Dataset Information

Biologically active isoforms of CobB sirtuin deacetylase in Salmonella enterica and Erwinia amylovora.

ABSTRACT: Sirtuins are NAD(+)-dependent protein deacylases that are conserved in all domains of life and are involved in diverse cellular processes, including control of gene expression and central metabolism. Eukaryotic sirtuins have N-terminal extensions that have been linked to protein multimerization and cellular localization. Here the first evidence of sirtuin isoforms in bacteria is reported. The enterobacterium Salmonella enterica synthesizes two isoforms of CobB sirtuin, a shorter 236-amino-acid isoform (here CobB(S)) and a longer 273-amino-acid isoform (here CobB(L)). The N-terminal 37-amino-acid extension of CobB(L) is amphipathic, containing 18 basic amino acids (12 of which are Arg) and 13 hydrophobic ones; both isoforms were active in vivo and in vitro. Northern blot and transcription start site analyses revealed that cobB is primarily expressed as two monocistronic cobB mRNAs from two transcription start sites, one of which was mapped within the neighboring ycfX gene and the other of which was located within cobB. Additionally, a low-abundance ycfX-cobB bicistronic mRNA was observed which could encode up to three proteins (YcfX, CobB(L), and CobB(S)). CobB(L) isoforms are common within the family Enterobacteriaceae, but species of the genus Erwinia (including the plant pathogen Erwinia amylovora) encode only the CobB(L) isoform. The CobB(L) isoform from E. amylovora restored growth of as S. enterica cobB mutant strain on low acetate.

SUBMITTER: Tucker AC

PROVIDER: S-EPMC2981217 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Biologically active isoforms of CobB sirtuin deacetylase in Salmonella enterica and Erwinia amylovora.

Tucker Alex C AC Escalante-Semerena Jorge C JC

Journal of bacteriology 20101001 23

Sirtuins are NAD(+)-dependent protein deacylases that are conserved in all domains of life and are involved in diverse cellular processes, including control of gene expression and central metabolism. Eukaryotic sirtuins have N-terminal extensions that have been linked to protein multimerization and cellular localization. Here the first evidence of sirtuin isoforms in bacteria is reported. The enterobacterium Salmonella enterica synthesizes two isoforms of CobB sirtuin, a shorter 236-amino-acid i ...[more]

PMID: 20889757

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Project description:Lysine acetylation is a protein posttranslational modification (PTM) that occurs on thousands of lysine residues in diverse organisms from bacteria to humans. Accurate measurement of acetylation stoichiometry on a proteome-wide scale remains challenging. Most methods employ a comparison of chemically acetylated peptides to native acetylated peptides, however, the potentially large differences in abundance between these peptides presents a challenge for accurate quantification. Stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry (MS) is one of the most widely used quantitative proteomic methods. Here we show that serial dilution of SILAC-labeled peptides (SD-SILAC) can be used to identify accurately quantified peptides and to estimate the quantification error rate. We applied SD-SILAC to determine absolute acetylation stoichiometry in exponentially-growing and stationary-phase wild-type and Sirtuin deacetylase CobB-deficient cells. To further analyze CobB-regulated sites under conditions of globally increased or decreased acetylation, we measured stoichiometry in phophotransacetylase (pta?) and acetate kinase (ackA?) mutant strains in the presence and absence of the Sirtuin inhibitor nicotinamide. We measured acetylation stoichiometry at 3,669 unique sites and found that the vast majority of acetylation occurred at a low stoichiometry. Manipulations that cause increased nonenzymatic acetylation by acetyl-phosphate (AcP), such as stationary-phase arrest and deletion of ackA, resulted in globally increased acetylation stoichiometry. Comparison to relative quantification under the same conditions validated our stoichiometry estimates at hundreds of sites, demonstrating the accuracy of our method. Similar to Sirtuin deacetylase 3 (SIRT3) in mitochondria, CobB suppressed acetylation to lower than median stoichiometry in WT, pta?, and ackA? cells. Together, our results provide a detailed view of acetylation stoichiometry in E. coli and suggest an evolutionarily conserved function of Sirtuin deacetylases in suppressing low stoichiometry acetylation.

Dataset Information

Biologically active isoforms of CobB sirtuin deacetylase in Salmonella enterica and Erwinia amylovora.

Publications

Biologically active isoforms of CobB sirtuin deacetylase in Salmonella enterica and Erwinia amylovora.

Similar Datasets

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