33 research outputs found
Cell wall proteome of sugarcane stems: comparison of a destructive and a non-destructive extraction method showed differences in glycoside hydrolases and peroxidases
Abstract\ud
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Background\ud
Sugarcane has been used as the main crop for ethanol production for more than 40Â years in Brazil. Recently, the production of bioethanol from bagasse and straw, also called second generation (2G) ethanol, became a reality with the first commercial plants started in the USA and Brazil. However, the industrial processes still need to be improved to generate a low cost fuel. One possibility is the remodeling of cell walls, by means of genetic improvement or transgenesis, in order to make the bagasse more accessible to hydrolytic enzymes. We aimed at characterizing the cell wall proteome of young sugarcane culms, to identify proteins involved in cell wall biogenesis. Proteins were extracted from the cell walls of 2-month-old culms using two protocols, non-destructive by vacuum infiltration vs destructive. The proteins were identified by mass spectrometry and bioinformatics.\ud
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Results\ud
A predicted signal peptide was found in 84 different proteins, called cell wall proteins (CWPs). As expected, the non-destructive method showed a lower percentage of proteins predicted to be intracellular than the destructive one (33Â % vs 44Â %). About 19Â % of CWPs were identified with both methods, whilst the infiltration protocol could lead to the identification of 75Â % more CWPs. In both cases, the most populated protein functional classes were those of proteins related to lipid metabolism and oxido-reductases. Curiously, a single glycoside hydrolase (GH) was identified using the non-destructive method whereas 10 GHs were found with the destructive one. Quantitative data analysis allowed the identification of the most abundant proteins.\ud
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Conclusions\ud
The results highlighted the importance of using different protocols to extract proteins from cell walls to expand the coverage of the cell wall proteome. Ten GHs were indicated as possible targets for further studies in order to obtain cell walls less recalcitrant to deconstruction. Therefore, this work contributed to two goals: enlarge the coverage of the sugarcane cell wall proteome, and provide target proteins that could be used in future research to facilitate 2G ethanol production
Cell wall proteome of sugarcane stems: comparison of a destructive and a non-destructive extraction method showed differences in glycoside hydrolases and peroxidases
RNase E and HupB dynamics foster mycobacterial cell homeostasis and fitness
International audienceRNA turnover is a primary source of gene expression variation, in turn promoting cellular adaptation. Mycobacteria leverage reversible mRNA stabilization to endure hostile conditions. Although RNase E is essential for RNA turnover in several species, its role in mycobacterial single-cell physiology and functional phenotypic diversification remains unexplored. Here, by integrating live-single-cell and quantitative-mass-spectrometry approaches, we show that RNase E forms dynamic foci, which are associated with cellular homeostasis and fate, and we discover a versatile molecular interactome. We show a likely interaction between RNase E and the nucleoid-associated protein HupB, which is particularly pronounced during drug treatment and infection, where phenotypic diversity increases. Disruption of RNase E expression affects HupB levels, impairing Mycobacterium tuberculosis growth homeostasis during treatment, intracellular replication, and host spread. Our work lays the foundation for targeting the RNase E and its partner HupB, aiming to undermine M. tuberculosis cellular balance, diversification capacity, and persistence
Cell wall proteomic datasets of stems and leaves of Brachypodium distachyon
International audienceThis article provides experimental data describing the cell wall protein profiles of stems and leaves of Brachypodium distachyon at two different stages of development. The cell wall proteomics data have been obtained from (i) stem internodes at young and mature stages of development, and (ii) leaves at young and mature stages of development. The proteins have been extracted from purified cell walls using buffers containing calcium chloride (0.2Â M) or lithium chloride (2Â M). They have been identified by LC-MS/MS and bioinformatics. These data allow deepening our knowledge of these cell wall proteomes. They are a valuable resource for people interested in plant cell wall biology to understand the roles of cell wall proteins during the growth of vegetative organs
