L-Phase variants ofAgromyces ramosus

Horwitz, Alexandra; Casida, L. E.

doi:10.1007/bf02565047

Cited by 7 publications

(2 citation statements)

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“…The effect of magnesium on L-form stability associated with serum was examined on agar medium no. 4 in experiments similar to those in Tables 2 and 3. The inoculum was grown on agar medium no.…”

Section: Resultssupporting

confidence: 56%

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Effects of magnesium, calcium, and serum on reversion of stable L-forms

Horwitz¹,

Casida²

1978

J Bacteriol

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The L-form of Agromyces ramosus was stable in the absence of penicillin when transferred on heart infusion agar containing NaCl and serum. It reverted to its bacterial form, however, when magnesium replaced the serum in this medium. On a dilute medium containing NaCl but lacking serum, the L-form died out unless calcium, magnesium, or serum was added. It grew as the L-form in the presence of calcium or serum but reverted to the bacterial form in the presence of magnesium. Reversion also occurred when magnesium was added to the dilute medium containing serum. Calcium interfered with or prevented the magnesiuminduced reversion. The revertant bacterial form resulting from these studies was not NaCl sensitive, as was the case of the bacterial revertant of this organism produced in soil (A. H. Horwitz and L. E. Casida, Jr., Can. J. Microbiol, 24:50-55, 1978).Horwitz and Casida (4) described L-form induction by penicillin or glycine for the filamentous soil bacterium Agromyces ramosus. These L-forms were extremely stable. Over 40 transfers were made during a 5-year period on media lacking penicillin and glycine without a single case of reversion. Moreover, no reversion occurred when conventional reversion techniques, such as gelatin treatment or hard agar (3), were used. In contrast to these results, Horwitz and Casida (5) obtained reversion of this L-form to a bacterial form by incubating it in soil. The bacterial form, however, differed from the original bacterial form in that it could be reinduced into the L-form merely by raising the NaCl content of the growth medium. This medium did not contain serum. The study did not indicate the nature of the agent that caused the reversion. Magnesium would appear to be suspect, however, since it is required as a cofactor in several steps of cell wall synthesis (2) and is an absolute requirement for the reversion of protoplasts of Bacillus subtilis (8) and Streptomyces coelicolor (9). The present study, therefore, considers the possible effects of magnesium, calcium, and serum on reversion of the stable Lform of A. ramosus. . nents, except fructose, were Difco products (Difco Laboratories, Detroit, Mich.). Medium no. 2 (Table 1) was heart infusion broth (2.5%, wt/vol) supplemented with 0.96% (wt/vol) NaCl. In combination with the NaCl already present in this medium, this resulted in a final NaCl concentration of 0.25 M. PPLO serum fraction at 1% (wt/vol) was added after sterilization. Medium no. 3 consisted of heart infusion broth (0.25%, wt/vol), yeast extract (0.1%, wt/vol), and 1.41% NaCl, which resulted in a final NaCl concentration of 0.25 M. The pH was adjusted to 7.4. Medium no. 4 was medium no. 3 supplemented with fructose (0.1%, wt/vol). Medium no. 8 was medium no. 4 without added NaCl. The fructose (crystalline; Sigma Chemical Co., St. Louis, Mo.) was autoclaved separately as a 10% (wt/vol) solution and added to the sterile medium at 1.0% (vol/vol). Difco agar was added to the above media at 1.5% (wt/vol). All inorganic chemicals were analytical reagent grade and wer...

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Section: Resultssupporting

confidence: 56%

“…Observation of L-form and reverted cells and colonies. The procedures for observation were those described by Horwitz and Casida (4,5).…”

