Ethylene Production by Pseudomonas syringae Pathovars In Vitro and In Planta

Significant amounts of ethylene were produced by Pseudomonas syringae pv. glycinea, pv. phaseolicola (which had been isolated from viny weed Pueraria lobata [Willd.] Ohwi [common name, kudzu]), and pv. pisi in synthetic medium. On the other hand, the bean strains of P. syringae pv. phaseolicola and strains of 17 other pathovars did not produce ethylene. P. syringae pv. glycinea and P. syringae pv. phaseolicola produced nearly identical levels of ethylene (about 5 x 10(sup-7) nl h(sup-1) cell(sup-1)), which were about 10 times higher than the ethylene level of P. syringae pv. pisi. Two 22-bp oligonucleotide primers derived from the ethylene-forming enzyme (efe) gene of P. syringae pv. phaseolicola PK2 were investigated for their ability to detect ethylene-producing P. syringae strains by PCR analysis. PCR amplification with this primer set resulted in a specific 0.99-kb fragment in all ethylene-producing strains with the exception of the P. syringae pv. pisi strains. Therefore, P. syringae pv. pisi may use a different biosynthetic pathway for ethylene production or the sequence of the efe gene is less conserved in this bacterium. P. syringae pv. phaseolicola isolated from kudzu and P. syringae pv. glycinea also produced ethylene in planta. It could be shown that the enhanced ethylene production in diseased tissue was due to the production of ethylene by the inoculated bacteria. Ethylene production in vitro and in planta was strictly growth associated.     

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.     

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     

An evaluation of 4-s-methyl-2-keto-butyric Acid as an intermediate in the biosynthesis of ethylene

Stimulation of ethylene production by cauliflower (Brassica oleracea var. botrytis L.) tissue in buffer solution containing 4-S-methyl-2-keto-butyric acid is not due to activation of the natural in vivo system. Increased ethylene production derives from an extra-cellular ethylene-forming system, catalyzed by peroxidase and other factors, which leak from the cauliflower tissue and cause the degradation of 4-S-methyl-2-keto-butyric acid. This exogenous ethylene-forming system is similar to the ethylene-forming horseradish peroxidase system which utilizes methional or 4-S-methyl-2-keto-butyric acid as substrate. We conclude that 4-S-methyl-2-keto-butyric acid is probably not an intermediate in the biosynthetic pathway between methionine and ethylene.     

Studies on transfer processes in mixing vessels: effect of particles on gas-liquid mass transfer using modified Rushton turbine agitators

The influence of carbon dioxide concentration in liquid medium on elemental sulphur oxidation by Thiobacillus thiooxidans bacteria presented in this paper can be divided into 3 differing relationships. First relationship shows increase of sulphur biooxidation rate with increase of carbon dioxide concentration in liquid medium. Second one shows decrease of S⁰ oxidation rate with increase of CO₂ concentration in nutrient and in the third relationship there is no influence of carbon dioxide concentration on sulphur oxidation by Thiobacillus thiooxidans bacteria. The influence of carbon dioxide concentration in liquid nutrient on alive bacteria concentration in liquid medium is similar to those described above.     

Leaching of Pyrite by Acidophilic Heterotrophic Iron-Oxidizing Bacteria in Pure and Mixed Cultures

Seven strains of heterotrophic iron-oxidizing acidophilic bacteria were examined to determine their abilities to promote oxidative dissolution of pyrite (FeS₂) when they were grown in pure cultures and in mixed cultures with sulfur-oxidizing Thiobacillus spp. Only one of the isolates (strain T-24) oxidized pyrite when it was grown in pyrite-basal salts medium. However, when pyrite-containing cultures were supplemented with 0.02% (wt/vol) yeast extract, most of the isolates oxidized pyrite, and one (strain T-24) promoted rates of mineral dissolution similar to the rates observed with the iron-oxidizing autotroph Thiobacillus ferrooxidans. Pyrite oxidation by another isolate (strain T-21) occurred in cultures containing between 0.005 and 0.05% (wt/vol) yeast extract but was completely inhibited in cultures containing 0.5% yeast extract. Ferrous iron was also needed for mineral dissolution by the iron-oxidizing heterotrophs, indicating that these organisms oxidize pyrite via the “indirect” mechanism. Mixed cultures of three isolates (strains T-21, T-23, and T-24) and the sulfur-oxidizing autotroph Thiobacillus thiooxidans promoted pyrite dissolution; since neither strains T-21 and T-23 nor T. thiooxidans could oxidize this mineral in yeast extract-free media, this was a novel example of bacterial synergism. Mixed cultures of strains T-21 and T-23 and the sulfur-oxidizing mixotroph Thiobacillus acidophilus also oxidized pyrite but to a lesser extent than did mixed cultures containing T. thiooxidans. Pyrite leaching by strain T-23 grown in an organic compound-rich medium and incubated either shaken or unshaken was also assessed. The potential environmental significance of iron-oxidizing heterotrophs in accelerating pyrite oxidation is discussed.     

