Dataset Information

Saccharopolyspora erythraea LC-MSMS

ABSTRACT: Lysine acetylation is a dynamic, reversible post-translational modification that is known to play an important role in regulating the activity of many key enzymes in bacteria. Acetylproteome studies have been performed on some bacteria. However, until now there have been no data on the Actinomycetes, which are the major producers of therapeutic antibiotics. In this study, we investigated the first acetylproteome of the erythromycin-producing Actinomycete Saccharopolyspora erythraea using a high-resolution mass spectrometry-based proteomics approach. Using immune-affinity isolation of acetyl-peptides with an anti-acetyllysine antibody followed by nano-UPLC/MS/MS analysis, we identified 664 unique lysine acetylated sites on 363 proteins.

INSTRUMENT(S):

ORGANISM(S): Saccharopolyspora Erythraea (strain Nrrl 23338)

TISSUE(S): Whole Body

SUBMITTER: huang dan

LAB HEAD: Bang-Ce Ye

PROVIDER: PXD001238 | Pride | 2025-11-27

REPOSITORIES: Pride

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Publications

Lysine acetylproteome analysis suggests its roles in primary and secondary metabolism in Saccharopolyspora erythraea.

Huang Dan D Li Zhi-Hai ZH You Di D Zhou Ying Y Ye Bang-Ce BC

Applied microbiology and biotechnology 20141210 3

Lysine acetylation is a dynamic, reversible posttranslational modification that is known to play an important role in regulating the activity of many key enzymes in bacteria. Acetylproteome studies have been performed on some bacteria. However, until now, there have been no data on Actinomycetes, which are the major producers of therapeutic antibiotics. In this study, we investigated the first acetylproteome of the erythromycin-producing actinomycete Saccharopolyspora erythraea using a high-reso ...[more]

PMID: 25487885

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Project description:Lipid intermediates derived from sphingolipid metabolism are crucial regulators of mitochondrial function from yeast to humans. Among these intermediates, trans-2-hexadecenal (t-2hex) within the sphingolipid degradation pathway exhibits remarkable efficiency in inducing mitochondria-mediated cell death. In yeast cell cultures, the addition of t-2-hex triggers complete disintegration of the mitochondrial network, leading to subsequent cell death. This effect is particularly pronounced in yeast cells lacking the activity of the t-2-hex degrading enzyme, Hfd1. However, the molecular mechanisms of t-2-hex induction of mitochondrial dysfunction are completely unknown. In this project, we want to exploit the unprecedented power of yeast genetics to unveil novel genetic determinants involved in t-2-hex's pro-apoptotic function. To accomplish this, we employed the SATAY method, which combines saturated transposon mutagenesis with high-throughput sequencing to functionally explore the yeast genome. In our screening approach, hfd1 mutant cells harboring a plasmid-encoded inducible MiniDs transposon were induced by galactose, resulting in extensive integration of the transposon throughout the yeast genome. Cells with the plasmid excised and the transposon genomically integrated were pooled together, creating a high-density transposon library comprising approximately 2.3E+06 independent insertion mutants. Subsequently, the pooled mutant library was subjected to treatment with the mitochondria-mediated death inducer, t-2-hexadecenal. As a control, cells were also incubated with the solvent dimethyl sulfoxide (DMSO), in which hexadecenal is dissolved. Following the treatments, cells were collected for genomic DNA extraction and digestion, using restriction enzymes with frequent four-base pair recognition sites. The resulting library fragments were circularized using T4 DNA ligase, and the transposon-genome junctions were selectively amplified through PCR with outward-facing primers specific to the transposon. Finally, the pooled and purified amplicons were subjected to massive sequencing on an Illumina MiSeq platform. The obtained sequences were then aligned to the reference genome of Saccharomyces cerevisiae, allowing for the mapping of transposon insertions and the calculation of transposon counts per gene. This project enabled the identification of genes required for the resistance and toxicity to t-2hex.

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Dataset Information

Saccharopolyspora erythraea LC-MSMS

Dataset's files

Publications

Lysine acetylproteome analysis suggests its roles in primary and secondary metabolism in Saccharopolyspora erythraea.

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