Computational protocol: Low Concentrations of Vitamin C Reduce the Synthesis of Extracellular Polymers and Destabilize Bacterial Biofilms

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Protocol publication

[…] Acquired MS spectra were processed with the MaxQuant software suite (version (), integrated with an Andromeda search engine. Database search was performed against a target-decoy database of B. subtilis 168 downloaded from UniProt (taxonomy ID 1423), containing 4,195 protein entries, and additionally including also 248 commonly observed laboratory contaminant proteins. Endoprotease Trypsin/P was set as the protease with a maximum missed cleavage of two. Carbamidomethylation (Cys) was set as a fixed modification. Label free quantification was enabled with a minimum ratio count of two. A false discovery rate of 1% was applied at the peptide and protein level individually for filtering identifications. Initial mass tolerance was set to 20 ppm. In case of the main search, the peptide mass tolerance of precursor and the fragment ions were set to 4.5 and 20 ppm, respectively. Downstream bioinformatics analysis was performed using Perseus version (). Grouping of proteins with similar expression profiles was achieved by hierarchical clustering analysis. Log10 transformation of mean LFQ intensities of proteins was performed for all the tested conditions. Missing values were replaced from the normal distribution via imputation. Hierarchical clustering was performed on Z-score transformed values using Euclidean as a distance measure and Average linkage cluster analysis. Significance B (p ≤ 0.05) was calculated to identify significantly regulated proteins in each of the ascorbate treatment conditions relative to the control. [...] Log10 LFQ protein intensities were used to generate sodium ascorbate concentration specific models by mapping the protein intensities to the genome-scale metabolic model of B. subtilis (Bs-iYO844) (). Log10 LFQ protein intensities from biological replicates (three replicates for each sodium ascorbate concentration and 2 for the control) were averaged and used to constrain the fluxes in the associated reaction using Gene Inactivity Moderated by Metabolism and Expression (GIMME) (). GIMME was run using 90% of the objective function threshold and 50th percentile of proteins expression level. Since properly constrained reactions do not demonstrate uniform distributions of feasible steady-state fluxes, the range and distribution of feasible metabolic flux for each reaction were determined by using Markov Chain Monte Carlo (MCMC) sampling (). To do this, a large number of feasible sets of metabolic fluxes were randomly moved within the solution space until they were well mixed, thereby sampling the entire solution space (). This sampling process yielded a distribution of feasible steady-state fluxes for each reaction. Sampling was done using gpSampler with default settings from the COBRA toolbox () using Gurobi (Gurobi Optimization, Inc., Houston, TX, United States) and MATLAB® (The MathWorks Inc., Natick, MA, United States). Averaged sampled predicted flux distributions for each reaction at each sodium ascorbate concentration were compared to those from the control model in order to identify reactions (and their associated genes) that have significantly altered flux rates upon adding sodium ascorbate to B. subtilis. […]

Pipeline specifications

Software tools GIMME, COBRA Toolbox
Application Metabolic engineering
Organisms Escherichia coli, Bacteria
Chemicals Ascorbic Acid