Genetic engineering of Staphylococcus haemolyticus : Overcoming Restriction-Modification Barriers and Targeting...

Area of Science:

• Microbiology
• Genomics
• Molecular Biology

Background:

• Staphylococcus haemolyticus is a multidrug-resistant nosocomial pathogen.
• Complex restriction-modification (RM) systems hinder genetic manipulation, limiting studies on its pathogenesis and immune evasion.

Purpose of the Study:

• To develop a molecular toolbox for precise genomic engineering of clinical S. haemolyticus isolates.
• To overcome RM barriers and enable functional studies of virulence and host adaptation.

Main Methods:

• Defined genomes and methylomes of nine isolates using PacBio SMRT and bisulfite sequencing.
• Implemented a dual strategy using SyngenicDNA and an engineered E. coli strain (JMC4) to bypass RM systems.
• Enabled targeted knockout of virulence genes (sraP, secA2, capA, capI) and allelic exchange of the capsule operon.

Main Results:

• Successfully engineered a molecular toolbox for precise genomic modification in S. haemolyticus.
• Identified Type II and Type III RM systems overrepresented in clinical isolates, suggesting a role in adaptation.
• Enhanced transformation efficiency and enabled targeted gene knockouts and large-scale chromosomal modifications.

Conclusions:

• This study establishes the first robust molecular tools for transformation and genome editing in S. haemolyticus.
• The developed tools lay the foundation for future functional studies of virulence and host adaptation in this pathogen.
• The approaches have broader utility for engineering other bacteria with RM barriers.