The biosynthesis and regulation of bacterial prodiginines

The red-pigmented prodiginines are bioactive secondary metabolites produced by both Gram-negative and Gram-positive bacteria. Recently, these tripyrrole molecules have received renewed attention owing to reported immunosuppressive and anticancer properties. The enzymes involved in the biosynthetic pathways for the production of two of these molecules, prodigiosin and undecylprodigiosin, are now known. However, the biochemistry of some of the reactions is still poorly understood. The physiology and regulation of prodiginine production in Serratia and Streptomyces are now well understood, although the biological role of these pigments in the producer organisms remains unclear. However, research into the biology of pigment production will stimulate interest in the bioengineering of strains to synthesize useful prodiginine derivatives.     

灵菌红素研究进展

灵菌红素是一类天然红色素的总称,是一些放线菌、沙雷菌及其他细菌的次级代谢产物,具有抗细菌、抗真菌、抗疟疾、免疫抑制和抗肿瘤等生物活性,因此受到人们的广泛关注.我们对灵菌红素的性质、生物活性及其生产现状进行了阐述,并预测了其发展趋势.     

Streptomyces sp. JS520 produces exceptionally high quantities of undecylprodigiosin with antibacterial, antioxidative, and UV-protective properties

A Gram-positive, red-pigment-producing bacterial strain, designated JS520 was isolated from the pristine sediment from the cave on mountain Miroc in Serbia. Strain was confirmed to belong to Streptomyces genus based on phenotypic and genetic analysis. Streptomyces sp. JS520 has the ability to produce exceptionally high amounts of deep red pigment into both solid and liquid media. Liquid chromatography and mass spectroscopy of the purified pigments revealed the major component to be undecylprodigiosin (93?%) with minor component being oxidatively cyclized derivative. The pigment production was affected by medium composition, temperature, pH, and the aeration rate. By medium optimization, yields of undecylprodigiosin of 138?mg?l^?1 were achieved, what is the highest level of undecylprodigiosin production reported for the members of Gram-positive Streptomyces genus. Purified pigment had antimicrobial properties against bacterial Bacillus and Micrococcus species (50?μg?ml^?1) and against Candida albicans species (100–200?μg?ml^?1 range). The ability to affect auto-oxidation of the linoleic acid was demonstrated for the purified undecylprodigiosin, suggesting antioxidative properties of this pigment. Multiple ecophysiological roles of the pigment were revealed by comparing cultures grown under pigment-producing and pigment-nonproducing conditions. Cells grown under undecylprodigiosin-producing conditions could tolerate presence of hydrogen peroxide exhibiting three times smaller zones of inhibition at 100?mM H₂O₂. Undecylprodigiosin-producing cells were also less susceptible to tetracycline, kanamycin, chloramphenicol, and 8-hydroxyquinoline. While the growth of the cells not producing pigment was completely inhibited by 15?min of exposure to ultraviolet light (254?nm), cells producing undecylprodigiosin and cells supplied with purified pigment in vitro showed survival rates at 22 and 8?%, respectively.     

A novel medium for the enhanced cell growth and production of prodigiosin from <Emphasis Type="Italic">Serratia marcescens</Emphasis> isolated from soil

Background

Prodigiosin produced by Serratia marcescens is a promising drug owing to its reported characteristics of having antifungal, immunosuppressive and antiproliferative activity. From an industrial point of view the necessity to obtain a suitable medium to simultaneously enhance the growth of Serratia marcescens and the pigment production was the aim of this work. The usage of individual fatty acid as substrate in industries would be cost-effective in the long run and this paved the way for us to try the effect of different fatty acid-containing seeds and oils of peanut, sesame and coconut as source of substrate.

Results

The addition of sugars only showed slight enhancement of prodigiosin production in nutrient broth but not in fatty acid containing seed medium. The powdered peanut broth had supported better growth of Serratia marcescens and higher yield of prodigiosin when compared with the existing nutrient broth and peptone glycerol broth. A block in prodigiosin production was seen above 30°C in nutrient broth, but the fatty acid seed medium used by us supported prodigiosin production upto 42°C though the yields were lower than what was obtained at 28°C. From the results, the fatty acid form of carbon source has a role to play in enhanced cell growth and prodigiosin production.

