Carbon source dependent reprograming of anaerobic metabolism in Staphylococcus aureus
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Synapt MS
ORGANISM(S): Staphylococcus Aureus (strain Col)
SUBMITTER: 
Daniela Zuehlke
LAB HEAD: Katharina Riedel
PROVIDER: PXD017966 | Pride | 2021-02-15
REPOSITORIES: Pride
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Project description:For many years now, Bacillus megaterium has served as a microbial industrial strain for high-level production of recombinant proteins in the g/L-scale. Nevertheless, the impact of process-related stress has only been poorly characterized so far. Taking advantage of the recent technical developments for quantifying the cell at various molecular levels, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome and fluxome analyses. Under osmotic upshift conditions, several stress response enzymes, iron scavenging, and reactive oxygen species (ROS) fighting proteins were upregulated. The downregulation of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The (NADH/NAD+) ratio indicating the redox state of the cell was also altered, which was partially compensated by the higher production of lactate accompanied by the reduction of acetate secretion. NADH was produced mainly within the tricarboxylic acid cycle (TCA) cycle elucidated from the higher mRNA and protein levels of enzymes involved within this pathway. This adaptation mainly focused on the massive de novo synthesis of the compatible solute proline recruiting an osmo-dependent pathway to fulfil this requirement. 13C flux analyses confirmed these findings. Giving the high flux towards acetyl-CoA and large pool of NADPH, B. megaterium cells redirected the produced acetyl-CoA to the polyhydroxybutyrate (PHB) biosynthetic pathway under non-limiting nutrient condition, amassing around 30% of the CDW as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.
2020-05-26 | PXD015605 | Pride
Project description:Absolute protein quantification was applied to follow the dynamics of the cytoplasmic proteome of Staphylococcus aureus in response to long-term oxygen starvation. For 1,168 proteins, the majority of all expressed proteins, molecule numbers per cell have been determined to monitor the cellular investments in single branches of bacterial life for the first time. In the presence of glucose the anaerobic protein pattern is characterized by increased amounts of glycolytic and fermentative enzymes such as Eno, GapA1, Ldh1, and PflB. Interestingly, the ferritin-like protein FtnA belongs to the most abundant proteins during anaerobic growth. Depletion of glucose finally leads to an accumulation of different enzymes such as ArcB1, ArcB2, and ArcC2 involved in arginine deiminase pathway. Concentrations of 29 exo- and 78 endometabolites were comparatively assessed and have been integrated to the metabolic networks. Here we provide an almost complete picture on the response to oxygen starvation, from signal transduction pathways to gene expression pattern, from metabolic reorganization after oxygen depletion to beginning cell death and lysis after glucose exhaustion. This experimental approach can be considered as a proof of principle how to combine cell physiology with quantitative proteomics for a new dimension in understanding simple life processes as an entity.
2016-07-11 | PXD000330 | Pride
Project description:Pathogens that grow during infections must cope with the reactive oxygen species (ROS) released by host immune cells. Among the different strategies to prevent oxidative damage during infections, pathogenic bacteria have evolved mechanisms to reduce respiration and other cellular processes that are particularly sensitive to free radicals, obtaining energy preferentially by undergoing to fermentative metabolism. As fermentation produces fewer ATP per glucose than respiration, bacteria ensure an appropriate supply of energy by increasing the glycolytic flux. The opportunistic human pathogen Staphylococcus aureus is one such microbe but the underlying mechanism that enables S. aureus to induce fermentation during infections is still unclear. Here we show that the ComK-like regulator of natural competence that is present in many gram-positive bacteria is crucial to redirect S. aureus metabolism to fermentation during infection. ComK is cryptic in laboratory conditions but highly induced during infections or in response to infection-related cues, such as ROS. ComK induces the glycolytic flux and glucose consumption rate, a key step to redirect ATP production to fermentation. This licenses fermenting S. aureus to reduce oxidative damage while increasing DNA uptake by natural transformation. As a consequence, a comK-defective mutant shows an accumulation of ROS as well as DNA mutations that lower bacterial survival. This mutant shows no distinctive phenotype in laboratory conditions but is unable to cause infection in vertebrates or invertebrate infection models. ComK-mediated synchronization of natural transformation to fermentative metabolism may allow S. aureus, and probably other gram-positive bacteria, to use the fermentation acid end products to eliminate bacterial competitors while assimilating their DNA. Assimilated DNA may serve as a source of nucleotides for DNA repair or to promote genetic variability, thereby enabling a successful host colonization of this bacterium.
