Morphology, Growth and Reversion in a Stable L-form of Escherichia coli K12

Abstract

An L-form isolated from Escherichia coli K 12 by sequential treatment with N-methyl-"-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaC12, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaClz was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KC1 replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaC12. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaC1. I N T R O D U C T I O NThe transformation of bacteria into L-forms is accompanied by various morphological, metabolic and antigenic changes (Hijimans, 1962;Reusch & Panos, 1976;Hayami et al., 1979). In bacterial L-forms, the cytoplasmic membrane is the only barrier between the cell and its environment. Stable bacterial L-forms, which can grow without cell walls, are expected to differ considerably from their original bacteria in the structure and function of their plasma membrane (Gilpin et al., 1973). Most stable L-forms require the addition of osmotic stabilizers to the culture medium : salts, sugars or serum components are commonly used. NaCl is not only a good osmotic stabilizer for the growth of bacterial L-forms, but it also affects several physiological processes (Linker & Wilson, 1985). Stable bacterial L-forms which have permanently lost their cell walls but multiply in liquid media (Burmeister & Hesseltine, 1968;Young et al., 1970;Gilpin et al., 1973;Eda et al., 1976Eda et al., , 1979, would prove useful in studies of bacterial transport systems and physiological mechanisms of bacterial cell membranes. The present paper describes some investigations concerning growth, morphological features and reversion of a stable L-form of Escherichia coli. METHODS Strains.The parent strain, Escherichia coli K12 strain 3301, the stable L-form NC7 derived from it, and strain RNC7-1, isolated as a revertant of NC7, were used.Induction of L-forms. E. coli was grown for 3 h on a rotary shaker (250 r.p.m.) in nutrient broth at 37 "C. The cells were centrifuged, washed three times with distilled water and suspended in the same medium containing 50 pg N-methyl-N'-nitro-N-nitrosoguanidine ml-l. The suspension was incubated for 30 min at 37 "C. The cells were harvested, washed once with brain heart infusion (BHI) broth containing 0.5 M-sucrose, and suspended in 15 ml of the same medium containing lysozyme (200pgml-l) and Na2EDTA (0.05%). After 60min at 30"C, the protoplasts were plated on BHI agar containing 0.5 M-sucrose, 1 % (v/v) horse serum, and 750 units penicillin G ml-l. Of the surviving three L-form col...

Molecules into Cells: Specifying Spatial Architecture

“…Take, for example, the peptidoglycan walls of bacteria. Cells stripped of their wall can often grow in highosmolarity medium; L-forms, as they are called, are spherical, devoid of any visible cell wall, and resistant to antibiotics that inhibit wall biosynthesis (123). Some strains can revert to the normal, walled state and bacillary form, and it has been proposed that the new wall must be laid down upon a foundation of peptidoglycan seed molecules.…”

Patch Clamp Studies on Ion Pumps of the Cytoplasmic Membrane ofEscherichia coli

“…It is not clear whether the respiratory chain components and F o F 1 -ATPase (H ϩ -translocating ATPase) are present in the membrane of such preparations, and therefore origin of the membrane is not clear. On the other hand, large protoplasts have been prepared from a penicillin-resistant mutant (7,8). Those protoplasts were still too small to apply for the patch clamp method.…”

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

“…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].…”