AN EXPORT-SPECIFIC REPORTER DESIGNED FOR GRAM-POSITIVE BACTERIA - APPLICATION TO LACTOCOCCUS-LACTIS

The identification of exported proteins by fusion studies, while well developed for gram-negative bacteria, is limited for gram-positive bac teria, in part due to drawbacks of available export reporters. In this work, we demonstrate the export specificity and use of the Staphyloco ccus aureus secreted nuclease (Nuc) as a reporter for gram-positive ba cteria. Nuc devoid of its export signal (called Delta(SP)Nuc) was used to create two fusions whose locations could be differentiated. Nuclea se activity was shown to require an extracellular location in Lactococ cus lactis, thus demonstrating the suitability of Delta(SP)Nuc to repo rt protein export. The shuttle vector pFUN was designed to construct D elta(SP)Nuc translational fusions whose expression signals are provide d by inserted DNA. The capacity of Delta(SP)Nuc to reveal and identify exported proteins was tested by generating an L. lactis genomic libra ry in pFUN and by screening for Nuc activity directly in L. lactis. Al l 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 signal s was confirmed by cell fractionation studies. The fusions analyzed in cluded long (up to 455-amino-acid) segments of the exported proteins, all previously unknown in L. lactis. Homology searches indicate that s everal of them may be implicated in different cell surface functions, such as nutrient uptake, peptidoglycan assembly, environmental sensing , and protein folding. Our results with L. lactis show that Delta(SP)N uc is well suited to report both protein export and membrane protein t opology.