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Pull down of enzymes using alpha-amylase activity based probes from Bacteroides thetaiotaomicron lysate

ABSTRACT: This experiment aimed to identify GH13 alpha-amylaases from Bacteroides thetaiotaomicron lysate using activity based probes.

INSTRUMENT(S):

ORGANISM(S): Bacteroides Thetaiotaomicron (strain Atcc 29148 / Dsm 2079 / Nctc 10582 / E50 / Vpi-5482)

SUBMITTER: Isabelle Pickles

LAB HEAD: Gideon J Davies

PROVIDER: PXD053850 | Pride | 2025-06-16

REPOSITORIES: Pride

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			Action	DRS
	11032024_Bt1_minus_S1-A1_1_5541.d.zip	Other
	11032024_Bt1_minus_S1-A1_1_5541_uncalibrated.mzML	Mzml
	11032024_Bt1_plus_S1-A3_1_5543.d.zip	Other
	11032024_Bt1_plus_S1-A3_1_5543_uncalibrated.mzML	Mzml
	11032024_Bt2_minus_S1-A2_1_5542.d.zip	Other

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Regulated expression of polysaccharide utilization and capsular biosynthesis loci in biofilm and planktonic Bacteroides thetaiotaomicron during growth in chemostats

Project description:We used Affymetrix GeneChips to determine the physiological differences between biofilm and planktonic cells of Bacteroides thetaiotaomicron strain VPI-5482 (ATCC 29148) by comparing gene expression. For this purpose, B. thetaiotaomicron cells were grown in sterile, continuous flow bioreactors fed with tryptone, yeast extract, glucose (TYG) medium. The bioreactors were controlled at a temperature of 37C using a water jacket and a recirculating water heater. After 8 hours post-inoculation, planktonic cells were harvested from the bulk solution in the bioreactor, and after 8 days post-inoculation, the biofilm was scraped from the carbon paper. RNA was harvested from both biofilm and planktonic populations. RNA was extracted by a phenol:chloroform method and purified with a Qiagen RNA Easy mini-kit. Overall growth conditions are summarized above. The experimental conditions were: biofilm (BF), and planktonic (PL).

2014-01-01 | E-GEOD-38534 | biostudies-arrayexpress

Arylsulfamates inhibit colonic Bacteroides growth through lipid kinases and not sulfatases

Project description:Excessive degradation of the colonic mucin layer by Bacteroides within the human gut microbiota drives inflammatory bowel disease in mice. Bacterial carbohydrate sulfatases are key enzymes in gut colonization, as they are elevated in human inflammatory bowel disease and correlate with disease severity. Selective inhibitors of carbohydrate sulfatases could function as sulfatase-selective drugs, allowing precise control of sulfatase activity while preserving these otherwise beneficial bacteria. Arylsulfamates are covalent inhibitors that target a catalytic formylglycine residue of steroid sulfatases, which is also conserved in carbohydrate sulfatases. Here, using a library of aryl- and carbohydrate sulfamates, we find that they are ineffective against Bacteroides carbohydrate sulfatases yet can inhibit human gut microbiota species grown on sulfated glycans. Leveraging thermal proteome profiling, we identify a lipid kinase as the target responsible for these effects. This work highlights the imperative for developing specific inhibitors targeting carbohydrate sulfatases and unveils the adverse effect that arylsulfamates have on Bacteroides species of the human gut microbiota.

2025-07-28 | PXD065411 | Pride

Investigation of Role of Eleven TonB Homologs in Carbohydrate Utilization by Bacteroides thetaiotaomicron

Project description:Key conserved features of these transporters include a 22 beta-strand barrel that traverses the outer membrane and houses a plug domain that occludes solute passage until the transporter is activated. Some B. theta TBDTs are also predicted to include two additional domains termed the N-terminal extension (NTE) and the Secretin and TonB N-terminus (STN) domain. The precise rearrangement of the plug domain to allow solute passage through the barrel is poorly understood but is facilitated by pairing to an inner membrane complex that harnesses proton motive force. This complex in E. coli includes the proteins TonB, ExbB, and ExbD. Recent structural analysis of this complex reveals the inner membrane spanning ExbB in a pentameric arrangement enclosed around a dimer of ExbD that extends into the periplasm. The structure of the complex with TonB has not been determined but other characterization of TonB suggests at least one copy of TonB interacts with the ExbBD complex via the TonB N-terminal membrane spanning -helix. This N-terminal -helix is followed by a linker region that spans the periplasm and a well-ordered C-terminal domain. The C-terminal domains of characterized TonB proteins share a common fold of three antiparallel -sheets with two -helices (28, 29). The final -strand at the C-terminus directly contacts the TBDT for -sheet pairing with the N-terminal extension of the TBDT plug domain called the TonBox.

