The Quantitative Measurement of Peptidoglycan Components Obtained from Acidic Hydrolysis in Gram-Positive and Gram-Negative Bacteria via Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry.

The high throughput in genome sequencing and metabolic model (MM) reconstruction has democratised bioinformatics approaches such as flux balance analysis. Fluxes' prediction accuracy greatly relates to the deepness of the MM curation for a specific organism starting from the cell composition. One component is the cell wall, which is a functional barrier (cell shape, exchanges) with the environment. The bacterial cell wall (BCW), including its thickness, structure, and composition, has been extensively studied in Escherichia coli but poorly described for other organisms. The peptidoglycan (PG) layer composing the BCW is usually thinner in Gram- bacteria than in Gram+ bacteria. In both bacteria groups, PG is a polymeric mesh-like structure of amino acids and sugars, including N-acetylglucosamine, N-acetylmuramic acid, and amino acids. In this study, we propose a high-throughput method to characterise and quantify PG in Gram-positive and Gram-negative bacteria using acidic hydrolysis and hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS). The method showed a relatively short time frame (11 min analytical run), low inter- and intraday variability (3.2% and 4%, respectively), and high sensitivity and selectivity (limits of quantification in the sub mg/L range). The method was successfully applied on two Gram-negative bacteria (Escherichia coli K12 MG1655, Bacteroides thetaiotaomicron DSM 2079) and one Gram-positive bacterium (Streptococcus salivarius ssp. thermophilus DSM20259). The PG concentration ranged from 1.6% w/w to 14% w/w of the dry cell weight. The results were in good correlation with previously published results. With further development, the PG concentration provided by this newly developed method could reinforce the curation of MM.

Genome-scale metabolic modeling reveals metabolic trade-offs associated with lipid production in Rhodotorula toruloides.

Rhodotorula toruloides is a non-conventional, oleaginous yeast able to naturally accumulate high amounts of microbial lipids. Constraint-based modeling of R. toruloides has been mainly focused on the comparison of experimentally measured and model predicted growth rates, while the intracellular flux patterns have been analyzed on a rather general level. Hence, the intrinsic metabolic properties of R. toruloides that make lipid synthesis possible are not thoroughly understood. At the same time, the lack of diverse physiological data sets has often been the bottleneck to predict accurate fluxes. In this study, we collected detailed physiology data sets of R. toruloides while growing on glucose, xylose, and acetate as the sole carbon source in chemically defined medium. Regardless of the carbon source, the growth was divided into two phases from which proteomic and lipidomic data were collected. Complemental physiological parameters were collected in these two phases and altogether implemented into metabolic models. Simulated intracellular flux patterns demonstrated the role of phosphoketolase in the generation of acetyl-CoA, one of the main precursors during lipid biosynthesis, while the role of ATP citrate lyase was not confirmed. Metabolic modeling on xylose as a carbon substrate was greatly improved by the detection of chirality of D-arabinitol, which together with D-ribulose were involved in an alternative xylose assimilation pathway. Further, flux patterns pointed to metabolic trade-offs associated with NADPH allocation between nitrogen assimilation and lipid biosynthetic pathways, which was linked to large-scale differences in protein and lipid content. This work includes the first extensive multi-condition analysis of R. toruloides using enzyme-constrained models and quantitative proteomics. Further, more precise kcat values should extend the application of the newly developed enzyme-constrained models that are publicly available for future studies.

Xylose Metabolism and the Effect of Oxidative Stress on Lipid and Carotenoid Production in Rhodotorula toruloides: Insights for Future Biorefinery.

The use of cell factories to convert sugars from lignocellulosic biomass into chemicals in which oleochemicals and food additives, such as carotenoids, is essential for the shift toward sustainable processes. Rhodotorula toruloides is a yeast that naturally metabolises a wide range of substrates, including lignocellulosic hydrolysates, and converts them into lipids and carotenoids. In this study, xylose, the main component of hemicellulose, was used as the sole substrate for R. toruloides, and a detailed physiology characterisation combined with absolute proteomics and genome-scale metabolic models was carried out to understand the regulation of lipid and carotenoid production. To improve these productions, oxidative stress was induced by hydrogen peroxide and light irradiation and further enhanced by adaptive laboratory evolution. Based on the online measurements of growth and CO2 excretion, three distinct growth phases were identified during batch cultivations. Majority of the intracellular flux estimations showed similar trends with the measured protein levels and demonstrated improved NADPH regeneration, phosphoketolase activity and reduced ß-oxidation, correlating with increasing lipid yields. Light irradiation resulted in 70% higher carotenoid and 40% higher lipid content compared to the optimal growth conditions. The presence of hydrogen peroxide did not affect the carotenoid production but culminated in the highest lipid content of 0.65 g/gDCW. The adapted strain showed improved fitness and 2.3-fold higher carotenoid content than the parental strain. This work presents a holistic view of xylose conversion into microbial oil and carotenoids by R. toruloides, in a process toward renewable and cost-effective production of these molecules.

