The physiology of L-methionine catabolism to the secondary metabolite ethylene by Escherichia coli

Catabolism of L-methionine by Escherichia coli strain B SPAO led to the formation of ethylene as a secondary metabolite (ethylenogenesis). Methionine was initially deaminated by a transamination reaction to the 2-oxo acid 2-oxo-4-methylthiobutyric acid (KMBA) which was then converted to ethylene. The utilization of L-methionine as an additional nitrogen source was investigated by examining ethylene synthesis under different nitrogen supply conditions. Ethylene formation in batch culture was unaffected by the concentration of the precursor L-methionine in the medium although increasing concentrations of NH4Cl resulted in progressively less ethylene formation. Cultures grown without L-methionine did not produce ethylene but were able to synthesize ethylene when L-methionine or KMBA was provided. Addition of L-tyrosine to batch cultures reduced the yield of ethylene after 42 h by 54%. Under these conditions the maximum transient level of KMBA was reduced by 32% and occurred later compared to when L-methionine was the only amino acid supplement. Continuous cultures grown under ammonia limitation produced both ethylene and KMBA. In contrast, when glucose was limiting, neither of these metabolites were produced. Cells harvested from continuous cultures grown under glucose or ammonia limitation were able to synthesize ethylene from either L-methionine or KMBA although their capacity for ethylene synthesis (ethylenogenic capacity) was optimal under ammonia limitation (C:N ratio = 20).     

Bacterial ethylene synthesis from 2-oxo-4-thiobutyric acid and from methionine

The ability of selected bacterial cultures to synthesize ethylene during growth in nutrient broth supplemented with methionine or 2-oxo-4-methylthiobutyric acid (KMBA) was examined. Although most cultures transformed KMBA into ethylene, only those of Escherichia coli SPAO and Chromobacterium violaceum were able to convert exogenously added methionine to ethylene. In chemically defined media, E. coli SPAO produced the highest amounts of ethylene from methionine and KMBA. This capability was affected by the nature of the carbon source and the type and amount of nitrogen source used for growth. When glutamate was used as sole source of carbon and nitrogen for growth, the activity of the ethylenogenic enzymes was reduced to 25% of that observed with cultures grown with glucose and NH4Cl. Neither methionine nor KMBA significantly affected the ethylenogenic capacity of E. coli SPAO. Menadione and paraquat, compounds that generate superoxide radicals, stimulated ethylene synthesis by harvested cells, but not by cell-free extracts of E. coli SPAO. In addition, cells of Pseudomonas aeruginosa, which produced no ethylene in culture in the presence of exogenously added KMBA, yet possessed the necessary enzymes in an active form, were able to synthesize ethylene from KMBA when incubated with menadione or paraquat.     

Ethylene formation by cultures of Escherichia coli

Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH₄Cl and methionine resulted in production of ethylene into the culture headspace. When methionine was excluded from the medium there was little formation of ethylene. Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth. Ethylene formation was stimulated by increasing the medium concentration of Fe³⁺ when it was chelated to EDTA. Lowering the medium phosphate concentration also appeared to stimulate ethylene formation. Ethylene formation was inhibited in cultures where NH₄Cl remained in the stationary phase. Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine. Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation. It is concluded that ethylene production by E. coli exhibits the typical properties of secondary metabolism.Abbreviations HMBA 2-Hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - KMBA 2-keto-4-methylthiobutyric acid - MOPS 3-[N-morpholino] propanesulphonic acid     

Ethylene production from l-methionine by Cryptococcus albidus

Cryptococcus albidus IFO 0939 was selected from microorganisms producing ethylene from l-methionine in a culture medium. When methionine was excluded from the culture medium of C. albidus, there was little production of ethylene. Ethylene production in a methionine-containing culture medium occurred for a brief period at the end of the growth phase. 2-oxo-4-methylthiobutyric acid (KMBA), a deaminated product of methionine, accumulated in the culture filtrate. An ethylene-forming enzyme was partially purified from C. albidus by means of DEAE-Sepharose CL-6B ion exchange chromatography, and a cell-free ethylene-forming system was constructed. Using this system, the precursor of ethylene was found to be KMBA and essential factors were NAD(P)H, Fe³⁺, EDTA and oxygen.     

Evaluation of the role of methional, 2-keto-4-methylthiobutyric acid and peroxidase in ethylene formation by Escherichia coli.

