Amino Acid Production by Submerged Cultivation ofPseudomonas fluorescens on Gasoline

During our screening program for hydrocarbon-degrading microorganismsPseudomonas fluorescens was isolated that grew on gasoline as the sole source of carbon and energy. During growth on gasoline it produced proline and tryptophan. Among the nitrogen salts tested, ammonium sodium hydrogen orthophosphate supported maximum amino acid production and growth. By optimizing the medium components a two-fold increase in proline and tryptophan production was achieved.     

Artificial Citrate Operon Confers Mineral Phosphate Solubilization Ability to Diverse Fluorescent Pseudomonads

Citric acid is a strong acid with good cation chelating ability and can be very efficient in solubilizing mineral phosphates. Only a few phosphate solubilizing bacteria and fungi are known to secrete citric acids. In this work, we incorporated artificial citrate operon containing NADH insensitive citrate synthase (gltA1) and citrate transporter (citC) genes into the genome of six-plant growth promoting P. fluorescens strains viz., PfO-1, Pf5, CHAO1, P109, ATCC13525 and Fp315 using MiniTn7 transposon gene delivery system. Comprehensive biochemical characterization of the genomic integrants and their comparison with plasmid transformants of the same operon in M9 minimal medium reveals the highest amount of ∼7.6±0.41 mM citric and 29.95±2.8 mM gluconic acid secretion along with ∼43.2±3.24 mM intracellular citrate without affecting the growth of these P. fluorescens strains. All genomic integrants showed enhanced citric and gluconic acid secretion on Tris-Cl rock phosphate (TRP) buffered medium, which was sufficient to release 200–1000 µM Pi in TRP medium. This study demonstrates that MPS ability could be achieved in natural fluorescent pseudomonads by incorporation of artificial citrate operon not only as plasmid but also by genomic integration.     

Characterization of a Cell Envelope-Associated Proteinase Activity from Streptococcus thermophilus H-Strains

The production and biochemical properties of cell envelope-associated proteinases from two strains of Streptococcus thermophilus (strains CNRZ 385 and CNRZ 703) were compared. No significant difference in proteinase activity was found for strain CNRZ 385 when cells were grown in skim milk medium and M17 broth. Strain CNRZ 703 exhibited a threefold-higher proteinase activity when cells were grown in low-heat skim milk medium than when grown in M17 broth. Forty-one percent of the total activity of CNRZ 385 was localized on the cell wall. The optimum pH for enzymatic activity at 37°C was around 7.0. Serine proteinase inhibitors, such as phenylmethylsulfonyl fluoride and diisopropylfluorophosphate, inhibited the enzyme activity in both strains. The divalents cations Ca²⁺, Mg²⁺, and Mn²⁺ were activators, while Zn²⁺ and Cu²⁺ were inhibitors. β-Casein was hydrolyzed more rapidly than α_s1-casein. The results of DNA hybridization and immunoblot studies suggested that the S. thermophilus cell wall proteinase and the lactococcal proteinase are not closely related.     

Optimization of the hide powder azure assay for quantitating the protease of Pseudomonas fluorescens

The extracellular protease of Pseudomonas fluorescens NC 3 was optimally active at 40°C in a reaction mixture containing: 50 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer (pH 6.6), 0.5 mM CaCl₂, and 25 mg hide powder azure in 5 ml total volume. Divalent cation chelators, i.e., EDTA, o-phenanthroline, citrate or phosphate, inhibited the enzyme. Protease production by P. fluorescens NC 3 was initiated during late-logarithmic-growth phase in a sodium caseinate medium and reached its maximum at the onset of the stationary phase.     

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h⁻¹ and a maximum prodigiosin concentration of 80 ng of prodigiosin · mg of cell protein⁻¹ were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h⁻¹), and maximum prodigiosin concentrations were higher (1,500 ng · mg of protein⁻¹). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na⁺; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na⁺ up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH₂PO₄ above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion · liter⁻¹, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng · mg protein⁻¹) was observed in chemostat cultures at a dilution rate of 0.057 h⁻¹. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h⁻¹, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.     

