Prodigiosin

A novel red pigment, 2,2V -[3-methoxy-1Vamyl-5V -methyl-4-(1U-pyrryl)] dipyrryl-methene (MAMPDM), which has properties similar to those of prodigiosins, has been isolated for the first time from a bacterium putatively identified as Micrococcus sp. Our studies showed that MAMPDM inhibited proliferation of both human T as well as B cells and murine T cells, in response to polyclonal mitogens, in a concentration-dependent manner while murine B cell proliferation induced by lipopolysaccharide was inhibited only at high concentration. The effect of MAMPDM on constitutive cell cycling was ascertained using four mouse and human tumour cell lines. At 100 nM, the concentration that inhibited con A induced proliferation of mouse spleen cells, the viability of these cell lines was not affected. At 10 -100-fold higher concentration of MAMPDM, however, there was a decrease in cell viability with T cell-derived cell lines being more sensitive. MAMPDM did not block the secretion of IL-2 or expression of CD25 though it inhibited the proliferation of con A stimulated T cells. The higher amount of IL-2 in the supernatant of the con A stimulated T cells, cultured in the presence of the immunomodulator, indicated accumulation of IL-2 due to its reduced utilisation. At inhibitory concentration, MAMPDM induced apoptosis in con A stimulated cells. Thus, MAMPDM may have considerable and selective T cell immunosuppressive potential and appears to act by a mechanism distinct from that of other known immunosuppressors. D

Since microorganisms normally co-exist with other species in nature, they have developed complex metabolic and physiological responses as a result of such interspecies interactions. We utilized some of these interactions by introducing heat-killed cells of Bacillus subtilis and Staphylococcus aureus to Streptomyces coelicolor cultures and, as a result, stimulated undecylprodigiosin production. Undecylprodigiosin is not only an antibiotic; it has also been attributed with antitumor activities, but, in a defined medium, pure cultures of S. coelicolor produced only low concentrations. Elicitation with B. subtilis increased the maximum undecylprodigiosin concentration by threefold and S. aureus by fivefold compared with the pure culture of S. coelicolor. Growth and glucose consumption of elicited S. coelicolor, however, remained similar to those observed in the pure culture. Furthermore, another positive outcome of the elicitation with both B. subtilis and S. aureus was the earlier onset of undecylprodigiosin production by 24 h compared with the pure culture of S. coelicolor. This is the first time that such a phenomenon has been seen in 2L bioreactors. Our work supports the use of biotic elicitation in order to enhance the production of secondary metabolites for industrial-scale applications.

Prodigiosin,  a  pigment  produced  by Serratia  marcescensinhibits  the  growth  of  different  microorganisms  and  the  proliferation  of some human cancer cell lines. Prodigiosin is usually produced by fermentations of substrates such as starch and proteins, andpig-ment yield depends on the concentration of the carbon source, stirring speed of cultures, temperature and time of incubation,nitro-gen  sources,  and  pH  of  medium.  In  the  present  study,  pigment  production  was  assessed  using  two  types  of  casein  as  substrate; pigment yield was greater when casein precipitated with vinegar was used as substrate than when high purity casein was. The maxi-mum prodigiosin production was achieved with 10 g/L casein at pH 8.0. Additionally, supplementation of culture media with glu-cose was found to considerably decrease prodigiosin production and growth inhibition of Staphylococcus aureus, which is directly related to pigment yield. Production in stirred-tank bioreactor at 0.75 vvm aeration was higher than that at 0.5 and 1.0 vvm. Sub-strate type, concentration and pH affected pigment production in Erlenmeyer flasks, whereas aeration rate influenced pigment pro-duction in a stirred-tank bioreactor.

