Separation of Prodigiosenes and Identification as Prodigiosin Syntrophic Pigment from Mutant Pairs of Serratia marcescens

Countercurrent distribution is capable of resolving mixtures of closely related prodigiosene pigments. Syntrophic pigment produced by several pairs of Serratia marcescens color mutants was identified as prodigiosin (2-methyl-3-amyl-6-methoxyprodigiosene) by countercurrent distribution, soda lime pyrolysis, and other techniques. The metabolic block of mutant strain H-462, derived from parent strain HY, was located between the blocks of mutant strains OF and WF, both derived from parent strain Nima.     

Strain improvement of Serratia marcescens ECU1010 and medium cost reduction for economic production of lipase

Industrial strain improvement plays a central role in the commercial development of microbial fermentation processes. The strain of Serratia marcescens ECU1010, a wild-type lipase-producer capable of stereospecific synthesis of a Diltiazem precursor, was subjected to physical mutation involving treatment by UV-irradiation for 30 s. A mutant strain, no. UV-01, showed enhanced lipase production, but lost the capability of producing red pigment (prodigiosin). The variant strain UV-01 had a 2.3-fold higher activity than the wild type and was stable in its enzyme production for ten serial transfers. For reduction of the fermentation medium cost, dried powder of corn steep liquor was used as an inexpensive substitute for beef extract in the medium. Dextrin as an organic carbon source and Tween-80 as an important element were further optimized, respectively. The high primary biodegradation of the Tween-80 by S. marcescens ECU1010 and its variant demonstrated their potential ability of degrading alkyl polyethoxylates to remove harmful nonionic surfactants from polluted effluents and streams. The optimal cultivation time for lipase biosynthesis was 24 h. These optimized compositions resulted in an economic production of lipase by S. marcescens ECU1010 var. UV-01, with a dramatically reduced cost (1/8–1/7 of the initial one) which is more suitable for industrial application.     

Cyclic-AMP inhibition of fimbriae and prodigiosin production by Serratia marcescens is strain-dependent

The cyclic-nucleotide 3′,5′-cyclic AMP (cAMP) is an ancient and widespread regulatory molecule. Previous studies have shown that fimbria production and secondary metabolite production are inhibited by cAMP in the prokaryote Serratia marcescens. This study used genetic manipulations to test the strain specificity of cAMP–cyclic-AMP receptor protein regulation of fimbria production and of the red pigment, prodigiosin. A surprising amount of variation was observed, as multicopy expression of the cAMP-phosphodiesterase gene, cpdS, conferred either an increase or decrease in fimbriae-dependent yeast agglutination and prodigiosin production depending upon the strain background. Mutation of crp, the gene coding for the cAMP-receptor protein, similarly conferred strain-dependent phenotypes. This study shows that three distinct biological properties, modulated by a conserved genetic regulatory molecule, can vary significantly among strains. Such variation can complicate the functional analysis of bacterial phenotypic properties which are dependent upon global genetic regulators such as cAMP.     

Kinetic analysis of growth rate, ATP, and pigmentation suggests an energy-spilling function for the pigment prodigiosin of Serratia marcescens

Serratia marcescens is a gram-negative environmental bacterium and opportunistic pathogen. S. marcescens expresses prodigiosin, a bright red and cell-associated pigment which has no known biological function for producing cells. We present here a kinetic model relating cell, ATP, and prodigiosin concentration changes for S. marcescens during cultivation in batch culture. Cells were grown in a variety of complex broth media at temperatures which either promoted or essentially prevented pigmentation. High growth rates were accompanied by large decreases in cellular prodigiosin concentration; low growth rates were associated with rapid pigmentation. Prodigiosin was induced most strongly during limited growth as the population transitioned to stationary phase, suggesting a negative effect of this pigment on biomass production. Mathematically, the combined rate of formation of biomass and bioenergy (as ATP) was shown to be equivalent to the rate of prodigiosin production. Studies with cyanide inhibition of both oxidative phosphorylation and pigment production indicated that rates of biomass and net ATP synthesis were actually higher in the presence of cyanide, further suggesting a negative regulatory role for prodigiosin in cell and energy production under aerobic growth conditions. Considered in the context of the literature, these results suggest that prodigiosin reduces ATP production by a process termed energy spilling. This process may protect the cell by limiting production of reactive oxygen compounds. Other possible functions for prodigiosin as a mediator of cell death at population stationary phase are discussed.     

