Biosynthesis of Prodigiosin, a Secondary Metabolite of Serratia marcescens

Prodigiosenes (prodigiosin and prodigiosin-like pigments) are known to be synthesized by only one genus of Eubacteriales and by two genera of Actinomycetales. Biosynthesis by Serratia marcescens occurs over a relatively narrow range of temperatures, although the bacteria grow over a broad range. When cultures of S. marcescens were incubated at 27 C in 1.0% casein hydrolysate, viable count and protein attained maximal values within 24 to 48 h, whereas prodigiosin did not reach a maximum until 96 h. The greatest amount of pigment was synthesized when cultures were in the senescent phase of growth. Suspensions of nonproliferating bacteria incubated at 27 C in only L-alanine also synthesized prodigiosin, although at a slower rate than growing cultures. Kinetics of growth for the wild-type, red S. marcescens and a white mutant were identical when incubated at 27 C, but the wild type produced abundant pigment. These results plus other data obtained from the literature suggest that prodigiosin is a secondary metabolite. The importance of this proposal to understanding the function of prodigiosin in S. marcescens is discussed.     

粘质沙雷氏菌产灵菌红素培养基的筛选

目的：确定菌株S418产生灵菌红素的最优培养基配方及其的分类地位。方法：以花生粉为基础培养基,通过单因素试验和四因素三水平正交试验筛选出了菌株S418产灵菌红素的最佳培养基配方;根据该菌株的16S rRNA基因序列系统发育树分析初步确定了菌株S418的分类地位。结果：培养基最优配方为：花生粉2%,花生油0.5%,L-脯氨酸1%,硫酸镁0.025%。在28℃、pH7.5、250r/min振荡培养24h,灵菌红素产量达67.92mg/L。菌株S418初步鉴定为粘质沙雷氏菌（Serratia marcescensS418）。结论：花生粉培养基是一种适合粘质沙雷氏菌产灵菌红素的优良培养基。     

Role of Methionine in Biosynthesis of Prodigiosin by Serratia marcescens

Methionine alone did not allow biosynthesis of prodigiosin (2-methyl-3-amyl-6-methoxyprodigiosene) in nonproliferating cells (NPC) of Serratia marcescens strain Nima. However, when methionine was added to NPC synthesizing prodigiosin in the presence of other amino acids, the lag period for synthesis of prodigiosin was shortened, an increased amount of the pigment was formed, and the optimal concentrations of the other amino acids were reduced. Less prodigiosin was synthesized when addition of methionine was delayed beyond 4 h. The specific activity of prodigiosin synthesized by addition of (14)CH(3)-methionine was 40 to 50 times greater than that synthesized from methionine-2-(14)C or (14)COOH-methionine. NPC of mutant OF of S. marcescens synthesized norprodigiosin (2-methyl-3-amyl-6-hydroxyprodigiosene), and the specific activity of this pigment synthesized in the presence of (14)CH(3)-methionine was only 5 to 13 times greater than that synthesized from methionine-2-(14)C or (14)COOH-methionine. A particulate, cell-free extract of mutant WF of S. marcescens methylated norprodigiosin to form prodigiosin. When the extract was added to NPC of mutant OF synthesizing norprodigiosin in the presence of (14)CH(3)-methionine, the prodigiosin formed had 80% greater specific activity than the norprodigiosin synthesized in the absence of the extract. The C6 hydroxyl group of norprodigiosin was methylated in the presence of the extract and methionine. Biosynthesis of prodigiosin by NPC of strain Nima also was augmented by addition of S-adenosylmethionine. Various analogues of methionine such as norleucine, norvaline, ethionine, and alpha-methylmethionine did not affect biosynthesis of prodigiosin by NPC either in the presence or absence of methionine.     

Inhibition of quorum sensing regulated biofilm formation in Serratia marcescens causing nosocomial infections

Serratia marcescens is an opportunistic pathogen causing severe urinary tract infections in hospitalized individuals. Infections of S. marcescens are of great concern because of its increasing resistance towards conventional antibiotics. Quorum sensing (QS)-a cell to cell communication-system of S. marcescens acts as a global regulator of almost all the virulence factors and majorly its biofilm formation. Since, the QS system of S. marcescens directly accords to its pathogenesis, targeting QS system will provide an improved strategy to combat drug resistant pathogens. In the present study, QS system of S. marcescens has been used as target and its inhibition has been studied upon exposure to bioactives from coral associated bacteria (CAB). This study also emphasises the potential of CAB in producing bioactive agents with anti-QS and antibiofilm properties. Two CAB isolates CAB 23 and 41 have shown to inhibit biofilm formation and the production of QS dependent virulence factors like prodigiosin, protease, lipase and swarming motility. The study, on the whole explicates the potential of QS system as a target to treat drug resistant bacterial infections.     

