Michael Lalk

Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis

Functional genomics of the Gram-positive model organism Bacillus subtilis reveals valuable insights into basic concepts of cell physiology. In this study, we monitor temporal changes in the proteome, transcriptome and extracellular metabolome of B. subtilis caused by glucose starvation. For proteomic profiling, a combination of in vivo metabolic labelling and shotgun mass spectrometric analysis was carried out for five different proteomic subfractions (cytosolic, integral membrane, membrane, surface and extracellular proteome fraction), leading to the identification of ∼52% of the predicted proteome of B. subtilis. Quantitative proteomic and corresponding transcriptomic data were analysed with Voronoi treemaps linking functional classification and relative expression changes of gene products according to their fate in the stationary phase. The obtained data comprise the first comprehensive profiling of changes in the membrane subfraction and allow in-depth analysis of major physiological processes, including monitoring of protein degradation.

Metaproteomics of a gutless marine worm and its symbiotic microbial community reveal unusual pathways for carbon and energy use

Low nutrient and energy availability has led to the evolution of numerous strategies for overcoming these limitations, of which symbiotic associations represent a key mechanism. Particularly striking are the associations between chemosynthetic bacteria and marine animals that thrive in nutrient-poor environments such as the deep sea because the symbionts allow their hosts to grow on inorganic energy and carbon sources such as sulfide and CO2. Remarkably little is known about the physiological strategies that enable chemosynthetic symbioses to colonize oligotrophic environments. In this study, we used metaproteomics and metabolomics to investigate the intricate network of metabolic interactions in the chemosynthetic association between Olavius algarvensis, a gutless marine worm, and its bacterial symbionts. We propose previously undescribed pathways for coping with energy and nutrient limitation, some of which may be widespread in both free-living and symbiotic bacteria. These pathways include (i) a pathway for symbiont assimilation of the host waste products acetate, propionate, succinate and malate; (ii) the potential use of carbon monoxide as an energy source, a substrate previously not known to play a role in marine invertebrate symbioses; (iii) the potential use of hydrogen as an energy source; (iv) the strong expression of high-affinity uptake transporters; and (v) as yet undescribed energy-efficient steps in CO2 fixation and sulfate reduction. The high expression of proteins involved in pathways for energy and carbon uptake and conservation in the O. algarvensis symbiosis indicates that the oligotrophic nature of its environment exerted a strong selective pressure in shaping these associations.

Redox sensing by a Rex-family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus

An alignment of upstream regions of anaerobically induced genes in Staphylococcus aureus revealed the presence of an inverted repeat, corresponding to Rex binding sites in Streptomyces coelicolor. Gel shift experiments of selected upstream regions demonstrated that the redox‐sensing regulator Rex of S. aureus binds to this inverted repeat. The binding sequence – TTGTGAAW4TTCACAA – is highly conserved in S. aureus. Rex binding to this sequence leads to the repression of genes located downstream. The binding activity of Rex is enhanced by NAD+ while NADH, which competes with NAD+ for Rex binding, decreases the activity of Rex. The impact of Rex on global protein synthesis and on the activity of fermentation pathways under aerobic and anaerobic conditions was analysed by using a rex‐deficient strain. A direct regulatory effect of Rex on the expression of pathways that lead to anaerobic NAD+ regeneration, such as lactate, formate and ethanol formation, nitrate respiration, and ATP synthesis, is verified. Rex can be considered a central regulator of anaerobic metabolism in S. aureus. Since the activity of lactate dehydrogenase enables S. aureus to resist NO stress and thus the innate immune response, our data suggest that deactivation of Rex is a prerequisite for this phenomenon.

The stringent response of Staphylococcus aureus and its impact on survival after phagocytosis through the induction of intracellular PSMs expression