The effects of resident bacteria in the stomach of 5th-instar larvae of Rhodnius prolixus on the erythrocyte lysis and Trypanosoma cruzi infection were studied. The bacteria population increased approximately 10,000-fold after feeding. Hemolysis rose to approximately 28% within 24 h postfeeding and then linearly grew until day 4 attaining almost 100%. The number of surviving Y strain of T. cruzi in the stomach declined drastically, while the infection with Dm28c clone was maintained stable. Five days after feeding, few different types of bacterial colonies were obtained when stomach content suspensions were spread onto BHI agar plates. The hemolytic bacteria were isolated and identified as Serratia marcescens biotype A1a (referenced as RPH), which produces the pigment prodigiosin. In vitro experiments, comparing incubations of RPH with S. marcescens SM365, a prodigiosin pigment producer, and S. marcescens DB11, a nonpigment variant, as a control, with erythrocytes and T. cruzi demonstrated that: (i) at 30°C, SM365 and RPH diminished the populations of Y strain, but not DM28c clone, and DB11 was unable to lyse both T. cruzi strains; (ii) at 0°C, SM263 and RPH killed the flagellates, but DB11 did not; and (iii) all three strains of S. marcescens were able to lyse erythrocytes. These results suggest that S. marcescens trypanolytic activity from the SM365 and RPH strains is distinct from the hemolytic activity and that prodigiosin is an important factor for the trypanolytic action of the bacteria.

An elegant way of forming carbonÀcarbon bonds is the inversion of the normal mode of reactivity of carbonyl compounds. The benzoin condensation (1,2-addition) and the Stetter reaction (1,4-addition) employ synthetically useful umpolung reactivity. The 1,2-addition of aldehydes to a,bunsaturated carbonyl compounds results in the construction of 2-hydroxy ketones, whereas the 1,4-addition of aldehydes provides an access to 1,4-bifunctional molecules. Owing to problems with chemoselectivity, such umpolung reactions are often limited to the homocoupling of aldehydes. Also, efforts to introduce stereoselectivity into the intermolecular Stetter reaction by using various chiral catalysts were mostly not successful. Although the organocatalytic transformations introduced by Enders et al. [4] and Rovis and co-workers offered access to the asymmetric intermolecular Stetter reaction, certain limitations concerning the substrate range, catalytic efficiency, and enantioselectivity remained. In fact, the intramolecular-, and especially the intermolecular asymmetric Stetter reaction is still a great challenge.

redD and actII-ORF4, regulatory genes required for synthesis of the antibiotics undecylprodigiosin and actinorhodin by Streptomyces coelicolor A3(2), were transcribed in vitro by an RNA polymerase holoenzyme containing σhrdD. Disruption of hrdD had no effect on antibiotic production, indicating that redD and actII-ORF4 are transcribed in vivo by at least one other RNA polymerase holoenzyme. These data provide the first

The bacterial growth and the production of pigment by bacteria might be increased in a stressful condition either in the presence or absent of nutrient factors. Many studies proved that nutrients in a certain amount might be able to give a respond to the bacterial growth and the production of secondary metabolites like pigment. However, there have been comparatively few studies on the potential of nutrients availability such as carbon, nitrogen, and inorganic source that can affect to the bacterial growth and pigments production. Therefore, this research is conducted to find out the effect of selected nutrient factors on the bacterial growth and the production of pigment by Serratia marcescens. Serretia marcescens cells were cultivated in media with the addition of difference nutritional factors such as sucrose, peptone and sodium nitrite separately and incubated at 28oC for 7 days. The bacterial density was measured at optical density (OD) at 600 nm for degree of pigmentation analysis. The pigments produced were extracted and characterized using UV-Vis Spectrophotometer and Thin Layer Chromatography (TLC). The growth of S. marcescens is maximum during fifth day of 7 days incubation period especially at stationary phase. The optical density of prodigiosin (OD600) by Biophotometer showed that 1% (w/v) peptone gave the highest value of OD600. The spectrophotometric analysis by UV-Vis Spectrophotometer showed that the red colour pigment was detected at 535.1 nm. The maximum absorption of prodigiosin is detected from the peptone by 0.506 abs, as well as showed the highest degree of pigmentation by 0.398. However, the analytical chromatography by Thin Layer Chromatography (TLC) showed that 1% (w/v) sodium nitrite produce the highest value of Retention factor (Rf) by 0.200 on the TLC plate. Further study on pigment stability as well as biological activity such as antioxidant, anticancer, toxicity and synergistic effects with food preservative should be performed in future research.