Effect of glucose concentration on the biosynthesis of prodigiosin by serratia marcescens (author's transl)]

Serratia marcescens is an enterobacteria which produces a characteristic red pigment denominated prodigiosin. To study the effect of glucose on the kinetics of this secondary metabolite, cultures of Serratia marcescens S10 were incubated at 30 degrees C in the mineral medium GL, with glucose (2 g/l) as the carbon source. Prodigiosin production in relation to glucose consumption is studied, and parallel-wise, the effect of various concentrations of glucose on prodigiosin production. The kinetics data show the close correlation between glucose consumption and the synthesis of prodigiosin. This substrate inhibits the synthesis of pigment in cultures grown on solid medium GL with concentrations of glucose up to 15 g/l.     

Isolation of Serratia marcescens in the midgut of Rhodnius prolixus: impact on the establishment of the parasite Trypanosoma cruzi in the vector

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 24h 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 degrees 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 degrees 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.     

Inhibition of quorum sensing in Serratia marcescens AS-1 by synthetic analogs of N-acylhomoserine lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C(6)-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C(9)-CPA), had a strong inhibitory effect on prodigiosin production. C(9)-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C(9)-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C(6)-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C(9)-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Cloning and characterization of the Pseudomonas fluorescens ATP-binding cassette exporter, HasDEF, for the heme acquisition protein HasA

Two ATP-binding cassette (ABC) exporters are present in Pseudomonas fluorescens no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system. P. fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa and Serratia marcescens. The P. fluorescens Has exporter secreted HasA proteins from P. fluorescens and P. aeruginosa but not S. marcescens HasA in Escherichia coli, whereas the Has exporter from S. marcescens allowed secretion of all three HasA proteins. The P. fluorescens HasDEF system also promoted the secretion of the lipase and alkaline protease of P. fluorescens. Hybrid exporter analysis demonstrated that the HasD proteins, which are ABC proteins, are involved in the discrimination of export substrates. Chimeric HasA proteins containing both P. fluorescens and S. marcescens sequences were produced and tested for secretion through the Has exporters. The C-terminal region of HasA was shown to be involved in the secretion specificity of the P. fluorescens Has exporter.     

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.     

Defects in Prodigiosin Formation by L-Forms of Serratia marcescens

An L-form of Serratia marcescens has previously been shown incapable of producing the red pigment, prodigiosin, characteristic of the parent bacteria. Mutants of S. marcesens, unable to form one or the other of the two prodigiosin precursors, 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde or 2-methyl-3-n-amylpyrrole, were used to test the nature of the L-form defect. The L-forms failed to form sufficient amounts of either precursor to be detected by the appropriate mutant, and, when furnished the precursors, failed to couple them to form prodigiosin.     

Leishmania (Leishmania) chagasi interactions with Serratia marcescens: ultrastructural studies, lysis and carbohydrate effects

Studies on the lysis of L. chagasi caused by the bacteria Serratia marcescens were carried out. In vitro experiments demonstrated that S. marcescens variant SM 365, a prodigiosin pigment producer, lysed this species of Leishmania but variant DB11, a nonpigmented bacteria, was unable to lyse the parasite. High concentrations of d-mannose were found to protect L. chagasi markedly diminishing the lysis by S. marcescens SM 365. Promastigotes of L. chagasi bound the lectin Concanavalin A conjugated with FITC, the fluorescence was intensely found at the base of the flagellum (flagellar pocket). Scanning electron microscopy revealed that the bacteria adherence occurred mainly in the flagellar pocket. S. marcescens SM 365 formed filamentous structures, identified as biofilms, which connect the protozoan to the developing bacterial clusters, in low concentrations of bacteria after 30 min incubation time. We suggest that bacterial mannose-sensitive (MS) fimbriae are relevant to S. marcescens SM 365 in the lysis of L. chagasi.     

Decrease in respiration activity related to prodigiosin synthesis in Serratia marcescens

Variation in the cell respiration rate of pigmented and nonpigmented strains of Serratia marcescens was exhibited. The respiration rate of a pigmented strain decreased earlier than that of nonpigmented strains in the late exponential or early stationary phase. However when prodigiosin synthesis was not induced by exchange of carbon sources in the medium, the decrease in the respiration rate of the pigmented strain was the same as that of nonpigmented strains. Measurement of the oxygen consumption rate in the sonicated cell membrane by adding NADH solution showed that the rate in the pigmented strain was lower than that in nonpigmented strains. Furthermore, the cell membrane of prodigiosin-induced organisms was more sensitive to respiration inhibitors than that of pigment-noninduced organisms of the pigmented strain. These results showed that the respiration activity was decreased by prodigiosin synthesis in S. marcescens.     

