Control of type III protein secretion using a minimal genetic system

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• Gram-negative bacteria utilize type III secretion systems (T3SS) for protein export, crucial for pathogenesis and motility.
• T3SS assembly involves complex genetic regulation within pathogenicity islands like Salmonella SPI-1.

Purpose of the Study:

• To simplify and re-engineer the Salmonella SPI-1 T3SS genetic locus.
• To identify essential regulatory components and assess the robustness of T3SS assembly.
• To explore the potential of a synthetic T3SS for biotechnological applications.

Main Methods:

• Genetic simplification of Salmonella SPI-1 by gene removal/recoding and replacement of non-coding DNA with synthetic parts.
• Construction of a de novo 16kb genetic cluster with altered organization and regulation.
• Investigation of essential internal start sites (SpaO) and small RNAs (InvR) in the simplified system.
• Application of synthetic regulatory circuits for T3SS control.

Main Results:

• A highly simplified 16kb T3SS genetic cluster was constructed, sharing no sequence identity or organizational principles with the native SPI-1.
• Essential roles for a novel internal start site (SpaO) and a small RNA (InvR) in T3SS assembly were discovered.
• The engineered T3SS demonstrated robust post-transcriptional control and could be regulated using synthetic circuits, even under repressing conditions.

Conclusions:

• The T3SS exhibits significant post-transcriptional robustness, allowing for genetic simplification and re-engineering.
• Synthetic biology approaches can be used to create functional and controllable T3SS variants.
• This work provides a foundation for utilizing T3SS as a controllable tool in biotechnology.