During growth of Escherichia coli strain SPA O in the presence of methionine, an intermediate accumulates in the medium. This intermediate reacts with 2,4-dinitrophenylhydrazine, and can be degraded to ethylene either enzymically or photochemically, the latter being stimulated by the addition of a flavin. The pH optimum for the photochemical degradation of this intermediate and 2-keto-4-methylthiobutyric acid (KMBA) is pH 3 whereas the optimum for methional is pH 6. The enzyme which converts the intermediate to ethylene also converts KMBA to ethylene and has many of the properties of a peroxidase including inhibition by catalase, cyanide, azide and anaerobiosis. The enzyme which synthesizes the intermediate is not known but requires oxygen and pyridoxal phosphate. A pathway for ethylene biosynthesis is proposed in which methionine is converted to KMBA which can be degraded either by peroxidase or in a flavin-mediated photochemical reaction. Its relevance to the properties of other ethylene-producing bacteria and to the proposed pathway of ethylene release by higher plants is discussed.     

Effect of substrate-dependent microbialy produced ethylene on plant growth

Various compounds have been identified as precursors/substrates for the synthesis of ethylene (C2H4) in soil. This study was designed to compare the efficiency of four substrates, namely L-methionine (L-MET), 2-keto-4-methylthiobutyric acid (KMBA), 1-aminocyclopropane-1-carboxylic acid (ACC), and calcium carbide (CaC2) for ethylene biosynthesis in a sandy clay loam soil by gas chromatography. The classic "triple" response in etiolated pea seedling was employed as a bioassay to demonstrate the effect of substrate-dependent microbialy produced ethylene on plant growth. Results revealed that an amendment with L-MET, KMBA, ACC (up to 0.10 g/kg soil) and CaC2 (0.20 g/kg soil) significantly stimulated ethylene biosynthesis in soil. Overall, ACC proved to be the most effective substrate for ethylene production (1434 nmol/kg soil), followed by KMBA, L-MET, and CaC2 in descending order. Results further revealed that ethylene accumulation in soil released from these substrates created a classic "triple" response in etiolated pea seedlings with different degrees of efficacy. A more obvious classic "triple" response was observed at 0.15, 0.10, and 0.20 g/kg soil of L-MET, KMBA/ACC, and CaC2, respectively. Similarly, direct exposure of etiolated pea seedlings to commercial ethylene gas also modified the growth pattern in the same way. A significant direct correlation (r = 0.86 to 0.97) between substrate-derived [C2H4] and the classic triple response in etiolated pea seedlings was observed. This study demonstrated that the presence of substrate(s) in soil may lead to increased ethylene concentration in the air of the soil, which may affect plant growth in a desired direction.     

Pathways that produce volatile sulphur compounds from methionine in Oenococcus oeni

Aims:  Determination of pathways involved in synthesis of volatile sulphur compounds (VSC) from methionine by Oenococcus oeni isolated from wine.
Methods and Results:  Production of VSC by O. oeni from methionine was investigated during bacterial cultures and in assays performed in the presence of resting cells or protein fractions. Cells of O. oeni grown in a medium supplemented with methionine produced methanethiol, dimethyl disulphide, methionol and 3-(methylthio)propionic acid. Methional was also detected, but only transiently during the exponential growth phase. It was converted to methionol and 3-(methylthio) propionic acid in assays. Although this acid could be produced alternatively from 2-oxo-4-(methylthio) butyric acid (KMBA) by oxidative decarboxylation. In addition, KMBA was a precursor for methanethiol and dimethyl disulphide synthesis. Interestingly, assays with resting cells and protein fractions suggested that a specific enzyme could be involved in this conversion in O. oeni .
Conclusion:  This work shows that methional and KMBA are the key intermediates for VSC synthesis from methionine in O. oeni . Putative enzymatic and chemical pathways responsible for the production of these VSC are discussed.
Significance and impact of the study:  This work confirms the capacity of O. oeni to metabolize methionine and describes the involvement of potential enzymatic pathways.     

Methionine-induced Ethylene Production by Penicillium digitatum

Shake cultures, in contrast to static cultures of Penicillium digitatum grown in liquid medium, were induced by methionine to produce ethylene. The induction was concentration-dependent, and 7 mM was optimum for the methionine effect. In the presence of methionine, glucose (7 mM) enhanced ethylene production but did not itself induce ethylene production. The induction process lasted several hours, required the presence of viable mycelium, exhibited a lag period for ethylene production, and was effectively inhibited by cycloheximide and actinomycin D. Thus, the methionine-induced ethylene production appeared to involve induction of an enzyme system(s). Methionine not only induced ethylene production but was also utilized as a substrate since labeled ethylene was produced from [¹⁴C]methionine.     