The effects of growth rate and nutrient feed rate on the production of actinorhodin (Act) and undecylprodigiosin (Red) were determined in Streptomyces coelicolor A3(2) and in a congenic relA null-mutant known to be deficient in ppGpp synthesis and antibiotic production under conditions of nitrogen limitation. In the relA strain, Act production was inversely related to specific growth rate in continuous cultures limited by glucose, ammonium, or phosphate, while Red biosynthesis was optimal at 0.05 h 3I regardless of the specific nutrient limitation. Production of Act and Red in the relA mutant was lower than that of the parental strain, particularly under conditions of glucose-and ammonium-limitation, indicating an important and general role for ppGpp in determining the onset of the antibiotic biosynthesis under conditions of nutrient limitation. At constant growth rate, but with varying nutrient feed rates, the specific rate of Act production was adversely influenced by increasing levels of glucose, ammonium, and phosphate, with phosphate having the greatest inhibitory effect. Under the same conditions, the specific rate of Red production was stimulated by increasing glucose levels, but markedly decreased by increased levels of phosphate. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. : S 0 3 7 8 -1 0 9 7 ( 9 8 ) 0 0 4 4 6 -7

Prodigiosin is a red pigment produced by Serratia marcescens with apoptotic activity. We examined the mechanism of action of this tripyrrole alkaloid, focusing on its interaction with DNA and its ability to inhibit both topoisomerase I and topoisomerase II. We also evaluated the DNA damage induced in cancer cell lines. Prodigiosin-DNA intercalation was analyzed using a competition dialysis assay with different DNA base sequences. Topoisomerase I and II inhibition was studied in vitro by a cleavage assay, and in cultured cells, by analysis of its ability to form covalent complexes. Furthermore, we analyzed DNA damage by pulse-field gel electrophoresis and by immunocytochemistry. Apoptosis inducing factor (AIF)/phospho-H2AX (p-H2AX) double labeling by confocal microscopy was performed to determine the possible implication of AIF in the prodigiosin-DNA damage. Finally, we studied the ability of this drug to induce copper-mediated DNA damage at different pH by a DNA cleavage assay. Our results demonstrate prodigiosin-DNA interaction in vitro and in cultured cells. It involves prodigiosin-DNA intercalation, with some preference for the alternating base pairs but with no discrimination between AT or CG sequences, dual abolition of topoisomerase I and II activity and, as consequence, DNA cleavage. Prodigiosin-DNA damage is independent of AIF. Furthermore, we found that copper-mediated cleavage activity is associated with pH (occurring at pH 6.8 rather than pH 7.4) and with the Cu 2+ ion concentration. These results indicate DNA a therapeutic target for prodigiosin and could explain the apoptosis mechanism of action induced by this antineoplastic drug.

Members of the ROK family of proteins are mostly transcriptional regulators and kinases that 23 generally relate to the control of primary metabolism, whereby its member glucose kinase 24 acts as the central control protein in carbon control in Streptomyces. Here we show that 25

The growing demand to fulfill the needs of presentday medicine in terms of novel effective molecules has lead to reexamining some of the old and known bacterial secondary metabolites. Bacterial prodigiosins (prodiginines) have a long history of being re markable multipurpose compounds, best examined for their anticancer and antimalarial activities. Production of prodigiosin in the most common producer strain Serratia marcescens has been described in great detail. However, few reports have discussed the ecophysiological roles of these molecules in the producing strains, as well as their antibiotic and UV-protective properties. This review describes recent advances in the production process, biosynthesis, properties, and applications of bacterial prodigiosins. Special emphasis is put on undecylprodigiosin which has generally been a less studied member of the prodigiosin family. In addition, it has been suggested that proteins involved in undecylprodigiosin synthesis, RedG and RedH, could be a useful addition to the biocatalytic toolbox being able to mediate regio-and stereoselective oxidative cyclization. Judging by the number of recent references (216 for the 2 0 0 7-2 0 1 3 p e r i o d) , i t h a s b e c o m e c l e a r t h a t undecylprodigiosin and other bacterial prodigiosins still hold surprises in terms of valuable properties and applicative potential to medical and other industrial fields and that they still deserve continuing research curiosity.

