Comparative transcriptome analysis of Ogataea parapolymorpha wild-type and met4 mutant under sulfur limitation
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ABSTRACT: We analyze the transcriptome of Ogataea parapolymorpha wild-type and met4 mutant under sulfur limited condition to investigate the function of Met4 protein in this yeast in the absence of any sulfur source. By sequencing the mRNAs of the wild-type and mutant grown in sulfur-limited B minimal medium with or without cysteine as a sole sulfur source, we find the role of OpMet4p as a master regulator for cell homeostasis under sulfur limitation.
Project description:All yeast strains used in this study (Table 1) are in the W303 background (ade2-1 can1-100, his3-1,15 leu2-3,112 trp1-1 ura3). For sulfur limitation microarray studies, WT, met4 delete, met31 delete met32 delete, cbf1 delete, and met28 delete strains were grown in minimal B-media [see Cherest, H., and Surdin-Kerjan, Y. (1992). Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway. Genetics 130, p51-58 for B-media composition] supplemented with 0.5mM methionine as the sole sulfur source. An aliquot of cells was harvested for a t=0 time point while the remainder were filtered through a .22um Stericup filter (Millipore), then washed and resuspended in pre-warmed (30 C) B-media lacking any source of sulfur. Cells were harvested after 20, 40, and 80 minutes.
Project description:Candidatus Pelagibacter ubique is the most abundant marine microorganism, but is unable to utilize inorganic sulfur compounds that are plentiful in the ocean. To investigate how these cells adapt to organic sulfur limitation, batch cultures were grown in defined media containing either limiting or non-limiting amounts of dimethylsulfoniopropionate (DMSP) as the sole sulfur source. Protein and mRNA expression were measured during exponential growth, immediately prior to stationary phase, and in late stationary phase. Two distinct responses were observed: one as DMSP approached exhaustion, and another after the DMSP supply was depleted. The first response was characterized by increased transcription and translation of all Ca. P. ubique genes downstream of previously confirmed S-adenosyl methionine (SAM) riboswitches: bhmT, mmuM, and metY. These genes were up to 33 times more abundant during low DMSP conditions and shunt all available sulfur to methionine. The osmotically inducible organic hydroperoxidase OsmC was the most up-regulated protein as DMSP (an osmolyte) became scarce. The second response, during sulfur-depleted stationary phase, saw increased transcription of the heme c shuttle ccmC and two small genes of unknown function (SAR11_1163 and SAR11_1164) which were 6-10 times higher in sulfur-starved cultures. No known membrane transporters were up-regulated in response to sulfur limitation, suggesting that this bacterium's strategy for coping with sulfur stress focuses on intracellularly redistributing, rather than importing, organic sulfur compounds. This supports the conclusion that the few organosulfur molecules that Ca. P. ubique is able to metabolize are rarely limiting in the marine environment. Batch cultures of P. ubique were grown in a defined arificial seawater media. Five cultures were amended with a limiting concentration of DMSP as the sole sulfur source and another four control cultures were amended with a non-limiting DMSP concentration. Cultures were harvested for microarray analyses at multiple timepoints for the purpose of observing differences in gene expression related to sulfur limitation. Proteomic analyses were conducted in parallel and are available at https://www.ebi.ac.uk/pride/archive/projects/PXD003672 .
Project description:Candidatus Pelagibacter ubique is the most abundant marine microorganism, but is unable to utilize inorganic sulfur compounds that are plentiful in the ocean. To investigate how these cells adapt to organic sulfur limitation, batch cultures were grown in defined media containing either limiting or non-limiting amounts of dimethylsulfoniopropionate (DMSP) as the sole sulfur source. Protein and mRNA expression were measured during exponential growth, immediately prior to stationary phase, and in late stationary phase. Two distinct responses were observed: one as DMSP approached exhaustion, and another after the DMSP supply was depleted. The first response was characterized by increased transcription and translation of all Ca. P. ubique genes downstream of previously confirmed S-adenosyl methionine (SAM) riboswitches: bhmT, mmuM, and metY. These genes were up to 33 times more abundant during low DMSP conditions and shunt all available sulfur to methionine. The osmotically inducible organic hydroperoxidase OsmC was the most up-regulated protein as DMSP (an osmolyte) became scarce. The second response, during sulfur-depleted stationary phase, saw increased transcription of the heme c shuttle ccmC and two small genes of unknown function (SAR11_1163 and SAR11_1164) which were 6-10 times higher in sulfur-starved cultures. No known membrane transporters were up-regulated in response to sulfur limitation, suggesting that this bacterium's strategy for coping with sulfur stress focuses on intracellularly redistributing, rather than importing, organic sulfur compounds. This supports the conclusion that the few organosulfur molecules that Ca. P. ubique is able to metabolize are rarely limiting in the marine environment.
