Single-Stranded DNA Uptake during Gonococcal Transformation

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Bacterial transformation facilitates genetic exchange, accelerating adaptation and DNA repair.
• DNA uptake through the outer membrane is the initial step in transformation for Gram-negative bacteria.
• This process is mediated by a powerful molecular machine with an incompletely understood mechanism.

Purpose of the Study:

• To investigate whether the DNA uptake machine in Neisseria gonorrhoeae imports single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) at comparable rates.
• To determine the role of the DNA uptake sequence (DUS) structure in DNA recognition and transport.
• To elucidate the fate of imported ssDNA within the bacterial cell.

Main Methods:

• Quantification of fluorescence signals from short DNA fragments labeled with a single fluorescent marker molecule during uptake.
• Analysis of DNA import and transformation efficiencies in wild-type and nuc deletion strains of Neisseria gonorrhoeae.
• Comparison of uptake rates for ssDNA and dsDNA with both double-stranded and single-stranded DUS.

Main Results:

• ssDNA with a double-stranded DNA uptake sequence (DUS) was imported with efficiency comparable to dsDNA.
• Imported ssDNA was rapidly degraded by the thermonuclease Nuc.
• In the absence of Nuc, both dsDNA and ssDNA with a double-stranded DUS were efficiently imported and utilized for transformation.
• ssDNA with a single-stranded DUS showed minimal import and transformation efficiency.

Conclusions:

• The DNA uptake machine in Neisseria gonorrhoeae requires a double-stranded DUS for efficient DNA recognition.
• The machine transports ssDNA and dsDNA with comparable efficiencies.
• The secondary structure of transforming DNA is crucial for self DNA recognition but not for the transport process across the outer membrane.