Transcriptomic and proteomic data in developing tomato fruit.

Transcriptomic and proteomic analyses were performed on three replicates of tomato fruit pericarp samples collected at nine developmental stages, each replicate resulting from the pooling of at least 15 fruits. For transcriptome analysis, Illumina-sequenced libraries were mapped on the tomato genome with the aim to obtain absolute quantification of mRNA abundance. To achieve this, spikes were added at the beginning of the RNA extraction procedure. From 34,725 possible transcripts identified in the tomato, 22,877 were quantified in at least one of the nine developmental stages. For the proteome analysis, label-free liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used. Peptide ions, and subsequently the proteins from which they were derived, were quantified by integrating the signal intensities obtained from extracted ion currents (XIC) with the MassChroQ software. Absolute concentrations of individual proteins were estimated for 2375 proteins by using a mixed effects model from log10-transformed intensities and normalized to the total protein content. Transcriptomics data are available via GEO repository with accession number GSE128739. The raw MS output files and identification data were deposited on-line using the PROTICdb database (http://moulon.inra.fr/protic/tomato_fruit_development) and MS proteomics data have also been deposited to the ProteomeXchange with the dataset identifier PXD012877. The main added value of these quantitative datasets is their use in a mathematical model to estimate protein turnover in developing tomato fruit.

Regulation of Pyridine Nucleotide Metabolism During Tomato Fruit Development Through Transcript and Protein Profiling.

Central metabolism is the engine of plant biomass, supplying fruit growth with building blocks, energy, and biochemical cofactors. Among metabolic cornerstones, nicotinamide adenine dinucleotide (NAD) is particularly pivotal for electron transfer through reduction-oxidation (redox) reactions, thus participating in a myriad of biochemical processes. Besides redox functions, NAD is now assumed to act as an integral regulator of signaling cascades involved in growth and environmental responses. However, the regulation of NAD metabolism and signaling during fruit development remains poorly studied and understood. Here, we benefit from RNAseq and proteomic data obtained from nine growth stages of tomato fruit (var. Moneymaker) to dissect mRNA and protein profiles that link to NAD metabolism, including de novo biosynthesis, recycling, utilization, and putative transport. As expected for a cofactor synthesis pathway, protein profiles failed to detect enzymes involved in NAD synthesis or utilization, except for nicotinic acid phosphoribosyltransferase (NaPT) and nicotinamidase (NIC), which suggested that most NAD metabolic enzymes were poorly represented quantitatively. Further investigations on transcript data unveiled differential expression patterns during fruit development. Interestingly, among specific NAD metabolism-related genes, early de novo biosynthetic genes were transcriptionally induced in very young fruits, in association with NAD kinase, while later stages of fruit growth rather showed an accumulation of transcripts involved in later stages of de novo synthesis and in NAD recycling, which agreed with augmented NAD(P) levels. In addition, a more global overview of 119 mRNA and 78 protein significant markers for NAD(P)-dependent enzymes revealed differential patterns during tomato growth that evidenced clear regulations of primary metabolism, notably with respect to mitochondrial functions. Overall, we propose that NAD metabolism and signaling are very dynamic in the developing tomato fruit and that its differential regulation is certainly critical to fuel central metabolism linking to growth mechanisms.

Modeling Protein Destiny in Developing Fruit.

Protein synthesis and degradation are essential processes that regulate cell status. Because labeling in bulky organs, such as fruits, is difficult, we developed a modeling approach to study protein turnover at the global scale in developing tomato (Solanum lycopersicum) fruit. Quantitative data were collected for transcripts and proteins during fruit development. Clustering analysis showed smaller changes in protein abundance compared to mRNA abundance. Furthermore, protein and transcript abundance were poorly correlated, and the coefficient of correlation decreased during fruit development and ripening, with transcript levels decreasing more than protein levels. A mathematical model with one ordinary differential equation was used to estimate translation (kt ) and degradation (kd ) rate constants for almost 2,400 detected transcript-protein pairs and was satisfactorily fitted for >1,000 pairs. The model predicted median values of ∼2 min for the translation of a protein, and a protein lifetime of ∼11 d. The constants were validated and inspected for biological relevance. Proteins involved in protein synthesis had higher kt and kd values, indicating that the protein machinery is particularly flexible. Our model also predicts that protein concentration is more strongly affected by the rate of translation than that of degradation.