2024-01-26 | PXD041518 | Pride

Bacteroides thetaiotaomicron OMV proteomics

Project description:Comparative proteomics of Bacteroides thetaiotaomicron samples comparing the total membrane (TM) and outer membrane vesicles (OMV) of WT B. thetaiotaomicron and delta 4364

2024-10-15 | PXD043219 | Pride

Transcription analysis of E. coli O103:H25 in co-culture with Bacteroides thetaiotaomicron CCUG 10774

Project description:Investigation of whole genome gene expression level changes in E. coli O103:H25 in co-culture with Bacteroides thetaiotaomicron CCUG 10774 compared to when E. coli is cultured individually. A study using total RNA recovered from three separate co-cultures (E. coli O103:H25 and B. thetaiotaomicron 1:100) at OD=0.5, compared to when E. coli is cultured individually (5 separate samples including 1-3 technical replicates of each sample). Three separate samples are also recovered when E. coli is cultured in supernatant from Bacteroides thetaiotaomicron at OD=0.5 compared to when E coli is cultured alone. A comparison is also done after treatment with Mitomycin C (MMC). MMC is added at OD=0.5. There are 3 separate samples from each group that are compared (co-culture versus E. coli grown individually, E. coli in supernatant from B. thetaiotaomicron versus E. coli grown individually).

2014-12-18 | E-GEOD-44790 | biostudies-arrayexpress

Comparative Proteomic Analysis of Fucosylated Glycoproteins in Bacteroides thetaiotaomicron under Different Polysaccharide Nutrition Conditions

Project description:Bacteroides thetaiotaomicron, one of the most eminent representative gut commensal Bacteroides species, is able to use the L-fucose in host-derived and dietary polysaccharides to modify its capsular polysaccharides and glycoproteins through a mammalian-like salvage metabolic pathway. This process is essential for the colonization of the bacteria and for symbiosis with the host. However, despite the importance of fucosylated proteins (FGPs) in Bacteroide thetaiotaomicron, their types, distribution, and functions remain unclear. In this project, the effects of different saccharide (glucose, corn starch, mucin, and fucoidan) nutrition conditions on FGP expressions and fucosylation are investigated using a chemical biological method based on metabolic labeling and bioorthogonal reaction. According to the results of label-free quantification, 559 FGPs (205 downregulated and 354 upregulated) are affected by the dietary conditions. Of these differentially expressed proteins, 65 proteins show extremely sensitive fucosylation levels. Specifically, the fucosylation of the chondroitin sulfate ABC enzyme, Sus proteins, and cationic efflux system proteins varies significantly upon the addition of mucin, corn starch, or fucoidan. Moreover, these polysaccharides can trigger an appreciable increase in the fucosylation level of the two-component system and ammonium transport proteins. These results highlight the efficiency of the combined metabolic glycan labeling and bio-orthogonal reaction in enriching the intestinal Bacteroide glycoproteins. Moreover, it emphasize the sensitivity of Bacteroides fucosylation to polysaccharide nutrition conditions, which allows for the regulation of bacterial growth.

2022-02-08 | PXD023756 | Pride

Characterizing the interactions between a naturally-primed IgA (mAb 260.8) and its conserved Bacteroides thetaiotaomicron species-specific epitope in gnotobiotic mice [array]

Project description:Comparisons of gnotobiotic Rag1-/- mice, with and without subcutaneous 260.8 hybridomas, disclosed that this IgA does not affect B. thetaiotaomicron population density or suppress 260.8 epitope production but does affect bacterial gene expression in ways that are emblematic of a diminished host innate immune response. C57BL/6 wildtype, C57BL/6J Rag1-/- , and C57BL/6J Rag1-/- mice harboring the 260.8 IgA producing hybridoma were colonized for 10 days with Bacteroides thetaiotaomicron VPI-5482.

2015-06-29 | E-GEOD-65455 | biostudies-arrayexpress

Bacteroides thetaiotaomicron grown on different sources of dietary protein and glucose

Project description:We previously observed in mice that Bacteroides thetaiotaomicron (B. theta) significantly increased in abundance in the gut microbiome of mice when mice were fed egg and yeast dietary protein sources. We also observed that B. theta was expressing proteins previously connected to growth on mucin glycans when mice were fed an egg-white diet. To confirm that the bacterium were actually responding to the protein sources, we grew it in vitro using a defined medium, where the sole carbon source was dietary protein, mucin, or glucose. Our controls were glucose and mucin, and our experimental protein sources were soy protein, egg-white protein, and Torula yeast protein. We grew four B. theta cultures per carbon source statically at 37°C in a Coy anaerobic chamber (2.5 % H2 /10 % CO2 /88.5 % N2) in minimal medium (100 mM KH2PO4, 8.5 mM [NH2]4SO4, 15 mM NaCl, 5.8 μM vitamin K3, 1.44 μM FeSO4⋅7H2O, 1 mM MgCl2, 1.9 μM hematin, 0.2 mM L-histidine, 3.69 nM vitamin B12, 208 μM L-cysteine, and 7.2 μM CaCl2⋅2H2O) with one of the above mentioned nutrients added at 0.5% (wt/v) concentration. In order to aid the solubilization of the dietary proteins, we pre-prepared the proteins in 200 mM NaOH at 37°C for four days, the glucose control was also dissolved in 200 mM NaOH. After 8 hours, we enumerated CFUs to confirm growth, and we pelleted cells by centrifuging at 4,000 g for 10 minutes. We then removed the supernatant and froze the pellets at -80°C within 30 minutes.

2024-12-18 | PXD050296 | Pride

RNAseq of the human gut commensal Bacteroides thetaiotaomicron wildtype, bt0700 and bt0700 bt3998 (ppGpp0) strains across three conditions: glucose minimal medium, 1h of carbon starvation, and mouse gut colonization

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