Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.
The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria. Introduction of the Mycobacterium fortuitum beta-lactamase gene (blaF*) into this vector led to the production in M. smegmatis of protein fusions with PhoA activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Sequences of the M. tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined. They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M. leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences. This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.
Lim
E M
EM
Unité de Génétique Mycobactérienne, Centre National de la Recherche Scientifique, URA 1300, Institut Pasteur, Paris, France.
Rauzier
J
J
Timm
J
J
Torrea
G
G
Murray
A
A
Gicquel
B
B
Portnoi
D
D
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D004269:DNA, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D015698:Genomic Library; D006899:Mixed Function Oxygenases / Q000235:genetics; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology / Q000235:genetics; D009166:Mycobacterium leprae / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics
C087864:19 kDa antigen, Mycobacterium; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D006899:Mixed Function Oxygenases; C018747:acyl-(acyl-carrier-protein)desaturase; D000469:Alkaline Phosphatase; D001618:beta-Lactamases