Clinical Rel mutations that increase basal (p)ppGpp promote conjugal transfer of staphylococcal resistance plasmids

Conjugative transfer of plasmids represents a major route through which antibiotic resistance genes are spread. In the case of the prevalent and deadly pathogen Staphylococcus aureus, more than 90% of clinical isolates carry at least one plasmid. While plasmid-encoded mechanisms [e.g. plasmid copy number (PCN)] can influence conjugation frequency, host factors and environmental stimuli can also affect transmission. In particular, stress responses like the stringent response have been associated with increased movement of mobile genetic elements. We have previously shown that clinical mutations in the stringent response controller, Rel, lead to elevated levels of the alarmones guanosine tetra- and pentaphosphate [(p)ppGpp] and antibiotic tolerance in S. aureus. Here, we report that elevated (p)ppGpp in these strains promotes the conjugal transfer of diverse staphylococcal resistance plasmids. We observed that clinical Rel mutations promote donation, but not receipt, of plasmids from the three families of staphylococcal plasmid and a mobilizable plasmid. This increased conjugation frequency could also be induced by chemical induction of the stringent response by mupirocin. Intriguingly, detailed experimental analysis revealed that the effect of elevated (p)ppGpp on plasmid donation was not due to CodY derepression, SOS response induction or increased PCN. Furthermore, comparative transcriptomics of wild-type and mutant donor did not highlight any putative plasmid- or host-derived mechanisms to explain this observation. Further investigations are required to explore the mechanistic link between (p)ppGpp and conjugation, given the pervasive transcriptional and post-translational effects of (p)ppGpp. Overall, the association between Rel mutation and increased plasmid donation is alarming, especially as Rel mutations are being increasingly identified among clinical isolates.