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Lysine Acetylome Profiling Reveals Diverse Functions of Acetylation in Deinococcus Radiodurans

ABSTRACT: D. radiodurans is distinguished by the most radioresistant organism identified to date. Lysine acetylation is a highly conserved post-translational modification that plays an essential role in the regulation of many cellular processes and may contribute to its extraordinary radioresistance. We integrate acetyl-lysine enrichment strategy, high-resolution mass spectrometry, and bioinformatics to profile the lysine acetylated proteins for the first time. It is striking that almost half of the total annotated proteins are identified as acetylated forms, which is the largest acetylome dataset reported in D. radiodurans to date. The acetylated proteins are involved in metabolic pathways, propanoate metabolism, carbon metabolism, fatty acid metabolism, and the tricarboxylic acid cycle. The results of this study reinforce the notion that acetylation plays critical regulatory roles in diverse aspects of the cellular process, especially in DNA damage repair and metabolism. It provides insight into the roles of lysine acetylation in the robust resistance to radiation.

INSTRUMENT(S): ultraflex, Q Exactive HF

ORGANISM(S): Deinococcus Radiodurans R1

SUBMITTER: Yongqian Zhang

LAB HEAD: Yongqian Zhang

PROVIDER: PXD035309 | Pride | 2022-08-09

REPOSITORIES: Pride

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Project description:This bacterial genome encodes pyrroloquinoline quinone (PQQ) synthase enzyme required for the synthesis of PQQ in bacteria. The gene pqqE encoding PQQ synthase was annotated in Deinococcus radiodurans R1 genome and synthesis of PQQ was shown in this bacterium. PQQ roles as an antioxidant and as an inducer of protein kinase in bacterial system was shown. The pqqE disuption mutant showed several folds decrease in gamma radiation tolerance of wild type cells and strong impairment of DSB repair. Homology search analysis of PQQ binding domains showed the presence of multiple PQQ binding domains in proteins encoded by five uncharacterized ORFs of this bacterial genome. Many of these proteins are having STK domain of proteins kinases. The effect of pqqE mutation on gene expression under normal and post irradiation conditions was monitored by transcriptome analysis of mutant and compared with wild type cells. All the genes subjected to microarray analysis are as described in the genome sequence of bacterium available at ftp://ftp.ncbi.nih.gov/genomes/bacteria/Deinococcus_radiodurans. Oligonucleotides designed to represent the respective predicted ORFs of Deinococcus radiodurans ((NC_001263, NC_001264, NC_000958, NC_000959) are synthesized and purified by hydrophobic column on HPLC. The oligonucleotides are deposited on to poly-L-Lysine slide in triplicate. Microarrays were scanned using Gene Pix 4000B and hybridization signals were quantified using GenePix Pro 5.1. Prior to channel normalization, microarray outputs were filtered to remove spots of poor signal quality by excluding those data points with a mean intensity by less that 2 standard deviation above background in both channels. Normalization of data and its statistical analysis were carried out in the R computing environment using linear models for microarray data package (Limma). Using this tool, the global LOESS normalization was carried out for each microarray. The 3- replicate spots per gene in each array were used to maximize the robustness of differential expression measurements of each gene via the âlmFitâ function within Limma. Two biological replicates were carried out for transcriptome analysis of pqqE mutant strain against wild type cells.

Project description:Abstract Background The extraordinarily resistant bacterium Deinococcus radiodurans withstands harsh environmental conditions present in outer space. Deinococcus radiodurans was exposed for one year outside the International Space Station within Tanpopo orbital mission to investigate microbial survival and space travel. In addition, a ground-based simulation experiment with conditions, mirroring those from Low Earth orbit, was performed. Methods We monitored Deinococcus radiodurans cells during early stage of recovery after Low Earth orbit exposure using electron microscopy tools. Furthermore, proteomic, transcriptomic and metabolomic analyses were performed to identify molecular mechanisms responsible for the survival of Deinococcus radiodurans in Low Earth orbit. Results D. radiodurans cells exposed to low Earth orbit conditions do not exhibit any morphological damage. However, an accumulation of numerous outer-membrane associated vesicles was observed. On levels of proteins and transcripts, a multifaceted response was detected to alleviate cell stress. The UvrABC endonuclease excision repair mechanism was triggered to cope with DNA damage. Defense against reactive oxygen species is mirrored by the increased abundance of catalases and is accompanied by the increased abundance of putrescine which works as scavenging molecule. In addition, several proteins and mRNAs, responsible for regulatory and transporting functions showed increased abundances. The decrease in primary metabolites indicate alternations in the energy status, which is needed to repair damaged molecules. Conclusion Low Earth orbit induced molecular rearrangements trigger multiple components of metabolic stress response and regulatory networks in exposed microbial cells. Presented results show that the non-sporulating bacterium Deinococcus radiodurans survived long-term Low Earth orbit exposure if wavelength below 200 nm are not present, which mirrors the UV spectrum of Mars, where CO2 effectively provides a shield below 190 nm. These results should be considered in the context of planetary protection concerns and the development of new sterilization techniques for future space missions.

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Lysine Acetylome Profiling Reveals Diverse Functions of Acetylation in Deinococcus Radiodurans

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