Isolation of an oxalate-resistant Ashbya gossypii strain and its improved riboflavin production

An oxalate-resistant strain of Ashbya gossypii was naturally isolated from spores grown on an oxalate-containing medium, and its medium was optimized to improve riboflavin production. Riboflavin production by the resistant strain was three-fold higher than that by the wild-type organism when grown in flask cultures. Medium optimization increased the riboflavin production by the resistant strain to 5 g l⁻¹, which was five-fold higher than that obtained by the wild-type strain. The productivity was reproduced in a 3-l bioreactor. During the early growth phase, the specific activity of isocitrate lyase in the oxalate-resistant strain was slightly higher than that in the wild-type strain. Proteomic analysis of the oxalate-resistant strain revealed that the expression of aldose reductase and cobalamin-independent methionine synthase decreased significantly. This is the first report that describes the natural isolation of a riboflavin producer using an antimetabolite-containing medium to enhance the riboflavin production level. This method should also be useful for improving the productivity of other bioproducts since it does not require any mutations or genetic modifications of the microorganism.     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system     

L-methionine as an ethylene precursor in Saccharomyces cerevisiae.

L-Methionine induced production of ethylene by Saccharomyces cerevisiae growing in lactate medium. The production induced by L-methionine was inhibited by pyruvate, and elevated by glucose. Labeled ethylene was produced when L-[U-14C]methionine, but not [U-14C]glucose, was fed to the yeast. The mutant S. cerevisiae G1332 (ade-, met-) did not produce significant amounts of ethylene unless L-methionine was added. Thus L-methionine acts as a precursor of ethylene in S. cerevisiae. The role of glucose appears to be other than as a precursor.     

Characterization and localization of prodiginines from Streptomyces lividans suppressing Verticillium dahliae in the absence or presence of Arabidopsis thaliana

The ascomycete Verticillium dahliae causes worldwide vascular wilt of many field and horticultural plants. The melanized resting structures of this fungus, so-called microsclerotia, survive for many years in soils and continuously re-infect plants. Due to the absence of known fungicides, Verticillium wilt causes immense crop losses. We discovered that the Gram-positive, spore-forming soil bacterium Streptomyces lividans expresses members of the prodiginine family during co-cultivation with V. dahliae. Using HPLC and LC-MS analysis of cultures containing S. lividans alone or grown together with V. dahliae, we found that undecylprodigiosin [394.4 M+H](+) is highly abundant, and streptorubin B [392.4 M+H](+) is present in smaller amounts. Within co-cultures, the quantity of undecylprodigiosin increased considerably and pigment concentrated at and within fungal hyphae. The addition of purified undecylprodigiosin to growing V. dahliae hyphae strongly reduced microsclerotia formation. Undecylprodigiosin was also produced when S. lividans grew on the roots of developing Arabidopsis thaliana plants. Furthermore, the presence of the undecylprodigiosin producer led to an efficient reduction of V. dahliae hyphae and microsclerotia on plant-roots. Based on these novel findings and previous knowledge, we deduce that the prodiginine investigated leads to multiple cellular effects, which ultimately impair specific pathways for signal transduction and apoptosis of the fungal plant pathogen.     

Effect of substrate-dependent microbial ethylene production on plant growth

Various compounds have been identified as precursors/substrates for the synthesis of ethylene (C₂H₄) in soil. This study was designed to compare the efficiency of four substrates, namely L-methionine (L-MET), 2-keto-4-methylthiobutyric acid (KMBA), 1-aminocyclopropane-1-carboxylic acid (ACC), and calcium carbide (CaC₂), for ethylene biosynthesis in a sandy clay loam soil by gas chromatography. The classic “triple” response in etiolated pea seedling was employed as a bioassay to demonstrate the effect of substrate-dependent microbial production of ethylene on plant growth. Results revealed that an amendment with L-MET, KMBA, ACC (up to 0.10 g/kg soil) and CaC₂ (0.20 g/kg soil) significantly stimulated ethylene biosynthesis in soil. Overall, ACC proved to be the most effective substrate for ethylene production (1434 nmol/kg soil), followed by KMBA, L-MET, and CaC₂ in descending order. Results further revealed that ethylene accumulation in soil from these substrates caused a classic “triple” response in etiolated pea seedlings with different degrees of efficacy. A more obvious classic “triple” response was observed at 0.15, 0.10, and 0.20 g/kg soil of L-MET, KMBA/ACC, and CaC₂, respectively. Similarly, direct exposure of etiolated pea seedlings to commercial ethylene gas also modified the growth pattern in the same way. A significant direct correlation (r = 0.86 to 0.97) between substrate-derived C₂H₄ and the classic triple response in etiolated pea seedlings was observed. This study demonstrated that the presence of substrate(s) in soil may lead to increased ethylene concentration in the air of the soil, which may affect plant growth in a desired direction. Published in Russian in Mikrobiologiya, 2006, Vol. 75, No. 2, pp. 277–283. The text was submitted by the authors in English.     

