Sandra Maaß

Adaptation of Bacillus subtilis carbon core metabolism to simultaneous nutrient limitation and osmotic challenge: a multi‐omics perspective

The Gram-positive bacterium Bacillus subtilis encounters nutrient limitations and osmotic stress in its natural soil ecosystem. To ensure survival and sustain growth, highly integrated adaptive responses are required. Here, we investigated the system-wide response of B. subtilis to different, simultaneously imposed stresses. To address the anticipated complexity of the cellular response networks, we combined chemostat experiments under conditions of carbon limitation, salt stress and osmoprotection with multi-omics analyses of the transcriptome, proteome, metabolome and fluxome. Surprisingly, the flux through central carbon and energy metabolism is very robust under all conditions studied. The key to achieve this robustness is the adjustment of the biocatalytic machinery to compensate for solvent-induced impairment of enzymatic activities during osmotic stress. Specifically, increased production of several enzymes of central carbon metabolism compensates for their reduced activity in the presence of high salt. A major response of the cell during osmotic stress is the production of the compatible solute proline. This is achieved through the concerted adjustment of multiple reactions around the 2-oxoglutarate node, which drives metabolism towards the proline precursor glutamate. The fine-tuning of the transcriptional and metabolic networks involves functional modules that overarch the individual pathways.

A comprehensive analysis of Bordetella pertussis surface proteome and identification of new immunogenic proteins

Whooping cough, caused by the gram negative pathogen Bordetella pertussis, is a worldwide acute respiratory disease that predominantly involves infants. In the present study, surface proteins of B. pertussis Tohama I and Saadet strains were identified by using 2DE followed by MALDI-TOF-MS/MS analysis and also geLC-MS/MS. With these approaches it was possible to identify 45 and 226 proteins, respectively. When surface proteins of the strains were separated by 2DE and analyzed by Western blotting for their reactivity, a total of 27 immunogenic spots which correspond to 11 different gene products were determined. Glutamine-binding periplasmic protein, leu/ile/val-binding protein, one putative exported protein, and iron-superoxide dismutase (Fe-SOD) were found as immunogenic for the first time in Bordetella. Of a total of 226 proteins identified, 16 were differentially expressed in B. pertussis Saadet and Tohama I strains. Five proteins were expressed only in Saadet (adhesin, chaperone protein DnaJ, fimbrial protein FimX, putative secreted protein Bsp22 and putative universal stress protein), and two (ABC transporter substrate-binding protein and a putative binding protein-dependent transport periplasmic protein) only in Tohama I.

The new horizon in 2D electrophoresis: New technology to increase resolution and sensitivity

A principally new type of an electrophoresis setup for the second dimension of 2DE named HPE (high performance electrophoresis) has recently become available that provides excellent reproducibility much superior to traditional 2DE. It takes up ideas from early beginnings of 2DE which could not be satisfactory realized at that time. The new HPE system is in contrast to all other established systems a horizontal electrophoresis that employs a new type of precast polyacrylamide gels on film‐backing and runs on a multilevel flatbed electrophoresis apparatus. In a systematic approach we compared its features to traditional 2DE for the cytosolic proteome of Bacillus subtilis. Not only the reproducibility is enhanced, but also nearly all qualitative parameters as resolution, sensitivity, the number of protein spots (25% more), and the number of different proteins (also additional 25%) are markedly increased. More than 200 proteins were exclusively found in HPE. This new electrophoresis system does not use buffer tanks. No glass plates are needed. Therefore handling of gels is greatly facilitated and very simple to use even for personnel with low technical skills. The new HPE system is technically at the beginnings and further development with increased performance can be expected.

Monitoring global protein thiol-oxidation and protein S- mycothiolation in Mycobacterium smegmatis under hypochlorite stress

Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes. Here, we used shotgun proteomics, OxICAT and RNA-seq transcriptomics to analyse protein S-mycothiolation, reversible thiol-oxidations and their impact on gene expression in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation was accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues using OxICAT revealed that 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%). The RseA and RshA anti-sigma factors and the Zur and NrdR repressors were identified as NaOCl-sensitive proteins and their oxidation resulted in an up-regulation of the SigH, SigE, Zur and NrdR regulons in the RNA-seq transcriptome. In conclusion, we show here that NaOCl stress causes widespread thiol-oxidation including protein S-mycothiolation resulting in induction of antioxidant defense mechanisms in M. smegmatis. Our results further reveal that MSH is important to maintain the reduced state of protein thiols.

The protein inventory of Clostridium difficile grown in complex and minimal medium.

The intestinal pathogen Clostridium difficile is causing an increasing number of infections often characterized by severity and high relapse rates. Profound knowledge of the physiology of the pathogen could help to develop new treatment strategies. Proteomics, a valuable tool to study bacterial physiology, was used in this work to establish a benchmark proteome of reference strain C. difficile 630Δerm with MS‐based details on all identified proteins. Our elaborate annotation and visualization of C. difficile 630Δerm 3764 ORFs will serve as a valuable base for researchers having to evaluate global expression studies. To exemplify expression variability, protein expression of late exponentially growing cells in complex brain–heart infusion medium and C. difficile minimal medium was compared. Noteworthy results of this comparison are as follows: (i) the higher expression of enzymes for the biosynthesis of some vitamins and purine and (ii) downregulation of proteins involved in butanoate fermentation in C. difficile minimal medium. However, the abundance of proteins involved in DNA metabolism, protein synthesis, and the cell envelope showed no variation between the two growth media.

Methods and applications of absolute protein quantification in microbial systems.

