Protoplast formation of selected Mycobacterium smegmatis mutants by lysozyme in combination with methionine

Yabu, Kunihiko; Takahashi, Shigetoshi

doi:10.1128/jb.129.3.1628-1631.1977

Cited by 11 publications

(4 citation statements)

References 9 publications

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“…The conversion of cells to protoplasts in S medium plus MgCl2 occurred over a period of several days. This slow process is similar to the case in Mycoplasma smegmatis where treatment with lysozyme and methionine led to protoplast formation over a period of 2 weeks (25).…”

Section: 0fsupporting

confidence: 72%

Spontaneous protoplast formation in Methanobacterium bryantii

Jarrell¹,

Colvin²,

Sprott³

1982

J Bacteriol

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Methanobacterium bryantii was found to undergo rapid lysis when grown in a prereduced chemically defined medium under H2-CO2 (4:1, vol/vol). The addition of 20 mM MgCl2 to the medium gave, rather than rapid lysis, a gradual formation of phase-dark spherical bodies which in thin section appeared as true protoplasts. In general, the protoplasts were stabilized by divalent but not monovalent cations and, unlike whole cells, were sensitive to lysis by Triton X-100. Electron microscopic examination revealed that protoplast formation was preceded by a general breakdown of the cell wall with an apparent squeezing out of the protoplast through the degraded wall. The growth of cells was greatly increased and not accompanied by detectable lysis in a medium modified by elevating the levels of nickel and ammonium.

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Section: 0fsupporting

confidence: 72%

Spontaneous protoplast formation in Methanobacterium bryantii

Jarrell¹,

Colvin²,

Sprott³

1982

J Bacteriol

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“…The investigators hypothesized that the spherical state is irreversible because DivIVA requires negative membrane curvature to localize and to form a new pole (Meniche et al, 2014;Lenarcic et al, 2009;Huang & Ramamurthi 2010). However, we noted that spherical mycobacterial cells generated by cell wall digestion (Rastogi & Venkitasubramanian 1979;Yabu and Takahashi 1977;Udou et al, 1982;Udou et al, 1983), rather than DivIVA depletion, have been reported to reform rods. Spheroplasting offered a means to track de novo morphogenesis from spherical cells that were formed independently of DivIVA status.…”

Section: Resultsmentioning

confidence: 68%

DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth

et al. 2018

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In many model organisms, diffuse patterning of cell wall peptidoglycan synthesis by the actin homolog MreB enables the bacteria to maintain their characteristic rod shape. In Caulobacter crescentus and Escherichia coli, MreB is also required to sculpt this morphology de novo. Mycobacteria are rod-shaped but expand their cell wall from discrete polar or subpolar zones. In this genus, the tropomyosin-like protein DivIVA is required for the maintenance of cell morphology. DivIVA has also been proposed to direct peptidoglycan synthesis to the tips of the mycobacterial cell. The precise nature of this regulation is unclear, as is its role in creating rod shape from scratch. We find that DivIVA localizes nascent cell wall and covalently associated mycomembrane but is dispensable for the assembly process itself. Mycobacterium smegmatis rendered spherical by peptidoglycan digestion or by DivIVA depletion are able to regain rod shape at the population level in the presence of DivIVA. At the single cell level, there is a close spatiotemporal correlation between DivIVA foci, rod extrusion and concentrated cell wall synthesis. Thus, although the precise mechanistic details differ from other organisms, M. smegmatis also establish and propagate rod shape by cytoskeleton-controlled patterning of peptidoglycan. Our data further support the emerging notion that morphology is a hardwired trait of bacterial cells.

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“…For measurement of the incorporation of labelled precursors into macromolecules, the method of Yabu & Takahashi (1977) was employed. Spheroplasts were suspended in stabilizing buffer at a concentration of 1 mg/ml protein.…”

Section: Macromolecular Synthesis In Spheroplastsmentioning

confidence: 99%

Preparation and Properties of Spheroplasts from Aspergillus parasiticus with Special Reference to the de novo Synthesis of Aflatoxins

Tyagi

Venkitasubramanian

1981

Journal of Applied Bacteriology

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Spheroplasts were prepared from Aspergillusparasiticus NRRL 3240using~-glucuronidase from Helix pornaria. They were osmotically fragile spherical structures which lysed when suspended in hypotonic buffers. Purity of the preparation was confirmed by phase-contrast microscopy. Maximal conversion of mycelia to spheroplasts was achieved with 48 and 72 h old cultures. Spheroplasts were metabolically active as indicated by the incorporation of labelled thymidine, uridine and leucine into DNA, RNA and proteins, respectively. A significant incorporation of [meth~l-~H] thymidine into trichloroacltic acid-insoluble material suggested the presence of thymidine kinase in this organism. Spheroplasts and lysates demonstrated the ability to incorporate labelled acetate into aflatoxins. Maximum incorporation was observed in those prepared from 96 h old cultures. Lysates were more efficient in de n o w aflatoxin synthesis as compared to intact mycelia and spheroplasts.AFLATOXINS are secondary metabolites produced by certain strains of Aspergillus ~~Q V U S and A . parasiticus. Attempts to prepare cell-free extracts, active in de novo aflatoxin synthesis, from A . parasiticus cultures, by grinding with sea sand in the presence of sucrose/EDTA and mannitol /EDTA buffers proved unsuccessful, indicating that the enzymes involved in aflatoxin biosynthesis may be part ofa complex whose native conformation is altered by drastic treatments. The conversion of mycelia to spheroplasts followed by osmotic lysis to yield lysates might preserve the integrity of the enzyme complex. Hence a study was made of gentler methods of disrupting the mycelium. In this paper, we report the method used for studying the incorporation of [1-I4C] acetate into aflatoxins in spheroplasts and their lysates prepared from A . parasiticus. Materials and Methods ChemicalsAsparagine, Tris (hydroxymethyl) aminomethane, fl-mercaptoethanol, 2,5-diphenyloxazole, 1,4-bis[2-(5-phenyloxazolyl)] benzene, bovine serum albumin and pglucuronidase (from Helix pornaria) were purchased from Sigma Chemical Co., St.

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Protoplast formation of selected Mycobacterium smegmatis mutants by lysozyme in combination with methionine

Abstract: Lysozyme-sensitive mutants of Mycobacterium smegmatis, isolated by nitrosoguanidine treatment, have been converted into protoplasts in a nutritionally enriched medium containing lysozyme and DL-methionine.

Cited by 11 publications

References 9 publications

Spontaneous protoplast formation in Methanobacterium bryantii

Spontaneous protoplast formation in Methanobacterium bryantii

DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth

Preparation and Properties of Spheroplasts from Aspergillus parasiticus with Special Reference to the de novo Synthesis of Aflatoxins

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