DNA adenine hypomethylation leads to metabolic rewiring in Deinococcus radiodurans Nayana S. Shaiwale a,b , Bhakti Basu a , Deepti D. Deobagkar b , Dileep N. Deobagkar b , Shree K. Apte a, ⁎ a Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India b Department of Zoology, Centre for Advanced Studies, University of Pune, Pune 411007, India abstract article info Article history: Received 16 February 2015 Received in revised form 22 May 2015 Accepted 30 May 2015 Available online 3 June 2015 Keywords: Deinococcus radiodurans DNA adenine methylation Adenine methyltransferase Metabolic rewiring Pyruvate dehydrogenase The protein encoded by DR_0643 gene from Deinococcus radiodurans was shown to be an active N-6 adenine- specific DNA methyltransferase (Dam). Deletion of corresponding protein reduced adenine methylation in the genome by 60% and resulted in slow-growth phenotype. Proteomic changes induced by DNA adenine hypome- thylation were mapped by two-dimensional protein electrophoresis coupled with mass spectrometry. As com- pared to wild type D. radiodurans cells, at least 54 proteins were differentially expressed in Δdam mutant. Among these, 39 metabolic enzymes were differentially expressed in Δdam mutant. The most prominent change was DNA adenine hypomethylation induced de-repression of pyruvate dehydrogenase complex, E1 component (aceE) gene resulting in 10 fold increase in the abundance of corresponding protein. The observed differential ex- pression profile of metabolic enzymes included increased abundance of enzymes involved in fatty acid and amino acid degradation to replenish acetyl Co-A and TCA cycle intermediates and diversion of phosphoenolpyruvate and pyruvate into amino acid biosynthesis, a metabolic rewiring attempt by Δdam mutant to restore energy gen- eration via glycolysis–TCA cycle axis. This is the first report of DNA adenine hypomethylation mediated rewiring of metabolic pathways in prokaryotes. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Post-replicative epigenetic modification of DNA is a dynamic signal universally used by both eukaryotes and prokaryotes to regulate gene expression [1,2]. This covalent modification occurs at the C-5 or N-4 po- sitions of cytosine or at the N-6 position of adenine and is catalyzed by enzymes known as DNA methyltransferases that use S-adenosyl methi- onine (SAM) as a methyl donor. The methylated adenine or cytosine mediated epigenetic modifications lead to gene expression or silencing, in both eukaryotes and prokaryotes [2,3]. In bacteria, DNA methyltrans- ferases associated with cognate endonucleases constitute a host- specific restriction/modification system that identifies and digests for- eign DNA based on methylation pattern [3]. At least two DNA adenine methyltransferases in bacteria, Dam and CcrM, lack a cognate restriction enzyme [2,3]. Constitutively active Dam methylates 5′-GATC-3′ DNA motif while CcrM methylates adenine in 5′-GANTC-3′ motif and its ac- tivity is tightly regulated during the cell cycle [2–4]. Although Dam me- diated methylation is dispensable in Escherichia and Salmonella [5,6], it is essential for the viability of Vibrio, Yersinia and Brucella [7,8]. Modulations in the DNA methylation status of cells lead to aberrant gene expression resulting in pleiotropic phenotypic effects. Hypermethylation or hypomethylation in eukaryotes sets in metabolic reprogramming, the famous ‘Warburg effect’, that has been claimed to aid in progression of cancer [1,9]. In prokaryotes, a dam mu- tant of Escherichia coli displayed upregulation of genes involved in aer- obic respiration, stress and SOS responses, amino acid metabolism and nucleotide metabolism while the genes involved in anaerobic respiration, flagella biosynthesis, chemotaxis and motility were down regulated [10,11]. Phenotypically, it resulted in increased mutation frequency, hyper-recombination and increased sensitivity to DNA- damaging agents [5]. Dam methylation also plays a crucial role in the virulence of several pathogenic organisms and deletion of dam gene results in loss of pathogenicity in Salmonella typhimurium, Yersinia pseudotuberculosis, Vibrio cholerae and Brucella abortus [7,8,12]. The Gram positive, non-pathogenic extremophile Deinococcus radiodurans R1 is well known for its extreme resistance to all DNA dam- aging agents owing to a rapid, efficient and accurate DNA repair capabil- ity [13,14]. The organism employs an inducible DNA repair mechanism that combines both prokaryotic type homologous recombination (HR) as well as eukaryotic type strand annealing (SA) and possibly non- homologous end joining (NHEJ) [14,15]. D. radiodurans also possesses a functional mismatch repair (MMR) system, comprising of MutS and MutL ATPases along with endonuclease VII (XseA) and UvrD helicase that ensures fidelity of replication and recombination [16]. Adenine methylated parent DNA strand is a prerequisite for correct recognition of unmethylated daughter strand which is then nicked and repaired by MMR [17]. Adenine residues in the genome of D. radiodurans are Journal of Proteomics 126 (2015) 131–139 ⁎ Corresponding author. E-mail addresses: aptesk@barc.gov.in, sksmbd@barc.gov.in (S.K. Apte). http://dx.doi.org/10.1016/j.jprot.2015.05.036 1874-3919/© 2015 Elsevier B.V. All rights reserved. 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