PMID: 9537391Apr 16, 1998Paper

An export-specific reporter designed for gram-positive bacteria: application to Lactococcus lactis

Journal of Bacteriology
I PoquetA Gruss

Abstract

The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bacteria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of the Staphylococcus aureus secreted nuclease (Nuc) as a reporter for gram-positive bacteria. Nuc devoid of its export signal (called delta(SP)Nuc) was used to create two fusions whose locations could be differentiated. Nuclease activity was shown to require an extracellular location in Lactococcus lactis, thus demonstrating the suitability of delta(SP)Nuc to report protein export. The shuttle vector pFUN was designed to construct delta(SP)Nuc translational fusions whose expression signals are provided by inserted DNA. The capacity of delta(SP)Nuc to reveal and identify exported proteins was tested by generating an L. lactis genomic library in pFUN and by screening for Nuc activity directly in L. lactis. All delta(SP)Nuc fusions displaying a strong Nuc+ phenotype contained a classical or a lipoprotein-type signal peptide or single or multiple transmembrane stretches. The function of some of the predicted signals was confirmed by cell fractionation studies. The fusions ...Continue Reading

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