Effects of Calcium and Calcium Chelators on Growth and Morphology of Escherichia coli L-Form NC-7

Abstract

Growth of a wall-less, L-form of Escherichia coli specifically requires calcium, and in its absence, cells ceased dividing, became spherical, swelled, developed large vacuoles, and eventually lysed. The key cell division protein, FtsZ, was present in the L-form at a concentration five times less than that in the parental strain. One interpretation of these results is that the L-form possesses an enzoskeleton partly regulated by calcium.Numerous roles for calcium in bacteria are now becoming apparent (10,12,19). We have proposed that calcium has a role as a "general reset" in cells (12) and that it participates in the regulation of the putative bacterial "enzoskeleton" (14). This enzoskeleton would comprise proteins such as the tubulin-like protein, FtsZ, which is the key player in cell division and which in vitro has a calcium-stimulated polymerization and GTPase activity (24). L-forms are wall-less derivatives of bacteria that grow and divide despite their lack of a normal peptidoglycan sacculus (7,8,15,18,22). This means that the morphology and progress through the cell cycle of L-forms must result from forces acting via some structure other than the sacculus. Membrane domains have been considered candidates for such structures (7), and these probably result from the coupled transcription, translation, and insertion (transertion) of proteins into and through membranes (1), processes that generate sufficient force to hold L-forms together (13). The L-form NC-7, which is a derivative of an Escherichia coli K-12 strain (16), possesses a secondary calcium/proton antiporter (17) and reveals a general inhibition of growth following addition of the calcium chelator EGTA (15 [but also see reference 23]). NC-7 is therefore an ideal model system for exploring the hypothesis of an enzoskeleton controlled by calcium.Effects of divalent ions on growth. First, the identity of the L-forms derived from E. coli (16) was confirmed by PCR amplification of ftsZ, hisS, and orf80 to obtain products of the expected M r (20), sequencing of 210 bp of the glnA gene (cloned at random), and N-terminal sequencing of Dps, YfiD, and the E1 component of the pyruvate dehydrogenase complex (4). Then, to study the effects of divalent ions, cells were preincubated in modified Na-Davis medium plus 1 mM EGTA and 2 M ionophore A23187 (to equilibrate internal and external calcium levels) for 3 h, harvested in the exponential phase of growth by centrifugation (1,000 ϫ g for 10 min), washed once with growth medium, and resuspended in modified Na-Davis medium containing either 0.2 or 0.5 mM BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid]. Modified Na-Davis medium contains 0.7 g of K 2 HPO 4 , 0.2 g of KH 2 PO 4 , 1 g of (NH 4 ) 2 SO 4 , 0.5 g of sodium succinate, 2 g of peptone, 1 g of yeast extract, 2 g of glucose, 0.34 M NaCl, 10 5 U of penicillin G, and 50 M (each) FeCl 2 , ZnCl 2 , MgCl 2 , CaCl 2 , and MnCl 2 . Cells were agitated at 45 rpm and 32°C, and plastic tubes were used throughout. Following preincubation, cells were trans...

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