Regulation of acetate metabolism in Escherichia coli BL21 by protein Nε-lysine acetylation
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Acetate production is one of the most striking differences between Escherichia coli K12 and BL21 strains. Transcription of acetate metabolism genes is regulated. Additionally, acetyl-CoA synthetase, which activates acetate to acetyl-CoA, is regulated by post-translational acetylation. The aim of this study was to understand the contribution of reversible protein lysine acetylation to the regulation of acetate metabolism in E. coli BL21. The phenotypic differences between both strains were especially important in the presence of acetate. The high expression of acetyl-CoA synthetase (acs) in glucose exponential phase in BL21 allows the simultaneous consumption of acetate and glucose. Lack of catabolite repression also affected its post-translational regulator, the protein acetyltransferase (patZ). The effect of the deletion of cobB (encoding a sirtuin-like protein deacetylase) and patZ genes depended on the genetic background. The deletion of cobB in both strains increased acetate production and decreased growth rate in acetate cultures. The deletion of patZ in BL21 suppressed acetate overflow in glucose medium and increased the growth rate in acetate cultures. Differences on acetate overflow between BL21 and K12 strains are caused by many overlapping factors. Two major contributing effects were identified: (1) the expression of acs during exponential growth is not repressed in the BL21 strain due to concomitant cAMP production and (2) the acetyl-CoA synthetase activity is more tightly regulated by protein acetylation in BL21 than in the K12. Altogether these differences contribute to the lower acetate overflow and the improved ability of E. coli BL21 to consume this metabolite in the presence of glucose.
KeywordsProtein acetylation Bacterial sirtuin Protein acetyltransferase Acetate BL21
We wish to thank José María Pastor (Dept. of Biochemistry and Molecular Biology B and Immunology) for helpful discussions, Marta Abrisqueta, Elena Martín-Orozco and David Cerezo (Dept. of Biochemistry and Molecular Biology B and Immunology, University of Murcia) for their help with western blotting and Professor Kerry Smith (Clemson University, South Carolina) for his assistance with the acetate kinase assay. S. Castaño-Cerezo is a recipient of a Ph.D. fellowship from Fundación Séneca (CARM, Murcia). V. Bernal acknowledges a post-doctoral contract from Universidad de Murcia (Programa Propio). This work has been partly funded by MICINN BIO2011-29233-C02-01 and Fundación Séneca-CARM 08660/PI/08 projects.
Conflict of interest
The authors declare that there are no conflicts of interest.
- Bergmeyer HU, Forster G, Bernt E (1974) Creatine kinase. Methods Enzym. Anal. pp 784–793Google Scholar
- Castaño-Cerezo S, Bernal V, Post H, Fuhrer T, Cappadona S, Sánchez-Díaz NC, Sauer U, Heck AJR, Altelaar AFM, Cánovas M (2014) Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli. Mol Syst Biol 10(11):762Google Scholar
- Crosby HA, Rank KC, Rayment I, Escalante-Semerena JC (2012a) Structural insights into the substrate specificity of the Rhodopseudomonas palustris protein acetyltransferase RpPat: identification of a loop critical for recognition by RpPat. J Biol Chem 287:41392–404. doi: 10.1074/jbc.M112.417360 CrossRefPubMedCentralPubMedGoogle Scholar
- Gardner JG, Escalante-Semerena JC (2009) In Bacillus subtilis, the sirtuin protein deacetylase, encoded by the srtN gene (formerly yhdZ), and functions encoded by the acuABC genes control the activity of acetyl coenzyme A synthetase. J Bacteriol 191:1749–1755CrossRefPubMedCentralPubMedGoogle Scholar
