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Genome-scale metabolic models (GEMs) have been widely used for quantitative exploration of the relation between genotype and phenotype. Streamlined integration of enzyme constraints and proteomics data into such models was first enabled by the GECKO toolbox, allowing the study of phenotypes constrained by protein limitations. Here, we upgrade the toolbox in order to enhance models with enzyme and proteomics constraints for any organism with a compatible GEM reconstruction. With this, enzyme-constrained models for the budding yeasts Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus are generated to study their long-term adaptation to several stress factors by incorporation of proteomics data. Predictions reveal that upregulation and high saturation of enzymes in amino acid metabolism are common across organisms and conditions, suggesting the relevance of metabolic robustness in contrast to optimal protein utilization as a cellular objective for microbial growth under stress and nutrient-limited conditions. The functionality of GECKO is expanded with an automated framework for continuous and version-controlled update of enzyme-constrained GEMs, also producing such models for Escherichia coli and Homo sapiens . In this work, we facilitate the utilization of enzyme-constrained GEMs in basic science, metabolic engineering and synthetic biology purposes.

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

Phospholipids are essential components of most cell membranes. In Staphylococcus aureus, PlsX acyltransferase is considered indispensable for initiating phospholipid synthesis, unless exogenous fatty acids (FAs) are available to bypass this requirement. We report that S. aureus can capture internal FA sources to overcome PlsX essentiality in a ∆plsX mutant via point mutations in either of two genes: fabF, which encodes the FA synthesis enzyme 3-oxoacyl-(acyl-carrier-protein) synthase II, or fadM, which encodes an understudied bifunctional acyl-CoA thioesterase and ACP binding protein. Despite growth rescue, both ∆plsX suppressors differ from the parental strain by producing phospholipids with shortened FA lengths, suggesting that both suppressors lead to premature FA release during synthesis. Additionally, both suppressors display increased sensitivity to β-lactam antibiotics. The similar behavior of both suppressors led us to show that fabF suppressors require the presence of fadM, indicative of FabF-FadM cooperation. We propose that reduced processivity of FabF suppressor variants, or greater availability of FadM for ACP binding in FadM variants, facilitates FA release from FabF-acyl-ACP intermediates. A FabF-FadM relay leading to FA release may contribute to homeostasis between FASII and phospholipid synthesis pathways.

Background Faecalibacterium duncaniae A2-165 is a rod-shaped, non-motile, and Extremely Sensitive to Oxygen microorganism, belonging to one of the most abundant genera in the human gut microbiome. A decreased abundance of Faecalibacterium species is correlated with Inflammatory Bowel Diseases (IBDs), highlighting this genus as a marker of gut health and a promising Next-Generation Probiotic. While the anti-inflammatory effects of F. duncaniae A2-165 are well known, current studies lack a global protein-level understanding of this species’ metabolism. To address this issue, this study investigated the proteome of F. duncaniae A2-165 during the stationary phase using a label-free LC-MS/MS proteomics approach. Results We quantified 1,280 proteins in total, corresponding to 44.7% of the in silico predicted proteome, with a clear distinction between insoluble and soluble protein abundances. The subcellular localization predictions for the quantified proteins identified 802 cytoplasmic proteins, 265 membrane proteins, six extracellular proteins, eight cell wall proteins, and 199 proteins with unknown localization. Functional analysis of the differentially abundant proteins between insoluble and soluble fractions showed a predominance of transporter proteins in the insoluble fraction. At the same time, the metabolism of amino acids, carbohydrates, and nucleotides was predominant in the soluble fraction. Further analysis of enriched pathways for each fraction showed that energy metabolism, carbon cycle, and amino acid metabolism were enriched in the soluble fraction. In contrast, ABC transporters, quorum sensing, and oxidative phosphorylation were enriched in the insoluble fraction. We identified proteins associated with anti-inflammatory effects, notably the key butyrate-production protein ButCoAT, the MAM protein and its ABC transporter, and shikimate pathway enzymes. Conclusion This study characterized, for the first time, the F. duncaniae A2-165 proteome in the stationary phase, profiling the subproteomes of soluble and insoluble fractions and identifying key proteins involved in F. duncaniae A2-165 metabolism at the protein level. The results presented here could provide new insights into the study of F. duncaniae A2-165.

The promising next-generation probiotic Faecalibacterium prausnitzii is one of the most abundant acetate-consuming, butyrate-producing bacteria in the healthy human gut. Yet, little is known about how acetate availability affects this bacterium's gene expression strategies. Here, we investigated the effect of acetate on temporal changes in the transcriptome of F. duncaniae A2-165 cultures using RNA sequencing. We compared gene expression patterns between two growth phases (early stationary vs. late exponential) and two acetate levels (low: 3 mM vs. high: 23 mM). Only in low-acetate conditions, a general stress response was activated. In high-acetate conditions, there was greater expression of genes related to butyrate synthesis and to the importation of B vitamins and iron. Specifically, expression was strongly activated in the case of the feoAABC operon, which encodes a FeoB ferrous iron transporter, but not in the case of the feoAB gene, which encodes a second putative FeoAB transporter. Moreover, excess ferrous iron repressed feoB expression but not feoAB. Lastly, FeoB but not FeoAB peptides from strain A2-165 were found in abundance in a healthy human fecal metaproteome. In conclusion, we characterized two early-stationary transcriptomes based on acetate consumption and this work highlights the regulation of feoB expression in F. duncaniae A2-165.