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.     

Development and Application of Small-Subunit rRNA Probes for Assessment of Selected Thiobacillus Species and Members of the Genus Acidiphilium

Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using ³²P radiolabels, probe specificity was characterized by hybridization dissociation temperature (T_d) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined T_ds. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris.     

Promotion by Ethylene of the Capability to Convert 1-Aminocyclopropane-1-carboxylic Acid to Ethylene in Preclimacteric Tomato and Cantaloupe Fruits

The intact fruits of preclimacteric tomato (Lycopersicon esculentum Mill) or cantaloupe (Cucumis melo L.) produced very little ethylene and had low capability of converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. When these unripe tomato or cantaloupe fruits were treated with ethylene for 16 hours there was no increase in ACC content or in ethylene production rate, but the tissue's capability to convert ACC to ethylene increased markedly. Such an effect was also observed in fruits of tomato mutants rin and nor, which do not undergo ripening and the climacteric increase in ethylene production during the senescence. The development of this ethylene-forming capability induced by ethylene increased with increasing ethylene concentration (from 0.1 to 100 microliters per liter) and duration (1 to 24 hours); when ethylene was removed this capability remained high for sometime (more than 24 hours). Norbornadiene, a competitive inhibitor of ethylene action, effectively eliminated the promotive effect of ethylene in tomato fruit. These data indicate that the development of the capability to convert ACC to ethylene in preclimacteric tomato and cantaloupe fruits are sensitive to ethylene treatment and that when these fruits are exposed to exogenous ethylene, the increase in ethylene-forming enzyme precedes the increase in ACC synthase.     

Use of Ethylene-Producing Bacteria for Stimulation of Striga spp. Seed Germination

Striga spp. are obligate root-parasitic flowering plants that threaten cereal and legume production, and consequently human well-being, in Africa. Successful control depends on eliminating the seed reserves of Striga spp. in soil and preventing parasitism. A proven method of eliminating these seed reserves is soil-injection of ethylene gas. This method was used successfully in the United States to control Striga asiatica, but injection of ethylene gas is potentially dangerous, very costly, and generally unsuitable in Africa. The bacterium Pseudomonas syringae pathovar glycinea synthesizes relatively large amounts of ethylene. In this study a laboratory procedure was developed for testing strains of P. syringae pv. glycinea for efficacy in stimulating germination of seeds of Striga spp. The procedure allows comparisons among bacteria, volatile compounds, root exudates, and synthetic stimulants for germination of Striga spp. seeds. Seeds of three Striga spp. were tested over a 10-month period. No seed germination was ever observed with sterile water. When compared across Striga spp. the bacterial strains were consistently better stimulators of germination of seeds of the parasites than ethylene gas or root pieces of a Vigna unguiculata cultivar known to stimulate germination of parasite seeds. The strains were as effective in germinating S. aspera and S. gesnerioides seeds as a synthetic germination stimulant. Our results showing that ethylene-producing bacteria are highly effective in promoting seed germination in Striga spp. suggest that these bacteria may provide a practical means of biological control of Striga spp. in Africa and other locations.     

Enhancement of germination rate of aged seeds by ethylene

Naturally and artificially aged seeds of rape, Brassica napus L., produced less ethylene than freshly harvested seed during the early stage of germination. With freshly harvested seeds one peak of ethylene production was observed during germination, which coincided with the emergence and elongation of root and cotyledon, accompanied by splitting of the seed coat. Application of exogenous ethylene was effective in accelerating germination in aged seeds but did not significantly improve the percentage of germination. Ethylene as a hormone was considered to serve as a stimulator of germination and growth. One of the factors causing seed aging might be the degeneration of an ethylene-producing system in the seed. Exogenous ethylene may be effective only for the seeds in which the ethylene-producing system is weakened but the following responding systems are still functional.     

Stimulation of ethylene production in tomato tissue by propionic Acid

Propionic acid (10⁻³m) increases ethylene production by about 30 to 60% in tissue from green and half-ripe tomatoes (Lycopersicon esculentum Mill. var. Homestead) but does not increase ethylene production in tissue from ripe fruit. Stimulation is not due to the conversion of propionic acid to ethylene but appears to be secondary in nature and to operate on the endogenous ethylene-forming system. Thus conversion of methionine to ethylene in green and half-ripe tomato tissue is increased in the presence of propionic acid. Inhibitors which affect the normal endogenous ethylene-forming system similarly affect the propionic acid-stimulated system. Endogenous propionic acid may play a role in the regulation of ethylene production in tomato tissues.     