Transcription of redO, the activator gene required for production of the red-pigmented antibiotic undecylprodigiosin by Streptomyces coelicolor A3(2), showed a dramatic increase during the transition from exponential to stationary phase. The increase in redO expression was followed by transcription of redX, a biosynthetic structural gene, and the appearance of the antibiotic in the mycelium, and coincided with the intracellular appearance of ppGpp. However, ppGpp production elicited either by nutritional shiftdown of, or addition of serine hydroxamate to, exponentially growing cultures had no stimulatory effect on redO transcription. The presence of redO on a multicopy plasmid resulted in elevated levels of the redO transcript and production of redX and undecylprodigiosin during exponential growth; the normal growth-phase-dependent production of undecylprodigiosin appeared to be mediated entirely through the redO promoter, which shows limited similarity to the consensus sequence for the major class of eubacterial promoters.

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.

Prodigiosin, a red linear tripyrrole pigment, has long been recognised for its antimicrobial property. However, the physiological contribution of prodigiosin to the survival of its producing hosts still remains undefined. Hence, the aim of this study was to investigate the biological role of prodigiosin from Serratia marcescens, particularly in microbial competition through its antimicrobial activity, towards the growth and secreted virulence factors of four clinical pathogenic bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa) as well as Staphylococcus aureus and Escherichia coli. Prodigiosin was first extracted from S. marcescens and its purity confirmed by absorption spectrum, high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). The extracted prodigiosin was antagonistic towards all the tested bacteria. A disc-diffusion...

Prodigiosin is a red pigment produced by Serratia marcescens with apoptotic activity. We examined the mechanism of action of this tripyrrole alkaloid, focusing on its interaction with DNA and its ability to inhibit both topoisomerase I and topoisomerase II. We also evaluated the DNA ...

The EepR transcription factor positively regulates secondary metabolites and tissue-damaging metalloproteases. To gain insight into mechanisms by which EepR regulates pigment and co-regulated factors, genetic suppressor analysis was performed. Suppressor mutations that restored pigment to the non-pigmented ∆eepR mutant mapped to the hexS ORF. Mutation of hexS also restored haemolysis, swarming motility and protease production to the eepR mutant. HexS is a known direct and negative regulator of secondary metabolites in Serratia marcescens and is a LysR family regulator and an orthologue of LrhA. Here, we demonstrate that HexS directly controls eepR and the serralysin gene prtS. EepR was shown to directly regulate eepR expression but indirectly regulate hexS expression. Together, these data indicate that EepR and HexS oppose each other in controlling stationary phase-associated molecules and enzymes.

The atypical two-component system (TCS) AbrC1/C2/C3 (encoded by SCO4598, SCO4597, and SCO4596), comprising two histidine kinases (HKs) and a response regulator (RR), is crucial for antibiotic production in Streptomyces coelicolor and for morphological differentiation under certain nutritional conditions. In this study, we demonstrate that deletion of the RR-encoding gene, abrC3 (SCO4596), results in a dramatic decrease in actinorhodin (ACT) and undecylprodiginine (RED) production and delays morphological development. In contrast, the overexpression of abrC3 in the parent strain leads to a 33% increase in ACT production in liquid medium. Transcriptomic analysis and chromatin immunoprecipitation with microarray technology (ChIPchip) analysis of the ⌬abrC3 mutant and the parent strain revealed that AbrC3 directly controls ACT production by binding to the actII-ORF4 promoter region; this was independently verified by in vitro DNA-binding assays. This binding is dependent on the sequence 5=-GAASGSGRMS-3=. In contrast, the regulation of RED production is not due to direct binding of AbrC3 to either the redZ or redD promoter region. This study also revealed other members of the AbrC3 regulon: AbrC3 is a positive autoregulator which also binds to the promoter regions of SCO0736, bdtA (SCO3328), absR1 (SCO6992), and SCO6809. The direct targets share the 10-base consensus binding sequence and may be responsible for some of the phenotypes of the ⌬abrC3 mutant. The identification of the AbrC3 regulon as part of the complex regulatory network governing antibiotic production widens our knowledge regarding TCS involvement in control of antibiotic synthesis and may contribute to the rational design of new hyperproducer host strains through genetic manipulation of such systems.