Exploring the bioactive potential of <Emphasis Type="Italic">Serriatia marcescens</Emphasis> VITAPI (Acc: 1933637) isolated from soil

BACKGROUND

Serratia is one of the most important groups of bacteria which produces proteolytic enzymes effectively and known to possess anti-inflammatory properties. The main focus of the current study was to extract the enzyme serratiopeptidase and pigment prodigiosin from Serratia mascescens. Prodigiosin is a red colored pigment produced by the bacterium Serratia marcescens. It is emerging as a valuable molecule because of its large applications. It has already been proved that pigmented strain of Serratia marcescens is less virulent than non-pigmented strains. Moreover the strain we have obtained is from farm soil which indicates that prodigiosin production can be carried safely using this strain.

METHODS

In the present study, the isolate VITASP strain was confirmed by morphological, biochemical and molecular studies. The enzyme and pigment were analyzed for anti-oxidant, anti-inflammatory and cytotoxic properties.

RESULTS

The isolate was further confirmed and identified as Serratia marcescens with 99% similarity. The extracted pigment showed potent radical scavenging effect with 86% and the enzyme was found to inhibit 83%, which was significant in comparison to ascorbic acid standard. The in vitro anti-inflammatory effect of pigment in controlled experimental conditions revealed its protection at 88% and the enzyme with 90%. Aspirin was used as the reference drug. The present findings exhibited a concentration dependent inhibition. The cytotoxic bioassay of pigment showed the IC₅₀ value as (50) μg/mL with 63% cytotoxicity which was statistically significant compared to positive control.

CONCLUSION

Therefore, it appears to be an essential remedial and application research. It may turn out to be highly beneficial to mankind in solving many problems associated with human health.

     

Bacterial pigment prodigiosin and its genotoxic effect

A pigment complex has been isolated from the biomass of bacteria Serratia marcescens ATCC 9986 and separated into single fractions by chromatography. An analysis of the fractions by spectrophotometry, thin layer chromatography, NMR spectroscopy and mass spectrometry enabled us to isolate the so-called red fraction, to confirm the identity of the product of the red fraction to the pigment prodigiosin, and to prove its purity. Some features of the toxic action of prodigiosin have been revealed. It has been found for the first time that the pigment is capable of inducing mutations in Salmonella typhimurium TA 100 cells (Ames test) and chromosomal damage in mammalian erythroblasts.     

Prodigiosin from <Emphasis Type="BoldItalic">Vibrio</Emphasis> sp. DSM 14379; A New UV-Protective Pigment

Pigments such as melanin, scytonemin and carotenoids protect microbial cells against the harmful effects of ultraviolet (UV) radiation. The role in UV protection has never been assigned to the prodigiosin pigment. In this work, we demonstrate that prodigiosin provides a significant level of protection against UV stress in Vibrio sp. DSM 14379. In the absence of pigment production, Vibrio sp. was significantly more susceptible to UV stress, and there was no difference in UV survival between the wild-type strain and non-pigmented mutant. The pigment’s protective role was more important at higher doses of UV irradiation and correlated with pigment concentration in the cell. Pigmented cells survived high UV exposure (324 J/m²) around 1,000-fold more successfully compared to the non-pigmented mutant cells. Resistance to UV stress was conferred to the non-pigmented mutant by addition of exogenous pigment extract to the growth medium. A level of UV protection equivalent to that exhibited by the wild-type strain was attained by the non-pigmented mutant once the prodigiosin concentration had reached comparable levels to those found in the wild-type strain. In co-culture experiments, prodigiosin acted as a UV screen, protecting both the wild-type and non-pigmented mutants. Our results suggest a new ecophysiological role for prodigiosin.     

