Sobemovirus RNA linked to VPg over a threonine residue

AbstractPositive sense ssRNA virus genomes from several genera have a viral protein genome-linked (VPg) attached over a phosphodiester bond to the 5′ end of the genome. The VPgs of Southern bean mosaic virus (SBMV) and Ryegrass mottle virus (RGMoV) were purified from virions and analyzed by mass spectrometry. SBMV VPg was determined to be linked to RNA through a threonine residue at position one, whereas RGMoV VPg was linked to RNA through a serine also at the first position. In addition, we identified the termini of the corresponding VPgs and discovered three and seven phosphorylation sites in SBMV and RGMoV VPgs, respectively. This is the first report on the use of threonine for linking RNA to VPg

Multi-omics approach to study the growth efficiency and amino acid metabolism in Lactococcus lactis at various specific growth rates

<p>Abstract</p> <p>Background</p> <p><it>Lactococcus lactis </it>is recognised as a safe (GRAS) microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth.</p> <p>Results</p> <p>Using glucose limited continuous cultivations, specific growth rate dependent metabolism of <it>L. lactis </it>including utilization of amino acids was studied based on extracellular metabolome, global transcriptome and proteome analysis. A new growth medium was designed with reduced amino acid concentrations to increase precision of measurements of consumption of amino acids. Consumption patterns were calculated for all 20 amino acids and measured carbon balance showed good fit of the data at all growth rates studied. It was observed that metabolism of <it>L. lactis </it>became more efficient with rising specific growth rate in the range 0.10 - 0.60 h^-1, indicated by 30% increase in biomass yield based on glucose consumption, 50% increase in efficiency of nitrogen use for biomass synthesis, and 40% reduction in energy spilling. The latter was realized by decrease in the overall product formation and higher efficiency of incorporation of amino acids into biomass. <it>L. lactis </it>global transcriptome and proteome profiles showed good correlation supporting the general idea of transcription level control of bacterial metabolism, but the data indicated that substrate transport systems together with lower part of glycolysis in <it>L. lactis </it>were presumably under allosteric control.</p> <p>Conclusions</p> <p>The current study demonstrates advantages of the usage of strictly controlled continuous cultivation methods combined with multi-omics approach for quantitative understanding of amino acid and energy metabolism of <it>L. lactis </it>which is a valuable new knowledge for development of balanced growth media, gene manipulations for desired product formation etc. Moreover, collected dataset is an excellent input for developing metabolic models.</p

Systems biology approach reveals that overflow metabolism of acetate in Escherichia coli is triggered by carbon catabolite repression of acetyl-CoA synthetase

<p>Abstract</p> <p>Background</p> <p>The biotechnology industry has extensively exploited <it>Escherichia coli </it>for producing recombinant proteins, biofuels etc. However, high growth rate aerobic <it>E. coli </it>cultivations are accompanied by acetate excretion <it>i.e</it>. overflow metabolism which is harmful as it inhibits growth, diverts valuable carbon from biomass formation and is detrimental for target product synthesis. Although overflow metabolism has been studied for decades, its regulation mechanisms still remain unclear.</p> <p>Results</p> <p>In the current work, growth rate dependent acetate overflow metabolism of <it>E. coli </it>was continuously monitored using advanced continuous cultivation methods (A-stat and D-stat). The first step in acetate overflow switch (at μ = 0.27 ± 0.02 h^-1) is the repression of acetyl-CoA synthethase (Acs) activity triggered by carbon catabolite repression resulting in decreased assimilation of acetate produced by phosphotransacetylase (Pta), and disruption of the PTA-ACS node. This was indicated by acetate synthesis pathways PTA-ACKA and POXB component expression down-regulation before the overflow switch at μ = 0.27 ± 0.02 h^-1with concurrent 5-fold stronger repression of acetate-consuming Acs. This in turn suggests insufficient Acs activity for consuming all the acetate produced by Pta, leading to disruption of the acetate cycling process in PTA-ACS node where constant acetyl phosphate or acetate regeneration is essential for <it>E. coli </it>chemotaxis, proteolysis, pathogenesis etc. regulation. In addition, two-substrate A-stat and D-stat experiments showed that acetate consumption capability of <it>E. coli </it>decreased drastically, just as Acs expression, before the start of overflow metabolism. The second step in overflow switch is the sharp decline in cAMP production at μ = 0.45 h^-1leading to total Acs inhibition and fast accumulation of acetate.</p> <p>Conclusion</p> <p>This study is an example of how a systems biology approach allowed to propose a new regulation mechanism for overflow metabolism in <it>E. coli </it>shown by proteomic, transcriptomic and metabolomic levels coupled to two-phase acetate accumulation: acetate overflow metabolism in <it>E. coli </it>is triggered by Acs down-regulation resulting in decreased assimilation of acetic acid produced by Pta, and disruption of the PTA-ACS node.</p

The Goblet Cell Protein Clca1 (Alias mClca3 or Gob-5) Is Not Required for Intestinal Mucus Synthesis, Structure and Barrier Function in Naive or DSS- Challenged Mice

The secreted, goblet cell-derived protein Clca1 (chloride channel regulator, calcium-activated-1) has been linked to diseases with mucus overproduction, including asthma and cystic fibrosis. In the intestine Clca1 is found in the mucus with an abundance and expression pattern similar to Muc2, the major structural mucus component. We hypothesized that Clca1 is required for the synthesis, structure or barrier function of intestinal mucus and therefore compared wild type and Clca1-deficient mice under naive and at various time points of DSS (dextran sodium sulfate)-challenged conditions. The mucus phenotype in Clca1-deficient compared to wild type mice was systematically characterized by assessment of the mucus protein composition using proteomics, immunofluorescence and expression analysis of selected mucin genes on mRNA level. Mucus barrier integrity was assessed in-vivo by analysis of bacterial penetration into the mucus and translocation into sentinel organs combined analysis of the fecal microbiota and ex-vivo by assessment of mucus penetrability using beads. All of these assays revealed no relevant differences between wild type and Clca1-deficient mice under steady state or DSS-challenged conditions in mouse colon. Clca1 is not required for mucus synthesis, structure and barrier function in the murine colon

Muc2-dependent microbial colonization of the jejunal mucus layer is diet sensitive and confers local resistance to enteric pathogen infection.

Intestinal mucus barriers normally prevent microbial infections but are sensitive to diet-dependent changes in the luminal environment. Here we demonstrate that mice fed a Western-style diet (WSD) suffer regiospecific failure of the mucus barrier in the small intestinal jejunum caused by diet-induced mucus aggregation. Mucus barrier disruption due to either WSD exposure or chromosomal Muc2 deletion results in collapse of the commensal jejunal microbiota, which in turn sensitizes mice to atypical jejunal colonization by the enteric pathogen Citrobacter rodentium. We illustrate the jejunal mucus layer as a microbial habitat, and link the regiospecific mucus dependency of the microbiota to distinctive properties of the jejunal niche. Together, our data demonstrate a symbiotic mucus-microbiota relationship that normally prevents jejunal pathogen colonization, but is highly sensitive to disruption by exposure to a WSD

Quantitative Proteomics of Escherichia coli: From Relative to Absolute Scale. Kvantitatiivne Escherichia coli proteoomika: relatiivsetest numbritest absoluutsete kogusteni

proteoomika, kvantitatiivne proteoomika, m\ue4rgisevaba kvantifitseerimine, Escherichia coli, dissertatsioonid, proteomics, quantitative proteomics, label-free quantification, Escherichia coli, dissertation

Magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of Semliki Forest virus

Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection