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Enhanced production of prodigiosin-like pigment from Serratia marcescens SMdeltaR by medium improvement and oil-supplementation strategies.

Serratia marcescens SMdeltaR, an SpnR-defective isogenic mutant of S. marcescens SS-1, was used to produce a prodigiosin-like pigment (PLP). Luria-Bertani (LB) broth, frequently used for prodigiosin biosynthesis with S. marcescens strains, was modified by increasing the concentrations of tryptone and yeast extract while completely removing NaCl from the medium. The resulting modified LB (MLB) medium achieved an almost 3.0-fold increase in PLP yield (152 mg l(-1)) when compared with the original LB broth. The addition of vegetable oils (2-6% [v/v]) to the fermentation broth markedly enhanced PLP production. PLP yields of 525, 579, and 790 mg l(-1) were obtained when the MLB medium was supplemented with 4% soybean oil, 4% olive oil and 6% sunflower oil, respectively. PLP production was found to be positively correlated with extracellular surface emulsification activity, suggesting a link between the PLP production and the presence of biosurfactant. This work shows that the optimal medium for PLP yield was sunflower oil (6%)-supplemented MLB medium, which resulted in an approximately 14-fold higher PLP yield than that in LB broth. Mass spectrometry and NMR analysis indicated that the PLP product is a prodigiosin derivative, called undecylprodigiosin.

Cell Culture Techniques↗

Scanning electron microscopy of Serratia marcescens producing prodigiosin.

Production of high concentrations of prodigiosin by growing cells of Serratia marcescens was accompanied by the formation of extracellular protrusions as was revealed by scanning electron microscopy. Prodigiosin extracted from the bacterium was compared with the extracellular material. Bacteria which did not produce prodigiosin showed no extracellular protrusions.

Microscopy, Electron, Scanning↗

PRODIGIOSIN-PRODUCING BACTERIA FROM MARINE SOURCES.

Two aerobic, gramnegative, red-pigmented, rod-shaped bacteria were compared morphologically and physiologically with Serratia species, which they resembled superficially. The pigment produced by the marine isolates was shown to be similar to prodigiosin, the red pigment of S. marcescens. The isolates had a single polar flagellum, were oxidative, and did not produce acetoin from glucose or reduce nitrates, which made them distinct from both S. marcescens and S. marinorubra. The latter conformed well to the descriptions of S. marcescens in Bergey's Manual. The marine isolates displayed an absolute growth requirement for sea water or its equivalent. The growth requirement for sea water was replaced by sea-water levels of sodium, potassium, and magnesium chloride. Pigment was produced only when this salt mixture was further supplemented with calcium chloride. Neither sea water nor a high salt level was required for growth or prodigiosin synthesis by the Serratia species examined.

Calcium↗

Synthesis and biological evaluation of nonylprodigiosin and macrocyclic prodigiosin analogues.

Nonylprodigiosin (4) and various of its analogues have been prepared by Suzuki cross-coupling reactions of a well accessible pyrrolyl triflate with (hetero)aryl boronic acid derivatives bearing alkenyl side chains. The resulting alkenes or dienes were subjected to metathesis dimerization or ring-closing metathesis (RCM) reactions, respectively, by using a ruthenium indenylidene complex as the catalyst. The biological activity of the products thus obtained was tested in two different assays monitoring i) the proliferation of murine spleen cells induced by lipopolysaccharides (LPS) and concanavalin A (Con A), and ii) the vacuolar acidification of baby hamster kidney (BHK) cells. Compounds 4 and 21 suppressed Con A-induced T-cell proliferation much more potently than LPS-induced B-cell proliferation. Furthermore, compounds 4 and 26 markedly inhibited vacuolar acidification, although other compounds exhibited no or only marginal effects. Thus, the immunosuppressive activity of prodigiosins toward T-cell proliferation seems to be mediated through cellular targets distinct from vacuolar acidification, and the prodigiosin analogues might be powerful tools to dissect these biological responses. The X-ray crystal structure of the macrocyclic product 25 has been determined, showing that the replacement of one pyrrole ring of the parent compound 4 by a phenyl group does not alter the overall electronic features of the remaining heterocyclic ring system of these alkaloids.

Animals↗

Photoinduced cytotoxicity and thioadduct formation by a prodigiosin analogue.

[reaction: see text] The prodigiosin alkaloid 1 and the synthetic analogue 2 show photoinduced cytotoxicity against HL-60 cancer cells. Photoirradiation of 1 and 2 causes photofading, photooxidation, and thioadduct formation. These results provide a model for the redox properties of prodigiosins that play a role in their biological activity and provide a new way to functionalize their pyrromethene entity with water-soluble thiol groups.

Alkaloids↗

Influence of the a-ring on the proton affinity and anticancer properties of the prodigiosins.

Prodigiosin (Prod, 1) is the parent member of a class of polypyrrole natural products that exhibit promising immunosuppressive and anticancer activities. They are known to act as H+/Cl- symporters possibly through electrostatic binding to Cl- that facilitates proton-coupled transmembrane transport of halides. This activity has been ascribed to their promotion of apoptosis by acidification of the intracellular pH (pHi). Since the protonated pyrromethene chromophore of Prod (1) is expected to play a critical role in pHi regulation, and the A-pyrrole ring is known to be important for anticancer activity, we prepared several Prod analogues with various A-ring systems to determine their proton affinity in 1:1 (v/v) acetonitrile (MeCN)/H(2)O and anticancer properties against HL-60 cancer cells. Our studies show that the A-ring strongly influences the proton affinity of the pyrromethene entity. Replacement of the C-2 methoxy group in 2,4-dimethoxy-pyrromethene 3 (apparent pK(a) = 4.95) with the A-pyrrole ring to generate the Prod analogue 5 raised the apparent pK(a) to 7.54 (increase by 2.59 pK units) and caused a 76 nm red shift in the UV-vis absorbance of the protonated species (AH+). The A-pyrrole NH atom plays an important role in stabilization of AH+, as its replacement with O or S atoms decreases the apparent pK(a) by 0.79 and 1.07 pK units, respectively. A 4-substituted phenyl series of Prod analogues 8-14 exhibited a linear correlation with the Hammett sigma(p) values. Within the phenyl series, two Prod analogues were found to inhibit colony formation of HL-60 cancer cells, although the inhibition did not correlate with the proton affinity of the pyrromethene entity. The implications of these findings with regard to the anticancer activities of the prodigiosins are discussed.

Antineoplastic Agents↗

Influence of the a-ring on the redox and nuclease properties of the prodigiosins: importance of the bipyrrole moiety in oxidative DNA cleavage.

Prodigiosin (Prod, 1) is the parent member of a class of polypyrrole natural products that exhibit promising immunosuppressive and cytotoxic activity. They can facilitate copper-promoted oxidative double-strand (ds) DNA cleavage through reductive activation of Cu(II). This is triggered by oxidation of the electron-rich Prod molecule and may provide a basis for the cytotoxicity of the prodigiosins. To gain an understanding of this activity, we prepared several Prod analogues with various A-ring systems to examine their electrochemical properties in acetonitrile (MeCN) as a means to establish a basis for structure-reactivity relationships in copper-promoted nuclease activity. The intact bipyrrole (BP) chromophore is critical for the copper-mediated nuclease properties of the Prods. In fact, simple BP systems are shown to facilitate oxidative single-strand (ss) DNA cleavage. Replacement of the Prod A-pyrrole ring with alternative arenes (phenyl, furan-2-yl, or thiophen-2-yl) inhibits DNA strand scission and raises the half-peak oxidation potential (E(p/2)) of the Prod free base [E(p/2) = 0.44 V vs saturated calomel electrode (SCE) in MeCN] by ca. 200 mV. The same effect was achieved through attachment of an electron-withdrawing group (acetyl) at the 5'-position of the A-pyrrole ring. The structural modifications that inhibit DNA cleavage correlate with known structure-reactivity relationships of Prods against leukemia and melanoma cancer cells. The implications of our findings with regard to the cytotoxicity of the Prods are discussed.

