Effect of Iron and Salt on Prodigiosin Synthesis in Serratia marcescens
Serratia marcescens wild-types ATCC 264 and Nima grew but did not synthesize prodigiosin in a glycerol-alanine medium containing 10 ng of Fe per ml. Wild-type 264 required the addition of 0.2 μg of Fe per ml for maximal growth and prodigiosin synthesis; Nima required 0.5 μg of Fe per ml. Three percent, but not 0.1%, sea salts inhibited prodigiosin synthesis in a complex medium containing up to 10 μg of Fe per ml. NaCl was the inhibitory sea salt component. The inhibition was not specific for NaCl; equimolar concentrations of Na2SO4, KCl, and K2SO4 also inhibited prodigiosin synthesis. Experiments with strains 264 and Nima and with mutant WF which cannot synthesize 4-methoxy-2-2′-bipyrrole-5-carboxyaldehyde (MBC), the bipyrrole moiety of prodigiosin, and with mutant 9-3-3 which cannot synthesize the monopyrrole moiety 2-methyl-3-amylpyrrole (MAP) showed that both MBC synthesis and the reaction condensing MAP and MBC to form prodigiosin were relatively more sensitive to NaCl inhibition than the MAP-synthesizing step. The capacity of whole cells to condense MAP and MBC was present, but inactive, in cells grown in NaCl; removal of the NaCl from non-proliferating salt-grown cells restored the activity. Other evidence suggests the existence of a common precursor to the MAP- and MBC-synthesizing pathways.
Induction of Pigmentation in Nonproliferating Cells of Serratia marcescens by Addition of Single Amino Acids
Addition of casein hydrolysate to suspensions of washed, nonpigmented, nonproliferating Serratia marcescens incubating at 27 C induced biosynthesis of prodigiosin. Four amino acids of casein hydrolysate, dl-aspartic acid, l-glutamic acid, l-proline, and l-alanine caused formation of pigment when added individually. dl-Ornithine also was effective. Optimal concentrations for maximal pigmentation were 5 to 10 mg/ml; at these high concentrations, d-serine also induced biosynthesis of some prodigiosin. dl-Alanine and -ornithine were as effective as the l-iosomers, but l-glutamic acid and l-proline gave better responses than their racemic mixtures. Kinetics of prodigiosin biosynthesis after addition of dl-alanine (20 mg/ml) were similar to those of cells suspended in 0.2% casein hydrolysate. The other amino acids were less effective. Addition of 5 mg of dl-alanine or casein hydrolysate per ml to minimal medium increased by 30% the amount of prodigiosin formed by growing cells after incubation for 7 days at 27 C. Cultures grown for 7 days at 27 C in 0.2% casein hydrolsate formed more prodigiosin than did suspensions of nonproliferating cells containing individual amino acids or casein hydrolysate. However, more pigment was produced by cells suspended in l-alanine (5 mg/ml) or l-proline (10 mg/ml) than when suspended in 0.4% natural or synthetic casein hydrolysate. Filtrates from suspensions of nonproliferating cells forming pigment in l-proline induced more rapid formation of prodigiosin, but filtrates from suspensions in dl-alanine did not. The data supported the hypothesis that pyrrole groups of prodigiosin may be synthesized from 5-carbon amino acids such as proline, ornithine, aspartic, and glutamic acids, but the role of alanine is unknown.
FACTORS INFLUENCING PIGMENT PRODUCTION IN A MUTANT STRAIN OF SERRATIA MARCESCENS
A high frequency of mutations affecting color production is characteristic of the HY strain of Serratia marcescens. After ultraviolet irradiation, mutants ranging from white, through pink and orange, to deep red shades darker than the wild type can be obtained (Labrum and Bunting, 1953). Many of the white and pink mutants emit diffusible substances which can be utilized by other color mutants growing in the same medium to produce red pigment (Rizki, 1954a, b). Some of these substances may be precursors of prodigiosin, the red pigment of the parent strain, as shown by the isolation and chemical analysis of one of these diffusible products from a mutant strain (Santer and Vogel, 1956). It is hoped that this approach to the biosynthesis of red pigment in S. marcescens will help to elucidate the pathways of its production. With this ultimate aim, numerous mutants of S. marcescens have been isolated and tested for their interaction with other mutants, i.e., whether they produce any substance which can be utilized by other mutants to form red pigment, or whether a particular mutant will respond to the presence of another mutant by developing red pigment. During this survey of mutant types, an unusual mutant was isolated and this report presents the conditions under which this mutant of S. marcescens will produce red pigment.