Catabolism of volatile sulfur compounds precursors by Brevibacterium linens and Geotrichum candidum, two microorganisms of the cheese ecosystem

Two Brevibacterium linens strains and the cheese-ripening yeast Geotrichum candidum were compared with regard to their ability to produce volatile sulfur compounds (VSCs) from three different precursors namely L-methionine, 4-methylthio-2-oxobutyric acid (KMBA) and 4-methylthio-2-hydroxybutyric acid (HMBA). All microorganisms were able to convert these precursors to VSCs. However, although all were able to produce VSCs from L-methionine, only G. candidum accumulated KMBA when cultivated on this amino acid, contrary to B. linens suggesting that the transamination pathway is not active in this microorganism. Conversely, a L-methionine gamma-lyase activity--which catalyses the one step L-methionine to methanethiol (MTL) degradation route--was only found in B. linens strains. Several other enzymatic activities involved in the catabolism of the precursors tested were investigated. KMBA transiently accumulated in G. candidum cultures, and was then reduced to HMBA by a KMBA dehydrogenase (KDH) activity. This activity was not detected in B. linens. Despite no HMBA dehydrogenase (HDH) was found in G. candidum, a strong HMBA oxidase (HOX) activity was measured in this microorganism. This latter activity was weakly active in B. linens. KMBA and HMBA demethiolating activities were found in all the microorganisms. Our results illustrate the metabolic diversity between cheese-ripening microorganisms of the cheese ecosystem.     

大丽轮枝菌毒素的分离、提纯及生物测定

试验用Czapek's培养液培养大丽轮枝菌(Verticillium dahliae Kleb.)不同致病力类型的5个菌株的培养滤液,经浓缩、离心和透析制成的粗毒素,再经DEAE-纤维素柱层析得到初提毒素,最后通过琼脂糖凝胶过滤层析获得纯毒素样品。生物活性测定表明初提毒素的最低生物活性浓度为5.0—5.5μg/ml。纯毒素的最低生物活性浓度为4.0—4.5μg/ml。配制相同浓度的不同致病力类型菌株的毒素液,它们的培养滤液、粗毒素或纯毒素对棉苗的致萎能力相同。     

Sequence tag analysis of gene expression during pathogenic growth and microsclerotia development in the vascular wilt pathogen Verticillium dahliae

Two cDNA libraries were constructed from cultures of the vascular wilt fungus Verticillium dahliae, grown either in simulated xylem fluid medium (SXM) or under conditions that induce near-synchronous development of microsclerotia. Expressed sequence tags (ESTs) were obtained for over 1000 clones from each library. Most sequences in the two EST collections were unique; nearly 55% of the translated ESTs had strong similarity to protein sequences in the NCBI nonredundant database. ESTs corresponding to melanin biosynthetic enzymes were exclusive to the developing microsclerotia (DMS) collection, and sequences corresponding to extracellular hydrolases (plant cell wall degrading enzymes) were more abundant in that collection. ESTs corresponding to proteins involved in transport and cell growth were more abundant in the SXM collection. The results of this preliminary analysis suggest that the in vitro growth conditions used here provide useful model systems that will facilitate studies of pathogenesis and microsclerotia development in V. dahliae.     

Ethylene Biosynthesis and Xylogenesis in Lactuca Pith Explants Cultured In Vitro in the Presence of Auxin and Cytokinin: The Effect of Ethylene Precursors and Inhibitors