Cytotoxic Effect of Prodigiosin,Natural Red Pigment,Isolated from Serratia marcescens UFPEDA 398

Prodigiosin is a secondary metabolite, with red pigmentation, produced by Serratia marcescens. Red pigment is a natural alkaloid whose chemical structure has three pyrrole rings. Prodigiosin has been described for several biological activities, including antitumor, inducing apotosis in T and B lymphocytes. This work aimed to evaluate the cytotoxic activity of prodigiosin in NCHI-292, HEp-2, MCF-7 and HL-60 tumor cell lines. The red pigment was isolated from Serratia marcescens UFPEDA 398 biomass whose fractions were previously separated by column chromatography, purified, identified and further characterized by GC–MS and compared with the computerized library of m/z values. The pigment corresponded to prodigiosin with maximum absorption at 534 nm, molecular weight 323 and structural formula C20H25N3O. During the prodigiosin purification process a purple absorbance fraction at 272.65 nm was also observed. Significant cytotoxic effects of prodigiosin were evidenced for NCHI-292, Hep-2, MCF-7 and HL-60 tumor cell lines. The isolated purple fraction had no cytotoxic effect (IC50 11.3 µg/mL) when compared to prodigiosin (IC50 3.4 µg/mL) for the tumor cell lines studied. The MCF-7 strain was slightly more pigment resistant (IC50 5.1 µg/mL). Therefore, further studies will be needed to elucidate the antitumor mechanisms of prodigiosin action against tumor strains from flow cytometry tests. However, although these data are preliminary, it was evidenced that prodigiosin showed cytotoxic activity in tumor cell lines suggesting promising antitumor properties. In this sense, future studies on the cytotoxic and genotoxic effects of prodigiosin produced by S. marcecsens UFPEDA 398 are suggested.     

Droplet enrichment factors of pigmented and nonpigmented Serratia marcescens: possible selective function for prodigiosin.

Drops produced by bursting bubbles provide a mechanism for the water-to-air transfer and concentration of matter. Bacteria can adsorb to air bubbles rising through bacterial suspensions and enrich the drops formed by the bubbles upon breaking, creating atmospheric biosols which function in dispersal. This bacterial enrichment can be quantified as an enrichment factor (EF), calculated as the ratio of the concentration of bacteria in the drop to that of the bulk bacterial suspension. Bubbles were produced in suspensions of pigmented (prodigiosin-producing) and nonpigmented cultures of Serratia marcescens. EFs for pigmented cultures were greater than EFs for nonpigmented cells. Pigmented cells appeared hydrophobic based on their partitioning in two-phase systems of polyethylene glycol 6000 and dextran T500. The surface hydrophobicity of pigmented cells may result from the hydrophobic nature of prodigiosin and could account for the greater ability of these bacteria to adsorb to air bubbles and enrich airborne droplets. Enhancement of the aerosolization of S. marcescens may be a selective function of the bacterial secondary metabolite prodigiosin.     

Existence of a Free Form of a Specific Membrane Lipoprotein in Gram-Negative Bacteria

The existence of a free form of a specific lipoprotein of molecular weight 7,200 was examined in the envelopes of several gram-negative bacteria. When the envelope proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, distinct peaks were observed in Salmonella typhimurium, Serratia marcescens, and Pseudomonas aeruginosa at the same position as the free form of the lipoprotein of Escherichia coli. However, the peak was not observed in Proteus mirabilis. The protein at the peak in S. typhimurium was shown to contain little or no histidine as expected from the amino acid composition of the lipoprotein. Furthermore, antiserum against the highly purified lipoprotein from E. coli was shown to react with the proteins from S. typhimurium and S. marcescens and to form the specific immunoprecipitates. In contrast, the protein from P. aeruginosa did not react with the antiserum at all. Thus, it is concluded that S. typhimurium and S. marcescens have the free form of the lipoprotein in their envelopes as does E. coli. P. aeruginosa contains a protein of the same size as the lipoprotein, but it is not certain whether the protein is the same structural protein as the lipoprotein from E. coli. P. mirabilis may not have any free form of the lipoprotein, may have it in a very small amount, or may have a lipoprotein of different molecular weight serving the same function.     

