A simple, rapid and non-destructive procedure to extract cell wall-associated proteins from Frankia

• A simple cell fractionation procedure was developed to extract cell wall-associated proteins from the nitrogen-fixing actinomycete Frankia. The method was based on washing Frankia mycelia in 62.5 mM Tris-HCl (pH 6.8) buffer supplemented with 0.1% Triton X-100 as solubilizing agent. Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5+/-7.44 pg protein per extraction procedure from exponentially growing cells, corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50+/-0.51%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. Growth of the treated Frankia cells (i.e. cells from which the cell wall-associated proteins were removed) in semi-solid media suggested that these cells were still viable. This technique is of importance for functionality studies of cell wall-associated proteins, particularly for bacteria where traditional cell fractionation methods are difficult to be applied. (C) 2000 Elsevier Science B.V. All rights reserved.

Nyckelord

actinomycetes

cell fractionation

Frankia

gram-positive cell wall

peptidoglycan hydrolases

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