The cloned SPI-1 type 3 secretion system can be functionally expressed outside Salmonella backgrounds

Area of Science:

• Microbiology
• Molecular Biology
• Bacterial Genetics

Background:

• The SPI-1 type 3 secretion system (T3SS) from Salmonella Typhimurium is crucial for bacterial pathogenesis and has potential in engineering applications.
• Previous attempts to express cloned SPI-1 T3SS in Gram-negative bacteria like Escherichia coli resulted in significant expression defects.

Purpose of the Study:

• To identify the cause of the SPI-1 T3SS expression defect in non-Salmonella backgrounds.
• To develop functional SPI-1 T3SS clones expressible in Escherichia coli.
• To compare different cloning techniques for T3SS gene transfer.

Main Methods:

• Comparative DNA sequencing of SPI-1 across different bacterial backgrounds.
• Full sequencing of the original SPI-1 clone.
• Cloning SPI-1 from alternative Salmonella Typhimurium strains.
• Evaluation of expression defects using homology-based capture versus FRT-Capture techniques.

Main Results:

• Identified a DNA homology-based capture method as the cause of the expression defect.
• Successfully obtained and expressed full-length SPI-1 T3SS clones in Escherichia coli.
• Demonstrated the FRT-Capture technique as superior to homology-based methods for SPI-1 cloning.

Conclusions:

• The cloning methodology significantly impacts the functional expression of T3SS genes in heterologous hosts.
• FRT-Capture is a preferred method for cloning SPI-1 T3SS for expression in Escherichia coli.
• This work facilitates future applications of SPI-1 and other T3SS in non-Salmonella bacterial systems.