Proteomics on extracellular fractions of polyphosphate accumulating organisms from wastewater treatment plant
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ABSTRACT: Polyphosphate accumulating organisms are responsible for enhanced biological phosphate removal from wastewater, where they grow embedded in a matrix of extracellular polymeric substances. Little is known about the composition and dynamics of those proteins and their production by the different microorganisms. Tomás-Martínez et al., (2022) studied the turnover of proteins and polysaccharides in extracellular polymeric fractions of an enrichment culture of polyphosphate accumulating organisms using an anaerobic-aerobic sequencing batch reactor simulating EBPR conditions. Finally, the carbon source was switched to 13C-labelled acetate to study the protein turnover. Samples were collected at the end of each aerobic phase.
Project description:Proteins secreted by marine cyanobacterium Synechococcus under phosphorus stress is largely uncharacterized. This dataset characterizes the exoproteins for both an open ocean (WH8102) and coastal (WH5701) Synechococcus strain and were collected as part of the study "Dissolved organic phosphorus bond-class utilization by Synechococcus". Study Abstract: Dissolved organic phosphorus (DOP) contains compounds with phosphoester (P-O-C), phosphoanhydride (P-O-P), and phosphorus-carbon (P-C) bonds. Despite DOP’s importance as a nutritional source for marine microorganisms, the bioavailability of each bond-class to the widespread cyanobacterium Synechococcus remains largely unknown. This study evaluates bond-class specific DOP utilization by cultures of an open ocean and a coastal ocean Synechococcus strain. Both strains exhibited comparable growth rates when provided phosphate, short-chain and long-chain polyphosphate (P-O-P), adenosine 5’-triphosphate (P-O-C and P-O-P), and glucose-6-phosphate (P-O-C) as the phosphorus source. However, growth rates on phosphomonoester adenosine 5’-monophosphate (P-O-C) and phosphodiester bis(4-methylumbelliferyl) phosphate (C-O-P-O-C) varied between strains, and neither strain grew on selected phosphonates. Consistent with the growth measurements, both strains preferentially hydrolyzed 3-polyphosphate, followed by adenosine 5’-triphosphate, and then adenosine 5’-monophosphate. The strains’ exoproteome contained phosphorus hydrolases, which combined with enhanced cell-free hydrolysis of 3-polyphosphate and adenosine 5’-triphosphate under phosphate deficiency, suggests active mineralization of short-chain polyphosphate by Synechococcus’ exoproteins. Synechococcus alkaline phosphatases presented broad substrate specificities, including activity towards short-chain polyphosphate, with varying affinities between the two strains. Collectively, these findings underscore the potentially significant role of compounds with phosphoanhydride bonds in Synechococcus phosphorus nutrition, thereby expanding our understanding of microbially-mediated DOP cycling in marine ecosystems.
Project description:This experiment studied the effect of FPP accumulation on E. coli. E. coli cells transformed with pMBIS (the S. cerevisiae mevalonate pathway enzymes converting mevalonate to FPP) and fed mevalonate produce large amounts of FPP, which causes toxicity when it accumulates. When coupled with an active amorphadiene synthase (pADS) the cells produce amorphadiene, a non-toxic isoprenoid. To accumulate FPP, but maintain similar protein burden, an amorphadiene synthase with 3 mutations to render it inactive was used (pADSmut) to accumulate FPP. E. coli was transformed with pMBIS and pADS or pMBIS and pADSMut and grown in LB and fed 10 mM mevalonate and induced with 0.5 mM IPTG, then sampled at subsequent time points. This comparison is between E. coli DH1 cells accumulating FPP via the heterologous mevalonate pathway (pMBIS/pADSmut) to cells producing amorphadiene via the same pathway (pMBIS/pADS). Samples were collected 2.5, 5, 7, and 29.5 hr after addition of IPTG and mevalonate. One biological replicate was used. Total RNA was extracted, reverse transcribed, labeled, and hybridized to multiple slides for technical replicates.
