Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase and acetate kinase

• Published in: Microbiology (Society for General Microbiology) Vol. 145; no. 2; pp. 503 - 513
• Main Authors: Reinscheid, Dieter J, Schnicke, Stephanie, Rittmann, Doris, Zahnow, Ulrike, Sahm, Hermann, Eikmanns, Bernhard J
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.02.1999; Society for General Microbiology
• Subjects: Acetate Kinase - chemistry; Acetate Kinase - genetics; Acetate Kinase - metabolism; Acetates - metabolism; Amino Acid Sequence; Bacteriology; Base Sequence; Biological and medical sciences; Blotting, Northern; Cloning, Molecular; Corynebacterium - enzymology; Corynebacterium - genetics; Corynebacterium - growth & development; Corynebacterium glutamicum; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genetics; Glucose - metabolism; Microbiology; Molecular Sequence Data; Operon; Phosphate Acetyltransferase - chemistry; Phosphate Acetyltransferase - genetics; Phosphate Acetyltransferase - metabolism; Restriction Mapping; Sequence Alignment; Sequence Analysis, DNA; Transcription, Genetic; Acyltransferases; Phosphate acetyltransferase; Nucleotide sequence; Transcription; Enzyme; Operon; Transferases; Homology; Gene organization; Acetate; Gene expression; Metabolism; Regulation(control); Enzymatic activity; Corynebacterium glutamicum; Bacteria; Actinomycetes; Corynebacteriaceae; Acetate kinase; Aminoacid sequence
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/13500872-145-2-503

Abteilung Angewandte Mikrobiologie, Universität Ulm, D-89069 Ulm, Germany Institut für Biotechnologie, Forschungszentrum Jülich, D-52425 Jülich, Germany Author for correspondence: Bernhard J. Eikmanns. Tel: +49 731 50 22707. Fax: +49 731 50 22719. e-mail: bernhard.eikmanns@biologie.uni-ulm.de ABSTRACT Summary: The Corynebacterium glutamicum ack and pta genes encoding the acetate-activating enzymes acetate kinase and phosphotransacetylase were isolated, subcloned on a plasmid and re-introduced into Corynebacterium glutamicum. Relative to the wild-type, the recombinant strains showed about tenfold higher specific activities of both enzymes. Sequence analysis of a 3657 bp DNA fragment revealed that the ack and pta genes are contiguous in the corynebacterial chromosome, with pta upstream and the last nucleotide of the pta stop codon (TAA) overlapping the first of the ack start codon (ATG). The predicted gene product of pta consists of 329 amino acids ( M r 35242), that of ack consists of 397 amino acids ( M r 43098) and the amino acid sequences of the two polypeptides show up to 60% (phosphotransacetylase) and 53% (acetate kinase) identity in comparison with respective enzymes from other organisms. Northern (RNA) blot hybridizations using pta- and ack -specific probes and transcriptional cat fusion experiments revealed that the two genes are transcribed as a 2·5 kb bicistronic mRNA and that the expression of this operon is induced when Corynebacterium glutamicum grows on acetate instead of glucose as a carbon source. Directed inactivation of the chromosomal pta and ack genes led to the absence of detectable phosphotransacetylase and acetate kinase activity in the respective mutants and to their inability to grow on acetate. These data indicate that no isoenzymes of acetate kinase and phosphotransacetylase are present in Corynebacterium glutamicum and that a functional acetate kinase/phosphotransacetylase pathway is essential for growth of this organism on acetate. Keywords: Corynebacterium glutamicum , acetate metabolism, acetate kinase, phosphotransacetylase, pta-ack operon The EMBL/GenBank/DDBJ accession number for the sequence reported in this paper is X89084 .