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Resuscitation of Viable But Not Culturable Sinorhizobium meliloti 41 pRP4-luc: Effects of Oxygen and Host Plant

Basaglia, Marina, Povolo, Silvana, Casella, Sergio
Current microbiology 2007 v.54 no.3 pp. 167-174
Medicago sativa, Sinorhizobium meliloti, ampicillin, bacteria, chloramphenicol, host plants, oxygen, reporter genes, soil, soil sampling
A plasmid-borne, firefly-derived, luciferase gene (luc) was inserted and stably inherited in Sinorhizobium meliloti 41 as a reporter gene. The strain obtained, S. meliloti 41/pRP4-luc, and its parental strain served as a model system for viable but not culturable (VBNC) resuscitation experiments in both in vitro and soil samples. Incubation under oxygen (O₂) concentrations varying from 1% to atmospheric levels did not result in resuscitation. A demonstration of recovery was attained through exposure to the appropriate concentrations of antibiotics, bacteriostatic chloramphenicol, and bactericidal ampicillin. The resuscitation ratio was 1 recovered VBNC cell in every 10⁵ 5-cyano-2,3-di-4-tolyl-tetrazolium chloride (CTC⁺) bacteria. Although isolated VBNC rhizobia were unable to nodulate Medicago sativa, which apparently did not enhance VBNC reversion, resuscitated bacteria maintained their symbiotic properties. Soil experiments showed that the lack of O₂ leads to onset of VBNC status as in liquid microcosm, but the number of recoverable and culturable cells decreased more drastically in soil.