The stringent response is initiated by rapid (p)ppGpp synthesis, which leads to a profound reprogramming of gene expression in most bacteria. The stringent phenotype seems to be species specific and may be mediated by fundamentally different molecular mechanisms. In Staphylococcus aureus, (p)ppGpp synthesis upon amino acid deprivation is achieved through the synthase domain of the bifunctional enzyme RSH (RelA/SpoT homolog). In several firmicutes, a direct link between stringent response and the CodY regulon was proposed. Wild-type strain HG001, rshSyn, codY and rshSyn, codY double mutants were analyzed by transcriptome analysis to delineate different consequences of RSH-dependent (p)ppGpp synthesis after induction of the stringent response by amino-acid deprivation. Under these conditions genes coding for major components of the protein synthesis machinery and nucleotide metabolism were down-regulated only in rsh positive strains. Genes which became activated upon (p)ppGpp induction are mostly regulated indirectly via de-repression of the GTP-responsive repressor CodY. Only seven genes, including those coding for the cytotoxic phenol-soluble modulins (PSMs), were found to be up-regulated via RSH independently of CodY. qtRT-PCR analyses of hallmark genes of the stringent response indicate that an RSH activating stringent condition is induced after uptake of S. aureus in human polymorphonuclear neutrophils (PMNs). The RSH activity in turn is crucial for intracellular expression of psms. Accordingly, rshSyn and rshSyn, codY mutants were less able to survive after phagocytosis similar to psm mutants. Intraphagosomal induction of psmα1-4 and/or psmβ1,2 could complement the survival of the rshSyn mutant. Thus, an active RSH synthase is required for intracellular psm expression which contributes to survival after phagocytosis.

Transcriptome and Functional Analysis of the Eukaryotic-Type Serine/Threonine Kinase PknB in Staphylococcus aureus

The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by performing transcriptome analysis using DNA microarray technology and biochemical assays. The transcriptional profile revealed a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. Functional activity of overexpressed and purified PknB kinase was demonstrated using the myelin basic protein as a surrogate substrate. Phosphorylation occurred in a time-dependent manner with Mn(2+) as a preferred cofactor. Furthermore, biochemical characterization revealed regulation of adenylosuccinate synthase (PurA) activity by phosphorylation. Phosphorylated PurA showed a 1.8-fold decrease in enzymatic activity compared to unphosphorylated PurA. Loss of PknB led to formation of larger cell clusters, and a pknB deletion strain showed 32-fold-higher sensitivity to the cell wall-active antibiotic tunicamycin. The results of this study strongly indicate that PknB has a role in regulation of purine biosynthesis, autolysis, and central metabolic processes in S. aureus.

Nitric Oxide Stress Induces Different Responses but Mediates Comparable Protein Thiol Protection in Bacillus subtilis and Staphylococcus aureus

The nonpathogenic Bacillus subtilis and the pathogen Staphylococcus aureus are gram-positive model organisms that have to cope with the radical nitric oxide (NO) generated by nitrite reductases of denitrifying bacteria and by the inducible NO synthases of immune cells of the host, respectively. The response of both microorganisms to NO was analyzed by using a two-dimensional gel approach. Metabolic labeling of the proteins revealed major changes in the synthesis pattern of cytosolic proteins after the addition of the NO donor MAHMA NONOate. Whereas B. subtilis induced several oxidative stress-responsive regulons controlled by Fur, PerR, OhrR, and Spx, as well as the general stress response controlled by the alternative sigma factor SigB, the more resistant S. aureus showed an increased synthesis rate of proteins involved in anaerobic metabolism. These data were confirmed by nuclear magnetic resonance analyses indicating that NO causes a drastically higher increase in the formation of lactate and butanediol in S. aureus than in B. subtilis. Monitoring the intracellular protein thiol state, we observed no increase in reversible or irreversible protein thiol modifications after NO stress in either organism. Obviously, NO itself does not cause general protein thiol oxidations. In contrast, exposure of cells to NO prior to peroxide stress diminished the irreversible thiol oxidation caused by hydrogen peroxide.

A protocol for the investigation of the intracellular Staphylococcus aureus metabolome.

Systems biology studies assume the acquisition of reliable and reproducible data sets. Metabolomics, in particular, requires comprehensive evaluated workflows to enable the analysis of hundreds of different compounds. Therefore, a protocol to elucidate the metabolome of the gram-positive pathogen, Staphylococcus aureus COL strain, grown in a chemically defined medium is introduced here. Different standard operating procedures in the field of metabolome experiments were tested for common pitfalls. These included suitable and fast sampling processes, efficient metabolite extraction, quenching effectiveness (energy charge), and estimation of leakage and recovery of metabolites. Moreover, a cell disruption protocol for S. aureus was developed and optimized for metabolome analyses, for the express purpose of obtaining reproducible data. We used complementary methods (e.g., gas chromatography and/or liquid chromatography coupled with mass spectrometry) to detect the highly chemically diverse groups of metabolites for a global insight into the intracellular metabolism of S. aureus.

