Evidence for different pathways during horizontal gene transfer in competent Bacillus subtilis cells

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• The Bacillus subtilis DNA uptake machinery is located at a single cell pole, internalizing single-stranded (ss) DNA.
• Homologous DNA integration into the recipient chromosome depends on RecA.
• Plasmid establishment, independent of RecA, requires RecO and RecU.

Purpose of the Study:

• To investigate the distinct pathways of chromosomal and plasmid DNA transformation in Bacillus subtilis.
• To elucidate the roles of RecA, RecO, RecU, and RecN in DNA uptake and recombination.
• To understand how the bacterial DNA recombination machinery responds to different types of incoming DNA.

Main Methods:

• Cytological and genetic analyses were employed.
• Recruitment and localization of key proteins (RecA, RecO, RecU, RecN) were observed in response to DNA addition.
• In vitro assays were performed to study RecO's ssDNA annealing activity.
• The role of the Ku protein in transformation was assessed.

Main Results:

• RecA and RecN colocalize at the polar DNA uptake machinery; RecA forms filamentous structures ('threads') with chromosomal DNA.
• RecU accumulates at the cell pole in competent cells, dependent on RecA, and dissipates upon DNA addition.
• RecO accumulates at the pole specifically with plasmid or phage DNA, not chromosomal DNA, and exhibits ssDNA annealing activity in vitro.
• The Ku protein is involved in viral and plasmid transformation.
• SPP1 phage DNA transfection utilizes functions from both chromosomal and plasmid transformation pathways.

Conclusions:

• Competent Bacillus subtilis cells possess a dynamic DNA recombination machinery that differentiates between incoming DNA types based on homology or self-annealing potential.
• Chromosomal transformation involves RecA-mediated homology search and strand invasion.
• Plasmid establishment requires RecO for single-strand annealing and RecU, potentially regulating RecA.
• Viral DNA transfection follows an intermediate pathway, integrating elements of both chromosomal and plasmid transformation.