Conclusion

We conclude by reporting that the powdered and sieved peanut seed of different quality grades were consistent in yielding a fourty fold increase in prodigiosin production over the existing media. A literature survey on the composition of the different media components in nutrient broth, peptone glycerol broth and the fatty acid containing seeds and oils enabled us to propose that the saturated form of fatty acid has a role to play in enhanced cell growth and prodigiosin production. This work has also enabled us to report that the temperature related block of prodigiosin biosynthesis varies with different media and the powdered peanut broth supports prodigiosin production at higher temperatures. The medium suggested in this work is best suitable from an industrial point of view in being economically feasible, in terms of the higher prodigiosin yield and the extraction of prodigiosin described in this paper is simple with minimal wastage.

     

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.     

New prodigiosin-like pigment from Alteromonas rubra.

The red prodigiosin-like pigment from Alteromonas rubra was shown to be a mixture of prodigiosin (pigment 1) and a new cyclic isomer (pigment 2). The new structure was elucidated by mass and nuclear magnetic resonance spectra. Careful examinations of the prodigiosins produced by Serratia marcescens, Vibrio psychoerythrus, and an unidentified red bacterium (LL-100-6) failed to disclose any of the new pigment, pigment 2.     

Genetic and biochemical characterization of the red gene cluster of Streptomyces coelicolor A3(2)

Production of the red antibiotic, undecylprodigiosin, by Streptomyces coelicolor A3(2) was studied by DNA cloning and biochemical analysis. Over 21 kb of genomic DNA were cloned, in several segments, into plasmid vectors. The cloned DNA 'complemented' several specific mutations in the red gene cluster. Four red genes (redA, B, E, and F) were mapped to different regions within the cloned DNA. Screening with redE probes for DNA homologies among various streptomycetes revealed hybridizing DNA in three strains, one of them not known to synthesize prodigiosin pigments. Biochemical studies using protoplasted cells revised our interpretation of the nature of redE and redF mutations. Two forms of undecylnorprodigiosin: S-adenosylmethionine O-methyltransferase activity on gel filtration columns were detected: a very high molecular mass peak (greater than 5 MDal) and a 49 kDal) and a 49 kDal peak. Analyses of extracts from red mutants suggested that these two forms are related, and that at least the redE and redF gene products are necessary for O-methyltransferase activity in vivo. Lack of activity of the redE gene in a heterologous host, S. glaucescens, is consistent with the necessity for a biosynthetic complex involving several red gene products for efficient expression. Experiments in liquid antibiotic production medium indicated that prodigiosin compounds in S. coelicolor are examples of 'secondary metabolites' whose synthesis lags behind that of cell mass. The peak of specific activity of O-methyltransferase coincided with the 'late exponential' phase of growth. Thus, understanding the genetic regulation of undecylprodigiosin biosynthesis in S. coelicolor may be relevant to other antibiotic production pathways, and perhaps to 'secondary' metabolism in general.     

New prodigiosin-like pigment from Alteromonas rubra.

The red prodigiosin-like pigment from Alteromonas rubra was shown to be a mixture of prodigiosin (pigment 1) and a new cyclic isomer (pigment 2). The new structure was elucidated by mass and nuclear magnetic resonance spectra. Careful examinations of the prodigiosins produced by Serratia marcescens, Vibrio psychoerythrus, and an unidentified red bacterium (LL-100-6) failed to disclose any of the new pigment, pigment 2.     

