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Investigation of the First Events Leading to Loss of Culturability during Escherichia coli Starvation: Future Nonculturable Bacteria Form a Subpopulation

Laboratoire de Chimie Bactérienne-UPR 9043-CNRS,¹ Laboratoire de Microbiologie Marine, INSU, UMR 6117, Université de la Méditerranée, Campus de Luminy Case 907, Marseille, France,² Department of Cell and Molecular Biology-Microbiology, Göteborg University, Göteborg, Sweden³

Received 28 July 2004/ Accepted 6 December 2004

In previous experiments we were able to separate, using a nondestructive separation technique, culturable and nonculturable bacteria, from a Luria-Bertani (LB) medium culture of Escherichia coli incubated for 48 h. We observed in the nonculturable bacterial population an increase in oxidative damage and up-induction of most defenses against reactive oxygen species (ROS), along with a decrease in cytoplasmic superoxide dismutases. In this study, using the same separation technique, we separated into two subpopulations a 10-h LB medium culture containing only culturable bacteria. For the first time, we succeeded in associating physical separation with physiological differences. Although the levels of defense against ROS (RpoS, RpoH, OxyR, and SoxRS regulons) and oxidative damage (carbonyl contents) were apparently the same, we found that bacteria in one subpopulation were more sensitive to LB medium starvation and to various stresses, such as phosphate buffer starvation, heat shock, and hydrogen peroxide exposure. Based on these results, we suggest that these physiological differences reflect uncharacterized bacterial modifications which do not directly involve defenses against ROS.