Anti-Bacterial Agents↗

luxS mutants of Serratia defective in autoinducer-2-dependent 'quorum sensing' show strain-dependent impacts on virulence and production of carbapenem and prodigiosin.

The enzyme LuxS is responsible for the production of autoinducer-2 (AI-2), a molecule that has been implicated in quorum sensing in many bacterial species. This study investigated whether there is a luxS-dependent signalling system in the Gram-negative bacteria Serratia spp. Serratia marcescens is a broad-host-range pathogen and an important cause of nosocomial infections. Production of AI-2 activity was detected in S. marcescens ATCC 274 and Serratia ATCC 39006 and their luxS genes were sequenced. luxS mutants were constructed in these strains and were analysed to determine which phenotypes are regulated by luxS and therefore, potentially, by AI-2. The phenotypes of the luxS mutants included decreased carbapenem antibiotic production in Serratia ATCC 39006 and decreased prodigiosin and secreted haemolysin production in S. marcescens ATCC 274. The luxS mutant of S. marcescens ATCC 274 was also found to exhibit modestly reduced virulence in a Caenorhabditis elegans model. Finally, it was shown that the culture supernatant of a wild-type strain contains a signal, presumably AI-2, capable of complementing the prodigiosin defect of the luxS mutant of another strain, even when substantially diluted. It is concluded that luxS modulates virulence and antibiotic production in Serratia, in a strain-dependent manner, and that, for at least one phenotype, this regulation is via extracellular signalling.

Animals↗

The Serratia gene cluster encoding biosynthesis of the red antibiotic, prodigiosin, shows species- and strain-dependent genome context variation.

The prodigiosin biosynthesis gene cluster (pig cluster) from two strains of Serratia (S. marcescens ATCC 274 and Serratia sp. ATCC 39006) has been cloned, sequenced and expressed in heterologous hosts. Sequence analysis of the respective pig clusters revealed 14 ORFs in S. marcescens ATCC 274 and 15 ORFs in Serratia sp. ATCC 39006. In each Serratia species, predicted gene products showed similarity to polyketide synthases (PKSs), non-ribosomal peptide synthases (NRPSs) and the Red proteins of Streptomyces coelicolor A3(2). Comparisons between the two Serratia pig clusters and the red cluster from Str. coelicolor A3(2) revealed some important differences. A modified scheme for the biosynthesis of prodigiosin, based on the pathway recently suggested for the synthesis of undecylprodigiosin, is proposed. The distribution of the pig cluster within several Serratia sp. isolates is demonstrated and the presence of cryptic clusters in some strains shown. The pig cluster of Serratia marcescens ATCC 274 is flanked by cueR and copA homologues and this configuration is demonstrated in several S. marcescens strains, whilst these genes are contiguous in strains lacking the pig cluster.

Bacterial Proteins↗

A protein associated with prodigiosin formation in Serratia marcescens.

A protein associated to prodigiosin formation was found in Serratia marcescens. The protein was not found in nonpigmented strains and was correlated with the pigment level. The protein was about 100 kilodaltons (kDa) and was also found in nonpigmented bacteria of the pigmented strain grown in glucose medium, at high temperature, or under anaerobic condition. The 100 kDa protein was found not in the outer membrane and the periplasm, but in the inner membrane and/or the cytoplasm. The protein was also found singly or dominantly in pigment-protein complexes and pigment-localizing vesicles described in previous reports. These results suggest that the 100 kDa protein is associated with prodigiosin formation.

Bacterial Proteins↗

Separation of the prodigiosin-localizing crude vesicles which retain the activity of protease and nuclease in Serratia marcescens.