Nutritional suitability of milk is not only related to gross composition, but is also strongly affected by the microheterogeniety of the protein fraction. Hence, to go further into the evaluation of the potential suitability of non-bovine milks in human/infant nutrition it is necessary to have a detailed characterization of their protein components. Combining proven proteomic approaches (SDS-PAGE, LC-MS/MS and LC-ESI-MS) and cDNA sequencing, we provide here in depth characterization of the milk protein fraction of dromedary and Bactrian camels, and their hybrids, from different regions of Kazakhstan. A total 391 functional groups of proteins were identified from 8 camel milk samples. A detailed characterization of 50 protein molecules, relating to genetic variants and isoforms arising from post-translational modifications and alternative splicing events, belonging to nine protein families (κ-, αs1-, αs2-, β-; and γ-CN, WAP, α-LAC, PGRP, CSA/LPO) was achieved by LC-ESI-MS. The presence of two unknown proteins UP1 (22,939 Da) and UP2 (23,046 Da) was also reported as well as the existence of a β-CN short isoform (946 Da lighter than the full-length β-CN), arising very likely in both genetic variants (A and B) from proteolysis by plasmin. In addition, we report, for the first time to our knowledge, the occurrence of a αs2-CN phosphorylation isoform with 12P groups within two recognition motifs, suggesting thereby the existence of two kinase systems involved in the phosphorylation of caseins in the mammary gland. Finally, we demonstrate that genetic variants, which hitherto seemed to be species- specific (e.g. β-CN A for Bactrian and β-CN B for dromedary), are in fact present both in Camel dromedarius and C. bactrianus.

Most currently used antibiotics originate from Streptomycetes and phosphate limitation is an important trigger of their biosynthesis. Understanding the molecular processes underpinning such regulation is of crucial importance to exploit the great metabolic diversity of these bacteria and get a better understanding of the role of these molecules in the physiology of the producing bacteria. To contribute to this field, a comparative proteomic analysis of two closely related model strains, Streptomyces lividans and Streptomyces coelicolor was carried out. These strains possess identical biosynthetic pathways directing the synthesis of three well-characterized antibiotics (CDA, RED and ACT) but only S. coelicolor expresses them at a high level. Previous studies established that the antibiotic producer, S. coelicolor, is characterized by an oxidative metabolism and a reduced triacylglycerol content compared to the none producer, S. lividans, characterized by a glycolytic metabolism. Our proteomic data support these findings and reveal that these drastically different metabolic features could, at least in part, due to the weaker abundance of proteins of the two component system PhoR/PhoP in S. coelicolor compared to S. lividans. In condition of phosphate limitation, PhoR/PhoP is known to control positively and negatively, respectively, phosphate and nitrogen assimilation and our study revealed that it might also control the expression of some genes of central carbon metabolism. The tuning down of the regulatory role of PhoR/PhoP in S. coelicolor is thus expected to be correlated with low and high phosphate and nitrogen availability, respectively and with changes in central carbon metabolic features. These changes are likely to be responsible for the observed differences between S. coelicolor and S. lividans concerning energetic metabolism, triacylglycerol biosynthesis and antibiotic production. Furthermore, a novel view of the contribution of the bio-active molecules produced in this context, to the regulation of the energetic metabolism of the producing bacteria, is proposed and discussed.

The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology. Fermentation parameters of industrial processes are often not the ideal growth conditions for industrial microbes. Here, the authors reveal that young genes are more responsive to environmental stress than ancient genes using a new gene age assignment method and provide targeted genes for metabolic engineering.

CodY acts as a key regulatory protein involved in adaptive responses in low‐G+C Gram‐positive bacteria. This global transcriptional regulator diagnoses the nutritional status of the cell and responds by regulating transcription of genes involved in metabolism, differenciation and virulence. Phosphoproteomic studies evidenced that CodY is phosphorylated on its serine 215 in Bacillus subtilis. In Bacillus cereus, CodY is also phosphorylated by the Hanks kinases PrkC and YbdM. CodY phosphorylation negatively affects its DNA‐binding properties. We constructed B. cereus mutant strains where the codY wild‐type allele has been replaced by codY‐S215D or codY‐S215A, encoding a phosphomimetic or a phosphoablative CodY derivative, respectively. We showed that the phosphomimetic mutation leads to a notable reduction in CodY control over several critical cellular processes, including motility, biofilm formation, cytotoxic effects and pathogenicity. Lack of CodY phosphorylation and CodY overphosphorylation have opposite repercussions on gene expression, showing that CodY phosphorylation contributes to the adaptation of B. cereus to diverse environmental conditions. S215 is strictly conserved in CodY orthologs in firmicutes, suggesting that gene regulation mediated by Hanks kinase‐dependent CodY phosphorylation could be a general regulatory mechanism in this phylum. CodY is a global regulator whose phosphorylation at serine 215 by PrkC/YbdM alters its DNA binding, thereby modulating gene expression and impacting motility, biofilm formation, cytotoxicity, and pathogenicity in Bacillus cereus, suggesting a conserved regulatory mechanism in Firmicutes

Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues.