Identification of Mycobacterium tuberculosis signal sequences that direct the export of a leaderless beta-lactamase gene product in Escherichia coli.
Proteins secreted by Mycobacterium tuberculosis may play a key role in virulence and may also constitute antigens that elicit the host immune response. However, the M. tuberculosis protein export machinery has not been characterized. A library of M. tuberculosis H37Rv genomic DNA fragments ligated into a signal sequence selection vector that contained a leaderless beta-lactamase gene and an upstream Tac promoter was constructed. Transformation of Escherichia coli with the M. tuberculosis DNA library and selection on plates containing 50-100 micrograms ampicillin ml-1 resulted in the identification of 15 Ampr clones out of a total of 14,000 transformants. Twelve of the beta-lactamase gene fusions conferred high levels of Ampr (up to 1 mg ampicillin ml-1); insert sizes ranged from 350 to 3000 bp. Of ten inserts that were completely sequenced, two were identified as fragments of the genes for M. tuberculosis antigens 85A and 85C, which are the major secreted proteins of this pathogen. Seven of the remaining inserts were > or = 97% identical to hypothetical ORFs in the M. tuberculosis genome, one of which encoded a protein with 35% identity to a low-affinity penicillin-binding protein (PBP) from Streptomyces clavuligerus. Four of the seven hypothetical ORFs encoded putative exported proteins with one or more membrane interaction elements, including lipoprotein attachment sites and type I and II transmembrane (TM) segments. All of the inserts encoded typical signal sequences, with the exception of a possible type II membrane protein. It is concluded that expression of beta-lactamase gene fusions in E. coli provides a useful system for the identification and analysis of M. tuberculosis signal-sequence-encoding genes.
Chubb
Anthony J
AJ
Department of Medical Biochemistry, University of Cape Town Medical School, Observatory 7925, South Africa.
Woodman
Zenda L
ZL
Department of Medical Biochemistry, University of Cape Town Medical School, Observatory 7925, South Africa.
da Silva Tatley
Fernanda M P R
FMPR
Department of Medical Biochemistry, University of Cape Town Medical School, Observatory 7925, South Africa.
Hoffmann
Hans Jürgen
HJ
Department of Medical Biochemistry, University of Cape Town Medical School, Observatory 7925, South Africa.
Scholle
Renate R
RR
Department of Medical Biochemistry, University of Cape Town Medical School, Observatory 7925, South Africa.
Ehlers
Mario R W
MRW
Department of Medical Biochemistry, University of Cape Town Medical School, Observatory 7925, South Africa.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000235:genetics*; D016415:Sequence Alignment; D014169:Transformation, Bacterial; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D001618:beta-Lactamases