensive intraspecies as well as interspecies cell-cell cross-communication is expected, but little is known about. Four ruminal Prevotella strains, Prevotella bryantii B14, TC1-1 and TF1-3, and P. ruminicola-like strain 223/M2/7A were examined for the presence of AI-2 type quorum sensing systems. Vibrio harveyi BB170 autoinducer bioassay was used in order to detect the production of AI-2 autoinducer in examined strains. Optimization of autoinducer bioassay was made to test the influence of sampling time and the substrate used in chemically defined medium CD on reporter strain luminescence signal. We discovered that AI-2 type qurum sensing system is present in P. ruminicola-like strain 223/M2/7A, which induced more than 74% of reporter strain bioluminescence, regardless of the presence of glucose in chemically defined medium CD. Such induction of bioluminescence was not observed in P. bryantii strains, however.

Medium- to large-sized xylo-oligosaccharides are responsible for xylanase induction in Prevotella bryantii B14

Experiments were done to define the nature of the xylan-derived induction signal for xylanase activity, and evaluate which xylanase genes among the three known ones (xynA, xynB and xynC) are induced by the presence of xylan in Prevotella bryantii B(1)4. During the later stages of exponential growth on glucose, addition of 0.05 % water-soluble xylan (WS-X) stimulated xylanase formation within 30 min. Xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, arabinose and glucuronic acid all failed to induce the xylanase activity. An acid-ethanol-soluble fraction of WS-X (approximate degree of polymerization 30) enhanced the activity significantly, whereas the acid-ethanol-insoluble fraction had no effect, unless first digested by the cloned P. bryantii XynC xylanase. These results indicate that medium- to large-sized xylo-oligosaccharides are responsible for induction. The transcription of all three known xylanase genes from P. bryantii was upregulated coordinately by addition of WS-X. There have been relatively few investigations into the regulation of xylanase activity in bacteria, and it appears to be unique that medium- to large-sized xylo-oligosaccharides are responsible for induction.

Transcriptional Regulation of β-Glucanase Activity in the Ruminal Bacterium, Prevotella bryantii B14

The ruminal bacterium, Prevotella bryantii B(1)4, grew more rapidly with glucose as an energy source than mannose (0.73 versus 0.47 h(-1)) and had 8-fold less beta-glucanase activity (50 versus 400 nmol reducing sugar mg protein(-1) min(-1)). Cultures that were provided with glucose and mannose had little beta-glucanase activity even though both sugars were utilized simultaneously. The observation that glucose and mannose were utilized simultaneously indicated that beta-glucanase expression was not merely a simple induction or inducer exclusion. When glucose was added to cultures growing on mannose, hexose flux through the glucomannokinase increased 1.5-fold, and this increase was associated with an almost immediate decrease in beta-glucanase mRNA. After only three generation (doubling) times, the amount of beta-glucanase mRNA was comparable to that observed in cells growing only with glucose. These results indicate that beta-glucanase activity is transcriptionally regulated. However, further work will be needed to define more precisely the nature of this regulation and to identify the intermediate in this response.

The effect of condensed tannins from Lotus pedunculatus and Lotus corniculatus on the growth of proteolytic rumen bacteria in vitro and their possible mode of action.

Five strains of proteolytic rumen bacteria were treated with condensed tannins (CT) purified from Lotus pedunculatus and Lotus corniculatus to investigate their effect on the growth of these bacteria in vitro. Streptococcus bovis NCFB 2476, Eubacterium sp. C124b, Prevotella bryantii B14, Butyrivibrio fibrisolvens H17c, and Clostridium proteoclasticum B316T were tested against 200, 400, and 600 microg CT x mL(-1) extracted from L. pedunculatus and L. corniculatus. In the absence of CT, all bacterial strains showed typical growth and reached maximum optical density (OD) after 6-8 h of incubation in a plant protein medium. Growth of Eubacterium sp., P. bryantii, and B. fibrisolvens was inhibited (P < 0.01-0.001) more by the CT from L. pedunculatus than by the CT from L. corniculatus. All strains continued to grow in the presence of 200 microg x mL(-1) of the CT from L. pedunculatus, but attained significantly (P < 0.05-0.01) lower maximum OD600 values than (minus CT) controls, except for S. bovis. At 400 and 600 microg x mL(-1), the addition of CT from L. pedunculatus inhibited (P < 0.05-0.001) the growth of all bacterial strains tested compared with controls. The growth of Eubacterium sp. and P. bryantii was stimulated for the first 4-6 h of incubation (P < 0.001) by 200 microg x mL(-1) of CT from L. corniculatus, but then declined leading to a significant difference in OD values compared with the controls. At 400 microg x mL(-1), the CT from L. corniculatus reduced (P < 0.05-0.01) the growth of all strains except S. bovis, while 600 microg x mL(-1) inhibited (P < 0.01-0.001) the growth of all strains. To study the mechanism of CT action, the degradation of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; Fraction 1 Leaf protein) was followed after bacterial cells or Rubisco were preincubated with CT extracted from L. corniculatus and L. pedunculatus. Both preincubations decreased LSU degradation, but they differed in their response to polyethylene glycol (PEG) addition. Addition of PEG to CT-Rubisco preincubations negated the effects of CT, while PEG addition to CT-bacteria preincubations did not. This implies that the CT-bacterial interaction is stronger than the CT-Rubisco interaction or the interaction is of a different type. Also, L. pedunculatus CT reduced the degradation of the LSU to a greater extent than the CT from L. corniculatus when preincubated with bacteria.