Prodigiosin is a red pigment produced by Serratia marcescens with apoptotic activity. We examined the mechanism of action of this tripyrrole alkaloid, focusing on its interaction with DNA and its ability to inhibit both topoisomerase I and topoisomerase II. We also evaluated the DNA damage induced in cancer cell lines. Prodigiosin-DNA intercalation was analyzed using a competition dialysis assay with different DNA base sequences. Topoisomerase I and II inhibition was studied in vitro by a cleavage assay, and in cultured cells, by analysis of its ability to form covalent complexes. Furthermore, we analyzed DNA damage by pulse-field gel electrophoresis and by immunocytochemistry. Apoptosis inducing factor (AIF)/phospho-H2AX (p-H2AX) double labeling by confocal microscopy was performed to determine the possible implication of AIF in the prodigiosin-DNA damage. Finally, we studied the ability of this drug to induce copper-mediated DNA damage at different pH by a DNA cleavage assay. Our results demonstrate prodigiosin-DNA interaction in vitro and in cultured cells. It involves prodigiosin-DNA intercalation, with some preference for the alternating base pairs but with no discrimination between AT or CG sequences, dual abolition of topoisomerase I and II activity and, as consequence, DNA cleavage. Prodigiosin-DNA damage is independent of AIF. Furthermore, we found that copper-mediated cleavage activity is associated with pH (occurring at pH 6.8 rather than pH 7.4) and with the Cu 2+ ion concentration. These results indicate DNA a therapeutic target for prodigiosin and could explain the apoptosis mechanism of action induced by this antineoplastic drug.

Streptomycetes are exploited for production of a wide range of secondary metabolites, and there is much interest in enhancing the level of production of these metabolites. Secondary metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of the primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by 13 C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely because of accumulation of glucose 6-phosphate and fructose 6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations, we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate. Integrated analysis of gene expression data using a genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate biosynthesis.

Alphaproteobacterium strain MOLA1416, related to Mycoplana ramosa DSM 7292 and Chelativorans intermedius CC-MHSW-5 (93.6% 16S rRNA sequence identity) was isolated from the marine lichen, Lichina pygmaea and its chemical composition was characterized by a metabolomic network analysis using LC-MS/MS data. Twenty-five putative different compounds were revealed using a dereplication workflow based on MS/MS signatures available through GNPS (https://gnps.ucsd.edu/). In total, ten chemical families were highlighted including isocoumarins, macrolactones, erythrinan alkaloids, prodiginines, isoflavones, cyclohexane-diones, sterols, diketopiperazines, amino-acids and most likely glucocorticoids. Among those compounds, two known metabolites (13 and 26) were isolated and structurally identified and metabolite 26 showed a high cytotoxic activity against B16 melanoma cell lines with an IC 0.6 ± 0.07 μg/mL.

The potential of three Serratia marcescens strains (CFFSUR-B2, CFFSUR-B3 and CFFSUR-B4) isolated from tropical regions in Mexico to inhibit the mycelial growth and conidial germination of Colletotrichum gloeosporioides, causal agent of fruit anthracnose, was evaluated. The ability of these strains to produce prodigiosin and chitinases when cultivated in oil seed-based media (peanut, sesame, soybean and castor bean) and in Luria-Bertani medium was determined. All of the strains exhibited similar fungal antagonistic activities and inhibited myceliar growth by more than 40% while inhibiting conidial germination by 81-89% (P = 0.01). The highest level of prodigiosin (40 μg/ml) was produced in the peanut-based medium while growth in soybean-based medium allowed the highest production of chitinases (56 units/ml), independent of the strain used. Strain CFFSUR-B2 grown in peanut medium was used to evaluate the effect of inoculum density and initial pH on metabolite production. The amount of...

