Bacterial culturability and the viable but non-culturable (VBNC) state studied by a proteomic approach using an artificial soil

Gram-negative bacteria in soil rapidly adapt to various stresses, including nutrient limitation and desiccation, by adopting the viable but non-culturable (VBNC) state as a survival strategy. Due to the physico-chemical and microbiological complexity of soils, little is understood on the effects of nutrient availability and moisture level on the transition from the VBNC state to culturability in soil. We evaluated the effects of gluconate or water on the transition of the soil borne bacterium C. metallidurans strain CH34 from the VBNC state to culturability by experiments of inoculation into artificial soils and bacterial metaproteomic analysis. Incubation without water or nutrients reduced the bacterial culturability to zero in 12 d, and addition of both water or gluconate restored the bacterial culturability to high levels within 24 h. The proteomic analysis showed that under water and nutrient limitation, proteins related to the cell shape and protein synthesis were rapidly down-regulated and stressrelated proteins were quickly up-regulated during the transition from culturability to VBNC state. Reversion from the VBNC state to a culturable state with water or gluconate led to highly different bacterial proteomic profiles of C. metallidurans. Gluconate availability restored main protein biosynthesis and energy metabolic pathways, whereas water addition led to up-regulation of only six proteins, one of which degrade sigma factors involved in expression of genes controlling bacterial resistance under nutrient limitation. Proteins regulated during the transition between culturable and VBNC states could also be involved in the phenotypic VBNC for other soil bacteria, and can highlight some of the microbial genetic mechanisms allowing the entering and exiting from the VBNC state. Implications of the VBNC in microbial diversity and soil functionality are discussed.