title: Expression and efficient export of enzymatically active Mycobacterium tuberculosis glutamine synthetase in Mycobacterium smegmatis and evidence that the information for export is contained within the protein.
abstract: We have investigated the expression and extracellular release of active, recombinant Mycobacterium tuberculosis glutamine synthetase (EC 6.3.1.2), an enzyme that is a potentially important determinant of M. tuberculosis infection and whose extracellular release is correlated with pathogenicity. The M. tuberculosis glutamine synthetase gene encodes a polypeptide of 478 amino acids; 12 such subunits comprise the active enzyme. Northern blot, nuclease S1, and primer extension analyses revealed glutamine synthetase specific transcripts of approximately 1,550 and 1,650 nucleotides produced under low and high nitrogen conditions, respectively. Expression of recombinant M. tuberculosis glutamine synthetase in Escherichia coli YMC21E, a glutamine synthetase deletion mutant, led to transcomplementation of the mutant but not to release of active enzyme. Expression in Mycobacterium smegmatis 1-2c, from the gene's own promoter, resulted in the release of >95% of all recombinant enzyme. No hybrid molecules containing M. tuberculosis and M. smegmatis glutamine synthetase subunits were detected. Native and recombinant exported and intracellular glutamine synthetase molecules were indistinguishable from one another by mass, N-terminal amino acid sequence, antibody reactivity, and enzymatic activity. Since M. tuberculosis glutamine synthetase is similar to other, strictly intracellular, bacterial glutamine synthetases and the DNA sequence upstream of the structural gene does not encode a leader peptide, the information to target the protein for export must be contained in its amino acid sequence and/or conformation.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, 37-121 CHS, School of Medicine, UCLA, Los Angeles, California 90095, USA.
authors.forename: G
authors.initials: G
authors.lastname: Harth
authors.forename: M A
authors.initials: MA
authors.lastname: Horwitz
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D005974:Glutamate-Ammonia Ligase