Project description:Several in vitro models have been developed to recapitulate mouse embryogenesis solely from embryonic stem cells (ESCs). Despite mimicking many aspects of early development, they fail to capture the interactions between embryonic and extraembryonic tissues. To overcome this difficulty, we have developed a mouse ESC-based in vitro model that reconstitutes the pluripotent ESC lineage and the two extra-embryonic lineages of the post-implantation embryo by transcription factor-mediated induction. This unified model recapitulates developmental events from embryonic day 5.5 to 8.5, including gastrulation, and formation of the anterior-posterior axis, brain, a beating heart structure, and the development of extraembryonic tissues, including yolk sac and chorion. Comparing single-cell RNA sequencing from individual structures with time-matched natural embryos identified remarkably similar transcriptional programs across lineages, but also showed when and where the model diverges from the natural program. Our findings demonstrate an extraordinary plasticity of ESCs to self-organize and generate a whole embryo-like structure.
Project description:We used three biological replicas of T. reesei (QM9414), a M-NM-^Tlae1 mutant and a lae1-overexpressing strain in the chemostat on glucose at two different growth rates (0.075 and 0.020 h-1). Two to four subsequently achieved, independent steady-states were sampled and analysed for each dilution rate and fungal strain.
Project description:According to the systems biology approach, genomics tells what can happen, transcriptomics what appears to be happening, metabolomics what has happened and proteomics what makes it happen. Thereby, to get a better what makes resurrection plants extremely tolerant to drought, we explored changes in the resurrection proteome and cellular ultrastructure of Haberlea rhodopensis in response to desiccation. Because genomic and proteomic data concerning resurrection plants are limited, particularly for H. rhodopensis, we identified proteins based on previous transcriptomic studies. For the identified proteins, fold changes and differences in transcript levels between fresh and dry plants were analyzed; proteins significantly enriched in various biological processes and metabolic pathways were detected using metabolomic metadata. The results confirmed that the transcription of different genes is regulated as previously described at the proteomic level; new genes were identified in desiccation tolerance, posttranscriptional regulation events, and posttranslational regulation events. We revealed new evidences about organelle and cell preservation, posttranscriptional and posttranslational regulation, photosynthesis, primary metabolism, and phagocytosis in H. rhodopensis. These findings can inform further genomic and evolutionary studies, as well as targeted genetic engineering to improve drought tolerance in crops as a response to climate change.
Project description:This experiment studied the effect of FPP accumulation on E. coli. E. coli cells transformed with pMBIS (the S. cerevisiae mevalonate pathway enzymes converting mevalonate to FPP) and fed mevalonate produce large amounts of FPP, which causes toxicity when it accumulates. When coupled with an active amorphadiene synthase (pADS) the cells produce amorphadiene, a non-toxic isoprenoid. To accumulate FPP, but maintain similar protein burden, an amorphadiene synthase with 3 mutations to render it inactive was used (pADSmut) to accumulate FPP. E. coli was transformed with pMBIS and pADS or pMBIS and pADSMut and grown in M9+glucose with varying magnesium concentrations and fed 20 mM mevalonate and induced with 0.5 mM IPTG, then sampled at subsequent time points. This comparison is between E. coli DH1 cells accumulating FPP via the heterologous mevalonate pathway (pMBIS/pADSmut) to cells producing amorphadiene via the same pathway (pMBIS/pADS). Samples were collected 6, 10, 14, and 26.5 hr after addition of IPTG and mevalonate. One biological replicate was used. Total RNA was extracted, reverse transcribed, labeled, and hybridized to multiple slides for technical replicates.
Project description:Honeybee health is a major ecological, agricultural and societal concern due to the critical role of these insects for plant reproduction and the massive losses of colonies observed within the last decade. The search for abiotic (e.g. pesticides) and biotic (e.g. pathogens) stressors is essential for understanding bee declines and design protection plans. Flagellated Trypanosomatid protozoan parasites and particularly Lotmaria passim, are widely distributed in honeybee colonies and have been associated with colony losses. However, little is known about their life cycles, routes for transmission and the strategies for survival inside and outside their hosts. Here we describe the proteome of L. passim Extracellular polymeric substances linked to secretion of Extracellular vesicles using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The EPS-EVs composition will provide clues on how these pathogens survive and spread in apiaries worldwide.