Crude vesicles in which prodigiosin is localized were separated from pigmented Serratia marcescens. The bacteria were grown on peptone-glycerol agar plate, suspended in saline, and fractionated into cells, vesicles, and supernatant by differential centrifugation. Electron microscopic observations showed that the fractionation was conducted properly and the separated vesicles were lysed in distilled water. The vesicles suspended in saline retained 100 kilodalton protein of which amount is correlated with prodigiosin level, but the 100 kDa protein was found in the supernatant when the vesicles were lysed in distilled water. The vesicle fraction retained few colony-forming units and little detectable activity of NADH oxidase, but showed much higher activities of protease and nuclease than the cell fraction. The profiles of the activities of the protease and the nuclease in the fractions were different from each other, that is, the protease activity in the vesicle fraction was lower than that in the supernatant fraction, whereas the nuclease activity in the vesicle fraction was higher than that in the supernatant fraction, suggesting that the two extracellular enzymes were released from the pigmented bacteria by different mechanisms.

Cell Fractionation↗

Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2-methyl-3-n-amyl-pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces.

The biosynthetic pathway of the red-pigmented antibiotic, prodigiosin, produced by Serratia sp. is known to involve separate pathways for the production of the monopyrrole, 2-methyl-3-n-amyl-pyrrole (MAP) and the bipyrrole, 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) which are then coupled in the final condensation step. We have previously reported the cloning, sequencing and heterologous expression of the pig cluster responsible for prodigiosin biosynthesis in two Serratia sp. In this article we report the creation of in-frame deletions or insertions in every biosynthetic gene in the cluster from Serratia sp. ATCC 39006. The biosynthetic intermediates accumulating in each mutant have been analysed by LC-MS, cross-feeding and genetic complementation studies. Based on these results we assign specific roles in the biosynthesis of MBC to the following Pig proteins: PigI, PigG, PigA, PigJ, PigH, PigM, PigF and PigN. We report a novel pathway for the biosynthesis of MAP, involving PigD, PigE and PigB. We also report a new chemical synthesis of MAP and one of its precursors, 3-acetyloctanal. Finally, we identify the condensing enzyme as PigC. We reassess the existing literature and discuss the significance of the results for the biosynthesis of undecylprodigiosin by the Red cluster in Streptomyces coelicolor A3(2).

Anti-Bacterial Agents↗

Characterization of Serratia marcescens isolates from subgingival biofilm, extraoral infections and environment by prodigiosin production, serotyping, and genotyping.

INTRODUCTION: Serratia marcescens is widely distributed in nature, and has emerged in the last years as an important nosocomial pathogen. The organism may also be found in subgingival biofilm in periodontitis patients. This study aimed to verify the subgingival prevalence of S. marcescens in different periodontal conditions and to evaluate whether the oral cavity would harbor strains similar to those causing infectious diseases. METHODS: The subgingival occurrence of S. marcescens was determined in 334 subjects. The phenotypic and genotypic diversity of 23 isolates from subgingival biofilm, 22 from extra-oral infections and 10 environmental strains, was compared by prodigiosin production, O and H serotyping and genotyping using polymorphic GC-rich repetitive sequences-polymerase chain reaction. RESULTS: S. marcescens was found more frequently in severe periodontitis patients (4.1%) than in gingivitis (3.2%) and healthy subjects (2.5%), but these differences were not statistically significant. Analysis of serotype distribution, prodigiosin production, and genotyping revealed that environmental strains were markedly different from most human isolates, either oral or extraoral. CONCLUSION: These data suggest that S. marcescens isolates from subgingival biofilm are not just contaminants from the environment, but that the oral cavity may act as a reservoir of strains able to promote human infections. However, further studies are needed to elucidate the role of this bacterium in the pathogenesis of periodontal diseases.

Adolescent↗

Incorporation of proline into prodigiosin by a Put mutant of Serratia marcesens.

A Put mutant of Serratia marcescens, deficient in proline oxidase and therefore unable to degrade proline, was used to assay for an enzymatic reaction responsible for incorporation of proline into prodigiosin. The reaction had a pH optimum of 7.5 and a Km of 1.1 X 10(-4) M at 27 C. At temperatures above 27 C, the velocity of the reaction decreased with increasing temperature and little activity was detected at 42 C. Activity of the enzyme was directly proportional to the quantity of pigment formed and was inhibited by thioproline, a substrate analog. These data suggested the presence of a unique and specific enzyme in the biosynthetic pathway for prodigiosin.

