High-efficiency gene inactivation and replacement system for gram-positive bacteria

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Homologous integration is crucial for genetic manipulation in bacteria.
• Developing efficient gene replacement systems for gram-positive bacteria remains a challenge.
• Rolling-circle replication has been observed to enhance recombination.

Purpose of the Study:

• To develop a high-efficiency system for single- and double-crossover homologous integration in gram-positive bacteria.
• To establish a gene replacement protocol utilizing a thermosensitive rolling-circle plasmid.
• To demonstrate the applicability of the system in Lactococcus lactis.

Main Methods:

• A thermosensitive rolling-circle plasmid (pG+host5) was employed in Lactococcus lactis.
• Chromosomal fragments of varying lengths were used to assess integration frequency.
• A two-step temperature shift protocol was utilized to achieve gene replacement via homologous recombination.

Main Results:

• Integration frequency showed a logarithmic relationship with homology length (0.35–2.5 kb).
• Homologous integration occurred across the entire Lactococcus lactis chromosome.
• Gene replacement efficiencies ranged from 1–50% with or without selection, yielding insertions and deletions.

Conclusions:

• The developed system provides a highly efficient method for gene replacement in gram-positive bacteria.
• The thermosensitive rolling-circle nature of the plasmid vector is key to the high efficiency.
• This protocol is adaptable for genetic engineering in a wide range of gram-positive species.