Effects of physiological self-crowding of DNA on shape and biological properties of DNA molecules with various levels of supercoiling

DNA in bacterial chromosomes and bacterial plasmids is supercoiled. DNA supercoiling is essential for DNA replication and gene regulation. However, the density of supercoiling in vivo is circa twice smaller than in deproteinized DNA molecules isolated from bacteria. What are then the specific advantages of reduced supercoiling density that is maintained in vivo? Using Brownian dynamics simulations and atomic force microscopy we show here that thanks to physiological DNA–DNA crowding DNA molecules with reduced supercoiling density are still sufficiently supercoiled to stimulate interaction between cis-regulatory elements. On the other hand, weak supercoiling permits DNA molecules to modulate their overall shape in response to physiological changes in DNA crowding. This plasticity of DNA shapes may have regulatory role and be important for the postreplicative spontaneous segregation of bacterial chromosomes.     

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.     

Induction of Prodigiosin Biosynthesis after Shift-Down in Temperature of Nonproliferating Cells of Serratia marcescens

Nonpigmented bacteria obtained by growth of Serratia marcescens at 38 C synthesized prodigiosin at 25 C if certain individual amino acids were added to cultures of nonproliferating cells. In order of effectiveness, the amino acids were: DL-histidine, L-proline, L-hydroxyproline, DL-alanine, L-alanine, DL-aspartic acid, D-alanine, DL-proline, L-serine, L-ornithine, L-glutamic acid, and D-proline. DL-Histidine at its optimal concentration (20 mg/ml) induced formation of prodigiosin (198 mug of prodigiosin per mg of bacterial protein) after incubation of cultures for 54 hr. Lower concentrations (10 mg/ml) of the other amino acids usually were optimum but less prodigiosin was synthesized, and the maximal amount of pigment occurred between 36 and 48 hr. DL-Methionine was not effective alone but at a low concentration (40 mug/ml) enhanced and accelerated biosynthesis of prodigiosin in the presence of other suitable amino acids. Addition of 2 mg of L-proline per ml at 0 hr induced formation of only 30 mug of prodigiosin after incubation for 42 hr, but addition at 36 hr of 5 mg more of L-proline per ml increased synthesis to 120 mug at 42 hr. Again, DL-methionine markedly augmented prodigiosin biosynthesis in these cultures. Synthesis of prodigiosin ceased if cultures were shifted from 25 to 38 C. Prodigiosin biosynthesis by the nonproliferating cells was maximum when cultures were aerated, the amount of bacterial protein was about 2.0 mg/ml, and amino acids were added at 0 hr. Bacteria synthesized prodigiosin most efficiently when they were harvested from aerated cultures grown at 38 C for 24 hr in a complete medium in a fermentor.     

Enhancement effects of cationic contaminants from bacteria on cake layer formation and biofouling on an RO membrane

The model cationic molecule prodigiosin interacted with a polyamide/polysulfone composite reverse osmosis (RO) membrane, resulting in a reduction of the membrane permeation rate. Prodigiosin is an antibacterial agent produced by Serratia marcescens that is frequently isolated from activated sludge of domestic or industrial wastewater. Such molecules respectively secreted or leaked from live or dead cells are thought to affect membrane biofouling. In this study, a cell suspension containing prodigiosin-producing S. marcescens AS-1 wild-type or the non-producing AS-1ΔspnI strain was fed to the thin RO membrane to determine the occlusion ratio on the membrane. Cationic prodigiosin enhanced membrane biofouling by clogging the pores and enhanced the accumulation of the cake layer. The effects remarkably recovered the occlusion ratio after removing the cake layer by feeding with water. After temporary pressure relief, the occlusion ratios for AS-1 and AS-1ΔspnI were recovered to stable levels from approximately 70 to 49% and 23%, respectively. Zetapotential analysis supported the neutralization effects leading to the accumulation of bacterial cells under applied high pressure for RO membrane permeation.     

