TheVibrio vulnificusstressosome is dispensable in nutrient-replete conditions

Abstract

ABSTRACTThe stressosome is a protein complex that has been demonstrated to sense environmental stresses and mediate the stress response in several Gram-positive bacteria, through the activation of the alternative sigma factor SigB. The in vivocharacterisation of this complex has never been performed inVibrio vulnificusor any other bacteria that do not possess SigB. The elucidation of the role of the stressosome inV. vulnificuswould provide elements to elaborate a functional model of the complex in a Gram-negative bacterium and identify the regulatory output in the absence of SigB. The stressosome locus is only found in 44% ofVibrio vulnificusisolates raising the question as to whether the role of stressosome is essential or modulatory in this bacterial species.In this work, the expression of the stressosome genes was proven in nutrient-replete conditions and the co-transcription as one operonic unit of the stressosome locus and its putative downstream regulatory locus was demonstrated.Moreover, the construction of a stressosome mutant lacking the four genes constituting the stressosome complex allowed us to examine the role of this complexin vivo. The initial established mutagenesis strategy relied on rifampicin-resistantV. vulnificusto select recombinant bacteria. Our data clearly showed that the influence of the RifRallele on stress and virulence characteristics overshadowed any effects of the stressosome. Therefore, we established an alternative mutagenesis strategy with a non-modifiedV. vulnificusparental strain and a DAP auxotrophicE. colidonor strain. Extensive phenotypic characterisation of the successfully-generated mutant in nutrient-replete conditions showed that the stressosome does not significantly contribute to the growth, ofV. vulnificus. The stressosome did not modulate the response ofV. vulnificusto the range of stresses tested – Ethanol, osmolarity, temperature, and salinity. Furthermore, the stressosome is dispensable for motility and exoenzyme production ofV. vulnificus.