The stringent response mediated by the signal molecule (p)ppGpp is involved in response to multiple environmental stresses and control of various physiological processes. Studies have revealed that (p)ppGpp strongly affects the formation and maintenance of several bacterial biofilms. However, the specific regulatory roles of (p)ppGpp in biofilms, especially in the expression of genes related to cell motility and exopolysaccharides (EPSs) production, remain poorly understood. We recently reported two (p)ppGpp synthetase genes relA and spoT from the epizootic pathogen Vibrio alginolyticus. Herein, we found that the (p)ppGpp synthetase genes of V. alginolyticus contributed to biofilm formation at low cell density and biofilm detachment at high cell density, respectively, in polystyrene microtiter plates. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that the expression levels of both EPSs and motility associated genes were consistent with the development of biofilms. Besides, the (p)ppGpp synthetase gene spoT was found to be closely involved in the regulation of flagellum, smooth/translucent colony morphology and spotty pellicle at the air-liquid interface. Interestingly, pleiotropic phenotypes of ΔrelAΔspoT were similar to that of the rpoN (σ54) deletion mutant. Meanwhile, the absence of (p)ppGpp synthetase genes significantly reduced the expression levels of rpoN at low cell density, suggesting that (p)ppGpp may mediate the formation via positively affecting the alternative sigma factor RpoN. These findings allow us to propose (p)ppGpp as a crucial regulator for biofilm development in V. alginolyticus, in view of the regulatory roles of relA and spoT in cell motility and EPSs production.

No abstract

Vibrio harveyi is one of the most serious bacterial pathogens to aquatic animals worldwide. Evidence is mounting that coinfections caused by multiple pathogens are common in nature and can alter the severity of diseases in marine animals. However, bacterial coinfections involving V. harveyi have received little attention in mariculture. In this study, the results of pathogen isolation indicated that bacterial coinfection was a common and overlooked risk for hybrid groupers (♀ Epinephelus polyphekadion × ♂ E. fuscoguttatus) reared in an industrialized flow-through pattern in Hainan Province. The artificial infection in hybrid groupers revealed that coinfections with V. harveyi strain GDH11385 (a serious lethal causative agent to groupers) and other isolated pathogens resulted in higher mortality (46.67%) than infection with strain GDH11385 alone (33.33%), whereas no mortality was observed in single infection with other pathogens. Furthermore, the intestine, liver and spleen of hybrid groupers are target organs for bacterial coinfections involving V. harveyi. Based on the infection patterns found in this study, we propose that V. harveyi may have a specific spatiotemporal expression pattern of virulence genes when infecting the host. Taken together, bacterial coinfection with V. harveyi is a neglected high-risk lethal causative agent to hybrid groupers in the industrialized flow-through aquaculture systems in Hainan Province.