Resuscitation of viable but nonculturable bacteria promoted by ATP-mediated NAD+ synthesis

[Display omitted] •Perturbing O-antigen ligation shortened the resuscitating lag phase.•Higher ATP levels of ΔrfaL VBNC cells promote resuscitation.•ATP consumption during lag phase was highly correlated with resuscitation efficiency.•ATP was utilized to activate the Handler and salvage pathways to synthesize NAD+.•VBNC cells using residual ATP recover metabolic activity, driving cells to exit dormancy. Entry into the viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria to survive harsh environment. Although VBNC cells still have metabolic activity, they lose the ability to form colonies on nonselective culture media. Thus, conventional bacterial detection methods, such as plate counting, are unable to detect the presence of VBNC cells. When the environmental conditions are appropriate, VBNC cells can initiate resuscitation, posing a great risk to the safety of public health. The study of the VBNC resuscitation mechanism could provide new insights into the prevention and control of VBNC resuscitation. Uncovering the molecular mechanism of VBNC cell resuscitation by investigating the role of O-antigen ligase (RfaL) in inhibiting the resuscitation of Escherichia coli O157:H7 in the VBNC state. RfaL was screened and verified as a resuscitation inhibitor of VBNC Escherichia coli O157:H7 by detecting resuscitation curve and time-lapse microscopy. The mechanism of RfaL impacts VBNC E. coli resuscitation was investigated by detecting the single cell ATP content, metabolomic changes, NAD(H) content and new protein biosynthesis of WT and ΔrfaL at different stage of resuscitation. Mutation of rfaL, which encoded an O-antigen ligase, markedly shortened the resuscitating lag phase. Further studies indicated that ΔrfaL VBNC cells contained higher ATP levels, and ATP consumption during the resuscitating lag phase was highly correlated with resuscitation efficiency. Metabolomic analysis revealed that ATP was utilized to activate the Handler and salvage pathways to synthesize NAD+, balancing redox reactions to recover cell activity and promote cell resuscitation. Our findings revealed a strategy employed by VBNC cells for revival, that is, using residual ATP to primarily recover metabolic activity, driving cells to exit dormancy. The synthesis pathway of lipopolysaccharide (LPS) in rfaL null mutant was inhibited and could supply more ATP to synthesis NAD+ and promote resuscitation.