Peptide filtering differently affects the performances of XIC-based quantification methods.

In bottom-up proteomics, data are acquired on peptides resulting from proteolysis. In XIC-based quantification, the quality of the estimation of protein abundance depends on how peptide data are filtered and on which quantification method is used to express peptide intensity as protein abundance. So far, these two questions have been addressed independently. Here, we studied to what extent the relative performances of the quantification methods depend on the filters applied to peptide intensity data. To this end, we performed a spike-in experiment using Universal Protein Standard to evaluate the performances of five quantification methods in five datasets obtained after application of four peptide filters. Estimated protein abundances were not equally affected by filters depending on the computation mode and the type of data for quantification. Furthermore, we found that filters could have contrasting effects depending on the quantification objective. Intensity modeling proved to be the most robust method, providing the best results in the absence of any filter. However, the different quantification methods can achieve similar performances when appropriate peptide filters are used. Altogether, our findings provide insights into how best to handle intensity data according to the quantification objective and the experimental design. SIGNIFICANCE: We believe that our results are of major importance because they address, as far as we know for the first time, the crossed-effects of peptide intensity data filtering and XIC-based quantification methods on protein quantification. While previous papers have dealt with peptide filtering independently of the quantification method, here we combined four peptide filters (based on peptide sharing between proteins, retention time variability, peptides occurrence and peptide intensity profiles) with five XIC-based quantification methods representing different modes of calculating protein abundances from peptide intensities. For these different combinations, we analyzed the quality of protein quantification in terms of precision, accuracy and linearity of response to increasing protein concentration using a spike-in experiment. We showed that not only filters effect on the estimation of protein abundances depend on the quantification methods but also that quantification methods can reach similar performances when appropriate peptide filters are used. Also, depending on the quantification objective, i.e. absolute or relative, filters can have contrasting effects and we demonstrated that protein quantification by the peptide intensity modeling was the most robust method.

Putting primary metabolism into perspective to obtain better fruits.

Background: One of the key goals of fruit biology is to understand the factors that influence fruit growth and quality, ultimately with a view to manipulating them for improvement of fruit traits. Scope: Primary metabolism, which is not only essential for growth but is also a major component of fruit quality, is an obvious target for improvement. However, metabolism is a moving target that undergoes marked changes throughout fruit growth and ripening. Conclusions: Agricultural practice and breeding have successfully improved fruit metabolic traits, but both face the complexity of the interplay between development, metabolism and the environment. Thus, more fundamental knowledge is needed to identify further strategies for the manipulation of fruit metabolism. Nearly two decades of post-genomics approaches involving transcriptomics, proteomics and/or metabolomics have generated a lot of information about the behaviour of fruit metabolic networks. Today, the emergence of modelling tools is providing the opportunity to turn this information into a mechanistic understanding of fruits, and ultimately to design better fruits. Since high-quality data are a key requirement in modelling, a range of must-have parameters and variables is proposed.

Slow potentials encode intercellular coupling and insulin demand in pancreatic beta cells.

AIMS/HYPOTHESIS: Ion fluxes constitute a major integrative signal in beta cells that leads to insulin secretion and regulation of gene expression. Understanding these electrical signals is important for deciphering the endogenous algorithms used by islets to attain homeostasis and for the design of new sensors for monitoring beta cell function. METHODS: Mouse and human islets were cultured on multielectrode arrays (MEAs) for 3-13 days. Extracellular electrical activities received on each electrode were continuously amplified and recorded for offline characterisation. RESULTS: Differential band-pass filtering of MEA recordings of mouse islets showed two extracellular voltage waveforms: action potentials (lasting 40-60 ms) and very robust slow potentials (SPs, lasting 800-1,500 ms), the latter of which have not been described previously. The frequency of SPs directly correlated with glucose concentration, peaked at 10 mmol/l glucose and was further augmented by picomolar concentrations of glucagon-like peptide-1. SPs required the closure of ATP-dependent potassium channels as they were induced by glucose or glibenclamide but were not elicited by KCl-induced depolarisation. Pharmacological tools and the use of beta cell specific knockout mice showed that SPs reflected cell coupling via connexin 36. Moreover, increasing and decreasing glucose ramps showed hysteresis with reduced glucose sensitivity during the decreasing phase. SPs were also observed in human islets and could be continuously recorded over 24 h. CONCLUSIONS/INTERPRETATION: This novel electrical signature reflects the syncytial function of the islets and is specific to beta cells. Moreover, the observed hysteresis provides evidence for an endogenous algorithm naturally present in islets to protect against hypoglycaemia.