Electron probe analysis, X-ray mapping, and electron energy-loss spectroscopy of calcium, magnesium, and monovalent ions in log-phase and in dividing Escherichia coli B cells

Abstract

The elemental composition of individual cells of rapidly frozen and cryosectioned Escherichia coli B was measured with electron optical microanalytic methods. The Ca content was high (26.2 mmol/kg) in a 10-nm-wide region of the cell envelope. Amounts of cytoplasmic Ca in actively dividing cells were significantly higher (32.6 mmol/kg [dry weight]) than in the log-phIse (1.5 mmol/kg) cells. Cellular Mg was 205 mmol/kg (dry weight) and it was uniformly distributed throughout the cell. Cells washed in distilled water before freezing lost monovalent ions (Na, Cl, and K), but the membrane-bound Ca and cellular Mg were not reduced, indicating that cellular Mg and membrane Ca are more tightly bound.The ability of bacteria to actively transport and concentrate inorganic ions is well known (11,19), but uncertainty exists about their intracellular distribution and exchange ability. For example, recent authors (17) found that the Mg content of Escherichia coli is higher than had been previously reported (14, 16) and suggested that it was bound to DNA in the nucleoid. Until now, bacterial electrolytes have been measured with bulk chemical methods, primarily atomic absorption spectroscopy. However, with such methods, it is not possible to determine the intracellular distribution and proportion of membrane-bound ions nor to detect fluxes in putative ionic messengers, such as Ca2+, that may occur in a small proportion of a partially synchronized population. In contrast, electron probe microanalysis (EPMA), X-ray mapping, and electron energy loss spectroscopy (EELS) of ultrathin cryosections are techniques uniquely suitable for measuring the subcellular concentration and distribution of various elements (28) and have been used previously to demonstrate the distribution of calcium and other elements in bacterial spores (1,30,31). We used these latter methods to quantitate and determine the subcellular distribution of Mg2+, Ca2+, Na+, K+, and other elements (P, S, and Cl) in E. coli. In addition to these measurements of cell composition and elemental distribution, we demonstrated the high Ca concentration in E. coli envelopes and showed that the cytoplasmic Ca2+ was increased in dividing cells. A preliminary report of some of these findings has been published (4). Rapid freezing. The centrifuged pellets were scraped with a spatula, and the material was transferred from the spatula to a bamboo stick 0.4 mm in diameter. Upon transferring the material onto the stick, the stick was immediately frozen by plunging it manually into supercooled Freon 22 (E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.) at -164°C. Frozen specimens were sectioned and were freeze-dried as described in detail by Somlyo and Silcox (29). A schematic diagram of the experiment described above is shown in Fig. 1. MATERIALS AND METHODSA representative electron micrograph of a cryosection of a dense region of a pellet is shown in Fig. 2a, and images of freeze-substituted bacteria (the latter not used for analysis) are shown in Fig. 2b. The latter specimen wa...

The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins

“…Although Ca 2ϩ was identified as the metal ion bound in the active site of syringacin M, both Mg 2ϩ and Ca 2ϩ have been shown to be relatively abundant in the periplasm of Gram-neg- ative bacteria, at least under the conditions tested, with Ca 2ϩ ions concentrated therein relative to the cytoplasm or extracellular environment (55,56). Because the ionic composition of the periplasm, where colicin M-like bacteriocins are active, is highly dependent on external conditions, flexibility in the use of metal ion cofactors could be advantageous, so this class of bacteriocins may have evolved to utilize both Mg 2ϩ and Ca 2ϩ as a cofactor.…”

The Nonbilayer/Bilayer Lipid Balance in Membranes

“…Mg 2ϩ binds strongly to oligo-and polyphosphates (42) and adsorbs to and lowers the bilayer surface potential of phospholipids like PG (6,43). The intracellular concentration of Mg 2ϩ is Ϸ100 mM in E. coli (44), and A. laidlawii membranes (lipids) bind Ϸ25% that in the E. coli cell envelope (114 mol/g) (44,45). Hence, Mg 2ϩ is present and important for the DGlcDAG synthase (Fig.…”

Diadenosine 5′,5′′′‐P1,P4‐tetraphosphate (Ap4A) controls the timing of cell division in Escherichia coli

“…Second, a single flux of calcium ions triggers cell cycle events, including cell division in E. coli (Norris et al 1988;Holland et al 1990). This speculation is based upon the following observation: the level of free calcium ions is highly regulated (Gangola & Rosen 1987), amounts of which are significantly greater in dividing cells (Chang et al 1986) and cell division is sensitive to inhibitors of calmodulin, which is often involved in calciummediating action in eukaryotes (Holland et al 1990). Third, a certain internal metabolite, that may be synthesized and accumulated to a critical threshold level, induces cell division (D'Ari et al 1990).…”