The PI3K/AKT/mTOR signaling pathway regulates cell proliferation, survival and angiogenesis. The mammalian target of rapamycin (mTOR) is a protein kinase ubiquitously expressed within cells that regulates cell growth and survival by integrating nutrient and hormonal signals. mTOR exists in two complexes, mTORC1 and mTORC2. Hyperactivation of the mTOR protein has been linked to development of cancer, raising mTOR as an attractive target for cancer therapy. Prodigiosin (PG) and Obatoclax (OBX), two members of the prodiginines family, are small molecules with anticancer properties which are currently under clinical trials. In the present paper, we demonstrate that mTOR is a molecular target of both prodiginines in melanoma, a highly drug-resistant cancer model. The inhibition of mTORC1 and mTORC2 complexes by PG or OBX resulted in a loss of AKT phosphorylation at S473, preventing its full activation, with no significant effect on T308. The strongest activity inhibition (89%) was induced by PG on mTORC2. Binding assays using Surface Plasmon Resonance (SPR) provide kinetic and affinity data of the interaction of these small molecules with mTOR. In addition, in silico modelling produced a detailed atomic description of the binding modes. These results provide new data to understand the mechanism of action of these molecules, and provide new structural data that will allow the development of more specific mTOR inhibitors for cancer treatment.

This study investigated the performance of two different strains of Serratia marcescens on the manganese (II) removal and the effects of the nutrient-rich (NB medium) and nutrient-poor culture (K medium) media on this process. In the NB medium, the pigmented LG1 strain and the non-pigmented CL11 strain exhibited improved growth and higher Mn(II) tolerance (0–2000 mg L−1). In addition, improved Mn(II) removal (64.25%) and increased formation of Mn oxides were observed in this medium, especially for the LG1 isolate. EELS analysis revealed Mn inside the cell for LG1 and EDX analysis revealed Mn outside of the cell for CL11, indicating that the two strains remove Mn(II) via distinct mechanisms. The biooxidation of Mn by the CL11 strain may involve indirect mechanisms which alter the pH of the medium, whereas the LG1 strain may use a direct mechanism for the biooxidation mediated by cellular components such as intracellular proteins. These results demonstrate the biotechnological potential of the two strains. In addition, is the first time the high potential of the NB medium for Mn removal is demonstrated. This medium was found to greatly influence the Mn removal and oxide formation, especially in the case of the pigmented LG1 isolate.

0 The equilibrium and relative rate of rotamer interconversion around the bond joining the 2,2′-bipyrrolyl and pyrromethene moieties in a synthetic analogue of immunosuppressant prodigiosin are investigated as a function of pHapp in a water/acetonitrile mixture (1/1 by volume). Two chromatographically separable isomeric forms are obtained in acid solutions (pHapp < 4), whereas rapid interconversion occurs above neutrality. Furthermore, pH modulates the conformational preference of the molecule according to nitrogen protonation on the three pyrrole rings system (pK a ) 7.2). At high pHapp (neutral form), the same conformer that is observed in pure acetonitrile prevails, whereas the other one is preferred by the protonated form. The nuclear magnetic resonance data indicate that the structures of the two conformers mainly differ in the value of the torsion angle around the aforementioned C−C bond. Kinetic and equilibrium data are quantitatively interpreted with a cyclic mechanism including two protonation (pK a1 ) 8.23 ± 0.03, pK a2 ) 5.4 ± 0.2) and two conformational rearrangement steps. A molecular interpretation of the observed behavior includes, for the preferred conformer at low pH, formation of a new hydrogen bond between the exocyclic oxygen and the neighboring pyrrole NH upon protonation of the three pyrrole rings system. of the Rand -conformers as deduced from NOESY data on PNU-156804 in CDCl3. The significant NOESY cross-peaks are indicated here with double arrows according to the following intensity-related code: heavy lines, strong, thin lines, moderate; and dashed lines, weak. Crosspeak 2 is not detected in this spectrum because of NH1a and NH1b signal overlapping. This NOE is detected in the NOESY spectrum of PNU-156804 (HCl salt) dissolved in CDCl 3, where only the -conformer is present.