Droplet enrichment factors of pigmented and nonpigmented Serratia marcescens: possible selective function for prodigiosin

Drops produced by bursting bubbles provide a mechanism for the water-to-air transfer and concentration of matter. Bacteria can adsorb to air bubbles rising through bacterial suspensions and enrich the drops formed by the bubbles upon breaking, creating atmospheric biosols which function in dispersal. This bacterial enrichment can be quantified as an enrichment factor (EF), calculated as the ratio of the concentration of bacteria in the drop to that of the bulk bacterial suspension. Bubbles were produced in suspensions of pigmented (prodigiosin-producing) and nonpigmented cultures of Serratia marcescens. EFs for pigmented cultures were greater than EFs for nonpigmented cells. Pigmented cells appeared hydrophobic based on their partitioning in two-phase systems of polyethylene glycol 6000 and dextran T500. The surface hydrophobicity of pigmented cells may result from the hydrophobic nature of prodigiosin and could account for the greater ability of these bacteria to adsorb to air bubbles and enrich airborne droplets. Enhancement of the aerosolization of S. marcescens may be a selective function of the bacterial secondary metabolite prodigiosin.     

First detection of OKP-A β-lactamase in two Serratia marcescens isolates in China

Two strains of Enterobacteriaceae producing prodigiosin were isolated from meat in the Sichuan province of China in 2010. The strains were identified by Vitek system, 16S rDNA, rpoB, pfs and luxS genes. Minimum inhibitory concentrations were determined using the broth microdilution method. The two strains were screened for the presence of β-lactamase genes (blaTEM, blaSHV, blaOKP, and blaCTX-M genes). Based on PCR amplification and 16S rDNA sequencing the analysed strains were identified as Serratia marcescens. In addition, morphological and biochemical identification showed that the two stains were definitely S. marcesens. Antimicrobial susceptibility test showed that both strains were resistant to ampicillin and first-generation cephalosporins while being susceptible to cefotaxime, ceftiofur, ceftriaxone, imipenem and aztreonam. It was found that blaOKP had been identified first from the two S. marcescens strains, ch1 and ch2. The isolates were closely related as shown by pulsed-field gel electrophoresis (PFGE). The narrow-spectrum OKP-A β-lactamase gene blaOKP-A-13 was found to be chromosomally located in S. marcescens. The isolates produced a β-lactamase with a pI of approximately 8.2, which corresponds to the OKPA family. Findings indicate that OKP enzymes are not Klebsiella pneumoniae-specific chromosomal ?-lactamases, and the first isolation of S. marcescens producing OKP-A ?-lactamase suggests that the blaOKP gene may be disseminated between different species.     

Adaptation and growth of Serratia marcescens in contact lens disinfectant solutions containing chlorhexidine gluconate.

Serratia marcescens (11 of 12 strains) demonstrated an ability to grow in certain chlorhexidine-based disinfecting solutions recommended for rigid gas-permeable contact lenses. For a representative strain, cells that were grown in nutrient-rich medium, washed, and inoculated into disinfecting solution went into a nonrecoverable phase within 24 h. However, after 4 days, cells that had the ability to grow in the disinfectant (doubling time, g = 5.7 h) emerged. Solutions supporting growth of S. marcescens were filter sterilized. These solutions, even after removal of the cells, showed bactericidal activity against Pseudomonas aeruginosa and a biphasic survival curve when rechallenged with S. marcescens. Adaptation to chlorhexidine by S. marcescens was not observed in solutions formulated with borate ions. For chlorhexidine-adapted cells, the MIC of chlorhexidine in saline was eightfold higher than that for unadapted cells. Cells adapted to chlorhexidine showed alterations in the proteins of the outer membrane and increased adherence to polyethylene. Cells adapted to chlorhexidine persisted or grew in several other contact lens solutions with different antimicrobial agents, including benzalkonium chloride.     

Regulatory roles of spnT, a novel gene located within transposon TnTIR

The transposon TnTIR contains spnIR quorum-sensing system regulating sliding motility and the production of nuclease, biosurfactant, and prodigiosin in Serratia marcescens. Within TnTIR, a gene named spnT is upstream of and co-transcribed with spnI. SpnT is a cytoplasmic protein and its level peaks during early stationary phase. spnT over-expression resulted in inhibition of sliding motility and synthesis of prodigiosin, and biosurfactant similar to spnR. spnT but not spnR over-expression induced cell elongation and aberrant DNA replication in S. marcescens and Escherichia coli strains. In comparison with wild-type E. coli strain, over-expression of spnT in an E. coli priA and dnaC double-mutant strain did not lead to the aberrant cell morphology phenotypes, suggesting SpnT may act through the recombination-dependent DNA replication system. As spnT over-expression inhibited swarming but not swimming motility, SpnT may indirectly function as a negative regulator of surface-dependent migration and secondary metabolite production.