Heat shock coupled transduction: a fast and facile method to circumvent the staphylococcal restriction barrier

As common members of the human microbiota, staphylococcal species colonize humans at various body sites. While some of these staphylococci, like Staphylococcus aureus, have the potential to cause a variety of devastating diseases, most of them have adopted a commensal lifestyle. Many of these staphylococcal species have been shown to produce various secondary metabolites, excluding more pathogenic bacteria from their niches, or modulating the host immune system to overcome infections by different pathogens. However, studying staphylococci in the laboratory context remains challenging, as many strains possess a strong restriction barrier, which hinders genetic manipulation. Even though significant progress has been made in recent years, the ability to study these bacteria and their genetic potential is still severely limited.

In this work, we describe a novel method combining heat shock and transduction to enable the genetic manipulation of previously inaccessible staphylococcal strains. We present evidence that the method is applicable to different plasmids, species, and different phages. Our experimental data indicates that different species show a varying temperature optimum for the heat shock and that restriction-competence is recovered over time, suggesting a temporary inactivation of restriction enzymes. We propose that this method is generally applicable to different bacteria, for which transducing phages are available, and is not limited to staphylococci. Overall, this method will enable the study of previously genetically inaccessible bacteria in a fast, easy, and cost-effective way.