灵杆菌合成灵菌红素的转录调控

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

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.     

Role of L-proline in the biosynthesis of prodigiosin.

Nonproliferating cells of Serratia marcescens, wild-type strain Nima, synthesized the pigment, prodigiosin, when saline suspensions were incubated with aeration at 27 degrees C in the presence of proline or alanine. Mutants PutS1 and PutS2 derived from strain Nima formed prodigiosin from alanine, but not from proline, unless alanine also was added. Strain Nima utilized proline as a sole source of carbon and of nitrogen for growth, whereas Put mutants did not. Investigation of enzymes degrading proline showed that the wild-type strain contained proline oxidase, which was absent in Put mutants. The wild type, as well as the mutants, utilized alanine as the sole source of carbon and nitrogen for growth. Although nonproliferating cells of Put mutants failed to synthesize prodigiosin from proline, addition of L-[U-14C]proline to suspensions metabolizing and synthesizing the pigment because of addition of alanine resulted in the incorporation of radioactive label into prodigiosin, as well as into cellular protein. Since Put mutants could not catabolize proline, the incorporation of [14C]proline into the prodigiosin molecule indicated that proline was incorporated directly into the pigment.     

Effect of Iron and Salt on Prodigiosin Synthesis in Serratia marcescens

Serratia marcescens wild-types ATCC 264 and Nima grew but did not synthesize prodigiosin in a glycerol-alanine medium containing 10 ng of Fe per ml. Wild-type 264 required the addition of 0.2 mug of Fe per ml for maximal growth and prodigiosin synthesis; Nima required 0.5 mug of Fe per ml. Three percent, but not 0.1%, sea salts inhibited prodigiosin synthesis in a complex medium containing up to 10 mug of Fe per ml. NaCl was the inhibitory sea salt component. The inhibition was not specific for NaCl; equimolar concentrations of Na(2)SO(4), KCl, and K(2)SO(4) also inhibited prodigiosin synthesis. Experiments with strains 264 and Nima and with mutant WF which cannot synthesize 4-methoxy-2-2'-bipyrrole-5-carboxyaldehyde (MBC), the bipyrrole moiety of prodigiosin, and with mutant 9-3-3 which cannot synthesize the monopyrrole moiety 2-methyl-3-amylpyrrole (MAP) showed that both MBC synthesis and the reaction condensing MAP and MBC to form prodigiosin were relatively more sensitive to NaCl inhibition than the MAP-synthesizing step. The capacity of whole cells to condense MAP and MBC was present, but inactive, in cells grown in NaCl; removal of the NaCl from non-proliferating salt-grown cells restored the activity. Other evidence suggests the existence of a common precursor to the MAP- and MBC-synthesizing pathways.     

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.     

Prodigiosin synthesis in mutants of Serratia marcesens

Morrison, D. A. (Harvard College, Cambridge, Mass.). Prodigiosin synthesis in mutants of Serratia marcescens. J. Bacteriol. 91:1509-1604. 1966.-Exchange of biosynthetic intermediates through the culture medium was used to characterize several hundred new color mutants of Serratia marcescens. The general scheme of prodigiosin synthesis as a bifurcated pathway, in which monopyrrole and bipyrrole precursors are synthesized separately and then coupled to form pigment, was confirmed and extended. Mutants of one new class excreted a product likely to be a new intermediate in monopyrrole synthesis, those of a second excreted a new product in the bipyrrole pathway, and those of a third were blocked at early steps in both pathways. Two novel classes of mutants were isolated, in each of which a lack of some product present in Serratia and Escherichia cultures resulted in loss of all steps in prodigiosin biosynthesis.     

Incorporation of methionine into prodigiosin

Addition of proline to suspensions of nonpigmented, nonproliferating cells of Serratia marcescens induced biosynthesis of the pigment, prodigiosin. If methionine was included with proline, 4 times as much prodigiosin was formed, although the amount synthesized in the presence of methionine alone was nil. Uniformly ¹⁴C-labelled proline and methionine were incorporated into prodigiosin to about 30% the extent of their incorporation into cellular protein. Experiments with [carboxy-¹⁴C]-, and [Me-¹⁴C] methionine established that isotope from the methyl group was utilized preferentially for biosynthesis of prodigiosin.     

