Abstract
Reproducible research is the bedrock of experimental science. To enable the deployment of large-scale proteomics, we assess the reproducibility of mass spectrometry (MS) over time and across ...instruments and develop computational methods for improving quantitative accuracy. We perform 1560 data independent acquisition (DIA)-MS runs of eight samples containing known proportions of ovarian and prostate cancer tissue and yeast, or control HEK293T cells. Replicates are run on six mass spectrometers operating continuously with varying maintenance schedules over four months, interspersed with ~5000 other runs. We utilise negative controls and replicates to remove unwanted variation and enhance biological signal, outperforming existing methods. We also design a method for reducing missing values. Integrating these computational modules into a pipeline (ProNorM), we mitigate variation among instruments over time and accurately predict tissue proportions. We demonstrate how to improve the quantitative analysis of large-scale DIA-MS data, providing a pathway toward clinical proteomics.
Fabry disease (FD) is a rare X-linked disease due to a multiverse of disrupting mutations within the GLA gene encoding lysosomal α-galactosidase A (AGAL). Absent AGAL activity causes the accumulation ...of complex glycosphingolipids inside of lysosomes in a variety of cell types and results in a progressive multisystem disease. Known disease-associated point mutations in protein-coding gene regions usually cause translational perturbations and result in premature chain termination, punctual amino acid sequence alterations or overall altered sequence alterations downstream of the mutation site. However, nucleotide exchanges at the border between introns and exons can affect splicing behavior and lead to abnormal pre-mRNA processing. Prediction with the Human Splicing Finder (HSF) revealed an indication of a significant change in splicing-relevant information for some known FD-associated GLA mutations. To experimentally determine the extent of the change, we made use of a minigene reporter assay and verified alternative splicing events for the exonic mutations c.194G>T and c.358C>G, which led to the usage of alternative donor splice sites at exon 1 and exon 2, respectively. In addition, the mutations c.548G>T and c.638A>T led to significant exon 4 skipping. We conclude that splicing phenotype analysis should be employed in the in vitro analysis of exonic GLA gene mutations, since abnormal splicing may result in a reduction of enzyme activity and alter the amenability for treatment with pharmacological chaperone (PC).
The pathogenicity of Staphylococcus aureus strains varies tremendously (as seen with animals). It is largely dependent on global regulators, which control the production of toxins, virulence, and ...fitness factors. Despite the vast knowledge of staphylococcal molecular genetics, there is still widespread dispute over what factors must come together to make a strain highly virulent. S. aureus NCTC8325 (RN1 and derivatives) is a widely used model strain for which an incomparable wealth of knowledge has accumulated in the almost 50 years since its isolation. Although RN1 has functional agr, sarA, and sae global regulators, it is defective in two regulatory genes, rsbU (a positive activator of SigB) and tcaR (an activator of protein A transcription), and is therefore considered by many to be a poor model for studies of regulation and virulence. Here, we repaired these genes and compared the resulting RN1 derivatives with other widely used strains, Newman, USA300, UAMS-1, and COL, plus the parental RN1, with respect to growth, extracellular protein pattern, hemolytic activity, protein A production, pigmentation, biofilm formation, and mouse lethality. The tcaR-repaired strain, showed little alteration in these properties. However, the rsbU-repaired strain was profoundly altered. Hemolytic activity was largely decreased, the exoprotein pattern became much more similar to that of typical wild-type (wt) S. aureus, and there was a surprising increase in mouse lethality. We note that each of the strains tested has a mutational alteration in one or more other regulatory functions, and we conclude that the repaired RN1 is a good model strain for studies of staphylococcal regulation and pathobiology; although strain Newman has been used extensively for such studies in recent years, it has a missense mutation in saeS, the histidine kinase component of the sae signaling module, which profoundly alters its regulatory phenotype. If this mutation were repaired, Newman would be considerably improved as a model strain.
Synthesis of acetate from carbon dioxide and molecular hydrogen is considered to be the first carbon assimilation pathway on earth. It combines carbon dioxide fixation into acetyl-CoA with the ...production of ATP via an energized cell membrane. How the pathway is coupled with the net synthesis of ATP has been an enigma. The anaerobic, acetogenic bacterium Acetobacterium woodii uses an ancient version of this pathway without cytochromes and quinones. It generates a sodium ion potential across the cell membrane by the sodium-motive ferredoxin:NAD oxidoreductase (Rnf). The genome sequence of A. woodii solves the enigma: it uncovers Rnf as the only ion-motive enzyme coupled to the pathway and unravels a metabolism designed to produce reduced ferredoxin and overcome energetic barriers by virtue of electron-bifurcating, soluble enzymes.
