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Quantitative Proteomics Reveals UGA-independent Mis-incorporation of Selenocysteine throughout Escherichia coli Proteome

ABSTRACT: LC-MS/MS based shotgun proteomics data of E. coli grew in standard M9 minimal medium, sulfur-depleted medium and selenite medium. We detected selenocysteine mis-incorporation in selenite group and compared its proteome changes with M9 and sulfur-depleted group. Files beginning with letter M, N and S are data sets of M9, sulfur-depleted, and selenite group, respectively. There are 4 biological replicates in each group, and 3 technical replicates in each biological replicate.

INSTRUMENT(S): LTQ Velos

ORGANISM(S): Escherichia Coli

SUBMITTER: Henry Lam Hao Chunlin

PROVIDER: MSV000086241 | MassIVE | Tue Oct 06 08:53:00 BST 2020

REPOSITORIES: MassIVE

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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. Chlamydomonas strains used for this study were D66 (wt strain; nit2 cw15 mt+), ars11 (snrk2.1cw15mt+), 21gr (wt strain; nit5 mt-) and sac1 (sac1mt+). The ars11 strain was designated as the snrk2.1 mutant throughout since the lesion is in the SNRK2.1 gene. Cells were cultured under continuous light of ~60 M-NM-<mol photon m-2s-1 at 23M-BM-:C in liquid and on solid Tris-Acetate-Phosphate (TAP) medium. To impose S deprivation, cells in mid-logarithmic growth phase were washed twice with liquid TAP medium without S (TAP-S), and equal numbers of cells were resuspended in TAP or TAP-S. Cell aliquots were collected for RNA isolation just prior to and 6 h after being transferred to TAP and TAP-S medium. Total RNA from wt (D66) and snrk2.1 mutant cells after 0 and 6 h of being transferred to -S medium were submitted to Illumina for sequencing using their proprietary Genome Analyzer. cDNA libraries were assembled according to the manufacturerM-bM-^@M-^Ys RNA-seq protocol, loaded and sequenced as 35-mers in a total of 11 Solexa lanes. Raw image files were collected by the sequencer and analyzed using the standard Solexa pipeline.

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Quantitative Proteomics Reveals UGA-independent Mis-incorporation of Selenocysteine throughout Escherichia coli Proteome

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