Growth Characteristics of Polyporales Mushrooms for the Mycelial Mat Formation

Mushroom strains of Polyporales from the genera Coriolus, Trametes, Pycnoporus, Ganoderma, and Formitella were explored in terms of mycelial growth characteristics for the application of mushroom mycelia as alternative sources of materials replacing fossil fuel-based materials. Among the 64 strains of Polyporales, G. lucidum LBS5496GL was selected as the best candidate because it showed fast mycelial growth with high mycelial strength in both the sawdust-based solid medium and the potato dextrose liquid plate medium. Some of the Polyporales in this study have shown good mycelial growth, however, they mostly formed mycelial mat of weak physical strength. The higher physical strength of mycelial mat by G. lucidum LBS5496GL was attributed to its thick hyphae with the diameter of 13 µm as revealed by scanning electron microscopic analysis whereas the hyphae of others exhibited less than 2 µm. Glycerol and skim milk supported the best mycelial growth of LBS5496GL as a carbon and a nitrogen source, respectively.     

Enhancement of linear gramicidin expression from Bacillus brevis ATCC 8185 by casein peptide

Bacillus brevis (Brevibacillus parabrevis) ATCC 8185 synthesizes two kinds of antibiotic peptides, cyclopeptide tyrocidine and linear gramicidin. The production of linear gramicidin can be induced by the standard method (using a skim milk medium for pre-culture and beef broth for the main culture) employed for the induction of tyrocidine. In this study, we tried to determine the optimal growth medium for B. brevis ATCC 8185 for synthesizing linear gramicidin. The yield of linear gramicidin produced by the standard method was 3.11 microg/ml. When beef broth was used both as the pre-medium and the main medium, the yield of the antibiotic was only 0.59 microg/ml. To confirm the influence of skim milk, the strain was grown in a 1% skim milk medium. As a result, the amount of linear gramicidin produced reached 20.3 microg/ml. These findings show the importance of skim milk in the production of linear gramicidin. In the skim milk medium, the cells produced an extracellular protease 2 h before the linear gramicidin was expressed. The 1% skim milk medium pretreated by this protease did not allow the induction of linear gramicidin into the cells, and protease activity was not detected in the supernatant of the culture. When the cells were cultivated in a 1% egg albumin medium, protease activity from the supernatant of the culture was detected, but production of linear gramicidin was not observed. Therefore, a 1% casein medium was used for production of linear gramicidin. As a result, the yield of linear gramicidin produced in the medium reached 6.69 microg/ml. We concluded that a digested product of the extracellular protease from casein enhances linear gramicidin production.     

Potential of the Virion-Associated Peptidoglycan Hydrolase HydH5 and Its Derivative Fusion Proteins in Milk Biopreservation

Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88], and three different fusion proteins created between HydH5 and lysostaphin has been assessed. The lytic activity of the five proteins (HydH5, HydH5Lyso, HydH5SH3b, CHAPSH3b and lysostaphin) was confirmed using commercial whole extended shelf-life milk (ESL) in challenge assays with 10⁴ CFU/mL of the strain S. aureus Sa9. HydH5, HydH5Lyso and HydH5SH3b (3.5 µM) kept the staphylococcal viable counts below the control cultures for 6 h at 37°C. The effect is apparent just 15 minutes after the addition of the lytic enzyme. Of note, lysostaphin and CHAPSH3b showed the highest staphylolytic protection as they were able to eradicate the initial staphylococcal challenge immediately or 15 min after addition, respectively, at lower concentration (1 µM) at 37°C. CHAPSH3b showed the same antistaphyloccal effect at room temperature (1.65 µM). No re-growth was observed for the remainder of the experiment (up to 6 h). CHAPSH3b activity (1.65 µM) was also assayed in raw (whole and skim) and pasteurized (whole and skim) milk. Pasteurization of milk clearly enhanced CHAPSH3b staphylolytic activity in both whole and skim milk at both temperatures. This effect was most dramatic at room temperature as this protein was able to reduce S. aureus viable counts to undetectable levels immediately after addition with no re-growth detected for the duration of the experiment (360 min). Furthermore, CHAPSH3b protein is known to be heat tolerant and retained some lytic activity after pasteurization treatment and after storage at 4°C for 3 days. These results might facilitate the use of the peptidoglycan hydrolase HydH5 and its derivative fusions, particularly CHAPSH3b, as biocontrol agents for controlling undesirable bacteria in dairy products.     