Metabolism of 1-Aminocyclopropane-1-Carboxylic Acid in Etiolated Maize Seedlings Grown under Mechanical Impedance

We investigated the metabolism of 1-aminocyclopropane-1-carboxylic acid (ACC) in etiolated maize (Zea mays L.) seedlings subjected to mechanical impedance by applying pressure to the growing medium. Total concentrations of ACC varied little in unimpeded seedlings, but impeded organs accumulated ACC. Roots had consistently higher concentrations of ACC than shoots or seeds, regardless of treatment. The concentration of ACC in the roots increased more than 100% during the first hour of treatment irrespective of the pressure applied; in shoots, total ACC concentration increased 46% at either low or high pressure during the first hour of treatment. The bulk of ACC synthesized under impeded and unimpeded conditions was present in a conjugated form, presumably, 1-(malonylamino)-cyclopropane-1-carboxylic acid. However, 1-(malonylamino)-cyclopropane-1-carboxylic acid increased 73% over controls after 10 hours at 25 kilopascals of pressure. Unimpeded tissue had about 77% ACC as the conjugate and 17% as free ACC, and less than 6% was used in ethylene production. Increased amounts of ACC were converted into ethylene under stress. In vivo ACC synthase activity in roots became six and seven times higher only 1 hour after initiation of treatment at 25 and 100 kilopascals of pressure, respectively, and remained high for at least 6 hours. However, the immediate and massive conjugation of mechanically induced ACC suggests that ACC N-malonyltransferase may play an important role in the regulation of mechanically induced ethylene production. After 8 hours, in vivo activity of the ethylene-forming enzyme complex increased 100 and 50% above normal level at 100 and 25 kilopascals, respectively. Furthermore, ethylene-forming enzyme complex activity was significantly greater at 100 kilopascals than in controls as early as 1 hour after treatment initiation. These data suggest that regulation of ethylene production under mechanical impedance involves the concerted action of ACC synthase, the ethylene-forming enzyme complex, and ACC N-malonyltransferase.     

Ethylene-forming Systems in Etiolated Pea Seedling and Apple Tissue

Auxin-induced ethylene formation in etiolated pea (Pisum sativum L. var. Alaska) stem segments was inhibited by inhibitors of RNA and protein synthesis. Kinetics of the inhibitions is described for actinomycin D, cordycepin, α-amanitin, and cycloheximide. α-Amanitin was the most potent and fast-acting inhibitor, when added before induction or 6 hours after induction of the ethylene-forming system. The ethylene-forming system of postclimacteric apple (Malus sylvestris L.) tissue, which is already massively induced, was not further stimulated by auxin. Ethylene production in apples was inhibited least by α-amanitin and most by actinomycin D. The relative responses of the ethylene system in apples to RNA inhibitors were different from the ethylene system of pea stems. However, the protein synthesis inhibitor, cycloheximide, appeared to act equally in both tissue systems. The effect of cycloheximide on ethylene production in postclimacteric apple tissue, already producing large quantities of ethylene, suggests a dynamic regulating system for the synthesis and degradation of the ethylene-forming system.     

Influence of calcium and magnesium on ethylene production by apple tissue slices

The decline in ethylene production in apple (Pyrus malus L. cv. Golden Delicious) tissue slices during 24 hours incubation in 600 millimolar sorbitol and 10 millimolar 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) is recognized as a senescent phenomenon. The inclusion of very high concentrations (100 millimolar) of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ severely inhibited ethylene production during the first 6 hours of incubation. However, after 6 hours and up to 24 hours the ethylene-forming system was stablized. These high concentrations of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ virtually eliminated lipid peroxidation and protein leakage from these slices. Also conversion of 1-aminocyclopropane-1-carboxylic-1-acid to ethylene and the influence of indoleacetic acid on ethylene production was stabilized after 24 hours of incubation by these high concentrations of Ca²⁺, Mg²⁺, and Ca²⁺ plus Mg²⁺. Addition of divalent ionophores severely inhibited ethylene production, but this inhibition was prevented by Ca²⁺ in concentrations greater than the ionophore. These data suggest that the loss of ethylene production by aging tissue slices results from degradation of membranes. They support previous work that indicates that the ethylene-forming system, perhaps the segment of the pathway from 1-aminocyclo-propane-1-carboxylic-1-acid to ethylene, resides in the plasma membrane.     

