Cu2+-induced VBNC state formation in Shewanella xiamenensis JL2 at wastewater-relevant levels

Heavy metals such as Cu²⁺ are widely prevalent in wastewater (typically 0.04-157.4 mM in typical treatment systems), threatening microbial communities critical for pollutant removal. However, the viable but nonculturable (VBNC) state, a dormant survival strategy under metal stress, remains poorly understood in these systems, potentially compromising treatment efficiency. This study first reveals Cu²⁺-induced VBNC state formation in Shewanella xiamenensis JL2 at environmentally relevant concentrations, supported by integrated physiological and transcriptomic analyses. Cu²⁺ triggered VBNC state entry via oxidative stress, evidenced by 3.41-fold elevated ROS levels and dynamic SOD/POD responses. This was accompanied by metabolic suppression, including 3.18-fold reduced ATP production and 11.12-fold decreased SDH activity. Transcriptomic profiling uncovered three previously unrecognized features in Bacterial VBNC state: (1) putative antioxidant roles of ohr; (2) upregulation of cytochrome bd oxidase coupled with downregulation of heme-copper oxidase; and (3) activation of the methylcitrate cycle, distinct from the glyoxylate cycle observed in Other bacteria. Furthermore, transcriptional reprogramming of carbon, sulfur, and histidine metabolism was accompanied by ribosomal dormancy. These findings collectively demonstrate a coordinated tripartite survival strategy involving oxidative stress defense, metabolic alteration, and transcriptional reprogramming. This work provides the first mechanistic model of Cu²⁺-induced VBNC state formation in Shewanella species, offering critical insights for optimizing microbial wastewater treatment systems under heavy metal stress.

Copper; Methylcitrate cycle; Viable but nonculturable; Wastewater; ohr.