The conidial ubiquitylome analysis in Arthrobotrys oligospora ATCC 24927
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ABSTRACT:
INSTRUMENT(S): Thermo Scientific Q Exactive HF-X
ORGANISM(S): Arthrobotrys Oligospora Atcc 24927 (ncbitaxon:756982)
SUBMITTER: 
Tong Liu
PROVIDER: MSV000084740 | MassIVE | Sun Dec 29 18:03:00 GMT 2019
REPOSITORIES: MassIVE
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Project description:Investigation of the whole genome gene expression level changes relative to exponential phase growth in Nitrosomonas europaea ATCC19718 after 12 hours ammonia starvation, 144 hours ammonia starvation, and 20 minutes following ammonia addition to starved cells. The ammonia monooxygenase of chemolithotrophic ammonia oxidizing bacteria (AOB) catalyzes the first step in ammonia oxidation by converting ammonia to hydroxylamine. The monooxygenase of Nitrosomonas europaea is encoded by two nearly identical operon copies (amoCAB1,2). Several AOB, including N. europaea, also posess a divergent monocistronic copy of amoC (amoC3) of unknown function. Previous work suggested a possible functional role for amoC3 in N. europaea during recovery from extended ammonia starvation as part of the σE- stress response regulon during the recovery of N. europaea from extended ammonia starvation, thus indicating its importance during the exit of cells from starvation. We here used global transcription analysis to show that expression of amoC3 is part of a general post-starvation cellular response system in N. europaea. We also found that amoC3 is required for efficient exit from prolonged ammonia starvation, as deleting this gene impaired growth at elevated temperatures and recovery following starvation under high oxygen tensions. Deletion of the σ32 global stress response regulator demonstrated that the heat shock regulon also plays a significant role in mediating the recovery of N. europaea from starvation. These findings provide the first described phenotype associated with the divergent AmoC3 subunit which appears to function as a stress responsive subunit capable of maintaining ammonia oxidation activity under stress conditions.
2012-08-03 | GSE35122 | GEO
Project description:Investigation of the whole genome gene expression level changes relative to exponential phase growth in Nitrosomonas europaea ATCC19718 after 12 hours ammonia starvation, 144 hours ammonia starvation, and 20 minutes following ammonia addition to starved cells. The ammonia monooxygenase of chemolithotrophic ammonia oxidizing bacteria (AOB) catalyzes the first step in ammonia oxidation by converting ammonia to hydroxylamine. The monooxygenase of Nitrosomonas europaea is encoded by two nearly identical operon copies (amoCAB1,2). Several AOB, including N. europaea, also posess a divergent monocistronic copy of amoC (amoC3) of unknown function. Previous work suggested a possible functional role for amoC3 in N. europaea during recovery from extended ammonia starvation as part of the σE- stress response regulon during the recovery of N. europaea from extended ammonia starvation, thus indicating its importance during the exit of cells from starvation. We here used global transcription analysis to show that expression of amoC3 is part of a general post-starvation cellular response system in N. europaea. We also found that amoC3 is required for efficient exit from prolonged ammonia starvation, as deleting this gene impaired growth at elevated temperatures and recovery following starvation under high oxygen tensions. Deletion of the σ32 global stress response regulator demonstrated that the heat shock regulon also plays a significant role in mediating the recovery of N. europaea from starvation. These findings provide the first described phenotype associated with the divergent AmoC3 subunit which appears to function as a stress responsive subunit capable of maintaining ammonia oxidation activity under stress conditions. A twelve chip study using total RNA recovered from four timepoints for each of three biological replicates of wild-type cultures of Nitrosomonas europaea ATCC 19718. Total RNA was obtained from each biological culture replicate during exponential growth, following 12 hours ammonia starvation, 144 hours ammonia starvations, and 20 minutes following ammonia addition to starved cells.
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Project description:Purpose: To explore the pathway cross-talk of endoplasmic reticulum (ER) stress response and cellulases synthesis in Neurospora crassa. Methods: To identify ER stress response targets(ESRTs), conidial suspensions of WT were obtained and 1 x 10^6 cells/ml incubated in cellulases induction medium (1x vogel’s salts , 1% w/v crystalline cellulose (Avicel PH-101; Sigma-Aldrich), 0.2% w/v NH4NO3 ,pH 7.0 )for 36 hours (25ºC, 200 rpm, constant light) followed by the addition of dithiothreitol (DTT, final concentration of 0.1mM for mild stress level while 5mM for acute stress level) or tunicamycin (TM, final concentration of 5ug/mL for mild stress level while 40ug/mL for acute stress level) and growth for 1 more hour.To identify which ESRTs regulated by IRE-1/Hac-1 mediated UPR pathway,conidial suspensions of the Ire-1 and Hac-1 KO mutants as well as their parental strains (FGSC#2489 and FGSC#9720, respectively) were obtained and 1 x 10^6 cells/ml incubated in minimal medium containing 2% sucrose for 16 hours, then young hyphae was harvested and transferred into cellulases induction medium as mentioned above culture for 4 hours followed by the addition of DTT (final concentration of 5mM)(or H2O as mock)and growth for 1 more hour.For Hac-1 KO mutant and FGSC#9720,cultures were plus with final concentration of 100ug/ml L-histidine.To explore the function of transcription factors RES-1, RES-2 and RRG-2 in response to ER stress and cellulases synthesis, each conidial suspension of the three strains was obtained and 1 x 10^6 cells/ml incubated in cellulases induction medium for 36 hours followed by the addition of dithiothreitol (DTT, final concentration of 5mM ) (or H2O as mock)and growth for 1 more hour.Cells were harvested by vacuum filtration with Whatman filter paper, frozen immediately in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. An additional clean-up including the on-column DNase I treatment was performed by using the RNeasy mini kit (Qiagen).The thirteen 49-nt single-end RNA-seq libraries were generated commercially at Beijing Genomics Institute (BGI, Shenzhen, China) while eight 90-nt paired-end RNA-seq libraries were generated at Genewiz Co. Ltd (Suzhou) by using the HiSeq™ platform.Sequencing data was handled essentially with Bowtie2, Tophat2, NOIseq. Expression levels are presented as Reads Per Kilobase of transcript per Million mapped reads (RPKM). Results: Transcriptional profiling of ER stress during cellulase synthesis in N.crassa uncovered a set of 766 genes was identified as the ER stress response target genes (ESRTs). Of these 766 genes, canonical UPR components IRE-1 and HAC-1 regulated 223 and 186 genes, respectively. In screening 527 available ESRT mutants, we found 249 that exhibited DTT sensibility, including 100 targets with unknown function. Disruption of ire-1 or hac-1 renders N. crassa cells seriously deficient in cellulase secretion. Further investigation of the 39 transcription factors (TFs) that showed differential expression under ER stress uncovered three novel TFs (RES-1, RES-2, RRG-2) that markedly affected cellulase secretion. Conclusions: Here, we determined the transcriptional scope of the ER stress response during lignocellulase synthesis in the model cellulolytic fungus N. crassa. Through genome-wide mutant screening and analysis, dozens of novel genes were discovered to be involved in the process. The findings of this work will be useful for strain improvement to facilitate lignocellulase and biomass-based chemical production.
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