Core‐like and Microtubular Structures in a Stable L‐form of Escherichia coli

Abstract

Although there have been a number of ultrastructural studies on the L-forms induced from various bacterial species, little is known about the presence of intracellular membranous structures in these organisms (2, 4). According to the reports concerning ultrastructures of the L-forms, the L-forms consist of various sizes of cells, and the L-form cells lack a cell wall and have nuclear materials and dispersed ribosomes in the cytoplasm. In general, it is said that intracellular membranous structures seem to be difficult to find in the L-forms and, if they are found, they are observed most frequently in the L-forms induced from gram-positive cocci (4). Only few reports have shown the presence of microtubular structures (4,6,8), and membrane structures (7, 9) in the L-forms.Cores are large, hollow, straight rod-shaped structures found in the cytoplasm of bacteria that were first observed by Abrams et al (1) in 1964. Thereafter, these structures in bacteria have been found almost exclusively in group D streptococci (5,11,12). In the L-forms or protoplasts, cores or core-like structures have been demonstrated in group D streptococcal protoplasts by Abrams et al (1) and Cohen et al (3), and in L-forms of Pseudomonas aeruginosa by Hubert et al (10). There have been no reports concerning cores in L-forms of Escherichia coli.We have already reported on intracellular membrane structures observed in the L-forms induced from Staphylococcus aureus (8), Escherichia coli (7), and Proteus mirabilis (9), and in this paper we demonstrate core-like and microtubular structures found in a stable L-form of Escherichia coli by electron microscopy.An L-form of Escherichia coli strain EcL (7) used was subcultured and maintained in L-form broth composed of 3.7% brain heart infusion broth (Difco), 0.5% yeast extract (Difco), 4% NaCl, and 10% horse serum (Bio Test) for several years. The L-form is stable in the absence of antibiotics and has been maintained for several years without any evidence of reversion during routine laboratory manipulations. This strain reached maximum growth at 24 hr in the L-form broth.The L-form, which was cultured at 37 C for 24 hr in the L-form broth, was harvested by centrifugation at 10,000 g for 30 min (Sorvall RC-2B) at 4 C. Sediments were prefixed for 2 hr at 4 C with chilled 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and were then harvested by centrifugation. The fixed cells were washed four times with the same buffer in the cold, followed by postfixation with 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) for 2 hr at 4 C. After dehydration in a graded acetone series, specimens were embedded in Epon 812 . Ultrathin sections were obtained with a Reichert OM U2 ultramicrotome and stained with 2% uranyl acetate and Reynolds lead citrate. Preparations were examined and photographed with a Hitachi HU-12 AS electron microscope at 75 kV acceleration.The E. coli L-form strain EcL consisted of a number of large and small cells measuring 250 nm to 3 pm in size. Each of the cells was spherical i...

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

“…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.…”

Bacteriophages in L form of Staphylococcus aureus

“…Tubular and other regular structures have been described in earlier electron microscopic studies of the L forms of various species of bacteria (6,8,9,11). Although some of these structures, occuring in the L form of Proteus mirabilis, were described as resembling incomplete phages (phage-like structures) (10,14), there has until now been no evidence for complete phages in thin-sectioned L-form colonies.…”