Expression and Efficient Export of Enzymatically ActiveMycobacterium tuberculosis Glutamine Synthetase in Mycobacterium smegmatis and Evidence That the Information for Export is Contained within the Protein

Günter Harth,Marcus A Horwitz

doi:10.1074/jbc.272.36.22728

Günter Harth, Marcus A Horwitz

Open Access

https://doi.org/10.1074/jbc.272.36.22728

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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.

Highlights

Mycobacterium tuberculosis, a facultative intracellular parasite, is one of the world’s most important pathogens, with new cases of pulmonary tuberculosis and deaths numbering many million annually (1)
Glutamine synthetase may influence the ammonia level within the phagosome containing the pathogen in host cells, profoundly altering physiological conditions in the phagosome, and the enzyme may be directly involved in the synthesis of poly-L-glutamic acid/glutamine found in abundance in the cell wall of pathogenic but not nonpathogenic mycobacteria (3)
In M. tuberculosis, the glnE gene encoding adenylyl transferase is located immediately downstream of the glutamine synthetase gene, whereas in E. coli and S. typhimurium, it is located far removed from the glnA locus

Summary

Introduction

Mycobacterium tuberculosis, a facultative intracellular parasite, is one of the world’s most important pathogens, with new cases of pulmonary tuberculosis and deaths numbering many million annually (1). E. coli YMC21E mutants that were stably complemented in trans by M. tuberculosis glutamine synthetase were grown in broth culture, and recombinant pKK233–2 plasmids were isolated and sequenced across the vector/insert junctions with glutamine synthetase gene-specific primers.

Results

Conclusion