High Resolution Analysis of Proteome Dynamics during Bacillus subtilis Sporulation

Abstract

Bacillus subtilis vegetative cells switch to sporulation upon nutrient limitation. To investigate the proteome dynamics during sporulation, high-resolution time-lapse proteomics was performed in a cell population that was induced to sporulate synchronously. Here, we are the first to comprehensively investigate the changeover of sporulation regulatory proteins, coat proteins, and other proteins involved in sporulation and spore biogenesis. Protein co-expression analysis revealed four co-expressed modules (termed blue, brown, green, and yellow). Modules brown and green are upregulated during sporulation and contain proteins associated with sporulation. Module blue is negatively correlated with modules brown and green, containing ribosomal and metabolic proteins. Finally, module yellow shows co-expression with the three other modules. Notably, several proteins not belonging to any of the known transcription regulons were identified as co-expressed with modules brown and green, and might also play roles during sporulation. Finally, levels of some coat proteins, for example morphogenetic coat proteins, decreased late in sporulation.

Nε-Lysine Acetylation of the Histone-Like Protein HBsu Regulates the Process of Sporulation and Affects the Resistance Properties of Bacillus subtilis Spores

“…The sporulation frequencies were significantly reduced when compared to wild-type in the sspA and sspB strains, with the most extreme phenotype observed for the sspA deletion (33-fold reduction, p -value < 0.0001, Figure 1C and Supplementary Table 2 ). The SASPs are synthesized and accumulate after the engulfment of the spore occurs ( Tu et al, 2021 ), so they are not involved in the early sporulation process. The SASPs play a role in spore heat resistance [see below, ( Setlow, 2006 )], and heat treatment is used during the determination of sporulation frequency, which could partially explain the observed phenotypes.…”

Whitefly effector G4 interacts with tomato proteins of which MIPDB141 affects whitefly performance

“…This analysis was performed as described in Tu et al (2021). Raw MS/MS data were searched in Maxquant (version: 1.6.14.0) (Cox and Mann, 2008) against an in house created B. tabaci proteome database and a S. lycopersicum database downloaded from Uniprot (06/2020).…”

Non-mechanical disruption ofBacillus subtilis sporesallows sensitive and deep characterization of the minimal proteome for resuming a cellular lifestyle

“…With the increasing demand for in-depth knowledge of the molecular physiology of spore-forming bacteria, further elucidation of the biological processes at hand is needed to completely comprehend the metabolic and adaptive networks operating within Bacillus cells and spores. This is also why the global analysis of proteins by proteomics is increasingly used in the sphere of research of bacterial sporulation, germination and outgrowth (4)(5)(6). Even though B. subtilis, a well-studied model of gram-positive bacteria, was among the first to have its entire genome sequenced, only around 50% of its theoretical proteome is routinely measured in proteomics studies while up to 80% has been identified when aggregating data from a large number of studies (7,8).…”

“…Proteins are of particular importance as some, such as small acid soluble proteins (SASPS) and highly cross-linked coat proteins, play a crucial role in spore resilience and structure (9,10). Because of this central role of proteins in the physical attributes of spores and concomitantly their resistance properties (11), analysis of protein content, quantity, crosslinking and complex formation and coat proteins using proteomics has been of the highest interest and multiple studies have been done over the past two decades (4)(5)(6)(12)(13)(14)(15)(16). Spore analysis through proteomics has multiple factors related to sample preparations that can influence the sensitivity, completeness and fidelity of the spore proteome: i) spore sample purity, ii) spore lysis efficiency, iii) protein extraction and solubilization efficiency and iv) completeness of protein digestion.…”

“…This approach (in essence an in-solution digestion approach) is also amenable to other lysis agents used for in solution digestion approaches such as guanidine hydrochloride (GnHCl), the bile salt sodium deoxycholate (SDC) or commercial mass spectrometry compatible surfactants like Rapigest SF and ProteaseMax. Other modern sample preparation techniques that utilize supplementary tools such as filters and beads, aimed at facilitating protein immobilization and/or purification like in-StageTip (iST) sample preparation using 18 discs (23) and filter assisted sample preparation (FASP) (24) with their ability to use harsher surfactants are also potentially useful and we previously optimized the use of single pot sample preparation for proteomics (25) (SP3) based on magnetic beads used in microbiology proteomics analysis (26–28) for spore proteomics in B. subtilis and B. cereus using the stronger surfactant SDS to improve solubilization of membrane bound proteins and less soluble proteins (5, 12). Although we used both OP and SP3 successfully to analyze spore proteomes and even performed the first true multi-omics study (29) of bacterial spores, a drawback of current approaches is the reliance on mechanic disruption techniques (bead milling) that require a relatively large sample input and are less amenable to state-of-the-art high throughput omics approaches using 96 or 384 well formats.…”