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

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Genetics

Background:

• Identifying exported proteins in gram-positive bacteria is challenging due to limitations of existing reporter systems.
• Existing methods for studying protein export are more developed for gram-negative bacteria compared to gram-positive counterparts.

Purpose of the Study:

• To establish and validate the use of Staphylococcus aureus secreted nuclease (Nuc) as a reporter for protein export in gram-positive bacteria.
• To develop a novel shuttle vector (pFUN) for constructing translational fusions with the Nuc reporter.
• To identify novel exported proteins and understand their cellular functions in Lactococcus lactis.

Main Methods:

• Created fusions using Nuc devoid of its export signal (delta(SP)Nuc) to differentiate protein locations.
• Demonstrated extracellular nuclease activity requirement in Lactococcus lactis to confirm reporter suitability.
• Constructed a Lactococcus lactis genomic library in the pFUN shuttle vector for screening Nuc activity.

Main Results:

• delta(SP)Nuc successfully reported protein export and membrane protein topology in Lactococcus lactis.
• Screening identified delta(SP)Nuc fusions containing signal peptides or transmembrane stretches, indicating export or membrane localization.
• Analysis revealed novel, potentially cell surface-associated proteins in Lactococcus lactis involved in nutrient uptake, sensing, and assembly.

Conclusions:

• The delta(SP)Nuc reporter system is effective for studying protein export in gram-positive bacteria like Lactococcus lactis.
• The pFUN vector facilitates the construction and screening of protein fusions for export and topology studies.
• This approach has identified previously unknown proteins with potential roles in diverse bacterial cell surface functions.