Physiological Conditions Conducive to High Cyanophycin Content in Biomass of Acinetobacter calcoaceticus Strain ADP1

The effects of the inorganic medium components, the initial pH, the incubation temperature, the oxygen supply, the carbon-to-nitrogen ratio, and chloramphenicol on the synthesis of cyanophycin (CGP) by Acinetobacter calcoaceticus strain ADP1 were studied in a mineral salts medium containing sodium glutamate and ammonium sulfate as carbon and nitrogen sources, respectively. Variation of all these factors resulted in maximum CGP contents of only about 3.5% (wt/wt) of the cell dry matter (CDM), and phosphate depletion triggered CGP accumulation most substantially. However, addition of arginine to the medium as the sole carbon source for growth promoted CGP accumulation most strikingly. This effect was systematically studied, and an optimized phosphate-limited medium containing 75 mM arginine and 10 mM ammonium sulfate yielded a CGP content of 41.4% (wt/wt) of the CDM at 30°C. The CGP content of the cells was further increased to 46.0% (wt/wt) of the CDM by adding 2.5 μg of chloramphenicol per ml of medium in the accumulation phase. These contents are by far the highest CGP contents of bacterial cells ever reported. CGP was easily isolated from the cells by using an acid extraction method, and this CGP contained about equimolar amounts of aspartic acid and arginine and no detectable lysine; the molecular masses ranged from 21 to 29 kDa, and the average molecular mass was about 25 kDa. Transmission electron micrographs of thin sections of cells revealed large CGP granules that frequently had an irregular shape with protuberances at the surface and often severely deformed the cells. A cphI::ΩKm mutant of strain ADP1 with a disrupted putative cyanophycinase gene accumulated significantly less CGP than the wild type accumulated, although the cells expressed cyanophycin synthetase at about the same high level. It is possible that the intact CphI protein is involved in the release of CGP primer molecules from initially synthesized CGP. The resulting lower concentration of primer molecules could explain the observed low rate of accumulation at similar specific activities.     

Propagation of Rhizopus javanicus Biosorbent

After propagation of Rhizopus javanicus in defined media containing glucose, urea, and mineral salts in deionized distilled water, the ability of the nonliving biomass to sequester cupric ion was assayed. Growth, uptake capacity (saturation uptake at >1 mM Cu²⁺ concentration in solution), and biosorptive yield (biomass concentration × uptake capacity) were increased by augmentation of the growth medium with mineral salts once growth was under way. In the stationary phase, the uptake capacity of mycelia, which were normally a poor biosorbent, was improved within 4 h of trace metal addition to the growth medium. Growth of the culture was inhibited by excessive concentrations (0.04 to 40 μM) of metals in the medium in the following order: Cu > Co ≥ Ni > Mn > Mo; zinc was not inhibitory at 40 μM, and chromium was stimulatory at 0.53 μM but slightly inhibitory at higher levels. Iron and potassium phosphate stimulated growth at levels of 0.53 and 40 mM, respectively. When R. javanicus was propagated in a medium with a high salt concentration, exponential growth (0.23 h⁻¹) to a biomass concentration of >3 g/liter and a biosorptive yield of >500 μmol/liter was achieved. It is evident that the powerful biosorbent characteristics of Rhizopus biomass led to depletion of available trace minerals in suspension culture, which in turn limited growth.     

Stability and Specificity of the Cell Wall-Associated Proteinase from Lactococcus lactis subsp. cremoris H2 Released by Treatment with Lysozyme in the Presence of Calcium Ions

The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris H2 (isolate number 4409) was released from the cells by treatment with lysozyme, even in the presence of 50 mM calcium chloride. Cell lysis during lysozyme treatment was minimal. The proteinase activity released by lysozyme treatment fractionated on ion-exchange chromatography as three main forms, the molecular masses of which were determined by gel exclusion chromatography and polyacrylamide gel electrophoresis. Two of the enzyme forms released, 137 and 145 kDa, were the same as those released by incubation of cells in calcium-free phosphate buffer. In the presence of calcium, lysozyme treatment also resulted in the release of a 180-kDa enzyme molecule. The total proteinase activity released by lysozyme treatment (in the presence or absence of calcium) was not only greater than that released by phosphate buffer but was also greater than that initially detectable on the surface of whole cells, suggesting an unmasking of enzyme on the cell surface. The presence of calcium during release treatment resulted in increased stability of the crude enzyme preparations. For the proteinase preparation released by using lysozyme with 50 mM CaCl₂, the half-life of proteinase activity at 37°C was 39 h, compared with 0.22 h for the calcium-free phosphate buffer-released preparation. In all cases, maximum stability was observed at pH 5.5. Comparison of β-casein hydrolysis by the three forms of the enzyme showed that the products of short-term (5- to 30-min) digestions were very similar, although subtle differences were detected with the 180-kDa form.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Enhancement and Cultural Characteristics of Leucomycin Production by Streptomyces kitasatoensis in the Presence of Magnesium Phosphate