2022-02-26 | GSE155016 | GEO
Project description:Background The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in S. aureus wild type and ccpA-deleted mutant, focussing on short-time effects of glucose under stable pH conditions. Results The addition of glucose to exponentially growing S. aureus increased enzymes of glycolytic pathway, indicating a higher glycolytic activity, while proteins required for the complete oxidation in the TCA cycle were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetylCoA to acetate with a concomitant substrate-level phosphorylation were neither regulated by glucose nor by CcpA. Most CcpA directly repressed genes were involved in utilization of amino acids as secondary carbon sources. More genes were found to be differentially expressed by CcpA in a glucose-independent manner than in the classical, glucose dependent way, suggesting that glucose-independent regulation by CcpA may be of particular importance in S. aureus. In the presence of glucose, CcpA was found to regulate expression of genes involved in metabolism, but that of genes coding for virulence determinants. Conclusions This study identified the CcpA regulon of exponentially growing S. aureus, for the first time. As in other bacteria, the CcpA-regulon of S. aureus comprised a large amount of metabolic genes but also some 50 genes associated with virulence. CcpA seemed to work in a glucose- as well as glucose-independent way. The transcriptomes of strain Newman and its isogenic ccpA-deleted mutant were determined in early exponential growth and 30 min after the addition of 10 mM glucose, under controlled pH conditions. In the absence of glucose, the wild type grew slightly faster than the mutant, reaching an OD600 of 1 approximately 20 min earlier than the mutant. Adding 10 mM glucose at OD600 1 increased the growth rate of the wild type but had only a minor effect on that of the mutant. 60 min after glucose addition, glucose was depleted down to 0.3 mM by the wild type, while still 3 mM glucose was left in the culture of the mutant. Despite increased glucose consumption rates in the wild type, acetate production was only slightly enhanced compared to the mutant. No lactate was excreted at any time point sampled. Acidification of the medium upon glucose metabolism was prevented by buffering, maintaining a pH of 7.5 for both strains and under both growth conditions for at least 2 h after glucose addition, allowing to rule out any pH effects.
2010-06-24 | E-GEOD-12614 | biostudies-arrayexpress
Project description:Background The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in S. aureus wild type and ccpA-deleted mutant, focussing on short-time effects of glucose under stable pH conditions. Results The addition of glucose to exponentially growing S. aureus increased enzymes of glycolytic pathway, indicating a higher glycolytic activity, while proteins required for the complete oxidation in the TCA cycle were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetylCoA to acetate with a concomitant substrate-level phosphorylation were neither regulated by glucose nor by CcpA. Most CcpA directly repressed genes were involved in utilization of amino acids as secondary carbon sources. More genes were found to be differentially expressed by CcpA in a glucose-independent manner than in the classical, glucose dependent way, suggesting that glucose-independent regulation by CcpA may be of particular importance in S. aureus. In the presence of glucose, CcpA was found to regulate expression of genes involved in metabolism, but that of genes coding for virulence determinants. Conclusions This study identified the CcpA regulon of exponentially growing S. aureus, for the first time. As in other bacteria, the CcpA-regulon of S. aureus comprised a large amount of metabolic genes but also some 50 genes associated with virulence. CcpA seemed to work in a glucose- as well as glucose-independent way.
2008-12-20 | GSE12614 | GEO
Project description:The engineering of Saccharomyces cerevisiae strains for lactose utilization has been attempted with the intent of developing high productivity processes for alcoholic fermentation of cheese whey. A recombinant S. cerevisiae flocculent strain that efficiently ferments lactose to ethanol was previously obtained by evolutionary engineering of an original recombinant that displayed poor lactose fermentation performance. In this study, we compared the transcriptomes of the original and the evolved recombinant strains (T1 and T1-E, respectively) growing in lactose, using cDNA microarrays. Microarray data revealed 173 genes whose expression levels differed more than 1.5-fold. About half of these genes were related to RNA mediated transposition. We also found genes involved in DNA repair and recombination mechanisms, response to stress, chromatin remodelling, cell cycle control, mitosis regulation, glycolysis and alcoholic fermentation. These transcriptomic data are in agreement with some of the previously identified physiological and molecular differences between the recombinants, and point to further hypotheses to explain those differences. Four samples, each corresponding to independent biological cultivations of the two strains (T1 and T1-E), were analysed. To reduce the bias due to unequal incorporation or differences in quantum efficiency of the two dyes, RNA samples from a second independent experiment were labeled by opposite dye to the first experiment (method called dye switch), and this procedure was repeated 4 times, leading to 4 independent intensity values for each gene (spots) on the microarray. This experimental design minimizes the intrinsic biological noise between identical culture conditions and the technical variations inherent to the DNA microarray technology.
