Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria

Area of Science:

• Microbiology
• Synthetic Biology
• Genetic Engineering

Background:

• Engineering robust bacteria for health applications requires overcoming challenges in genetic manipulation.
• Undomesticated bacteria are difficult to genetically modify due to limited DNA transfer methods.

Purpose of the Study:

• To develop a novel method for efficient DNA transfer into undomesticated bacteria.
• To engineer a donor strain (XPORT) capable of delivering genetic material via miniaturized integrative and conjugative elements (mini-ICEBs1).

Main Methods:

• Constructed XPORT donor strain with inducible conjugation and chromosomal integration capabilities.
• Utilized D-alanine auxotrophy for simplified isolation of engineered bacteria.
• Separated the type IV secretion system from transferred DNA to restrict heterologous DNA spread.

Main Results:

• Demonstrated efficient DNA transfer (10^-1 to 10^-7 events per donor) to 35 Gram-positive strains from human and soil environments.
• Successfully characterized isopropyl-β-D-thiogalactoside (IPTG)-inducible reporter performance across multiple strains.
• Transferred nitrogen fixation genes to four Bacillus species and showed DNA transfer in soil conditions.

Conclusions:

• The XPORT system provides a versatile platform for genetic engineering of diverse, wild bacteria.
• This method significantly advances the potential for engineering microorganisms for human and plant health.
• The developed approach is robust and effective even under challenging environmental conditions.