The possible involvement of ethylene in the induction of xylemdifferentiation was studied in lettuce (Lactuca saliva L. cv.Romaine) pith parenchyma explants. The addition of the ethyleneprecursors L-methionine (0.25 µM), S-adenosylmethionine(25 µM) and 1-aminocyclopropane-l-carboxylic acid (0.01µM), or the ethylene-releasing agent 2-chloroethylphosphonicacid (1.0 µM), to a standard IAA-kinetin-containing mediumenhanced xylogenesis compared to control explants cultured inthe absence of these compounds. In the presence of the ethyleneinhibitors aminoethoxyvinylglycine, Co(NO₃)₂ and AgNO₃, xylogenesiswas inhibited. Inhibition of xylogenesis by aminoethoxyvinylglycine(75 µM), Co(NO₃)₂ (50 µM) and AgNO₃ (6.0 µM)was reversed by exogenous 1-aminocyclopropane-l-carboxylic acid(0.01 µM), 2-chloroethylphosphonic acid (5.0 µM)and L-methionine (0.25 µM), respectively. Ethylene productionby explants cultured on media containing L-methionine or 1-aminocyclopropane-l-carboxylicacid was greater than the biosynthesis of ethylene by explantscultured in the absence of these compounds. The incorporationof 2-chloroethylphosphonic acid into the culture medium resultedin higher rates of ethylene production compared to explantscultured on the IAA-kinetin medium. The presence of either aminoethoxyvinylglycineor Co(NO₃)₂ inhibited ethylene production by explants culturedon the IAA-kinetin medium. The data support the hypothesis thatethylene plays a positive role in the initiation of xylem differentiation. Key words: Xylogenesis, Differentiation, Ethylene, IAA, Kinetin, Lactuca sativa     

Rhizogenesis and root growth ofCarica papaya L.in vitro in relation to auxin sensitive phases and use of riboflavin

High rooting percentages and high-quality adventitious root systems for papaya (Carica papaya L.) were obtainedin vitro by appropriate auxin source, duration of exposure to auxin and use of riboflavin. Root initiation of papaya shoots was higher using IBA than IAA, NAA or PCPA. Maximum rooting percentage (96%) was achieved by exposure of shoots to a medium containing 10 µM IBA for 3 days before transfer to a hormone-free medium. However, the resultant plants had small shoots and callused roots. Shoot and root growth were improved when shoots were transferred after 2 days from medium containing 10 µM IBA to hormone-free medium containing 10 µM riboflavin. Good root initiation, and root and shoot growth were also obtained when shoots were incubated for 2 days in darkness on a medium containing 10 µM IBA and 31 µM riboflavin before transfer to light. Alternatively, cultures could be placed in the light on medium containing 10 µM IBA, and after 1 day the medium overlaid with 300 µM riboflavin (1 ml over 10 ml of medium).     

Ethylene production by Agrobacterium rhizogenes strains in vitro and in vivo

Research was initiated to determine whether Agrobacteriumrhizogenes strains, used for plant transformation, are a source ofethylene and which compound these bacteria use for its production. A4 and LBAstrains produced ethylene on solid MG medium, used for culture of bacteria, andon MS medium used for plant in vitro culture. The enhancedethylene production in the presence of methionine and2-keto-4-methylthiobutyricacid (KMBA), but not in the presence of glutamic acid and1-aminocyclopropano-1-carboxylic acid (ACC), was observed. The removing ofethylene from the culture atmosphere caused the inhibition of bacterial growthand indicates, that these strains need this gas for their growth. Theinoculation of petunia explants with A. rhizogenes causedincreased ethylene production by the explants. Ethylene present in flasksduringthe growth of hairy roots of Petunia hybrida inhibitedtheir growth. These results indicate that in the flasks where the planttransformation takes place the source of ethylene, affecting the root growth,ispossibly not only the plant explant but also bacteria and pathogenesis.     

Effects of Fungal Elicitors on Cell Growth and Artemisinin Accumulation in Hairy Root Cultures of Artemisia annua

The artemisinin accumulation in the hairy root cultures of Artemisia annua L. was enhanced via a treatment of three fungal elicitors separately ( Verticillium dahliae Kleb., Rhizopus stolonifer (Ehrenb. ex Fr. ) Vuill and Colletotrichum dematium (Pers.) Grove). Among these three elicitors, V. dahliae had the highest inducing efficiency, but none of them manifests any noticeable effects on the cell growth of the hairy root cultures. The artemisinin content of the hairy root cultures treated with V. dahliae elicitor was 1.12 mg/g DW, which was 45% higher than the control (0.77 mg/g DW). The results showed that elicitation was dependent on the elicitor concentration, the incubation period and the physiological stage at which the hairy root cultures were treated. In addition, the authors found that for V. dahliae, the optimum concentration was 0.4 mg carbohydrate per millilitre medium, the strongest response of A. annua hairy root cultures to the elicitation was at the late exponential growth stage, and the highest artemisinin content of the hairy root cultures was on the 4th day post treatment.