2008-10-31 | E-GEOD-12433 | biostudies-arrayexpress
Project description:Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments a broad range of substrates to mainly acetate, CO2, and hydrogen gas (H2). Its high hydrogen-producing capacity make this bacterium an attractive candidate for microbial biohydrogen production. However, increased H2 levels tend to inhibit hydrogen formation and leads to the formation of other reduced end products like lactate and ethanol. To investigate the organismM-bM-^@M-^Ys strategy for dealing with elevated H2 levels and to identify alternative pathways involved in the disposal of the reducing equivalents, the effect of the hydrogen partial pressure (PH2) on fermentation performance was studied. For this purpose cultures were grown under high and low PH2 in a glucose limited chemostat setup. Transcriptome analysis revealed the up-regulation of genes involved in the disposal of reducing equivalents under high PH2, like lactate dehydrogenase and alcohol dehydrogenase as well as the NADH-dependent and ferredoxin-dependent hydrogenases. These findings were in line with the observed shift in fermentation profiles from acetate production under low PH2 to a mixed production of acetate, lactate and ethanol under high PH2. In addition, differential transcription was observed for genes involved in carbon metabolism, fatty acid biosynthesis and several transport systems. The presented transcription data provides experimental evidence for the involvement of the redox sensing Rex protein in gene regulation under high PH2 cultivation conditions. Overall, these findings indicate that the PH2 dependent changes in the fermentation pattern of C. saccharolyticus are, in addition to the known regulation at the enzyme/metabolite level, also regulated at the transcription level. Two conditions: low H2 partial pressure and high H2 partial pressure, both at steady state growth were harvested for a dye-flip microarray experimental design. Biological replicates were harvested for both conditions and combined prior to cDNA synthesis. Both conditions were labeled with cy3 and cy5 dyes allowing for a technical replicate of hybridization in addition to the biological replicates.
2013-08-28 | E-GEOD-40430 | biostudies-arrayexpress
Project description:Volatile fatty acids found in effluents of the dark fermentation of biowastes can be used for mixotrophic growth of microalgae, improving productivity and reducing the cost of the feedstock. Microalgae can use the acetate in the effluents very well, but butyrate is poorly assimilated and can inhibit growth above 1 gC.L-1. The non-photosynthetic chlorophyte alga Polytomella sp. SAG 198.80 was found to be able to assimilate butyrate fast. To decipher the metabolic pathways implicated in butyrate assimilation, a large-scale differential proteomics study was developed comparing Polytomella sp. cells grown on acetate and butyrate at 1 gC.L-1.
2022-11-02 | PXD035155 | Pride
Project description:We comprehensively profiled intracellular and ECM proteomes of S. aureus flow biofilms and complemented these data by metabolic footprint analysis and phenotypic assays. We show that S. aureus biofilms produce high amounts of secreted, moonlighting virulence factors stabilizing the ECM. Mechanistically, we propose that these alkaline virulence factors get protonated in an acidified ECM (due to the release of acids upon fermentation) mediating electrostatic interactions with anionic cell surface components and metabolites, which leads to cell aggregation and ECM stabilization.
2019-03-13 | PXD011157 | Pride
Project description:Background Lignocellulosic biomass is a promising renewable feedstock for biofuel production. Acetate is one of the major inhibitors liberated from hemicelluloses during hydrolysis. An understanding of the toxic effects of acetate on the fermentation microorganism and the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production. Results A new microarray was designed including both coding sequences and intergenic regions to investigate the acetate stress responses of Zymomonas mobilis 8b when using single carbon sources of glucose or xylose, or mixed sugars of both glucose and xylose. With the supplementation of exogenous acetate, 8b can utilize all the glucose with a similar ethanol yield, although the growth, final biomass, and ethanol production rate were reduced. However, xylose utilization was inhibited in both media containing xylose or a mixed sugar of glucose and xylose, although the performance of 8b was better in mixed sugar than xylose-only media. The presence of acetate caused genes related to biosynthesis, the flagellar system, and glycolysis to be downregulated, and genes related to stress responses and energy metabolism to be upregulated. Unexpectedly, xylose seems to pose more stress on 8b, recruiting more genes for xylose utilization, than does acetate. Several gene candidates based on transcriptome results were selected for genetic manipulation, and a TonB-dependent receptor knockout mutant was confirmed to have a slight advantage regarding acetate tolerance. Conclusions Our results indicate Z. mobilis utilized a different mechanism for xylose utilization, with an even more severe impact on Z. mobilis than that caused by acetate treatment. Our study also suggests redox imbalance caused by stressful conditions may trigger a metabolic reaction leading to the accumulation of toxic intermediates such as xylitol, but Z. mobilis manages its carbon and energy metabolism through the control of individual reactions to mitigate the stressful conditions. We have thus provided extensive transcriptomic datasets and gained insights into the molecular responses of Z. mobilis to the inhibitor acetate when grown in different sugar sources, which will facilitate future metabolic modeling studies and strain improvement efforts for better xylose utilization and acetate tolerance.
2014-09-08 | GSE57553 | GEO