Genetic and Immunochemical Characterization of Thiocyanate-Degrading Bacteria in Lake Water

Natural aquatic and soil samples were screened for the presence of thiocyanate-degrading bacteria. Using thiocyanate supplementation, we established an enrichment culture containing such bacteria from lake water. The dominant bacteria had the scnC-LS5 gene encoding thiocyanate hydrolase, which was closely related to the enzyme found previously in Thiobacillus thioparus THI115 isolated from activated sludge.     

Genetic improvement of acidophilic bacteria for biohydrometallurgical applications

Abstract

Recent success in introducing foreign genetic information into Acidiphilium and Thiobacillus ferrooxidans have opened the possibility of the improvement of the bioleaching properties of acidophilic bacteria through genetic means. The use of electroporation to transform acidophilic bacteria, while effective for diverse genera of bacteria, is potentially limited by observed strain dependence within a given species. Bacterial mating or conjugation may prove more widely applicable, but has not yet been demonstrated in T. ferrooxidans. An arsenic‐resistant, broad‐host‐range plasmid has been constructed and introduced by conjugation into Acidiphilium. This recombinant organism is being examined to assess whether it might lead to improved arsenopyrite leaching rates in mixed cultures with chemolithotrophs such as T. ferrooxidans and Leptospirillum ferrooxidans.     

Production of Acylated Homoserine Lactones by Psychrotrophic Members of the Enterobacteriaceae Isolated from Foods

Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic Enterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5°C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 10⁶ CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of Enterobacteriaceae stored at 5°C under an N₂ atmosphere when mean cell densities increased to 10⁶ CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous Enterobacteriaceae reached 10⁶ CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity.     

Grazing of acidophilic bacteria by a flagellated protozoan

A biflagellated protozoan was isolated from an acidic drainage stream located inside a disused pyrite mine. The stream contained copious amounts of acid streamer bacterial growths, and the flagellate was observed in situ apparently grazing the streamer bacteria. The protozoan was obligately acidophilic, growing between pH 1.8 and 4.5, but not at pH 1.6 or 5.0, with optimum growth between pH 3 and 4. It was highly sensitive to copper, molybdenum, silver, and uranium, but tolerated ferrous and ferric iron up to 50 and 25 mM, respectively. In the laboratory, the protozoan was found to graze a range of acidophilic bacteria, including the chemolithotrophs Thiobacillus ferrooxidans, Leptospirillum ferrooxidans, and the heterotroph Acidiphilium cryptum. Thiobacillus thiooxidans and Thiobacillus acidophilus were not grazed. Filamentous growth of certain acidophiles afforded some protection against being grazed by the flagellate. In mixed cultures of T. ferrooxidans and L. ferrooxidans, the protozoan isolate displayed preferential grazing of the former. The possibility of using acidophilic protozoa as a means of controlling bacteria responsible for the production of acid mine drainage is discussed.Offprint requests to: Dr. D. B. Johnson.     

Prevalence of Enterotoxigenic Clostridium perfringens in Meats in San Luis,Argentina

Clostridium perfringens is an important pathogen agent causing, among other diseases, enteritis in humans and enterotoxemia in domestic animals. This bacterium can produce more than 15 toxins, one of which is its enterotoxin (CPE), that causes human food poisoning. The aim of this work was (i) to determine the prevalence of C. perfringens in some non-industrial meat foods in San Luis, Argentina, (ii) to characterize the C. perfringens enterotoxigenic strains by PCR, RPLA and the slide reverse passive latex agglutination test, (iii) to type the C. perfringens strains isolated and identification by PCR and (iv) to develop a slide RPLA test. A total of 515 samples of meat food (315 fresh sausages, 100 hamburgers and 100 samples of minced meat) were studied. A 126 C. perfringens strains (24.46%) were isolated and characterized. Of these C. perfringens -positive samples, 48 contained counts higher than 2 log/g. No significant differences were observed between counts performed in iron–milk medium and tryptose–sulfite–cycloserine agar (r= 0.99). Twelve samples (9.52%) exhibited counts with MPN >5log bacteria/g. Modified Tórtora medium (Tm) with thiotone replaced by proteose peptone turned out to be the most useful medium for both sporulation and enterotoxin production. Of the 126 samples tested by PCR and RPLA, nine strains (7.14%) were enterotoxigenic. Similar results were obtained by Slide RPLA, which exhibited a sensitivity of 8 ng/mL. Of the 126 C. perfringens strains , 123 were of type A (97.20%), two were of type C (1.59%) and one of type E (0.79%). All enterotoxigenic strains were classified as type A.