The glucomannokinase of Prevotella bryantii B(1)4 and its potential role in regulating beta-glucanase expression.

Prevotella bryantii B(1)4 has a transport system for glucose and mannose, but beta-glucanase expression is only catabolite-repressed by glucose. P bryantii B(1)4 cell extracts had ATP-dependent gluco- and mannokinase activities, and significant phosphoenolpyruvate- or GTP-dependent hexose phosphorylation was not observed. Mannose inhibited glucose phosphorylation (and vice versa), and activity gels indicated that a single protein was responsible for both activities. Glucose was phosphorylated at a faster rate than was mannose [V(max) 280 nmol hexose (mg protein)(-1) min(-1) versus 60 nmol hexose (mg protein)(-1) min(-1), respectively] and glucose was a better substrate for the kinase (K(m) 0.12 mM versus 1.2 mM, respectively). The purified glucomannokinase (1250-fold) had a molecular mass of 68 kDa, but SDS-PAGE gels indicated that it was a dimer (monomer 34.5 kDa). The N-terminus (25 residues) had an 8 amino acid segment that was homologous to other bacterial glucokinases. The glucomannokinase was competitively inhibited by the nonmetabolizable glucose analogue 2-deoxyglucose (2DG), and cells grown with glucose and 2DG had lower rates of glucose consumption than did cells given only glucose. When the ratio of 2DG to glucose was increased, the glucose consumption rate decreased and the beta-glucanase activity increased. The glucose consumption rate and the glucomannokinase activity of cells treated with 2DG were highly correlated (r(2)=0.98). This result suggested that glucomannokinase activity was either directly or indirectly regulating beta-glucanase expression.

Molecular cloning of the rRNA genes of Prevotella bryantii B14

Parts of the rRNA operons of anaerobic rumen bacteria Prevotella bryantii B14 were cloned. 350 colonies carrying pBluescriptII with HindIII DNA fragments were screened with 16S rDNA specific probe and six positive clones were obtained. Inserts with 16S rRNA genes were identified by hybridisation with digoxigenin labelled Prevotella-Bacteroides 16S rRNA specific probe. All six inserts were approximately 2 kb long and contained large parts of the 16S rRNA gene, 16S-23S rRNA internal spacer region and approximately 100 bp of 23S rRNA gene. The origin of 16S rRNA genes were confirmed by PCR with P. bryantii 16S rRNA specific primer.

XYLANOLYTIC ENZYME SYSTEM OF RUMEN BACTERIUM Prevotella bryantii B14

Prevotella spp. are recognised as one of the most numerous strictly anaerobic bacteria inhabiting the rumen. Potentially significant activities include the degradation of plant cell wall polysaccharides, starch, proteins and peptides. P. bryantii B14 is not cellulolytic but actively degrades hemicellulose xylan and carries multiple xylanase genes. Four regions encoding xylanase activity have been isolated, one of which encodes a previously isolated CMC-ase. Of the remaining regions, one encodes activities against p-nitrophenyl-b -xyloside and p-nitrophenyl-a -L-arabinofuranoside (genes xyn A and xynB). The gene xynC encodes another endoxylanase. SDS PAGE xylanograms revealed four endoxylanolytic bands at 29 kDa, 45 kDa, 66 kDa and 88 kDa. The majority of endoxylanase and CMC-ase activity was found in periplasmic cell fraction while most of the a -L-arabinofuranosidase and b -xylosidase activities were found in the crude membrane fraction. HPLC separation of periplasmic proteins by CIM DEAE 8 tubes resulted in partial isolation of CMC-ase and 66-kDa endoxylanase.

On the localization of xylanolytic enzymes in Prevotella Bryantii B14

Prevotella bryantii B14 is a strictly anaerobic, Gram-negative, polysaccharides-degrading ruminal bacterium. EDTA/sphaeroplasting and osmotic shock procedure were used in present work to localise endoxylanolytic, b -xylosidase and a -L-arabinofuranosidase activities of P. bryantii B14. Late exponential phase cells released most of the endoxylanolytic and CMC-ase activity by osmotic shock, showing the periplasmic location of both enzymatic activities, while b -xylosidase and a -L-arabinofuranosidase activities were recovered largely in membrane cell fraction. About 23 % of the total culture endoxylanase activity and about 30 % of the CMC-ase activity were found to be extracellular. No b -xylosidase and a -L-arabinofuranosidase activities were detected in culture supernatant of P. bryantii B14.