An assessment of the role of intracellular free Ca2+ in E-coli

We have previously proposed that fluctuations in Ca2+ levels should play an important role in bacteria as in eukaryotes in regulating cell cycle events (Norris et al., J. Theor. Biol. 134 (1998) 341-350). This proposal implied the presence of Ca2+ uptake systems in bacteria, cell cycle mutants simultaneously defective in Ca2+-homeostasis, and perturbation of cell cycle processes when cellular Ca2+ levels are depleted. We review the properties of new cell cycle mutants in E. coli and B. subtilis resistant to inhibitors of calmodulin, PKC or Ca2+-channels; the evidence for Ca2+-binding proteins including Acp and FtsZ; and Ca2+-transporters. In addition, the effects of EGTA and verapamil (a Ca2+ channel inhibitor) on growth, protein synthesis and cell cycle events in E. coli are described. We also describe new measurements of free Ca2+-levels, using aequorin, in E. coli. Several new cell cycle mutants were obtained using this approach, affecting either initiation of DNA replication or in particular cell division at non-permissive temperature. Several of the mutants were also hypersensitive to EGTA and or Ca2+. However, none of the mutants apparently involved direct alteration of a drug target and surprisingly in some cases involved specific tRNAs or a tRNA synthetase. The results also indicate that the expression of several genes in E. coli may be regulated by Ca2+. Cell division in particular appears very sensitive to the level of cell Ca2+, with the frequency of division clearly reduced by EGTA and by verapamil. However, whilst the effect of EGTA was clearly correlated with depletion of cellular Ca2+ including free Ca2+, this was not the case with verapamil which appears to change membrane fluidity and the consequent activity of membrane proteins. Measurement of free Ca2+ in living cells indicated levels of 200-300 nM, tightly regulated in wild type cells in exponential phase, somewhat less so in stationary phase, with apparently La2+-sensitive PHB-polyphosphate complexes involved in Ca2+ influx. The evidence reviewed increasingly supports a role for Ca2+ in cellular processes in bacteria, however, any direct link to the control of cell cycle events remains to be established. (C) 1999 Societe francaise de biochimie et biologie moleculaire/Editions scientifiques et medicales Elsevier SAS.