The Adenylate Cyclase (CyaA) Toxin from <i>Bordetella pertussis</i> Has No Detectable Phospholipase A (PLA) Activity In Vitro
The adenylate cyclase (CyaA) toxin produced in Bordetella pertussis is the causative agent of whooping cough. CyaA exhibits the remarkable capacity to translocate its N-terminal adenyl cyclase domain (ACD) directly across the plasma membrane into the cytosol of eukaryotic cells. Once translocated, calmodulin binds and activates ACD, leading to a burst of cAMP that intoxicates the target cell. Previously, Gonzalez-Bullon et al. reported that CyaA exhibits a phospholipase A activity that could destabilize the membrane to facilitate ACD membrane translocation. However, Bumba and collaborators lately reported that they could not replicate these results. To clarify this controversy, we assayed the putative PLA activity of two CyaA samples purified in two different laboratories by using two distinct fluorescent probes reporting either PLA2 or both PLA1 and PLA2 activities, as well as in various experimental conditions (i.e., neutral or negatively charged membranes in different buffers.) However, we could not detect any PLA activity in these CyaA batches. Thus, our data independently confirm that CyaA does not possess any PLA activity
Loss of CorA, the primary magnesium transporter of Salmonella, is alleviated by MgtA and PhoP-dependent compensatory mechanisms.
In many Gram-negative bacteria, the stress sigma factor of RNA polymerase, ÏS/RpoS, remodels global gene expression to reshape the physiology of stationary phase cells and ensure their survival under non-optimal growth conditions. In the foodborne pathogen Salmonella enterica serovar Typhimurium, ÏS is also required for biofilm formation and virulence. We have recently shown that a ÎrpoS mutation decreases the magnesium content and expression level of the housekeeping Mg2+-transporter CorA in stationary phase Salmonella. The other two Mg2+-transporters of Salmonella are encoded by the PhoP-activated mgtA and mgtB genes and are expressed under magnesium starvation. The ÏS control of corA prompted us to evaluate the impact of CorA in stationary phase Salmonella cells, by using global and analytical proteomic analyses and physiological assays. The ÎcorA mutation conferred a competitive disadvantage to exit from stationary phase, and slightly impaired motility, but had no effect on total and free cellular magnesium contents. In contrast to the wild-type strain, the ÎcorA mutant produced MgtA, but not MgtB, in the presence of high extracellular magnesium concentration. Under these conditions, MgtA production in the ÎcorA mutant did not require PhoP. Consistently, a ÎmgtA, but not a ÎphoP, mutation slightly reduced the magnesium content of the ÎcorA mutant. Synthetic phenotypes were observed when the ÎphoP and ÎcorA mutations were combined, including a strong reduction in growth and motility, independently of the extracellular magnesium concentration. The abundance of several proteins involved in flagella formation, chemotaxis and secretion was lowered by the ÎcorA and ÎphoP mutations in combination, but not alone. These findings unravel the importance of PhoP-dependent functions in the absence of CorA when magnesium is sufficient. Altogether, our data pinpoint a regulatory network, where the absence of CorA is sensed by the cell and compensated by MgtA and PhoP- dependent mechanisms
Amphiregulin mediates non-cell-autonomous effect of senescence on reprogramming
International audienceCellular senescence is an irreversible growth arrest with a dynamic secretome, termed the senescence-associatedsecretory phenotype (SASP). Senescence is a cell-intrinsic barrier for reprogramming, whereas theSASP facilitates cell fate conversion in non-senescent cells. However, the mechanisms by which reprogramming-induced senescence regulates cell plasticity are not well understood. Here, we investigate how the heterogeneityof paracrine senescence impacts reprogramming. We show that senescence promotes in vitro reprogrammingin a stress-dependent manner. Unbiased proteomics identifies a catalog of SASP factorsinvolved in the cell fate conversion. Amphiregulin (AREG), frequently secreted by senescent cells, promotesin vitro reprogramming by accelerating proliferation and the mesenchymal-epithelial transition via EGFRsignaling. AREG treatment diminishes the negative effect of donor age on reprogramming. Finally, AREG enhances in vivo reprogramming in skeletal muscle. Hence, various SASP factors can facilitate cellular plasticityto promote reprogramming and tissue repair
Protein-protein interaction network of the proteins significantly less abundant in the Î<i>corA</i>Î<i>phoP</i> mutant, but not found less abundant in the Î<i>corA</i> and Î<i>phoP</i> strains, when comparing to the wild-type strain.