Stimulation of leucomycin production with Streptomyces kitasatoensis by magnesium phosphate (MgP), and a concomitant decrease of ammonium ion concentration in the culture supernatant have been reported from this laboratory. Cultural characteristics of the MgP-supplemented culture were further examined in relation to the stimulation of leucomycin production. When the leucomycin titer increased 4- to 5-fold in the presence of MgP, mycelial growth level and assimilation rate of a major carbon source (glycerol) increased about 2-fold. MgP-grown mycelia were not appreciably different in shape or in carbon and nitrogen contents from the control mycelia. Magnesium and phosphate ions were released from MgP into the medium, while the addition of magnesium or phosphate salts to the control medium was rather inhibitory for leucomycin production. The pH values of MgP-supplemented cultures were lower than those of the control culture. When modifications of the basal media and culture conditions were done so as to permit the pH-change almost identical with that of the control media, the leucomycin titer was still higher in the presence of MgP than in its absence. When uric acid, an insoluble compound, was used as the sole nitrogen source, the leucomycin production increased to a high level even in the absence of MgP, which was comparable to that seen when MgP was added to the ammonium lactate-containing medium. The ammonium ion concentration in the culture supernatant of the uric acid-containing medium was maintained at a considerably low level. While in the ammonium lactate-containing medium, leucomycin production was low and ammonium ion concentration remained at a high level. Thus, it was demonstrated that the stimulation of leucomycin production by MgP is related to the lowering of ammonium ion level, but not to the other factors examined such as growth level, carbon or phosphate regulation and pH-effect.     

Possible role of calcium in the formation of active extracellular proteinase by Pseudomonas fluorescens

The requirement for calcium during synthesis of extracellular proteinase by Pseudomonas fluorescens B52 was examined. Synthesis was monitored using cells resuspended at high density in fresh growth medium. Optimum enzyme production was found with cells grown to mid-logarithmic phase in mineral salts medium containing calcium chloride (1.0 mmol/l). Inhibition of synthesis by EDTA addition was rapid, similar to the effect produced by chloramphenicol, an inhibitor of translation. Appearance of enzyme initiated by calcium addition to depleted cells was rapid and was dependent on de novo protein synthesis. Sephadex G-100 chromatography of L-[4,5-3H]leucine-labelled cell-free supernatant liquids revealed that, in the absence of calcium, a low molecular weight (12000-14000 daltons) irreversibly inactive 'precursor' of the proteinase was formed. The results are consistent with the hypothesis that calcium is required for structural integrity of the proteinase as well as for activity.     

Growth optimization of a probiotic candidate,<Emphasis Type="Italic">Bifidobacterium pseudocatenulatum</Emphasis> G4, in milk medium using response surface methodology

Bifidobacterium pseudocatenulatum G4, a wild strain isolated from infant stools that has previously exhibited probiotic characteristics, was used in this study. The aim of this research was to improve the growth potential of this strain in milk-based medium. An initial screening study using a 2³ full factorial design was carried out to identify the impact on biomass production of the various components of the medium which were skim milk, yeast extract, and glucose. Statistical analysis suggested that yeast extract had a significant positive effect on viable cell count whereas glucose had a negative effect. Response surface methodology (RSM) was then applied to optimize the use of skim milk and yeast extract. A quadratic model was derived using a 3² face-centered central composite design to represent cell mass as a function of the two variables. The optimized medium composition was found to be 2.8% skim milk and 2.2% yeast extract, w/v. The optimized medium allowed a maximum biomass of 9.129 log₁₀ cfu/mL, 3.329 log units higher than that achieved with 10% skim milk, which is the amount commonly used. The application of RSM resulted in an improvement in the biomass production of this strain in a more cost-effective milk medium, in which skim milk use was reduced by 71.8%.     

Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by <Emphasis Type="Italic">Bacillus pumilus</Emphasis> KMM 62

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46–48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46–48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth.     