Role of the (p)ppGpp Synthase RSH, a RelA/SpoT Homolog, in Stringent Response and Virulence of Staphylococcus aureus

ABSTRACT In most bacteria, nutrient limitations provoke the stringent control through the rapid synthesis of the alarmones pppGpp and ppGpp. Little is known about the stringent control in the human pathogen Staphylococcus aureus, partly due to the essentiality of the major (p)ppGpp synthase/hydrolase enzyme RSH (RelA/SpoT homolog). Here, we show that mutants defective only in the synthase domain of RSH (rshsyn) are not impaired in growth under nutrient-rich conditions. However, these mutants were more sensitive toward mupirocin and were impaired in survival when essential amino acids were depleted from the medium. RSH is the major enzyme responsible for (p)ppGpp synthesis in response to amino acid deprivation (lack of Leu/Val) or mupirocin treatment. Transcriptional analysis showed that the RSH-dependent stringent control in S. aureus is characterized by repression of genes whose products are predicted to be involved in the translation machinery and by upregulation of genes coding for enzymes involved in amino acid metabolism and transport which are controlled by the repressor CodY. Amino acid starvation also provoked stabilization of the RNAs coding for major virulence regulators, such as SaeRS and SarA, independently of RSH. In an animal model, the rshsyn mutant was shown to be less virulent than the wild type. Virulence could be restored by the introduction of a codY mutation into the rshsyn mutant. These results indicate that stringent conditions are present during infection and that RSH-dependent derepression of CodY-regulated genes is essential for virulence in S. aureus.

Depletion of thiol‐containing proteins in response to quinones in Bacillus subtilis

Quinones are highly toxic naturally occurring thiol‐reactive compounds. We have previously described novel pathways for quinone detoxification in the Gram‐positive bacterium Bacillus subtilis. In this study, we have investigated the extent of irreversible and reversible thiol modifications caused in vivo by electrophilic quinones. Exposure to toxic benzoquinone (BQ) concentrations leads to depletion of numerous Cys‐rich cytoplasmic proteins in the proteome of B. subtilis. Mass spectrometry and immunoblot analyses demonstrated that these BQ‐depleted proteins represent irreversibly damaged BQ aggregates that escape the two‐dimensional gel separation. This enabled us to quantify the depletion of thiol‐containing proteins which are the in vivo targets for thiol‐(S)‐alkylation by toxic quinone compounds. Metabolomic approaches confirmed that protein depletion is accompanied by depletion of the low‐molecular‐weight (LMW) thiol cysteine. Finally, no increased formation of disulphide bonds was detected in the thiol‐redox proteome in response to sublethal quinone concentrations. The glyceraldehyde‐3‐phosphate dehydrogenase (GapA) was identified as the only new target for reversible thiol modifications after exposure to toxic quinones. Together our data show that the thiol‐(S)‐alkylation reaction with protein and non‐protein thiols is the in vivo mechanism for thiol depletion and quinone toxicity in B. subtilis and most likely also in other bacteria.

Genome-wide responses to carbonyl electrophiles in Bacillus subtilis: control of the thiol-dependent formaldehyde dehydrogenase AdhA and cysteine proteinase YraA by the MerR-family regulator YraB (AdhR)

Quinones and α,β‐unsaturated carbonyls are naturally occurring electrophiles that target cysteine residues via thiol‐(S)‐alkylation. We analysed the global expression profile of Bacillus subtilis to the toxic carbonyls methylglyoxal (MG) and formaldehyde (FA). Both carbonyl compounds cause a stress response characteristic for thiol‐reactive electrophiles as revealed by the induction of the Spx, CtsR, CymR, PerR, ArsR, CzrA, CsoR and SigmaD regulons. MG and FA triggered also a SOS response which indicates DNA damage. Protection against FA is mediated by both the hxlAB operon, encoding the ribulose monophosphate pathway for FA fixation, and a thiol‐dependent formaldehyde dehydrogenase (AdhA) and DJ‐1/PfpI‐family cysteine proteinase (YraA). The adhA–yraA operon and the yraC gene, encoding a γ‐carboxymuconolactone decarboxylase, are positively regulated by the MerR‐family regulator, YraB(AdhR). AdhR binds specifically to its target promoters which contain a 7‐4‐7 inverted repeat (CTTAAAG‐N4‐CTTTAAG) between the −35 and −10 elements. Activation of adhA–yraA transcription by AdhR requires the conserved Cys52 residue in vivo. We speculate that AdhR is redox‐regulated via thiol‐(S)‐alkylation by aldehydes and that AdhA and YraA are specifically involved in reduction of aldehydes and degradation or repair of damaged thiol‐containing proteins respectively.