- Gardner JG, Grundy FJ, Henkin TM, Escalante-Semerena JC (2006) Control of acetyl-coenzyme a synthetase (AcsA) activity by acetylation/deacetylation without NAD+ involvement in Bacillus subtilis. J Bacteriol 188:5460–5468Google Scholar
- Jeong H, Barbe V, Lee CH, Vallenet D, Yu DS, Choi S-H, Couloux A, Lee S-W, Yoon SH, Cattolico L, Hur C-G, Park H-S, Ségurens B, Kim SC, Oh TK, Lenski RE, Studier FW, Daegelen P, Kim JF (2009) Genome sequences of Escherichia coli B strains REL606 and BL21(DE3). J Mol Biol 394:644–52. doi: 10.1016/j.jmb.2009.09.052 CrossRefPubMedGoogle Scholar
- Karp PD, Paley SM, Krummenacker M, Latendresse M, Dale JM, Lee TJ, Kaipa P, Gilham F, Spaulding A, Popescu L, Altman T, Paulsen I, Keseler IM, Caspi R (2010) Pathway Tools version 13.0: integrated software for pathway/genome informatics and systems biology. Brief Bioinform 11:40–79. doi: 10.1093/bib/bbp043 CrossRefPubMedCentralPubMedGoogle Scholar
- Keseler IM, Mackie A, Peralta-Gil M, Santos-Zavaleta A, Gama-Castro S, Bonavides-Martínez C, Fulcher C, Huerta AM, Kothari A, Krummenacker M, Latendresse M, Muñiz-Rascado L, Ong Q, Paley S, Schröder I, Shearer AG, Subhraveti P, Travers M, Weerasinghe D, Weiss V, Collado-Vides J, Gunsalus RP, Paulsen I, Karp PD (2013) EcoCyc: fusing model organism databases with systems biology. Nucleic Acids Res 41:D605–12. doi: 10.1093/nar/gks1027 CrossRefPubMedCentralPubMedGoogle Scholar
- Lara AR, Caspeta L, Gosset G, Bolívar F, Ramírez OT (2008) Utility of an Escherichia coli strain engineered in the substrate uptake system for improved culture performance at high glucose and cell concentrations: an alternative to fed-batch cultures. Biotechnol Bioeng 99:893–901. doi: 10.1002/bit.21664 CrossRefPubMedGoogle Scholar
- Lin H, Castro N, Bennett G, San K-Y (2006) Acetyl-CoA synthetase overexpression in Escherichia coli demonstrates more efficient acetate assimilation and lower acetate accumulation: a potential tool in metabolic engineering. Appl Microbiol Biotechnol 71:870–874. doi: 10.1007/s00253-005-0230-4 CrossRefPubMedGoogle Scholar
- Marisch K, Bayer K, Scharl T, Mairhofer J, Krempl PM, Hummel K, Razzazi-Fazeli E, Striedner G (2013) A comparative analysis of industrial Escherichia coli K-12 and B strains in high-glucose batch cultivations on process-, transcriptome- and proteome level. PLoS ONE 8:e70516. doi: 10.1371/journal.pone.0070516 CrossRefPubMedCentralPubMedGoogle Scholar
- Nielsen J (2006) Microbial process kinetics. In Basic biotechnology. Ratledge, C.(ed.). Cambridge: Cambridge University Press, pp. 127-149Google Scholar
- Phue JN, Noronha SB, Hattacharyya R, Wolfe AJ, Shiloach J (2005) Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and northern blot analyses. Biotechnol Bioeng 90:805–820CrossRefPubMedGoogle Scholar
- Renilla S, Bernal V, Fuhrer T, Castaño-Cerezo S, Pastor JM, Iborra JL, Sauer U, Cánovas M (2012) Acetate scavenging activity in Escherichia coli: interplay of acetyl-CoA synthetase and the PEP-glyoxylate cycle in chemostat cultures. Appl Microbiol Biotechnol 95:2109–2124. doi: 10.1007/s00253-011-3536-4 CrossRefGoogle Scholar
- Starai VJ, Escalante-Semerena JC (2004) Acetyl-coenzyme A synthetase (AMP forming). Cell Mol Life Sci 61:2020–2030Google Scholar
- Thao S, Escalante-semerena JC (2011) Biochemical and thermodynamic analyses of Salmonella enterica Pat, a multidomain, multimeric N(ε)-lysine acetyltransferase involved in carbon and energy metabolism. MBio 2:1–8. doi: 10.1128/mBio.00216-11.Editor
- Waegeman H, Beauprez J, Moens H, Maertens J, De Mey M, Foulquie-Moreno M, Heijnen J, Charlier D, Soetaert W (2011) Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K-12 and its implications on understanding the metabolism of Escherichia coli BL21 (DE3). BMC Microbiol 11:70Google Scholar