DNA damage-associated vesicle production in Stenotrophomonas maltophilia is mediated by the maltocin endolysin

ABSTRACT In this study, we examined the effects of antibiotic-mediated DNA damage on vesiculation in a clinical strain of Stenotrophomonas maltophilia (strain 44/98, also referred to as LMG 26284). Although genomic analysis of this strain revealed multiple prophage clusters, subsequent proteomic analysis showed that, among the proteins most significantly affected by ciprofloxacin exposure, the majority belonged to the previously described maltocin gene cluster. We showed that deletion of the gene coding for the maltocin endolysin ( mal ) reduces cell lysis and, in turn, the vesiculation capacity of S. maltophilia . Furthermore, using a combination of fluorescence and cryogenic electron microscopy, we provided evidence that the spontaneous re-arrangement of shattered membrane fragments attributable to mal -induced explosive cell lysis results in the formation of predominantly single-layered explosive outer membrane vesicles, while only a minor portion are double-layered outer-inner membrane vesicles. We also detected putative cytoplasmic vesicles in these samples, suggesting an expanded role of cell lysis in vesicle formation. Finally, we showed that the maltocin endolysin (LysSM) is upregulated rapidly in response to DNA damage stress and accumulates at the cell poles, which are likely the sites of subsequent cell lysis. Our findings expand on the involvement of (cryptic) tailocins in vesicle biogenesis under stress conditions. IMPORTANCE In their natural environment, bacterial cells are able to communicate among themselves in multiple ways, and this communication has important implications for their ability to deal with unfavorable conditions. One key consequence is the spread of antimicrobial resistance from resistant cells to the remaining population, and such transfer can be mediated by membrane vesicles. In the highly multidrug-resistant bacterium Stenotrophomonas maltophilia , there exists a prophage that produces an endolysin capable of causing cell lysis and atypical vesicle production. The significance of this study lies in the description of such less characterized types of membrane vesicles produced in response to DNA damage, thereby providing insight into how bacteria respond to stress that can be brought about by antibiotic exposure.