Swarming motility and hemolysis are virulence-associated determinants for a wide array of pathogenic bacteria. The broad host-range opportunistic pathogen Serratia marcescens produces serratamolide, a small cyclic amino-lipid, that promotes swarming motility and hemolysis. Serratamolide is negatively regulated by the transcription factors HexS and CRP. Positive regulators of serratamolide production are unknown. Similar to serratamolide, the antibiotic pigment, prodigiosin, is regulated by temperature, growth phase, HexS, and CRP. Because of this co-regulation, we tested the hypothesis that a homolog of the PigP transcription factor of the atypical Serratia species ATCC 39006, which positively regulates prodigiosin biosynthesis, is also a positive regulator of serratamolide production in S. marcescens. Mutation of pigP in clinical, environmental, and laboratory strains of S. marcescens conferred pleiotropic phenotypes including the loss of swarming motility, hemolysis, and severely reduced prodigiosin and serratamolide synthesis. Transcriptional analysis and electrophoretic mobility shift assays place PigP in a regulatory pathway with upstream regulators CRP and HexS. The data from this study identifies a positive regulator of serratamolide production, describes novel roles for the PigP transcription factor, shows for the first time that PigP directly regulates the pigment biosynthetic operon, and identifies upstream regulators of pigP. This study suggests that PigP is important for the ability of S. marcescens to compete in the environment. Citation: Shanks RMQ, Lahr RM, Stella NA, Arena KE, Brothers KM, et al. (2013) A Serratia marcescens PigP Homolog Controls Prodigiosin Biosynthesis, Swarming Motility and Hemolysis and Is Regulated by cAMP-CRP and HexS. PLoS ONE 8(3): e57634.

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/m 2 ) 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 nonpigmented 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.

Prodigiosins (PrGs) are a family of promising therapeutic molecules, isolated mostly from Gram-negative bacteria and characterized by a common pyrryldipyrrylmethene structure with varying side chains. They show a broad spectrum of activities such as anti-microbial, anti-malarial, anti-cancer and immunosuppressive. PrGs are attracting increasing attention due to the ongoing research for less toxic, but effective agents for cancer chemotherapy and immunosuppression for preventing allograft rejection and autoimmunity. Different analogues have been synthesized and evaluated. This review discusses the immunosuppressive and anti-cancer activities of this class of compounds, as both involve inhibition of cell proliferation. The main focus is on the in vitro and in vivo immunosuppressive activity of the different PrGs and the mechanisms involved. PrGs primarily target the T cells, though some effects are observed on other cell types also. Unlike the well-known immunosuppressant cyclosporin A, PrGs do not inhibit the secretion of IL-2 but inhibit the mitogenic signaling from IL-2, suggesting a different mechanism of action. Janus tyrosine kinase 3 (Jak3) that associates with IL-2R upon activation is considered as the molecular target for PrGs. Its restricted expression makes Jak3 as an attractive target for immunosuppressive therapy. However, the available literature suggests that some other pathways are also influenced by the PrGs. These may be important for the anti-cancer activity, as well as immunosuppressive action. Therefore, PrGs appear to be potential candidates for pharmaceutical development as immunosuppressants and also as anti-cancer agents.

High levels of SKP2 are a poor prognostic factor in multiple human cancers and mostly correlate with low p27 KIP1 levels. Prodigiosin is a bacterial tripyrrole pigment with strong pro-apoptotic activity. Induction of cell cycle blockade underlies one of its anticancer actions but the mechanisms involved are unclear. The aim of this study was to explore the role of the SKP2-p27 KIP1 axis in prodigiosin's cytostatic effect on human lung adenocarcinoma cells.

