Medium dependent factors govern the functionality of engineered type III secretion systems

Area of Science:

• Synthetic Biology
• Microbiology
• Molecular Biology

Background:

• The type III secretion system (T3SS) is a bacterial machine for injecting effector proteins.
• T3SS engineering for biotechnological applications is promising.
• The effect of bacterial host culture conditions on T3SS performance is largely unstudied.

Purpose of the Study:

• To investigate how bacterial host culture conditions influence engineered T3SS performance.
• To characterize an IPTG-inducible, refactored T3SS (iT3SS) circuit in Salmonella enterica.
• To determine the impact of different growth media on iT3SS functionality.

Main Methods:

• Constructed an IPTG-inducible T3SS circuit (iT3SS) using a PrgH-GFP fusion in Salmonella.
• Assessed PrgH-GFP expression dynamics and SptP effector secretion in LB and glucose minimal media.
• Utilized RNA-sequencing (RNA-seq) to analyze gene expression under varying IPTG concentrations and media conditions.

Main Results:

• Secretion efficiency was maintained in glucose minimal medium but not in rich LB medium across IPTG concentrations.
• In LB medium, secretion and invasion efficiencies did not scale proportionally with IPTG, with reduced efficiency at high inducer levels.
• RNA-seq indicated that transcription was not limiting in LB medium; translational/post-translational burdens and stress responses likely limited iT3SS function.

Conclusions:

• Bacterial growth medium significantly impacts engineered T3SS performance, potentially due to host resource allocation affecting translational efficiency.
• Impaired translation and assembly of iT3SS components compromise their membrane insertion.
• A medium-aware framework is essential for optimizing engineered secretion systems in synthetic biology.