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

Staphylococcus haemolyticus is an emerging multidrug-resistant nosocomial pathogen noted for robust biofilm formation and complex restriction-modification (RM) systems that hinder genetic manipulation. These barriers have severely limited mechanistic studies into its pathogenesis and immune evasion. Here, we report the development of a molecular toolbox that enables precise genomic engineering of clinical S. haemolyticus isolates. Using PacBio Single Molecule Real Time (SMRT) and bisulfite sequencing, we defined the complete genomes and methylomes of nine isolates, generating a functional readout of the active RM defences present in each strain. Among the RM systems identified, a Type II (PDLC03279) and a Type III (PDL3649/PDLC03643) system were significantly overrepresented in clinical isolates, suggesting a potential role in adaptation to host or hospital-associated environments. To bypass these RM barriers, we implemented a dual strategy: first, applying SyngenicDNA-based approaches to eliminate RM target motifs from genetic tools; and second, engineering a surrogate E. coli strain (JMC4) to mimic conserved S. haemolyticus methylation patterns. These tools significantly enhanced transformation efficiency and enabled targeted knockout of four putative virulence genes ( sraP, secA2, capA , and capI ), as well as allelic exchange of the native capsule operon with the corresponding region from a non-encapsulated isolate. To our knowledge, this is the first report of precise genomic modifications in S. haemolyticus . The establishment of robust molecular tools for transformation and genome editing lays a foundation for future functional studies of virulence and host adaptation in this resilient opportunistic pathogen.

All genomic data were deposited in the European Nucleotide Archive and are available in NCBI under the project accession number PRJEB2705. Supporting data are provided in supplementary data files. Accession numbers are given in Table 1.

Staphylococcus haemolyticus is a multidrug-resistant opportunistic pathogen which causes disease in vulnerable patients. Previously, genetic modification of this pathogen was prevented due to the prevalence of strain-specific restriction modification systems, which use DNA methylation to identify and foreign DNA (including plasmids) when it enters the cells, and then destroy it using restriction enzymes. We analysed the methylomes of a selection of S. haemolyticus strains and used this data to tailor molecular tools to evade the RM systems. This enabled us to improve transformation efficiency and perform genome editing, including large-scale chromosomal modifications and targeted deletion of suspected virulence genes (a first for S. haemolyticus ). Beyond its immediate relevance to researchers studying this pathogen, the tools and approaches developed here have broader utility for genetic engineering of other coagulase-negative staphylococci and bacteria with similar RM barriers. Additionally, we found that two RM systems were enriched in clinical strains, suggesting they may have a function in virulence, in addition to their roles as bacterial phage defence systems.