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Intragenic DNA inversions expand bacterial coding capacity

ABSTRACT: Bacteria can generate heterogeneity through phase variation, including enzyme-mediated inversion of specific intergenic regions of genomic DNA. We developed a computation tool that identifies invertible elements using long-read datasets, PhaVa. We identified over 300 ‘intragenic invertons,’ a new class of invertible elements entirely within genes, in both bacteria and archaea. In the gut commensal Bacteroides thetaiotaomicron, we experimentally characterized an intragenic inverton in the thiamine biosynthesis protein thiC. We used mass spectrometry in data-independent acquisition mode to identify and quantify B. theta proteins, including thiC inverton expression at the protein level.

INSTRUMENT(S): timsTOF Ultra

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

SUBMITTER: Krystal Lum

LAB HEAD: Ileana M. Cristea

PROVIDER: PXD054577 | Pride | 2024-08-06

REPOSITORIES: Pride

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Project description:Although current mass spectrometry (MS)-based proteomics identifies and quantifies thousands of proteins and (modified) peptides, only a minority of them are subjected to in-depth downstream analysis. With the advent of automated processing workflows, biologically or clinically important results within a study are rarely validated by visualization of the underlying raw information. Although several tools for this are in principle available, they are often not integrated into the overall analysis nor readily extendable with new approaches. To remedy this, we developed AlphaViz, an open-source Python package to superimpose output from common analysis workflows on the raw data for quick and easy visualization and validation of protein and peptide identifications. AlphaViz takes advantage of recent breakthroughs in the deep learning-assisted prediction of experimental peptide properties to allow manual assessment of the expected and measured peptide result deviation. We focused on the visualization of the 4-dimensional data cuboid provided by Bruker TimsTOF instruments, where the ion mobility dimension, besides intensity and retention time, can be predicted and used for verification. We illustrate how AlphaViz can quickly validate or invalidate peptide identifications regardless of the score given to them by automated workflows. Furthermore, we provide a â€˜predict modeâ€™ that can locate peptides present in the raw data but not reported by the search engine. This is illustrated with dilution series and the recovery of missing values from experimental replicates. Applied to phosphoproteomics of the EGF-signaling pathway, we show how key signaling nodes can be validated to enhance confidence for downstream interpretation or follow-up experiments. AlphaViz follows accepted standards for open-source software development, including extensive documentation, testing and continuous integration. It features an easy-to-install graphical user interface for end-users and a modular Python package for bioinformaticians. We hope that AlphaViz can help to make validation of critical proteomics results a standard feature in MS-based proteomics.

Dataset Information

Intragenic DNA inversions expand bacterial coding capacity

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