Influence of Serratia marcescens Pigmentation on Cell Concentrations in Aerosols Produced by Bursting Bubbles

For eight strains of Serratia marcescens, increased cell concentrations were found in aerosols produced from bursting bubbles, with concentrations ranging from a maximum of ca. 80 times the bulk concentration for pigmented strains 4180, 933, and 274 to a minimum approximately equal to the bulk concentration for nonpigmented strain 8100. The increased cell concentration in the aerosol was suppressed when pigmented strains were grown at 37°C, a temperature at which the pigment prodigiosin is not synthesized, resulting in lower concentrations similar to those of nonpigmented strains. Strains that produce higher concentrations of prodigiosin after 1, 2, 4, and 8 days of growth show increasing concentrations in bubble-produced drops; duplicate cultures grown at 37°C did not show any increases. In four concurrent experiments, cells starved for 24 h showed greater concentrations than nonstarved cells for chromogenic strain NIMA, whereas for nonchromogenic strain WF, starved cells showed greater concentrations in three cases and a decreased concentration in the fourth. Bacterial concentrations in aerosol drops from bursting bubbles appear to be predominantly influenced by the surface condition of the bacterial cell.     

Apoptosis of human lung adenocarcinoma A549 cells induced by prodigiosin analogue obtained from an entomopathogenic bacterium Serratia marcescens

An entomopathogenic bacterial strain SCQ1 was isolated from silkworm (Bombyx mori) and identified as Serratia marcescens via 16S rRNA gene analysis. This strain produces a red pigment that causes acute septicemia of silkworm. The red pigment of strain SCQ1 was identified as prodigiosin analogue (PGA) with various reported biological activities. In this study, we found that low concentration of PGA showed significant anticancer activity in human lung adenocarcinoma A549 cells, but has little effect in human bone marrow stem cells, in vitro. By exposure to different concentrations of PGA for 24 h, morphological changes and the MTT assay showed that A549 cell line was very sensitive to PGA, with IC₅₀ value about 2.2 mg/L. Early stage of apoptosis was detected by flow cytometry while A549 cells were treated with PGA for 4 and 12 h, respectively. The proportion of dead cells was increased with treatment time or the concentrations of PGA, but it was inversely proportional to that of apoptotic cells. These results indicate that PGA obtained from strain SCQ1 induces apoptosis in A549 cells, but the molecular mechanisms of cell death are complicated, and the S. marcescens strain SCQ1 may serve as a source of the anticancer compound, PGA.     

灵杆菌合成灵菌红素的转录调控

灵菌红素是一种具有多种生物活性的红色素,具有巨大的经济价值和广阔的应用前景。灵杆菌是灵菌红素的生产菌株,同时也是研究灵菌红素合成的模式菌株。本文综述了转录水平上调控灵杆菌合成灵菌红素的研究进展,总结了双(多)组分调控系统、群体感应系统、σ因子和转录因子在调控灵杆菌合成灵菌红素过程中发挥的作用,并对未来的研究方向进行了展望。     

A prodiginine pigment toxic to embryos and larvae of Crassostrea virginica

The red pigment produced by a marine Pseudomonas sp. which causes abnormal development and mortality in developing embryos of the American oyster, Crassostrea virginica, was analyzed. A comparative study of a nonpigmented and two pigmented mutants of the red parental strain indicated that virulence was associated and varied with pigmentation. The use of sonicated cells supported lysing of the pseudomonad cells as the most probable means of pigment release. Crude pigment extracted from the red parental strain and its yellow mutant was toxic to developing oyster embryos. Neither the “pigment” extracted from the white mutant nor dimethyl sulfoxide, used for dissolving the extract, was toxic. Three pigment fractions were demonstrated by thin-layer chromatography even after purification. Studies indicate that only the fraction corresponding to R₁ 0.41 was necessary for virulence. The virulent pigment fraction was identified as belonging to the prodiginine group.     

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.     

Role of prodigiosin and chitinases in antagonistic activity of the bacterium <Emphasis Type="Italic">Serratia marcescens</Emphasis> against the fungus <Emphasis Type="Italic">Didymella applanata</Emphasis>

The molecular features of antagonism of the bacterium Serratia marcescens against the plant pathogenic fungus Didymella applanata have been studied. The chitinases and the red pigment prodigiosin (PG) of S. marcescens were isolated and characterized. Specific antifungal activity of the purified PG and chitinases against D. applanata was tested in vitro. The antagonistic properties of several S. marcescens strains exhibiting different levels of PG and chitinase production were analyzed in vitro with regard to D. applanata. It was found that the ability of S. marcescens to suppress the vital functions of D. applanata depends mainly on the level of PG production, whereas chitinase production does not provide the bacterium with any competitive advantage over the fungus.     

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.     

