Computational protocol: Glycan complexity dictates microbial resource allocation in the large intestine

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Protocol publication

[…] Potential surface-located enzymes (containing either a predicted lipoprotein signal sequence or uncleaved TM anchor) encoded by the B. ovatus xylan PULs were initially identified using the LipoP 1.0 (http://www.cbs.dtu.dk/services/LipoP/) and SigP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) servers. Note, the database N-term methionine for BACOVA_03425 GH43 is mis-annotated; the correct N-terminus actually starts 24aa downstream at the sequence MKN.Proteinase K treatment. Cultures of B. ovatus (50 ml) were grown in minimal media on WAX (0.5% w/v) as a sole carbon source, to mid-exponential growth phase (OD600 ∼0.8). Cells were harvested by centrifugation and washed twice in 10 ml phosphate buffered saline pH 7.2 (PBS), before being resuspended in 2.5 ml of the buffer. The cells were split into four 0.5 ml aliquots. To three of the aliquots 2 mg ml−1 Proteinase K was added and incubated at 37 °C for up to 16 h, the fourth sample was left as an untreated control for 16 h. Following incubation with the proteinase the samples were centrifuged at 5,000 g for 10 min and the supernatant discarded. The cell pellets were resuspended in 1 ml PBS and remaining ProteinaseK precipitated by the addition of 200 μl trichloroacetic acid and incubation on ice for 30 min. The cells were pelleted by centrifugation and washed four times in 1 ml ice cold acetone. The cell pellets were resuspended in 250 μl Laemmli buffer and subjected to SDS–PAGE. Proteins were transferred to Whatman Protran BA 85 nitrocellulose membrane using a wet transfer system (BioRad Mini Trans-Blot). Proteins of interest were detected using anti-sera raised in rats (Eurogentec) against the corresponding recombinant form of the protein, diluted 1/1,000 or 1/2,000 in 1% milk powder in TBS buffer. The secondary antibody used was a chicken anti-rat conjugated to horseradish peroxidase (Santa Cruz) diluted 1/5,000 in 1% milk powder in TBS buffer. Antibodies were detected by chemi-luminescence using Biorad Clarity Western ECL Substrate.Immunofluorescence microscopy. B. ovatus suspensions of mid-exponential phase cells (0.5 ml in PBS, produced as for Proteinase K treatment above) were fixed with an equal volume of 2 × formalin (9% formaldehyde in PBS), and rocked for 90 min at 25 °C in Eppendorf tubes. The cells were then pelleted by centrifugation for 3 min at 7,000 g and washed twice with 1 ml of PBS. The bacterial cell pellet was resuspended in 1 ml of blocking solution (2% goat serum, 0.02% NaN3 in PBS) and incubated at 4 °C for 16 h. After incubation cells were centrifuged again at 7,000 g and the supernatant discarded. For labelling, the bacteria were incubated with 0.5 ml of primary rat IgG (1/500 dilution of IgG in 1% milk powder in PBS, blocking solution) for 2 h at 25 °C. The cells were then pelleted, washed in 1 ml of PBS and resuspended in 0.4 ml goat anti-rat IgG Alexa-Fluor 488 (Sigma-Aldrich), diluted 1/500 in blocking solution and incubated 1 h at 25 °C in the dark. The cells were again pelleted, washed with PBS, resuspended in 1 ml of PBS containing ProLong Gold antifade reagent (Life Technologies). Aliquots (50 μl) of labelled bacterial cells were mounted onto glass slides and secured with coverslips. Fluorescence was visualized using a Leica SP2 UV microscope (Leica Microsystems, Heidelberg, GmbH) with × 63 NA1.32 lens. Alexa-Fluor 488-labelled bacteria were visualized under a UV view and compared with bright-field phase-contrast of the same image. […]

Pipeline specifications

Software tools LipoP, SignalP
Application Protein sequence analysis
Organisms Homo sapiens, Bacteroides ovatus
Chemicals Carbohydrates