Glyoxylate cycle maintains the metabolic homeostasis of Pseudomonas aeruginosa in viable but nonculturable state induced by chlorine stress.

Bacteria enter a viable but non-culturable (VBNC) state with low metabolic activity to cope with environmental stress (e.g., chlorine disinfection). Elucidating the mechanism and key pathway of VBNC bacteria maintaining low metabolic competence is of great significance to realize their effective control and reduce their environmental and health risks. This study discovered that the glyoxylate cycle is a key metabolic pathway for VBNC bacteria, but not for culturable bacteria. And blocking the glyoxylate cycle pathway inhibited the reactivation and led to the death of VBNC bacteria. The main mechanisms involved the breakdown of material and energy metabolism and the antioxidant system. Gas chromatography-tandem mass spectrometry analysis showed that blocking the glyoxylate cycle led to a disruption of carbohydrate metabolism and fatty acid catabolism in VBNC bacteria. As a result, the energy metabolism system of VBNC bacteria collapsed and the abundance of energy metabolites (ATP, NAD and NADP) decreased significantly. Moreover, the decrease in the level of quorum sensing signaling molecules (quinolinone and N-Butanoyl-D-homoserine lactone) inhibited the synthesis of extracellular polymeric substances (EPSs) and biofilm formation. And the downregulation of glycerophospholipid metabolic competence increased the permeability of cell membranes, leading to the entry of large amounts of hypochlorous acid (HClO) into the bacteria. In addition, the down-regulation of nucleotide metabolism, glutathione metabolism, and the reduction of antioxidant enzyme content resulted in the inability to scavenge reactive oxygen species (ROS) generated by chlorine stress. The large production of ROS and the reduction of antioxidants together led to the breakdown of the antioxidant system of VBNC bacteria. In short, the glyoxylate cycle is the key metabolism pathway of VBNC bacteria for stress resistance and maintaining cellular metabolic balance, and targeting the glyoxylate cycle represents an attractive strategy for developing new and efficient disinfection methods for the control of VBNC bacteria.