Project description:transcriptome changes in pea leaves with sulfur deficency/sufficiency during reproductive phase.-Characterization of transcriptome changes in leaves of wild-type and PsSultr4 mutant lines (for a sulfur transporter) subjected or not to sulfur deficiency during the reproductive phase 4plex_pea_2014_01 - transcriptome changes in pea leaves with sulfur deficency/sufficiency during reproductive phase. - Role of sulfur and of the sulfate store in leaf metabolism. - Comparison of: 1- The leaf transcriptome of pea subjected or not to sulfur deficiency during the reproductive phase (S+ versus S –) 2- The leaf transcriptome of wild-type and mutant lines for a sulfur transporter (two TILLING alleles) grown under sulfur sufficient conditions : WT1/Mut1 S+ et WT2/Mut2 S+ 3- The leaf transcriptome of wild-type and mutant lines for a sulfur transporter (two TILLING alleles) grown under sulfur deficient conditions : WT1/Mut1 S+ et WT2/Mut2 S+
Project description:The batch fermentation of Clostridium acetobutylicum is characterized by an acetogenic growth phase during exponential growth when mainly acetate and butyrate are fermentation products. Then, at the end of exponential growth and during stationary phase, the organism switches to solventogenic growth and large amounts of acetone, ethanol and butanol are produced. These growth phases can be studied independent from each other in a phosphate-limited continuous culture. In transcription analysis of continuous cultures using DNA microarrays it became evident that, among others, operons involved in sulfur assimilation are strongly up-regulated during solventogenesis. Using the ClosTron technique we constructed two knock-out mutants in the genes CAC0105 and CAC0930 annotated as involved in sulfate reduction and cysteine biosynthesis. Complementation experiments were carried out with sulfite and cysteine to prove the predicted function. The fermentation experiments of wild type and mutants using phosphate-limited and sulfur-limited continuous culture demonstrated that less sulfur source was consumed in solventogenic phase and the efficiency of cysteine uptake became lower. DNA microarrays were performed to study the difference of transcriptional expression when the wild type was challenged with insufficient sulfur source and the mccB (CAC0930) mutant was inactivated in the continuous culture. The result provided insights into understanding the sulfur metabolism regulatory.
Project description:Transcriptome profiling analysis of the Hansenula polymorpha MET4 gene deletion strain have been carried out to obtain comprehensive information on the HpMet4p-mediated regulatory networks in association with the cadmium (Cd) detoxification and sulfur regulation in H. polymorpha. Total RNA samples were collected from H. polymorpha wild type, and HpMET4 deletion strain, under sulfur starvation or Cd (0.6 mM) stress conditions. The differential fluorescence intensities of each RNA sample were measured after labeling with Cy3 or Cy5. For all analyses, we performed dye swapping experiments to avoid dye bias. Thus, four intensity values were generated for each ORF and averaged for analysis.
Project description:Transcriptome profiling analysis of the Hansenula polymorpha MET4 gene deletion strain have been carried out to obtain comprehensive information on the HpMet4p-mediated regulatory networks in association with the cadmium (Cd) detoxification and sulfur regulation in H. polymorpha.
Project description:The Chlamydomonas reinhardtii transcriptome was characterized from nutrient-replete and sulfur-depleted wild-type and snrk2.1 mutant cells; the mutant is null for the regulatory serine-threonine kinase SNRK2.1, which is required for acclimation to sulfur deprivation. The transcriptome analyses involved microarray hybridization and RNA-seq technology; RT-qPCR evaluation of the data obtained by these techniques showed that RNA-seq is significantly more quantitative than microarray hybridizations. Sulfur-deprivation-responsive transcripts included those encoding proteins involved in sulfur acquisition and assimilation, recycling of sulfur-containing amino acids, synthesis of reduced sulfur metabolites and cofactors, and modification of cellular structures such as the cell wall and complexes associated with the photosynthetic apparatus. Moreover, the data suggest that cells deprived of sulfur favors accumulation of proteins with fewer sulfur-containing amino acids. Most of these sulfur-deprivation responses are controlled by the SNRK2.1 kinase. Furthermore, the snrk2.1 mutant exhibits a set of unique responses during both sulfur-replete and sulfur-depleted conditions that are not observed in wild-type cells. Many of these responses are likely to be elicited by singlet O2 accumulation in the mutant cells. The transcriptome results for the wild-type and mutant cells strongly suggest the occurrence of massive changes in cellular physiology and metabolism as the cells become depleted for sulfur, and reveal aspects of acclimation that are likely critical for cell survival. The three supplementary files GSE17970_supplemental_table_*.xls below include results of the differential expression analysis (expression estimates, fold changes and p-values), and different clusters of functionally related genes.
Project description:P. ubique was investigated to understand the response to limited sulfur in seawater. Sulfur absorption was found to be localized to a rapidly evolving region of the genome.