Prodigiosin Biosynthesis Research Articles

The relationship between prodigiosin biosynthesis and cyclic depsipeptides in Serratia marcescens.

SUMMARY: The concentrations of lipid-bound serine (in the form of cyclic depsipeptide) and prodigiosin, in Serratia marcescens respond similarly to changes in environmental or cultural conditions so that highly pigmented cells always have a relatively large amount of lipid-bound serine. In addition, when prodigiosin synthesis is induced in non-pigmented cells, a parallel rapid synthesis of cyclic depsipeptide occurs concomitantly. Their simultaneous occurrence may indicate a common control.

Influence of temperature of incubation and type of growth medium on pigmentation in Serratia marcescens.

Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize prodigiosin when suspended in saline at 27 C when casein hydrolysate was added. These suspensions produced less prodigiosin at a slower rate than did cultures growing in casein hydrolysate at 27 C without prior incubation at 38 C. Optimal concentration of casein hydrolysate for pigment formation by suspensions was 0.4%; optimal temperature was 27 C. Anaerobic incubation, shift back to 38 C, killing cells by heating, or chloramphenicol (25 mug/ml) inhibited pigmentation. Suspensions of washed cells forming pigment reached pH 8.0 to 8.3 rapidly and maintained this pH throughout incubation for 7 days. Measurements of viable count and of protein, plus other data, indicated that cellular multiplication did not occur in suspensions of washed cells during pigment formation. By this procedure utilizing a shift down in temperature, biosynthesis of prodigiosin by washed cells could be separated from multiplication of bacteria.

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.

Incorporation of methionine into prodigiosin

Addition of proline to suspensions of nonpigmented, nonproliferating cells of Serratia marcescens induced biosynthesis of the pigment, prodigiosin. If methionine was included with proline, 4 times as much prodigiosin was formed, although the amount synthesized in the presence of methionine alone was nil. Uniformly 14C-labelled proline and methionine were incorporated into prodigiosin to about 30% the extent of their incorporation into cellular protein. Experiments with [ carboxy- 14C]-, and [ Me- 14C] methionine established that isotope from the methyl group was utilized preferentially for biosynthesis of prodigiosin.

Thiamine-induced formation of the monopyrrole moiety of prodigiosin.

Thiamine stimulates the production of a red pigment, which is chromatographically and spectrophotometrically identical to prodigiosin, by growing cultures of Serratia marcescens mutant 9-3-3. This mutant is blocked in the formation of 2-methyl-3-amylpyrrole (MAP), the monopyrrole moiety of prodigiosin, but accumulates 4-methoxy-2,2,'-bipyrrole-5-carboxaldehyde (MBC) and can couple this compound with MAP to form prodigiosin. Addition of thiamine caused production of MAP, and as little as 0.02 mg of thiamine per ml in a peptone-glycerol medium stimulated production of measurable amounts of prodigiosin. Phosphate salts and another type of peptone decreased the thiamine-induced formation of prodigiosin; yeast extract and glycerol enhanced the formation of this substance. Thiamine also enhanced production of prodigiosin by wild-type strain Nima of S. marcescens. The thiamine antagonists, oxythiamine and pyrithiamine, inhibited thiamine-induced production of MAP and of prodigiosin by the mutant strain 9-3-3, formation of prodigiosin by the wild-type strain Nima, and production of MAP by another mutant, strain WF. The pyrimidine moiety of thiamine was only 10% as effective as the vitamin; the thiazole moiety, only 4%; and the two moieties together, 25%. Various other vitamins tested did not stimulate formation of prodigiosin by strain 9-3-3. Thiamine did not stimulate production of prodigiosin by a single-step mutant that showed the same phenotypic block in prodigiosin biosynthesis as strain 9-3-3. This is not surprising since strain 9-3-3 originated as a result of two mutational events. One event may involve thiamine directly, and the other may involve the biosynthesis of MAP. Thiamine is probably involved in the regulation of the biosynthesis of MAP, because the vitamin or inhibitory antagonists must be added during the early phases of growth in order to be effective.

Prodigiosin synthesis in mutants of Serratia marcesens.

Morrison, D. A. (Harvard College, Cambridge, Mass.). Prodigiosin synthesis in mutants of Serratia marcescens. J. Bacteriol. 91:1509-1604. 1966.-Exchange of biosynthetic intermediates through the culture medium was used to characterize several hundred new color mutants of Serratia marcescens. The general scheme of prodigiosin synthesis as a bifurcated pathway, in which monopyrrole and bipyrrole precursors are synthesized separately and then coupled to form pigment, was confirmed and extended. Mutants of one new class excreted a product likely to be a new intermediate in monopyrrole synthesis, those of a second excreted a new product in the bipyrrole pathway, and those of a third were blocked at early steps in both pathways. Two novel classes of mutants were isolated, in each of which a lack of some product present in Serratia and Escherichia cultures resulted in loss of all steps in prodigiosin biosynthesis.

Inhibition by temperature of the terminal step in biosynthesis of prodigiosin

This chapter presents a discussion on the effects and control of temperature on micro-organisms and microbiological studies. The pioneer microbiologists realized that the rate of growth of a microbe is affected by the environmental temperature, and several studies on the effect of temperature on growth of pure cultures of microorganisms have been made. In a given medium, a microbe grows most rapidly at a particular temperature (or over a small range of temperatures) that is referred to as the ‘optimum temperature’ for growth. In most studies, the optimum temperature for growth is based upon the rate of growth, although it is occasionally based instead on the size of the crop of micro-organisms produced. It is common practice to store stock cultures of micro-organisms on, or in, solidified nutrient media at a temperature near or below the minimum for growth of the organisms on that medium. Anhydric incubators satisfy the requirements of most microbiological laboratories when the need is often only to incubate static cultures under conditions where variations in temperature are not critical. Temperature-gradient incubators have been used to study the effect of temperature on growth of bacteria, yeasts, and algae. A contact thermometer and electronic relay are now almost universally used for control of bath temperature in apparatus used for manometric work.

Inhibition by streptomycin of the biosynthesis of prodigiosin

Inhibition by streptomycin of the biosynthesis of prodigiosin