| More than 98%of the bacteria in the environment are viable but nonculturable(VBNC)bacteria,which have low metabolic activity and cannot be isolated and cultured on standard media.Environmental stresses such as antibiotics,low temperature,high pressure,starvation,oxidative stress and other stress factors can also induce bacteria into the VBNC state.VBNC bacteria induced by environmental stress can recover their ability to grow and reproduce on the medium under suitable conditions,and their pathogenicity is also restored.Research in genetic and molecular biology have shown that VBNC bacteria have the characteristics of low metabolic activity,but due to the differences among genotype,phenotype,quantity and function,the specific metabolic mechanism of VBNC bacteria is still poorly understood.This affects the accurate identification and complete removal of VBNC bacteria,thus inducing serious,pests and diseases of agricultural products and human health risks.Therefore,clarifying the regulatory mechanism of low metabolic activity of VBNC bacteria can enhance the understanding of the ecological function of VBNC bacteria,health risk assessment and effective control of harmful VBNC bacteria.As the most commonly used disinfection technology,chlorine disinfection has been shown to induce bacteria into the VBNC state.Pseudomonas aeruginosa as one of the most common conditional pathogenic microorganisms and still capable of producing virulence proteins after entering the VBNC state,thus posing a major threat to public health and drinking water safety.Therefore,this study used VBNC Pseudomonas aeruginosa induced by chlorine stress as a model to study the metabolic characteristics and biomarkers of the VBNC bacteria at the population level and the single cell level,and to explore the key pathways for the VBNC bacteria to maintain activity based on their metabolic regulation strategies.The specific research and results are as follows:(1)The metabolic characteristics of VBNC bacteria were studied at the population and single cell levels.Untargeted metabolomics-based GC-MS/MS studies analyze metabolic profiles and biomarkers at the population level of VBNC bacteria,and analyze their metabolic regulation strategies to maintain activity.By isotopic tracing(D,15N,13C)coupled Raman spectroscopy was used to analyze the metabolic differences of VBNC bacteria at the single cell level,and the metabolic fingerprints of individual VBNC bacteria were obtained.The results showed that the overall metabolic activity of VBNC bacteria was down-regulated,and the anabolic capacity of biomacromolecule was reduced,but the competence for the glyoxylate cycle,fatty acid and glycerophospholipid metabolism was up-regulated.In addition,the VBNC bacteria showed more significant metabolic heterogeneity.(2)The mechanism by which the glyoxylate cycle maintains the metabolic homeostasis of VBNC bacteria was elucidated.The key role of the glyoxylate cycle in maintaining the low metabolic activity of VBNC bacteria was investigated by assessing the respiratory activity,reactivation and biofilm formation capacity of VBNC bacteria after blocking the glyoxylate cycle,and the specific mechanisms by which the glyoxylate cycle maintains the intracellular metabolic homeostasis of VBNC bacteria were investigated based on metabolomics.The results showed that the glyoxylate cycle was the key pathway to maintain the metabolic homeostasis of VBNC cells,and blocking the glyoxylate cycle could completely inactivate VBNC bacteria.The main mechanism involved is that blocking the glyoxylate cycle leads to the disruption of carbohydrate and fatty acid metabolism,thereby disrupting the energy metabolism system of VBNC bacteria.And leads to a reduction in the abundance of antioxidants and the extracellular polymer synthesis,which together lead to the collapse of the antioxidant system of the bacteria.(3)The mechanism of the glycerophospholipid-fatty acid axis regulating regulates the adaptive morphogenesis of VBNC bacteria was explored.Scanning electron microscopy revealed that the cell size of bacteria decreased but the cell membrane was intact after entering the VBNC state.Based on metabonomics,the specific mechanism of VBNC bacteria forming an adaptive morphology by regulating the glycerophospholipid-fatty acid axis was analyzed.The results show that the VBNC bacteria reduce cell size by regulating the glycerophospholipid-fatty acid metabolic axis while maintaining a low level of energy metabolism and antioxidant system homeostasis to form an adaptive morphology.The primary mechanisms are as follows.VBNC bacteria minimize cell size by degrading fatty acids while replenishing the glyoxylate cycle to maintain intracellular energy metabolism at the necessary level.At the same time,the glycerophospholipid metabolism replenished the fatty acids and maintained cell membrane integrity,preventing HClO from entering the cell.In addition,regulation of the glycerophospholipid-fatty acid axis maintains the abundance of intracellular antioxidants,preventing VBNC bacteria lipid peroxidation.The result of this study helps to achieve accurate identification and effective control of VBNC bacteria,and provides ideas for the development of clean and efficient new disinfection methods.Thus reducing the environmental health risks posed by VBNC bacteria,which is of great significance for maintaining public health and drinking water safety. |