In the last years the scientific community faced an increased need to provide high-quality data on the concentration of single proteins within a cell. Especially against the background of the fast evolving field of systems biology this does not only apply for a few proteins but preferably for the whole proteome of the organism. Therefore there has been a rapid development from pure identification of proteins via characterization of changes between different conditions by relative protein quantification towards determination of absolute protein amounts for hundreds of protein species in a cell. This review aims for discussion of different small-scale and large-scale approaches for absolute protein quantification in bacterial cells to picture biological processes and explore life in deeper detail. The presented advantages and limitations of various methods may provide interested researchers help to appraise available methods, select the most appropriate technique and avoid common pitfalls during determination of protein concentration in a complex sample.

The glyceraldehyde-3-phosphate dehydrogenase GapDH of Corynebacterium diphtheriae is redox-controlled by protein S -mycothiolation under oxidative stress

Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes and functions in post-translational thiol-modification by protein S-mycothiolation as emerging thiol-protection and redox-regulatory mechanism. Here, we have used shotgun-proteomics to identify 26 S-mycothiolated proteins in the pathogen Corynebacterium diphtheriae DSM43989 under hypochlorite stress that are involved in energy metabolism, amino acid and nucleotide biosynthesis, antioxidant functions and translation. The glyceraldehyde-3-phosphate dehydrogenase (GapDH) represents the most abundant S-mycothiolated protein that was modified at its active site Cys153 in vivo. Exposure of purified GapDH to H2O2 and NaOCl resulted in irreversible inactivation due to overoxidation of the active site in vitro. Treatment of GapDH with H2O2 or NaOCl in the presence of MSH resulted in S-mycothiolation and reversible GapDH inactivation in vitro which was faster compared to the overoxidation pathway. Reactivation of S-mycothiolated GapDH could be catalyzed by both, the Trx and the Mrx1 pathways in vitro, but demycothiolation by Mrx1 was faster compared to Trx. In summary, we show here that S-mycothiolation can function in redox-regulation and protection of the GapDH active site against overoxidation in C. diphtheriae which can be reversed by both, the Mrx1 and Trx pathways.

Enrichment of Cell Surface-Associated Proteins in Gram-Positive Bacteria by Biotinylation or Trypsin Shaving for Mass Spectrometry Analysis

In microbial cells surface-exposed proteins represent a physiologically important class of molecules as they enable these cells to interact with their environment both as free-living organisms and during interactions with a host. However, the characteristics of these proteins are quite divergent, which makes attempts to enrich, analyze, and quantify these molecules a challenging task. In this chapter two complementary methods for the enrichment and identification of cell surface-associated proteins, namely the biotinylation and the shaving approaches, are presented. Both protocols have been optimized for Gram-positive bacteria, and we provide a step-by-step guide for sample generation. Possible pitfalls during protein preparation are discussed.

Toward the Quantitative Characterization of Arginine Phosphorylations in Staphylococcus aureus

The Gram-positive bacterium Staphylococcus aureus plays an important role as an opportunistic pathogen and causative agent of nosocomial infections. As pathophysiological research gained insights into host-specific adaptation and a broad range of virulence mechanisms, S. aureus evolved as a model organism for human pathogens. Hence the investigation of staphylococcal proteome expression and regulation supports the understanding of the pathogenicity and relevant physiology of this organism. This study focused on the analysis of protein regulation by reversible protein phosphorylation, in particular, on arginine residues. Therefore, both proteome and phosphoproteome of S. aureus COL wild type were compared with the arginine phosphatase deletion mutant S. aureus COL ΔptpB under control and stress conditions in a quantitative manner. A gel-free approach, adapted to the special challenges of arginine phosphorylations, was applied to analyze the phosphoproteome of exponential growing cells after oxidative stress caused by sublethal concentrations of H2O2. Together with phenotypic characterization of S. aureus COL ΔptpB, this study disclosed first insights into the physiological role of arginine phosphorylations in Gram-positive pathogens. A spectral library based quantification of phosphopeptides finally allowed us to link arginine phosphorylation to staphylococcal oxidative stress response, amino acid metabolism, and virulence.

A Metabolic Labeling Strategy for Relative Protein Quantification in Clostridioides difficile

Clostridioides difficile (formerly Clostridium difficile) is a Gram-positive, anaerobe, spore-forming pathogen, which causes drug-induced diseases in hospitals worldwide. A detailed analysis of the proteome may provide new targets for drug development or therapeutic strategies to combat this pathogen. The application of metabolic labeling (ML) would allow for accurate quantification of significant differences in protein abundance, even in the case of very small changes. Additionally, it would be possible to perform more accurate studies of the membrane or surface proteomes, which usually require elaborated sample preparation. Such studies are therefore prone to higher standard deviations during the quantification. The implementation of ML strategies for C. difficile is complicated due to the lack in arginine and lysine auxotrophy as well as the Stickland dominated metabolism of this anaerobic pathogen. Hence, quantitative proteome analyses could only be carried out by label free or chemical labeling methods so far. In this paper, a ML approach for C. difficile is described. A cultivation procedure with 15N-labeled media for strain 630Δerm was established achieving an incorporation rate higher than 97%. In a proof-of-principle experiment, the performance of the ML approach in C. difficile was tested. The proteome data of the cytosolic subproteome of C. difficile cells grown in complex medium as well as two minimal media in the late exponential and early stationary growth phase obtained via ML were compared with two label free relative quantification approaches (NSAF and LFQ). The numbers of identified proteins were comparable within the three approaches, whereas the number of quantified proteins were between 1,110 (ML) and 1,861 (LFQ) proteins. A hierarchical clustering showed clearly separated clusters for the different conditions and a small tree height with ML approach. Furthermore, it was shown that the quantification based on ML revealed significant altered proteins with small fold changes compared to the label free approaches. The quantification based on ML was accurate, reproducible, and even more sensitive compared to label free quantification strategies.