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.

     

Crystal structure of the catalytic domain of PigE: A transaminase involved in the biosynthesis of 2-methyl-3-n-amyl-pyrrole (MAP) from Serratia sp. FS14

Prodigiosin, a tripyrrole red pigment synthesized by Serratia and some other microbes through a bifurcated biosynthesis pathway, MBC (4-methoxy-2,2′-bipyrrole-5-carbaldehyde) and MAP (2-methyl-3-n-amyl-pyrrole) are synthesized separately and then condensed by PigC to form prodigiosin. MAP is synthesized sequentially by PigD, PigE and PigB. PigE catalyzes the transamination of an amino group to the aldehyde group of 3-acetyloctanal, resulting in an aminoketone, which spontaneously cyclizes to form H₂MAP. Here we report the crystal structure of the catalytic domain of PigE which involved in the biosynthesis of prodigiosin precursor MAP for the first time to a resolution of 2.3 Å with a homodimer in the asymmetric unit. The monomer of PigE catalytic domain is composed of three domains with PLP as cofactor: a small N-terminal domain connecting the catalytic domain with the front part of PigE, a large PLP-binding domain and a C-terminal domain. The residues from both monomers build the PLP binding site at the interface of the dimer which resembles the other PLP-dependent enzymes. Structural comparison of PigE with Thermus thermophilus AcOAT showed a higher hydrophobic and smaller active site of PigE, these differences may be the reason for substrate specificity.     

Streptomycin and syntrophic prodigiosin synthesis inSerratia marcescens

The syntrophic synthesis of prodigiosin was studied using the non-pigmenting mutants ofSerratia marcescens, strains 9-3-3 and WF. Prodigiosin started to form syntrophically after the two strains were grown near one another for ca. 10 hr. Maximum pigmentation required ca. 20 hr. The lag period of 10 hr was used for forming the mono- and bipyrrole precursors of prodigiosin, a tripyrrole.Streptomycin, 10–25 µg/ml, completely inhibited syntrophic pigmentation but had no visible effect on cellular growth. Inhibition was inversely proportional to the delay in application of the drug during the 10-hr lag preceding pigmentation. Streptomycin inhibited neither the synthesis of monopyrrole by strain WF nor the condensation of monopyrrole and bipyrrole to form prodigiosin. Rather streptomycin interfered with syntrophic prodigiosin synthesis by inhibiting the synthesis of the bipyrrole part of the pigment by strain 9-3-3.This material was taken from a thesis submitted in 1964 by M. A. Q. Siddiqui in partial fulfilment of the requirements for an M. S. degree at the University of Houston, Houston, Texas.     

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

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

Impaired nitrogen fixation and glutamine synthesis in methionine sulfoximine sensitive (MSs) mutants of Rhizobium phaseoli

Summary Random Tn5 mutagenesis of antibiotic-resistant derivatives of Rhizobium phaseoli CFN42 yielded several independent mutants that were sensitive to methionine sulfoximine (MS^s), a specific inhibitor of glutamine synthetase (GS). These MS^s mutants were analyzed for GSI and GSII activities and for their symbiotic properties. Four classes of MS^s mutants have been distinguished. Class I strains are impaired in their synthesis of glutamine and in their symbiotic properties. Class II strains have wild type levels of GSI and GSII activities but have a reduced capacity to fix nitrogen. Class III strains have lost GSII activity, but their symbiotic properties are wild type. In class IV mutants neither glutamine synthesis nor symbiotic properties are affected. Mutants of classes I, III, and IV all have the Tn5 inserted into the chromosome, whereas in class II mutants the Tn5 is located in plasmid p42e, a plasmid different from the previously identified symbiotic plasmid p42d.     