Genome-Wide Screening and Identification of Factors Affecting the Biosynthesis of Prodigiosin by Hahella chejuensis,Using Escherichia coli as a Surrogate Host

A marine bacterium, Hahella chejuensis, recently has attracted attention due to its lytic activity against a red-tide dinoflagellate. The algicidal function originates from its red pigment, prodigiosin, which also exhibits immunosuppressive or anticancer activity. Genome sequencing and functional analysis revealed a gene set contained in the hap gene cluster that is responsible for the biosynthesis of prodigiosin. To screen for the factors affecting the prodigiosin biosynthesis, we constructed a plasmid library of the H. chejuensis genomic DNA, introduced it into Escherichia coli strains harboring the hap cluster, and observed changes in production of the red pigment. Among the screened clones, hapXY genes whose products constitute a two-component signal transduction system were elucidated as positive regulators of the pigment production. In addition, an Hfq-dependent, noncoding region located at one end of the hap cluster was confirmed to play roles in regulation. Identification of factors involved in the regulation of prodigiosin biosynthesis should help in understanding how the prodigiosin-biosynthetic pathway is organized and controlled and also aid in modulating the overexpression of prodigiosin in a heterologous host, such as E. coli, or in the natural producer, H. chejuensis.Harmful algal blooms (HABs), commonly called red tide, are a phenomenon in which toxin-producing marine algae rapidly proliferate in the offshore area. The HAB-causing phytoplanktons are reported to interact with other organisms such as bacteria and fungi. Among them, the marine bacteria are known to play important roles in decreasing or developing HABs (3, 5, 14). For instance, Hahella chejuensis, isolated from the coastal area of Marado in South Korea (15), is capable of killing Cochlodinium polykrikoides (12). C. polykrikoides is a major microalga that causes HABs, especially in the Northeast Pacific coastal area (8). The bacterial determinant that kills C. polykrikoides was further characterized as a red pigment referred to as prodigiosin (12). Prodigiosin belongs to a family of tripyrrole antibiotic molecules called prodiginines, which have potential as anticancer agents or immunosuppressants (24). The prodigiosin congener isolated from H. chejuensis also exerts an immunosuppressive effect (11).Through completed genome sequencing of H. chejuensis and its functional analysis, the genomic region involved in biosynthesis of prodigiosin was elucidated (12). This complete set of prodigiosin-biosynthetic genes was named the hap gene cluster. The red pigment prodigiosin was further characterized structurally, and the biosynthetic pathway was proposed by Kim and colleagues (13, 14). Genes of the hap cluster share homology with those in the pig cluster and the red cluster which are involved in prodiginine-biosynthetic intermediates of Serratia marcescens and Streptomyces coelicolor, respectively (7, 23, 25). Enzymes encoded by the genes in the pig and red clusters have been characterized (24). However, gene expression of the hap cluster can be tightly controlled, based on the observation that heterologous expression of the hap cluster alone failed to produce the pigment in Escherichia coli. The recombinant E. coli was able to produce the pigment only when the culture filtrate of H. chejuensis was added to the growth media (12). This result indicates that another regulatory cue is needed for prodigiosin biosynthesis, which prompted us to search for regulatory factors that modulate prodigiosin biosynthesis in H. chejuensis.In this study, regulatory factors for biosynthesis of prodigiosin in H. chejuensis were identified by functional screening. To search for such factors, a plasmid library derived from the genomic DNA of H. chejuensis was constructed and transformed into E. coli strains carrying the hap cluster. In the cases of Serratia marcescens and Streptomyces coelicolor, molecular inputs, such as cell-produced quorum-sensing signal molecules or two-component systems (TCSs) for signal transduction, have been verified as key regulatory signals for prodigiosin biosynthesis so far (4, 9, 10, 20-22). Similarly, some clones of interest uncovered in this study include molecular factors such as those that belong to the TCS. Also, we elucidated that an apparently noncoding region in the hap cluster functions as a key factor of prodigiosin biosynthesis.     

Optimization of the culture condition for an antitumor bacterium Serratia proteamacula 657 and identification of the active compounds