Top enriched functional categories of proteins are colored: flagellum-dependent motility in blue, chemotaxis in red, T3SS-dependent secretion in pink and cobalamin biosynthetic pathway in green. See also S2 Dataset for details.</p
The Î<i>corA</i> mutation impairs <i>Salmonella</i> growth in the absence of PhoP or MgtA.
(A) The ÎcorA mutation impairs Salmonella growth in the absence of phoP. The Salmonella wild-type, ÎcorA, ÎphoP and ÎcorAÎphoP strains were grown 18 h in LB and in LB supplemented with MgCl2 10 mM at 37°C. Cultures were spread on LB plates supplemented or not with MgCl2 10 mM which were incubated at 37°C and colony size was examined overnight. (B) The phoQ*R16S allele improves growth of the ÎcorAÎmgtA mutant. The Salmonella wild-type, ÎcorA, and ÎcorAÎmgtA large and small colony variants (LCV and SCV, respectively) carrying or not a ÎphoQ mutation were grown 18 h in LB at 37°C. Cultures were spread on LB plates which were incubated at 37°C and colony size was examined overnight. Compared to the wild-type strain, growth of the ÎcorAÎmgtA SCV was affected (B), but to a lesser extent than that of the ÎcorAÎphoP strain (A). The ÎcorAÎmgtA LCV carrying the mutated phoQ allele (phoQ*R16S, see text) did not show visible growth defect compared to the wild-type strain.</p
S12 Raw images -
In many Gram-negative bacteria, the stress sigma factor of RNA polymerase, ÏS/RpoS, remodels global gene expression to reshape the physiology of stationary phase cells and ensure their survival under non-optimal growth conditions. In the foodborne pathogen Salmonella enterica serovar Typhimurium, ÏS is also required for biofilm formation and virulence. We have recently shown that a ÎrpoS mutation decreases the magnesium content and expression level of the housekeeping Mg2+-transporter CorA in stationary phase Salmonella. The other two Mg2+-transporters of Salmonella are encoded by the PhoP-activated mgtA and mgtB genes and are expressed under magnesium starvation. The ÏS control of corA prompted us to evaluate the impact of CorA in stationary phase Salmonella cells, by using global and analytical proteomic analyses and physiological assays. The ÎcorA mutation conferred a competitive disadvantage to exit from stationary phase, and slightly impaired motility, but had no effect on total and free cellular magnesium contents. In contrast to the wild-type strain, the ÎcorA mutant produced MgtA, but not MgtB, in the presence of high extracellular magnesium concentration. Under these conditions, MgtA production in the ÎcorA mutant did not require PhoP. Consistently, a ÎmgtA, but not a ÎphoP, mutation slightly reduced the magnesium content of the ÎcorA mutant. Synthetic phenotypes were observed when the ÎphoP and ÎcorA mutations were combined, including a strong reduction in growth and motility, independently of the extracellular magnesium concentration. The abundance of several proteins involved in flagella formation, chemotaxis and secretion was lowered by the ÎcorA and ÎphoP mutations in combination, but not alone. These findings unravel the importance of PhoP-dependent functions in the absence of CorA when magnesium is sufficient. Altogether, our data pinpoint a regulatory network, where the absence of CorA is sensed by the cell and compensated by MgtA and PhoP- dependent mechanisms.</div