Section: Methodsmentioning

confidence: 99%

Effects of magnesium, calcium, and serum on reversion of stable L-forms

Horwitz¹,

Casida²

1978

J Bacteriol

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A gromyces

Evtushenko¹,

Ariskina²,

Prisyazhnaya³

et al. 2017

Bergey's Manual of Systematics of Archaea and Bacteria

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Ag.ro.my'ces. Gr. n. agros field or soil; N.L. masc. n. myces (from Gr. masc. n. mykes ‐etis ) fungus; N.L. masc. n. Agromyces soil fungus. Actinobacteria / Actinobacteria / Micrococcales / Microbacteriaceae / Agromyces Young cultures usually produce thin ( mostly 0.3–0 . 6 μ m in diameter ) branched vegetative hyphae, short branching filaments, and occasionally form irregular rods that subsequently break up into diphtheroid, rod like to coccoid elements . Scant aerial hyphae occur on rare occasions. Nonmotile. Nonsporeforming. Gram‐stain‐positive type of cell wall. Non‐acid fast. Lysozyme sensitive. Colonies are generally yellow or white , circular, 1–2 mm in diameter, opaque, often penetrating into the agar. Chemo‐organotrophs , having a respiratory type of metabolism. Aerobic to microaerophilic . Catalase and oxidase test reactions intensities vary among species. Most strains grow well on standard laboratory media and use a wide range of organic compounds as sole sources of carbon for growth and energy. Some species are nutritionally exacting. Mesophilic; optimal growth at ∼24–30°C; growth range ∼7–40°C, some species show weak growth at 1–5°C. Grow optimally at near neutral or slightly alkaline pH, some can grow at initial pH values up to pH 12. Generally nonhalophilic, some species prefer low salt concentrations. The cell‐wall peptidoglycan is a group B type, based on 2,4‐diaminobutyric acid ( l ‐isomer predominating). Menaquinones are the sole respiratory quinones; the predominant component is typically an unsaturated menaquinone with 12 isoprene units ( MK‐12 ); the next most common components are MK‐11 and MK‐13. Major cellular fatty acids are C 15:0 anteiso, C 17:0 anteiso, and C 16:0 iso Mycolic acids are absent. Principal polar lipids are diphosphatidylglycerol, phosphatidylglycerol, and characteristic glycolipids. The usual natural habitat is soil; occur in other diverse terristrial and aquatic environments and in microbial assemblages associated with plants, animals, and humans. Isolation from unequivocally pathological materials is uncommon; a single case of bacteremia associated with Agromyces mediolanus is reported. DNA G + C content ( mol% ): 65 ( T m )–73 (HPLC). Type species : Agromyces ramosus Gledhill and Casida 1969, 346 AL .

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Isolation of a protoplast‐type L‐form from Streptomyces hygroscopicus

Baudler

Gumpert

1979

J of Basic Microbiology

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Although conversion of rod-shaped or coccal bacterial cells into spheroplasts and protoplasts and the subsequent growth in the L-form state are well-known for many species of different genera (HIJMANS et al. 1969) there are only a few reports about L-forms in Actinomycetales (HORWITZ and CASIDA 1975, BOURGEOIS and BEAMAN 1974, 1976, BEAMAN et al. 1978 and no information about L-forms in Streptomyces strains.I n a previous paper (GUMPERT 1978) we mentioned the induction of L-form growth in Streptomyces hygroscopicus on agar media. I n the following report isolation, growth conditions, and some morphological data will be described.Induction of L-form growth: In contrast to gram-negative and most other grampositive bacteria in which L-forms are induced by penicillin or lysozyme treatment of normal rod-shaped or coccal cells, isolated protoplasts had to be used for L-form induction in 8. hygroscopicus. For obtaining the protoplasts, S. hygroscopicus was grown in a complex glucose-peptone medium supplemented with yeast extract (0.4%) and glycine (lye). After 16-20 hrs shaking at 28 "C mycelial aggregates were harvested by centrifugation and resuspended in a protoplasting medium according to OKANISHI et al. (1954; PM, Tab. 1). Protoplasts formed during a 30-60 min period were purified by ultrafiltration through a glas filter (SCHOTT Jena, pore diameter about 20 pin), centrifuged and inoculated onto an L-induction medium (LIM, Tab. 1). Typical L-colonies are formed during 7 days incubation a t 28 "C under aerobic conditions. Cultivation of L-forms: A subsequent permanent growth in the L-form state could be obtained by transferring L-form colonies onto fresh L-medium (LM, Tab. 1) supplemented with glycine and penicillin or only glycine (1%).L-form growth was also possible in liquid L-medium. Induction of L-form growth and subsequent cultivation are reproducible. Some isolates were cultivated for more than 20 transfers. Until now a stable L-form growing without penicillin and glycine could not be isolated either on plates or in liquid media. Both substances are known as inhibitors of murein biosynthesis, and if they are lowered to a certain concentration (less than I : / , glycine) regeneration to normal filamentously growing hyphal cells takes place.Morphology of L-form colonies: On agar media L-forms grow as characteristic friedegg colonies with a dark centre and a brighter periphery (Fig. 1). The centre consists of L-form cells growing into the agar. I n the peripherical zone there are intact and lysed cells ranging in size from 0.3 to 15 pm in diameter (Fig. 2). L-form cells grow in liquid media as small aggregates consisting of spherical cells often arranged in chains.

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L-Phase variants ofAgromyces ramosus

Cited by 7 publications

References 30 publications

Effects of magnesium, calcium, and serum on reversion of stable L-forms

Effects of magnesium, calcium, and serum on reversion of stable L-forms

A gromyces

Isolation of a protoplast‐type L‐form from Streptomyces hygroscopicus

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