Alanine↗

Cloning of a pleiotropic gene that positively controls biosynthesis of A-factor, actinorhodin, and prodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans.

A-factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone), an autoregulating factor originally found in Streptomyces griseus, is involved in streptomycin biosynthesis and cell differentiation in this organism. A-factor production is widely distributed among actinomycetes, including Streptomyces coelicolor A3(2) and Streptomyces lividans. A chromosomal pleiotropic regulatory gene of S. coelicolor A3(2) controlling biosynthesis of A-factor and red pigments was cloned with a spontaneous A-factor-deficient strain of S. lividans HH21 and plasmid pIJ41 as a host-vector system. The restriction endonuclease KpnI-digested chromosomal fragments were ligated into the plasmid vector and introduced by transformation into the protoplasts of strain HH21. Three red transformants thus selected were found to produce A-factor and to carry a plasmid with the same molecular weight, and a 6.4-megadalton fragment was inserted in the KpnI site of pIJ41. By restriction endonuclease mapping and subcloning, a restriction fragment (1.2 megadaltons, approximately 2,000 base pairs) bearing the gene which causes concomitant production of A-factor and red pigments was determined. The red pigments were identified by thin-layer chromatography and spectroscopy to be actinorhodin and prodigiosin, both of which are the antibiotics produced by S. coelicolor A3(2). The cloned fragment was introduced into the A-factor-negative mutants (afs) of S. coelicolor A3(2) by using pIJ702 as the vector, where it complemented one of these mutations, afsB, characterized by simultaneous loss of A-factor and red pigment production. We conclude that the cloned gene pleiotropically and positively controls the biosynthesis of A-factor, actinorhodin, and prodigiosin.

4-Butyrolactone↗

Enzyme polymorphism, prodigiosin production, and plasmid fingerprints in clinical and naturally occurring isolates of Serratia marcescens.

Enzyme polymorphism and genetic relationship among 99 Serratia marcescens isolates obtained from clinical and environmental sources were determined by analysis of electromorphs in nine enzyme loci encoded by chromosomal genes. Seven of the loci were polymorphic, and 33 distinctive electrophoretic types (ETs) representing multilocus genotypes were identified. Cluster analysis, based on the proportion of mismatches between multilocus genotypes, revealed two clearly differentiated groups of ETs in S. marcescens. One was represented exclusively by isolates with nonchromogenic biotypes recovered almost entirely (97.3%) from clinical samples. The other group comprised all isolates characterized by the production of prodigiosin or by belonging to a chromogenic biotype. Absolute correlation was found between the ability to produce prodigiosin and the absence of plasmids. In contrast, 24% of the nonchromogenic isolates contained plasmids. Results obtained by analysis of multilocus genotypes were related to those obtained by biotyping and plasmid fingerprinting. However, more groups could be distinguished by analysis of ETs than by biotyping. Plasmid fingerprinting was a limited typing system because many isolates lacked plasmids. Although the results of this study did not permit a definitive correlation between ETs and pathogenicity of the isolates, more detailed studies of these groups will help to understand the different clinical significances of the nonchromogenic and chromogenic isolates of S. marcescens.

Animals↗

Prodigiosin 25-C and metacycloprodigiosin suppress the bone resorption by osteoclasts.

Prodigiosin 25-C and metacycloprodigiosin were found to suppress PTH-stimulated pit formation by cultured osteoclasts on bone slices. They also inhibited the acidification of vacuolar organelles in intact osteoclastic cells. Since the acidic pH in these organelles is generated by the action of proton-pumping ATPases of the organelle, these results indicate that the proton-pumping activity of V-ATPase in osteoclastic cells is essential in bone resorption and that the inhibition of the acidification of vacuolar organelles by prodigiosins results in suppression of PTH-stimulated bone resorption.

Animals↗