In-depth analysis of Bacillus subtilis proteome identifies new ORFs and traces the evolutionary history of modified proteins

Abstract

Bacillus subtilis is a sporulating Gram-positive bacterium widely used in basic research and biotechnology. Despite being one of the best-characterized bacterial model organism, recent proteomics studies identified only about 50% of its theoretical protein count. Here we combined several hundred MS measurements to obtain a comprehensive map of the proteome, phosphoproteome and acetylome of B. subtilis grown at 37 °C in minimal medium. We covered 75% of the theoretical proteome (3,159 proteins), detected 1,085 phosphorylation and 4,893 lysine acetylation sites and performed a systematic bioinformatic characterization of the obtained data. A subset of analyzed MS files allowed us to reconstruct a network of Hanks-type protein kinases, Ser/Thr/Tyr phosphatases and their substrates. We applied genomic phylostratigraphy to gauge the evolutionary age of B. subtilis protein classes and revealed that protein modifications were present on the oldest bacterial proteins. Finally, we performed a proteogenomic analysis by mapping all MS spectra onto a six-frame translation of B. subtilis genome and found evidence for 19 novel ORFs. We provide the most extensive overview of the proteome and post-translational modifications for B. subtilis to date, with insights into functional annotation and evolutionary aspects of the B. subtilis genome.

Comparison of Proteomic Responses as Global Approach to Antibiotic Mechanism of Action Elucidation

“…To build the proteomic response library, we chose the gram-positive model organism B. subtilis 168, which is susceptible to most antibiotics, limiting constraints due to resistance, compound uptake, or efflux, which can impede this type of analysis for pathogens, and, in particular, multi-resistant pathogens. The proteome of on November 1, 2020 by guest http://aac.asm.org/ Downloaded from B. subtilis has been investigated in-depth 20 and an extensive knowledgebase exists on its regulatory circuits and protein functions, which provide an optimal basis for a thorough interpretation of proteomic responses.…”

Evolutionary Analysis of the Bacillus subtilis Genome Reveals New Genes Involved in Sporulation

“…Based on this criterion, a manual literature search of phenotype data for all entries in the two sporulation databases yielded 361 known sporulation genes ( supplementary table S1 , Supplementary Material online). Next, we examined the distribution of these sporulation genes in the available B. subtilis phylostratigraphy map ( Ravikumar et al. 2018 ).…”

“…1 A ). It should be noted that the B. subtilis genome follows a well-established trend: most genes belong to the old PS, whereas younger PS contain progressively fewer genes ( Ravikumar et al. 2018 ).…”

“…By this criterion, we performed a manual literature search of phenotype data for all entries in the two sporulation databases. Distribution of resulted genes in the previously published B. subtilis phylostratigraphy map ( Ravikumar et al. 2018 ) was analyzed.…”

“…In this study, we asked whether B. subtilis sporulation genes, by analogy to developmental genes of Metazoa, might also cluster in terms of evolutionary age. By examining the B. subtilis whole genome phylostratigraphy map ( Ravikumar et al. 2018 ), we found that B. subtilis sporulation genes arose at several time-points in evolution (significantly enriched in four different PS), suggesting that the sporulation process underwent several stages of expansion.…”