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Background <p>Functional gene analysis of <i>Staphylococcus aureus</i> across different strains requires the development of genetic engineering tools. In studies on phage therapy, such analysis is especially important for understanding the genomic differences between strains, underlying the differences in sensitivity to phages. Meanwhile, difficulties in introducing DNA constructs for genetic manipulations into non-laboratory <i>S. aureus</i> strains hinder the elucidation of staphylococcal anti-phage defense strategies.</p> Results <p>We used a transducing <i>S. aureus</i> bacteriophage and an <i>E. coli</i> -<i>S. aureus</i> shuttle plasmid with a DNA fragment of this phage to develop a toolset for recombinational gene replacement in difficult-to-transform <i>S. aureus</i> isolates. The plasmid carried an erythromycin-resistance cassette flanked by unique restriction sites for cloning the flanking regions of target DNA to be replaced. Plasmid loss from recombinants with the desired gene replacement was achieved by removing the plasmid <i>par</i> gene, which is essential for plasmid inheritance. Recombinants that acquired the cassette but lost the plasmid were resistant to erythromycin and susceptible to chloramphenicol selective for plasmid backbone. We applied our system to inactivate the predicted anti-phage defense gene <i>avs2</i> in a clinical <i>S. aureus</i> isolate and inactivated the identical gene in a laboratory strain. While the mutation sensitized the laboratory strain to three phages, it did not affect the clinical strain’s sensitivity to phages, demonstrating the complexity of phage-bacterial interactions and limitations of bioinformatic phage susceptibility predictions without experimental validation. The large terminase subunits of Avs2-sensitive phages were predicted to form complexes with Avs2, suggesting their direct involvement in triggering Avs2.</p> Conclusions <p>Our new, simple toolset for introducing plasmids carrying donor DNA for genetic manipulation into <i>S. aureus</i> strains that are difficult to transform proved suitable for generating chromosomal gene knockouts in clinical <i>S. aureus</i> isolates. Its use to knock out an exemplary anti-phage defense gene, <i>avs2</i>, in a clinical <i>S. aureus</i> strain and comparative phenotypic analysis of the obtained mutant with that of a laboratory strain revealed differences resulting presumably from different genomic backgrounds of target genes in each strain. This supports the validity of our approach and shows the importance of empirical phage sensitivity testing of strains in native backgrounds.</p>

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A versatile toolset for Staphylococcus aureus gene inactivation enables testing of anti-phage defense systems in native genetic backgrounds

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月17日

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  • Łukasz Kałuski,
  • Stephen Amankwah,
  • Laura Wicke,
  • Ján Bíňovský,
  • Emil Stefańczyk,
  • Ivana Mašlaňová,
  • Lucie Kuntová,
  • Roman Pantůček,
  • Małgorzata Łobocka

摘要

Background

Functional gene analysis of Staphylococcus aureus across different strains requires the development of genetic engineering tools. In studies on phage therapy, such analysis is especially important for understanding the genomic differences between strains, underlying the differences in sensitivity to phages. Meanwhile, difficulties in introducing DNA constructs for genetic manipulations into non-laboratory S. aureus strains hinder the elucidation of staphylococcal anti-phage defense strategies.

Results

We used a transducing S. aureus bacteriophage and an E. coli -S. aureus shuttle plasmid with a DNA fragment of this phage to develop a toolset for recombinational gene replacement in difficult-to-transform S. aureus isolates. The plasmid carried an erythromycin-resistance cassette flanked by unique restriction sites for cloning the flanking regions of target DNA to be replaced. Plasmid loss from recombinants with the desired gene replacement was achieved by removing the plasmid par gene, which is essential for plasmid inheritance. Recombinants that acquired the cassette but lost the plasmid were resistant to erythromycin and susceptible to chloramphenicol selective for plasmid backbone. We applied our system to inactivate the predicted anti-phage defense gene avs2 in a clinical S. aureus isolate and inactivated the identical gene in a laboratory strain. While the mutation sensitized the laboratory strain to three phages, it did not affect the clinical strain’s sensitivity to phages, demonstrating the complexity of phage-bacterial interactions and limitations of bioinformatic phage susceptibility predictions without experimental validation. The large terminase subunits of Avs2-sensitive phages were predicted to form complexes with Avs2, suggesting their direct involvement in triggering Avs2.

Conclusions

Our new, simple toolset for introducing plasmids carrying donor DNA for genetic manipulation into S. aureus strains that are difficult to transform proved suitable for generating chromosomal gene knockouts in clinical S. aureus isolates. Its use to knock out an exemplary anti-phage defense gene, avs2, in a clinical S. aureus strain and comparative phenotypic analysis of the obtained mutant with that of a laboratory strain revealed differences resulting presumably from different genomic backgrounds of target genes in each strain. This supports the validity of our approach and shows the importance of empirical phage sensitivity testing of strains in native backgrounds.