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Deacetylation of topoisomerase I is an important physiological function of E. coli CobB.

ABSTRACT: Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The NAD+-dependent protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modification in E. coli. Here, we show that the absence of CobB in a ΔcobB mutant reduces intracellular TopA catalytic activity and increases negative DNA supercoiling. TopA expression level is elevated as topA transcription responds to the increased negative supercoiling. The slow growth phenotype of the ΔcobB mutant can be partially compensated by further increase of intracellular TopA level via overexpression of recombinant TopA. The relaxation activity of purified TopA is decreased by in vitro non-enzymatic acetyl phosphate mediated lysine acetylation, and the presence of purified CobB protects TopA from inactivation by such non-enzymatic acetylation. The specific activity of TopA expressed from His-tagged fusion construct in the chromosome is inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. These findings demonstrate that E. coli TopA catalytic activity can be modulated by lysine acetylation-deacetylation, and prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the CobB protein deacetylase.

SUBMITTER: Zhou Q

PROVIDER: S-EPMC5605244 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Deacetylation of topoisomerase I is an important physiological function of E. coli CobB.

Zhou Qingxuan Q Zhou Yan Ning YN Jin Ding Jun DJ Tse-Dinh Yuk-Ching YC

Nucleic acids research 20170501 9

Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The NAD+-dependent protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modification in E. coli. Here, we show that the absence of CobB in a ΔcobB mutant reduces intracellular TopA catalytic activity and increases negative DNA supercoiling. TopA expression level is elevated as topA transcription responds to the increased negative sup ...[more]

PMID: 28398568

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Project description:Acetylation of lysine residues is conserved in all three kingdoms; however, its role in prokaryotes is unknown. Here we demonstrate that acetylation enables the reference bacterium Escherichia coli to withstand environmental stress. Specifically, the bacterium reaches higher cell densities and becomes more resistant to heat and oxidative stress when its proteins are acetylated, as shown by deletion of the gene encoding acetyltransferase YfiQ and the gene encoding deacetylase CobB, as well as by overproducing YfiQ and CobB. Furthermore, we show that the increase in oxidative stress resistance with acetylation is due to the induction of catalase activity through enhanced katG expression. We also found that two-component system proteins CpxA, PhoP, UvrY, and BasR are associated with cell catalase activity and may be responsible as the connection between bacterial acetylation and the stress response. This is the first demonstration of a specific environmental role of acetylation in prokaryotes. Overnight cultures of cobB/pCA24N-cobB and cobB/pCA24N were cultured to a turbidity of 0.05 at 600 nm and grown 2 h. Then, 0.1 mM IPTG was added for another 4 h to induce cobB expression, and then the cells were exposed to 20 mM H2O2 for 10 min. Cell pellets were collected and resuspended in RNAlater (Ambion Inc., Austin, TX), and total RNA was isolated using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA). The E. coli GeneChip Genome 2.0 array (Affymetrix, P/N 900551) was used, and cDNA synthesis, fragmentation, and hybridizations were performed as described previously. If the gene with the larger transcription rate did not have a consistent transcription rate based on the 11-15 probe pairs (P-value less than 0.05), these genes were discarded. A gene was considered differentially expressed when the P-value for comparing two chips was lower than 0.05 (to assure that the change in gene expression was statistically significant and that false positives arise less than 5%) and if their fold change is higher than the standard deviation for the whole genome.

Dataset Information

Deacetylation of topoisomerase I is an important physiological function of E. coli CobB.

Publications

Deacetylation of topoisomerase I is an important physiological function of E. coli CobB.

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