Stress adaptation and virulence in Vibrio alginolyticus is mediated by two (p)ppGpp synthetase genes, relA and spoT

Vibrio alginolyticus belongs to gram-negative opportunistic pathogen realm infecting humans and aquatic animals causing severe economic losses. The (p)ppGpp-mediated stringent response is corroborated to stress adaptation and virulence of pathogenic mechanisms. Limited reports are documented for the intricate assessment of (p)ppGpp synthetase genes in combating various stress adaptation and elucidation of virulence in V. alginolyticus remains unraveled. The present assessment comprises of generation of deletion mutants in the (p)ppGpp-deficient strains, ΔrelA (relA gene single mutant) and ΔrelAΔspoT (relA and spoT genes double mutant), and the complemented strains, ΔrelA+ and ΔrelAΔspoT+, were constructed to investigate the pivotal roles of (p)ppGpp synthetase genes in V. alginolyticus, respectively. Amino acid sequence alignment analysis initially revealed that RelA and SpoT possess relatively conserved domains and synthetase activity. Hydrolase activity was emancipated by SpoT alone showing variant mode of action. Compared with the wild type and complemented strains, the relA-deficient strain was more sensitive to amino acid starvation and mupirocin. Interestingly, the deletion of spoT resulted in a significant growth deficiency supplemented with bile salts, 3 % ethanol and heat shock. Rapid growth was observed in the stationary phase upon exposure to cold stress and lower doses of ethanol. Subsequently, disruption of (p)ppGpp synthetase genes caused the decline in swimming motility, enhanced biofilm formation, cell aggregation of V. alginolyticus, and reduced mortality of Litopenaeus vannamei. The expression levels of some virulence-associated genes were quantified affirming consistency established by pleiotropic phenotypes. The results are evident for putative roles of (p)ppGpp synthetase genes attributing essential roles for environmental adaption and virulence regulation in V. alginolyticus.