Exploration of novel active anti-tumor compounds from marine microbes for pharmaceutical applications has been a continuously hot spot in natural product research. Bacterial growth and metabolites may greatly vary under different culture conditions. In this study, the effects of different culture conditions and medium components on the growth and bioactive metabolites of Serratia proteamacula 657, an anti-tumor bacterium found in our previous study, were investigated. The results showed that lower temperature, weak acidic condition and solid fermentation favored the bacterial growth and the production of active compounds. Four components in the culture medium, NaCl, peptone, yeast extract and MgSO₄, were found important to the bacterial growth and active compounds production in medium optimization. Under the optimized condition of solid state fermentation at pH 6.0–7.0, 23–25 °C, with the MgSO₄-free medium containing 10.0 g/L peptone, 1.0 g/L yeast extract and 19.45 g/L NaCl, the antitumor activity of S. proteamacula 657 and the yield of crude extracts increased about 15 times and 6 times than the sample obtained in the original liquid fermentation, respectively. The active components in the metabolites of S. proteamacula 657 were identified as a homolog of prodigiosin, a red bacterial pigment, based on the analysis of the NMR and GC–MS. The bacterium S. proteamacula 657, which is adapted to lower temperature, produced prodigiosin-like pigments with highly antitumor activity, suggesting the bacterium is a potential new source for prodigiosin production.     

Recombinant antibodies: engineering and production in yeast and bacterial hosts

After the appearance of the first FDA-approved antibody 25 years ago, antibodies have become major therapeutic agents in the treatment of many human diseases, including cancer and infectious diseases, and the use of antibodies as therapeutic/diagnostic agents is expected to increase in the future. So far, a variety of strategies have been devised for engineering of these fascinating molecules to develop superior properties and functions. Recent progress in systems biology has provided more information about the structures and cellular networks of antibodies, and, in addition, recent development of biotechnology tools, particularly in regard to high-throughput screening, has made it possible to perform more intensive engineering on these substances. Based on a sound understanding and new technologies, antibodies are now being developed as more powerful drugs. In this review, we highlight the recent, significant progress that has been made in antibody engineering, with a particular focus on Fc engineering and glycoengineering for improved functions, and cellular engineering for enhanced production of antibodies in yeast and bacterial hosts.     

Interactions between amyloidophilic dyes and their relevance to studies of amyloid inhibitors

Amyloid fibrils are filamentous aggregates of peptides and proteins implicated in a range of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. It has been known almost since their discovery that these β-sheet-rich proteinacious assemblies bind a range of specific dyes that, combined with other biophysical techniques, are convenient probes of the process of amyloid fibril formation. Two prominent examples of such dyes are Congo red (CR) and Thioflavin T (ThT). It has been reported that in addition to having a diagnostic role, CR is an inhibitor of the formation of amyloid structures, and these two properties have both been explained in terms of the same specific noncovalent interactions between the fibrils and the dye molecules. In this article, we show by means of quartz-crystal microbalance measurements that the binding of both ThT and CR to amyloid fibrils formed by the peptide whose aggregation is associated with Alzheimer's disease, Aβ(1-42), can be directly observed, and that the presence of CR interferes with the binding of ThT. Light scattering and fluorescence measurements confirm that an interaction exists between these dyes that can interfere with their ability to reflect accurately the quantity of amyloid material present in a given sample. Furthermore, we show that CR does not inhibit the process of amyloid fibril elongation, and therefore demonstrate the ability of the quartz-crystal microbalance method not only to detect and study the binding of small molecules to amyloid fibrils, but also to elucidate the mode of action of potential inhibitors.     

Enhanced production of insecticidal prodigiosin from Serratia marcescens TKU011 in media containing squid pen

Using fishery-processing wastes of squid pen powder (SPP) as the sole carbon and nitrogen (C/N) source, Serratia marcescens TKU011 produced prodigiosin. The culture was incubated in 50 mL of medium in an Erlenmeyer flask (250 mL) containing 1.5% SPP at 30 °C for 1 day and then changed to 25 °C for 2 more days. The culture broth had high prodigiosin (0.978 mg/mL). S. marcescens TKU011 grown under illumination conditions in a shaking culture exhibited higher prodigiosin production than when grown under dark conditions contrary to previous reports. The culture supernatant reduced surface tension of water, and the surfactant activity increased when prodigiosin production increased. In this study, the fishery-processing waste, squid pen, was used to produce prodigiosin at greater quantities than reported in other studies, and we found that the prodigiosin had a novel property of insecticidal activity. This method has the potential for developing mass production of prodigiosin.