Possible role of calcium in the formation of active extracellular proteinase by Pseudomonas fluorescens

The requirement for calcium during synthesis of extracellular proteinase by Pseudomonas fluorescens B52 was examined. Synthesis was monitored using cells resuspended at high density in fresh growth medium. Optimum enzyme production was found with cells grown to mid-logarithmic phase in mineral salts medium containing calcium chloride (1.0 mmol/l). Inhibition of synthesis by EDTA addition was rapid, similar to the effect produced by chloramphenicol, an inhibitor of translation. Appearance of enzyme initiated by calcium addition to depleted cells was rapid and was dependent on de novo protein synthesis. Sephadex G-100 chromatography of L-[4,5-³H]leucine-labelled cell-free supernatant liquids revealed that, in the absence of calcium, a low molecular weight (12000–14000 daltons) irreversibly inactive 'precursor' of the proteinase was formed. The results are consistent with the hypothesis that calcium is required for structural integrity of the proteinase as well as for activity.     

Yield enhancement strategies for the production of picroliv from hairy root culture of Picrorhiza kurroa Royle ex Benth.

Fast-growing hairy root cultures of Picrorhiza kurroa induced by Agrobacterium rhizogenes offers a potential production system for iridoid glycosides. In present study we have investigated the effects of various nutrient medium formulations viz B5, MS, WP and NN, and sucrose concentrations (1–8%) on the biomass and glycoside production of selected clone (14-P) of P. kurroa hairy root. Full strength B5 medium was found to be most suitable for maximum biomass yield on the 40th day of culture (GI = 32.72 ± 0.44) followed by the NN medium of the same strength (GI = 22.9 ± 0.43). Secondary metabolite production was 1.1 and 1.3 times higher in half strength B5 medium respectively in comparison to MS medium. Maximum biomass accumulation along with the maximum picroliv content was achieved with 4% sucrose concentration in basal medium. RT vitamin and Thiamine-HCl effected the growth and secondary metabolite production of hairy roots growing on MS medium but did not show any effect on other media. The pH of the medium played significant role in growth and secondary metabolite production and was found to be highest at pH 6.0 while lowest at pH 3.0 and pH 8.0. To enhance the production of biomass and Picroliv 5 liter working capacity bioreactor was used, 27-fold (324 g FW) higher growth was observed in bioreactor than shake flask and secondary metabolite production was similarly enhanced.     

Thermal resistance of partially purified proteinase of Pseudomonas fluorescens P-26

Heat resistance of the Pseudomonas fluorescens P-26 proteinase in terms of D -value was studied in whole milk, skim milk, whey and 0.05 mol 1^-1 phosphate buffer at 72.5, 130, 135, 140, 145 and 150°C subsequent to its partial purification through (NH₄)₂ SO₄ precipitation (45–65% saturation) and solvent fractionation with 1.0 to 2.0 volumes of isopropanol. The D -value was maximum for the proteinase at all temperatures when determined in whole milk ( D ₁₅₀= 0.088).     

Effects of Environmental and Nutritional Factors on Production of the Polyketide Phytotoxin Coronatine by Pseudomonas syringae pv. Glycinea

Pseudomonas syringae pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The effects of environmental, nutritional, and host factors on growth and coronatine production by PG4180 were examined by varying the components of a defined basal medium which contained the following nutrients per liter: glucose (10 g), NH₄Cl (1 g), MgSO₄ · 7H₂O (0.2 g), KH₂PO₄ (4.1 g), K₂HPO₄ · 3H₂O (3.6 g), and FeCl₃ (2 μM). Bacterial growth was recorded as dry weight, and coronatine production was measured by high-performance liquid chromatography. Both growth and the quantity of coronatine synthesized were significantly affected by carbon source, nutrient levels (glucose, NH₄Cl, phosphate, Mg, and SO₄), amino acid supplements, and the presence of complex carbon and nitrogen sources. The yield of coronatine generally declined when conditions were varied from those in the basal medium. Coronatine production and growth were not affected when the pH was adjusted from 6.5 to 7.8. Increases in the osmolarity of the basal medium significantly decreased coronatine production without affecting growth. The addition of plant extracts, plant-derived secondary metabolites, or zinc did not affect growth or coronatine production, while the addition of millimolar levels of KNO₃ or micromolar levels of FeCl₃ significantly enhanced coronatine production. The yield of coronatine was maximized after a 7-day incubation at 18°C and 280 rpm. The results of the present study were used to formulate a medium which allowed for enhanced coronatine production in nearly all strains of P. syringae tested. A rapid method for extracting coronatine from small volumes of culture supernatant was also developed.