Abstract
It is shown here that a plasmid (p29) derived from the transducing phage λaspC2 (Christiansen and Pedersen 1981) codes for pyruvate formate-lyase. The identity of the 80 kilodaltons (kd) gene product of plasmid p29 with the pyruvate formate-lyase polypeptide was proven (i) by comigration of the gene product expressed in the maxicell system with purified enzyme on O'Farrell gels, and (ii) by comparison of the peptide maps obtained from limited proteolysis. In vivo the 80 kd form of the enzyme was proteolytically converted to a 78 kd polypeptide. The two polypeptides (80 kd and 78 kd) and their charge isomers present in purified enzyme preparations are therefore products of a single gene.
Aerobically grown cells of Escherichia coli contained a basal level of pyruvate formate-lyase which was derepressed 5-to 10-fold under anaerobiosis. Derepression also occurred during anaerobic growth on glycerol plus fumarate. Presence of plasmid p29 caused overproduction of pyruvate formatelyase, 11-fold upon anaerobic growth on glucose, 14-fold upon aerobic growth on glucose and 33-fold upon aerobic growth at the expense of D-lactate.
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Abbreviations
- MOPS:
-
4-morpholine-propane sulfonic
References
Bloch PL, Philips TA, Neidhardt FC (1980) Protein identification on O'Farrell two-dimensional gels: locations of 81 Escherichia coli proteins. J Bacteriol 141:1409–1420
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Christiansen L, Pedersen S (1981) Cloning, restriction endonuclease mapping and post-transcriptional regulation of rpsA, the structural gene for ribosomal protein S1. Mol Gen Genet 181:548–551
Cleveland DW, Fischer SG, Kirschner MW, Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 252:1102–1106
Clewell DB, Helsinki DR (1969) Supercoiled DNA protein complex in Escherichia coli: Purification and induced conversion to an open circular DNA form. Proc Natl Acad Sci USA 62:1159–1166
Cohen SN, Chang ACY, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of E. coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114
Cole ST, Guest JR (1979) Amplification and aerobic synthesis of fumarate reductase in ampicillin-resistant mutants of Escherichia coli K-12. FEMB Microbiol Letters 5:65–67
Cole ST, Guest JR (1980) Amplification of fumarate reductase synthesis with λfrdA transducing phages and orientation of frdA gene expression. Mol Gen Genet 179:377–385
Fröhler J, Rechenmacher A, Thomale J, Nass G, Böck A (1980) Genetic analysis of mutations causing borrelidin resistance by overproduction of threonyl-transfer ribonucleic acid synthetase. J Bacteriol 143:1135–1141
Geyl D, Böck A (1977) Synthesis of ribosomal proteins in merodiploid strains and in minicells of Escherichia coli. Mol Gen Genet 154:327–334
Henning U (1963) Ein Regulationsmechanismus beim Abbau der Brenztraubensäure durch Escherichia coli. Biochem Z 337:490–504
Hom SSM, Hennecke H, Shanmugam KT (1980) Regulation of nitrogenase biosynthesis in Klebsiella pneumoniae: effect of nitrate. J Gen Microbiol 117:169–169
Knappe J, Schacht J, Möckel W, Höpner Th, Vetter H, Edenharder R (1969) Pyruvate formate-lyase reaction in Escherichia coli. The enzymatic system converting an inactive form of the lyase into the catalytically active enzyme. Eur J Biochem 11:316–327
Knappe J, Blaschkowski HP, Edenharder R (1972) Enzyme-dependent activation of pyruvate formate-lyase of Escherichia coli. In: Wieland O, Helmreich E, Holzer H (eds) Metabolic interconversion of enzymes. Springer, Berlin Heidelberg New York, pp 319–329
Knappe J, Blaschkowski HP, Gröbner P, Schmitt T (1974) Pyruvate formate-lyase of Escherichia coli: the acetyl enzyme intermediate. Eur J Biochem 50:253–263
Knappe J, Blaschkowski HP (1975) Pyruvate formate-lysase from Escherichia coli and its activation system. In: Wood WA (ed) Methods in Enzymology 41. Academic Press, New York San Francisco London, pp 508–518
Knappe J, Schmitt T (1976) A novel reaction of S-adenosyl-L-methionine correlated with the activation of pyruvate formate-lyase. Biochem Biophys Res Commun 71:1110–1117
Knappe J (1978) Pyruvate formate-lyase, mechanism and regulation by interconversion. Hoppe-Seyler's Z Physiol Chem 359:286
Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor
Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119:736–747
O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021
Philips TA, Bloch PL, Neidhardt FC (1980) Protein identifications on O'Farrell two-dimensional gels: Locations of 55 additional Escherichia coli proteins. J Bacteriol 144:1024–1033
Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693
Schreyer R, Böck A (1980) Phosphoglucose isomerase from Escherichia coli K10: Purification, properties and formation under aerobic and anaerobic condition. Arch Microbiol 127:289–298
Takahashi S, Abbe K, Yamada T (1982) Purification of pyruvate formate-lyase from Streptococcus mutans and its regulatory properties. J Bacteriol 149:1034–1040
Varenne S, Casse F, Chippaux M, Pascal MC (1975) A mutant of Escherichia coli deficient in pyruvate formate-lyase. Mol Gen Genet 141:181–184
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Pecher, A., Blaschkowski, H.P., Knappe, K. et al. Expression of pyruvate formate-lyase of Escherichia coli from the cloned structural gene. Arch. Microbiol. 132, 365–371 (1982). https://doi.org/10.1007/BF00413390
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DOI: https://doi.org/10.1007/BF00413390