From the outer space to the inner cell: deconvoluting the complexity of Bacillus subtilis disulfide stress responses by redox state and absolute abundance quantification of extracellular, membrane, and cytosolic proteins

Abstract

Understanding cellular mechanisms of stress management relies on omics data as a valuable resource. However, the lack of absolute quantitative data on protein abundances remains a significant limitation, particularly when comparing protein abundances across different cell compartments. In this study, we aimed to gain deeper insights into the proteomic responses of the Gram-positive model bacterium Bacillus subtilis to disulfide stress. We determined proteome-wide absolute abundances, focusing on different sub-cellular locations (cytosol and membrane) as well as the extracellular medium, and combined these data with redox state determination. To quantify secreted proteins in the culture medium, we developed a simple and straightforward protocol for the absolute quantification of extracellular proteins in bacteria. We concentrated extracellular proteins, which are highly diluted in the medium, using StrataClean beads along with a set of standard proteins to determine the extent of the concentration step. The resulting data set provides new insights into protein abundances in different sub-cellular compartments and the extracellular medium, along with a comprehensive proteome-wide redox state determination. Our study offers a quantitative understanding of disulfide stress management, protein production, and secretion in B. subtilis.

Importance: Stress responses play a crucial role in bacterial survival and adaptation. The ability to quantitatively measure protein abundances and redox states in different cellular compartments and the extracellular environment is essential for understanding stress management mechanisms. In this study, we addressed the knowledge gap regarding absolute quantification of extracellular proteins and compared protein concentrations in various sub-cellular locations and in the extracellular medium under disulfide stress conditions. Our findings provide valuable insights into the protein production and secretion dynamics of B. subtilis, shedding light on its stress response strategies. Furthermore, the developed protocol for absolute quantification of extracellular proteins in bacteria presents a practical and efficient approach for future studies in the field. Overall, this research contributes to the quantitative understanding of stress management mechanisms and protein dynamics in B. subtilis, which can be used to enhance bacterial stress tolerance and protein-based biotechnological applications.

Keywords: Bacillus subtilis; absolute protein quantification; disulfide stress; redox proteomics; subcellular fractionation.

Fig 1

(A) Workflow for absolute protein quantification of extracellular proteins. Shortly, extracellular proteins present in the cell supernatant were bound on StrataClean resin together with a known amount of concentration standards (spiked-in). Then, bound proteins were electroeluted by SDS-PAGE and separated together with UPS2 standards for absolute quantification. Finally, in-gel digestion of proteins was performed prior to liquid chromatography-tandem mass spectrometry measurement. (B) Concentration factors calculated with the concentration standards in four biological replicates. (C) Calibration curve for UPS2 quantification standards.

Fig 2

(A) Difference of secreted molecules per milliliter for secreted proteins [log₂ fold change (FC)] between exponential and stationary phases. Blue means higher abundance in the stationary phase; red means higher abundance in the exponential phase. (B) Dynamics of the top 10 major components of the exoproteome with their concentrations indicated in femtomoles per milliliter (average ± SD). Connecting lines visualize the change in the ranking position if the protein is still among the 10 most secreted proteins. The change in protein abundance between both growth phases is summarized in the log₂FC column, in which fold change is calculated as the difference of log-transformed secreted proteins in femtomole per milliliter in the stationary compared to the exponential phase for the top 10 most abundant proteins in stationary phase.

Fig 3

(A) Quantified molecules per cell assigned to proteins from each cellular location. Cytosolic proteins (115,000 ± 15,000 molecules per cell) were excluded from membrane fraction plotting to emphasize proteins assigned to the membrane proteome and translocated proteins. (B) Top 10 proteins with highest secretion rates. (C) Tracking of absolute protein abundances in molecules per cell at sub-cellular resolution for CwlO, TasA, and WprA. Protein copy numbers per cell for control samples are represented inside each box for the three different locations. Box color represents the relative abundance compared to other extracellular proteins in protein fractions from each location. Red indicates an abundance higher than the median abundance of extracellular proteins in the cellular fraction, whereas blue indicates lower abundant proteins. For the cytosol fraction, accumulation rates per minute were calculated from the difference between samples taken before induction and as control and are represented with a “ribosome-shaped” box, coded in green for active accumulation in the cytosol. For the membrane fraction, the transporter color is given in green for efficient secretion (here defined for those proteins whose secretion rate is higher than the median secretion rate quantified as 1.8 molecules/min) or in gray for standby secretion (proteins with a secretion rate lower than 1.8 molecules/min). For the extracellular fraction, secretion rates per minute are given for each protein.

Fig 4

Main quantified changes in the cytosol and membrane proteome in response to diamide stress. (A) Quantified cytosolic proteins involved in the oxidative stress response and protein homeostasis showing significant changes in their abundance (ng/µg of protein) under diamide stress. (B) Protein functions of which the molar proportion was changed under diamide stress in the cytosolic fraction. Quantified proteins within the functional category are listed below each plot. (C) Protein functions in the membrane of which the molar proportion was changed under diamide stress. Quantified proteins within the functional category are listed below each plot. Significance levels are expressed as * (adj. P ≤ 0.05), ** (adj. P ≤ 0.01), *** (adj. P ≤ 0.001) and **** (adj. P ≤ 0.0001). (D) Fatty acid biosynthetic pathway with quantified changes in protein abundance.

Fig 5

(A) Significantly changed (adjusted P value of <0.05) cysteines in the cellular fractions obtained from cytosol and membrane-enriched samples. In-box annotation (percent oxidized) and color reflect the average redox state for the quantified cysteine. (B) Fe-S cluster synthesis pathway describing protein complex stoichiometries under disulfide stress. In the table, percentage of oxidations (average ± SD) is described for each protein identified in the Fe-S cluster synthesis pathway for the samples taken before induction (BI), as well as 1 h after addition of diamide (DM) or medium [control (CT)].

Fig 6

Extracellular proteome response to diamide stress. (A) Total molecules per cell of quantified proteins in the supernatant for each condition grouped for cytosolic and extracellular proteins. (B) Sum of molecules per cell for proteins in the supernatant with other predicted cellular locations or unknown location. The inner pattern of each bar describes in further detail the predicted locations for proteins included in OTHER according to GP4 prediction (41). (C) Redox state of the one cysteine peptides from proteins in the extracellular space quantified for each condition. (D) Reduced cysteines per quantified molecules of protein in the supernatant for each condition grouped by their GP4-predicted cellular location.