Mechanical properties of elytra from Tribolium castaneum wild-type and body color mutant strains

Cuticle tanning in insects involves simultaneous cuticular pigmentation and hardening or sclerotization. The dynamic mechanical properties of the highly modified and cuticle-rich forewings (elytra) from Tribolium castaneum (red flour beetle) wild-type and body color mutant strains were investigated to relate body coloration and elytral mechanical properties. There was no statistically significant variation in the storage modulus E′ among the elytra from jet, cola, sooty and black mutants or between the mutants and the wild-type GA-1 strain: E′ averaged 5.1 ± 0.6 GPa regardless of body color. E′ is a power law function of oscillation frequency for all types. The power law exponent, n, averaged 0.032 ± 0.001 for elytra from all genotypes except black; this small value indicated that the elytra are cross-linked. Black elytra, however, displayed a significantly larger n of 0.047 ± 0.004 and an increased loss tangent (tan δ), suggesting that metabolic differences in the black mutant strain result in elytra that are less cross-linked and more pigmented than the other types. These results are consistent with the hypothesis that black elytra have a β-alanine-deficient and dopamine-abundant metabolism, leading to greater melanin (black pigment) production, probably at the expense of cross-linking of cuticular proteins mediated by N-β-alanyldopamine quinone.     

Induced mutation in β-CAROTENE HYDROXYLASE results in accumulation of β-carotene and conversion of red to orange color in pepper fruit

Pepper fruit is typically red, but green, orange and yellow cultivars are gaining consumer acceptance. This color variation is mainly due to variations in carotenoid composition. Orange color in pepper can result from a number of carotenoid profiles, but its genetic basis is only partly known. We identified an EMS-induced orange-fruited mutant using the wild-type blocky red-fruited cultivar ‘Maor’ as progenitor. This mutant accumulates mainly β-carotene in its fruit, instead of the complex pattern of red and yellow carotenoids in ‘Maor’. We identified an A⁷⁰⁹ to G transition in the cDNA of β-CAROTENE HYDROXYLASE2 in the orange pepper and complete co-segregation of this single-nucleotide polymorphism with the mutated phenotype. We therefore hypothesized that β-CAROTENE HYDROXYLASE2 controls the orange mutation in pepper. Interestingly, the expression of β-CAROTENE HYDROXYLASE2 and additional carotenogenesis genes was elevated in the orange fruit compared with the red fruit, indicating possible feedback regulation of genes in the pathway. Because carotenoids serve as precursors for volatile compounds, we compared the volatile profiles of the two parents. The orange pepper contained more volatile compounds than ‘Maor’, with predominant elevation of norisoprenoids derived from β-carotene degradation, while sesquiterpenes predominated in the red fruit. Because of the importance of β-carotene as a provitamin A precursor in the human diet, the orange-fruited mutant might serve as a natural source for pepper fruit biofortification. Moreover, the change in volatile profile may result in a fruit flavor that differs from other pepper cultivars.     

Relationship between Pigment Producibility and Drug Resistance in Serratia marcescens

Among the clinical isolates of Serratia marcescens, non-pigmented cells appeared more frequently from pigmented, drug-resistant strains than from pigmented, drug-sensitive strains. Transfer of R plasmid from Escherichia coli to pigmented strains caused spontaneous loss of pigment producibility, whereas such spontaneous loss never occurred in fresh cultures of drug-sensitive strains. The non-pigmented strain was a better recipient of R plasmid from E. coli than was the pigmented strain. R plasmid was transferred from the non-pigmented strain to the pigmented strain at a higher frequency than from E. coli to the pigmented strain. The results of the present investigation suggest that transfer of R plasmid may be one of the reasons for the significant increase of non-pigmented, drug-resistant strains of S. marcescens in nature.     

Production of Extracellular Pigment by a Mutant of Monascus kaoliang sp. nov

A hyperpigment-producing mutant, R-10847, was derived from Monascus kaoliang F-2 (ATCC 26264) through a series of mutagenesis steps. The mutant produced a large quantity of Monascus pigment when grown in mantou (steamed bread) by solid culture. The mutant produced pigments extracellularly by extruding the pigments outside the cell in a lump together with some viscous substances. The productivity of pigment was about 100-fold greater than that of the wild type. The mutant lost the capability of spore formation, the growth rate decreased, and both the size and quantity of conidia were reduced. The color of the pigment produced by the mutant changed from orange to deep red.