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).     

Disruption of the copper efflux pump (CopA) of Serratia marcescens ATCC 274 pleiotropically affects copper sensitivity and production of the tripyrrole secondary metabolite, prodigiosin

The prodigiosin biosynthetic gene cluster (pig cluster) of Serratia marcescens ATCC 274 (Sma 274) is flanked by cueR/copA homologues. Inactivation of the copA homologue resulted in an increased sensitivity to copper, confirming that CopA is involved in copper homeostasis in Sma 274. The effect of copper on the biosynthesis of prodigiosin in Sma 274 and the copA mutant strain was investigated. Increased levels of copper were found to reduce prodigiosin production in the wild type Sma 274, but increase production in the copA mutant strain. The physiological implications for CopA mediated prodigiosin production are discussed. We also demonstrate that the gene products of pigB–pigE of Sma 274 are sufficient for the biosynthesis of 2-methyl-3-n-amyl-pyrrole and condensation with 4-methoxy-2,2′-bipyrrole-5-carboxyaldehyde to form prodigiosin, as we have shown for Serratia sp. ATCC 39006.     

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

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

Organization of genes necessary for growth of the hydrogen-methanol autotroph Xanthobacter sp. strain H4-14 on hydrogen and carbon dioxide

Mutants unable to grow on H2 and CO2 were isolated in the hydrogen-methanol autotroph Xanthobacter sp. strain H4-14 and complemented with a clone bank constructed in a broad-host-range cosmid vector. The mutants fell into two classes. Class I mutants (Cfx-) cannot grow on hydrogen or methanol and are deficient in one or more of the key enzymes of the Calvin Cycle. Class II mutants (Hox-) can grow on methanol but not on hydrogen and lack hydrogenase activity. Restriction maps of the complementing clones show that each class is not linked to the other. Subcloning and Tn5 mutagenesis have localized the regions of DNA complementing these mutants. The region complementing a class I mutant which is deficient in ribulosebisphosphate carboxylase activity is approximately 3.2 kilobase pairs in size. Expression of this enzyme activity from cloned DNA gave evidence for the orientation of an operon containing the structural genes for this enzyme. The region complementing most of the class II mutants is 3 to 4.5 kilobase pairs in size.     

Pigmentation and Acriflavine Resistance in Serratia marcescens

Stable, orange, acriflavine-resistant variants were selected by treatment of a wild-type, red, acriflavine-sensitive strain of Serratia marcescens with acriflavine. Visible, ultraviolet, infrared, and nuclear magnetic resonance spectra of purified pigment from the red strain were identical to those of the pigment from the orange strain, and the orange mutant was not due to a mutation affecting the structure of the pigment, prodigiosin. The color of the red strain was not affected by variations in pH between 5.0 and 8.0, whereas the color of the orange mutant changed from pink to orange over the same pH range. This variation was mimicked by the pH-induced variation in color of prodigiosin purified from either the red, wild-type or the orange, mutant strains. Density-gradient centrifugation of cell fragments after ultrasonic disintegration resulted in characteristic pigmented bands. Biochemical characterization of these pigmented bands showed that they contained pigment and a protein component, but no lipids, polysaccharides, sugars, glucosamine, or phosphates were detected. Further fractionation of these pigmented bands by zone electrophoresis on a sucrose density gradient indicated that some pigment in S. marcescens was specifically attached to protein components.     

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

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

Genetic analyses ofCochliobolus heterostrophus albino mutant with deficiencies at two loci

A new class of albino mutant ofCochliobolus heterostrophus was isolated. Its colony color was indistinguishable from that of albino mutants previously reported. Application of the melanin intermediate scytalone induced this mutant to pigment slightly, but not completely. Genetic analyses showed that the mutant had two deficient genes. When only one of these genes was deficient, the colony color was indistinguishable from the wild type, whereas deficiency of both genes resulted in the albino phenotype. These deficiencies lie upstream of scytalone biosynthesis. These genes were designated asCal1 andCal2.