Predicting resource allocation between cell processes is the primary step towards decoding the evolutionary constraints governing bacterial growth under various conditions. Quantitative prediction at ...genome-scale remains a computational challenge as current methods are limited by the tractability of the problem or by simplifying hypotheses. Here, we show that the constraint-based modeling method Resource Balance Analysis (RBA), calibrated using genome-wide absolute protein quantification data, accurately predicts resource allocation in the model bacterium Bacillus subtilis for a wide range of growth conditions. The regulation of most cellular processes is consistent with the objective of growth rate maximization except for a few suboptimal processes which likely integrate more complex objectives such as coping with stressful conditions and survival. As a proof of principle by using simulations, we illustrated how calibrated RBA could aid rational design of strains for maximizing protein production, offering new opportunities to investigate design principles in prokaryotes and to exploit them for biotechnological applications.
•We introduced the constraint-based modeling method Resource Balance Analysis (RBA).•We measured absolute abundances of most of cytosolic proteins in 5 conditions.•We identified the apparent catalytic rates of cytosolic proteins.•We accurately predicted the resource allocation of B. subtilis using RBA.•RBA performs realistic simulations of protein excretion.
Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different ...conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria.
Splicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can ...be examined by gene expression analyses, by computational tools for predicting the effects of genetic variants on splicing, and by splicing reporter minigene assays for studying alternative splicing events under defined conditions. The minigene assay is based on transient transfection of cells with a vector containing a genomic region of interest cloned between two constitutive exons. Cloning can be accomplished by the use of restriction enzymes or by site-specific recombination using Gateway cloning. The vectors pDESTsplice and pSpliceExpress represent two minigene systems based on Gateway cloning, which are available through the Addgene plasmid repository. In this review, we describe the features of these two splicing reporter minigene systems. Moreover, we provide an overview of studies in which determinants of alternative splicing were investigated by using pDESTsplice or pSpliceExpress. The studies were reviewed with regard to the investigated splicing regulatory events and the experimental strategy to construct and perform a splicing reporter minigene assay. We further elaborate on how analyses on the regulation of RNA splicing offer promising prospects for gaining important insights into disease mechanisms.
The undefined poly(3-hydroxybutyrate)- (PHB-) negative mutant R. eutropha PHB-4 was generated in 1970 by 1-nitroso-3-nitro-1-methylguanidine (NMG) treatment. Although being scientific relevant, its ...genotype remained unknown since its isolation except a recent first investigation. In this study, the mutation causing the PHA-negative phenotype of R. eutropha PHB-4 was confirmed independently: sequence analysis of the phaCAB operon identified a G320A mutation in phaC yielding a stop codon, leading to a massively truncated PhaC protein of 106 amino acids (AS) in R. eutropha PHB-4 instead of 589 AS in the wild type. No other mutations were observed within the phaCAB operon. As further mutations probably occurred in the genome of mutant PHB-4 potentially causing secondary effects on the cells' metabolism, the main focus of the study was to perform a 2D PAGE-based proteome analysis in order to identify differences in the proteomes of the wild type and mutant PHB-4. A total of 20 differentially expressed proteins were identified which provide valuable insights in the metabolomic changes of mutant PHB-4. Besides excretion of pyruvate, mutant PHB-4 encounters the accumulation of intermediates such as pyruvate and acetyl-CoA by enhanced expression of the observed protein species: (i) ThiJ supports biosynthesis of cofactor TPP and thereby reinforces the 2-oxoacid dehydrogenase complexes as PDHC, ADHC and OGDHC in order to convert pyruvate at a higher rate and the (ii) 3-isopropylmalate dehydrogenase LeuB3 apparently directs pyruvate to synthesis of several amino acids. Different (iii) acylCoA-transferases enable transfer reactions between organic acid intermediates, and (iv) citrate lyase CitE4 regenerates oxaloacetate from citrate for conversion with acetyl-CoA in the TCC in an anaplerotic reaction. Substantial amounts of reduction equivalents generated in the TCC are countered by (v) synthesis of more ubiquinones due to enhanced synthesis of MenG2 and MenG3, thereby improving the respiratory chain which accepts electrons from NADH and succinate.
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