1887

Abstract

Proteins secreted by may play a key role in virulence and may also constitute antigens that elicit the host immune response. However, the protein export machinery has not been characterized. A library of H37Rv genomic DNA fragments ligated into a signal sequence selection vector that contained a leaderless β-lactamase gene and an upstream Tac promoter was constructed. Transformation of with the DNA library and selection on plates containing 50-100 μg ampicillin ml resulted in the identification of 15 Amp clones out of a total of 14000 transformants. Twelve of the β-lactamase gene fusions conferred high levels of Amp (up to 1 mg ampicillin ml); insert sizes ranged from 350 to 3000 bp. Of ten inserts that were completely sequenced, two were identified as fragments of the genes for antigens 85A and 85C, which are the major secreted proteins of this pathogen. Seven of the remaining inserts were ≥97% identical to hypothetical ORFs in the genome, one of which encoded a protein with 35% identity to a low-affinity penicillin-binding protein (PBP) from . 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 β-lactamase gene fusions in provides a useful system for the identification and analysis of signal-sequence-encoding genes.

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1998-06-01
2021-05-06
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