Members of the ROK family of proteins are mostly transcriptional regulators and kinases that 23 generally relate to the control of primary metabolism, whereby its member glucose kinase 24 acts as the central control protein in carbon control in Streptomyces. Here we show that 25

To date, prodigiosin and its analogues which have been shown to have anticancer, cytotoxic and immunosuppressive activities have been isolated from Serratia, Pseudomonas and Streptomyces species, and chemically synthesized. In a previous study, the red pigment content in Serratia sp. KH-95 was enhanced using a casein-enriched medium. Recently, an integrated bioreactor with an internal adsorbent has been developed to increase the production yield and allow easy recovery of the pigment. Thus, this study focused on both purifying and identifying a single red pigment from several pigments attached to the adsorbent in an integrated bioreactor. The red pigment was extracted directly from the internal adsorbent using acidified methanol and phase separation. Subsequently, it was purified by silica gel chromatography and high performance liquid chromatograph (HPLC). As a result, pure prodigiosin was identified by structural studies as a pigment. Also, this downstream procedure that uses the integrated bioreactor can be applied to the direct production and purification of other prodigiosin analogues and hydrophobic alkaloid compounds from several microorganisms.

Prodigiosin,  a  pigment  produced  by Serratia  marcescensinhibits  the  growth  of  different  microorganisms  and  the  proliferation  of some human cancer cell lines. Prodigiosin is usually produced by fermentations of substrates such as starch and proteins, andpig-ment yield depends on the concentration of the carbon source, stirring speed of cultures, temperature and time of incubation,nitro-gen  sources,  and  pH  of  medium.  In  the  present  study,  pigment  production  was  assessed  using  two  types  of  casein  as  substrate; pigment yield was greater when casein precipitated with vinegar was used as substrate than when high purity casein was. The maxi-mum prodigiosin production was achieved with 10 g/L casein at pH 8.0. Additionally, supplementation of culture media with glu-cose was found to considerably decrease prodigiosin production and growth inhibition of Staphylococcus aureus, which is directly related to pigment yield. Production in stirred-tank bioreactor at ...

Serratia peptidase is active proteolytic enzyme which has the potential of cleaving peptide bond.  Present investigation deals about the Microbial production of serratia peptidase using Serratia marcescens in small scale fermentor. Batch fermentor has been run continuously throughout the night to analyze the production of protein as well as kinetics. Culture broth was maintained at 150rpm for 72 hrs. Protein sample was isolated by centrifuging at 3000rpm for 10mints. The result revealed that Serratia marcescens showed the enormous production of protein in fed batch fermentor compared to the small scale level.  Different substrates were been used for the production of enzyme. Among all cysteine showed the better activity as 2 units/ml of enzyme. Enzymatic assay of Serratia peptidase was done at different time interval of crude broth. Enzyme activity showed that maximum at 40oC for 72hrs. It was observed that 0.65 units/ml of enzyme. Fed batch pilot scale production of Serratia peptid...

Prodigiosin is a red pigment produced by Serratia marcescens with apoptotic activity. We examined the mechanism of action of this tripyrrole alkaloid, focusing on its interaction with DNA and its ability to inhibit both topoisomerase I and topoisomerase II. We also evaluated the DNA damage induced in cancer cell lines. Prodigiosin–DNA intercalation was analyzed using a competition dialysis assay with different DNA base sequences. Topoisomerase I and II inhibition was studied in vitro by a cleavage assay, and in cultured cells, by analysis of its ability to form covalent complexes. Furthermore, we analyzed DNA damage by pulse-field gel electrophoresis and by immunocytochemistry. Apoptosis inducing factor (AIF)/phospho-H2AX (p-H2AX) double labeling by confo-cal microscopy was performed to determine the possible implica-tion of AIF in the prodigiosin–DNA damage. Finally, we studied the ability of this drug to induce copper-mediated DNA damage at different pH by a DNA cleavage assay. Ou...

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/m 2) 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 nonpigmented 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.

The occurrence of poly-3-hydroxybutyric acid (PHB) in 12 different strains of the genus Streptomyces was investigated. Gas chromatographic estimation indicated that all the strains produced PHB and the range of maximum PHB accumulation was between 1.5 and 11.8% dry cell weight. PHB was isolated from Streptomyces coelicolor A3(2) M145 and characterized using Fourier transform-infrared (FT-IR) spectroscopy. The correlation between PHB utilization and antibiotic production in S. coelicolor A3(2) M145, was studied; results indicated a possible role of PHB as a carbon reserve material used for antibiotic production.