Pigment Formation in L-forms of Serratia Marcescens

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SUMMARY L-forms of Serratia marcescens were produced and serially transferred in osmotically stabilized agar with penicillin. The bacterial form was pigmented, while the L-form colony was not. Lack of colour in the L-form colony was not due to pigment diffusion into the agar; an extract of agar with L-form growth did not show an absorption spectrum for prodigiosin. INTRODUCTION Although considerable information has accumulated about the chemical steps in the synthesis of the red pigment 'prodigiosin' by Serratia marcescens, little is known of the site of synthesis. Purkayastha & Williams (1960) found that acetylhexosamine and pigment content of various fractions of the organism paralleled each other and concluded that this was evidence that the pigment was contained in the cell 'envelope'. From their results, the authors were unable to determine whether the pigment was located in the plasma membrane or wall portion of the cell envelope. However, since lysozyme treatment resulted in the release of a clear supernatant fluid containing hexosamine, they suggested that the 'pigment might be located in the cell or plasma membrane'. L-forms are microbial forms with part or all of their cell wall missing and would, therefore, be a suitable tool for determining the importance of cell wall in pigment production and/or localization. The present studies were designed to prepare L-forms of S. marcescens in order to determine whether they could produce pigment. METHODS Bacteria. A pigmented strain of Serratia marcescens, originally obtained from a throat culture of a patient, was used throughout. Media. Several different osmotically stabilized media were tried for L-form produc- tion and propagation. The best was brain heart infusion agar (Difco)+ 1.8 % sodium chloride + 0.001 % MgS04. 7H20, inactivated (56" for 30 min.) 20 % (v/v) agamma horse serum (Hyland), and 10 % (w/v) yeast extract (Robbins and Fleischmann were both satisfactory). The bacterial form was routinely maintained on brain heart infusion agar. The bacterial form was grown on the medium used for growing the L-form for experiments involving pigment production. Production of L-forms. Bacteria were inoculated as either a pour or streak plate on media containing various amounts of penicillin and incubated aerobically. The plates were examined daily; when L-forms were noted after several days, serial transfers

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