Structural determinants for alcohol substrates to be oxidized to formaldehyde by rat liver microsomes.

Glycerol can be oxidized to formaldehyde by rat liver microsomes and by cytochrome P450. The ability of other alcohols to be oxidized to formaldehyde was determined to evaluate the structural determinants of the alcohol which eventually lead to this production of formaldehyde. Monohydroxylated alcohols such as 1- or 2-propanol did not produce formaldehyde when incubated with NADPH and microsomes. Geminal diols such as 1,3-propanediol, 1,3-butanediol, or 1,4-butanediol also did not yield formaldehyde. However, vicinal diols such as 1,2-propanediol or 1,2-butanediol produced formaldehyde. With 1,2-propanediol, the residual two-carbon fragment was found to be acetaldehyde, while with 1,2-butanediol, the residual three-carbon fragment was propionaldehyde. Oxidation of 1,2-propanediol to formaldehyde plus acetaldehyde involved interaction with an oxidant derived from H2O2 plus nonheme iron, since production of the two aldehydic products was completely prevented by catalase or glutathione plus glutathione peroxidase and by chelators such as desferrioxamine or EDTA. The oxidant was not superoxide or hydroxyl radical. Product formation was fivefold lower when NADH replaced NADPH, and was inhibited by substrates, ligands, and inhibitors of cytochrome P450. A charged glycol such as alpha-glycerophosphate (but not the geminal beta-glycerophosphate) was readily oxidized to formaldehyde, suggesting that interaction of the glycol with the oxidant was occurring in solution and not in a hydrophobic environment. These results indicate that the carbon-carbon bond between 1,2-glycols can be cleaved by an oxidant derived from microsomal generated H2O2 and reduction of non-heme iron, with the subsequent production of formaldehyde plus an aldehyde with one less carbon than the initial glycol substrate.     

An examination and correction of plant tissue culture basal medium formulations

The inorganic formulations of fourteen common plant tissue culture basal media were examined from the primary literature. Inaccuracies and errors were found for molecular formulae, chemical hydrations, and molar equivalences for iron/EDTA complexation. A comparison with published basal medium formulations from six commercial suppliers uncovered additional inaccuracies, modifications, and errors, thereby emphasizing the need for investigators to examine and describe medium formulations precisely in future publications.     

Specification of the effect of chelating complex of iron ions in androgenesisin vitro by means of cation-free minimal medium

The addition of Na₂EDTA to a minimal medium (agar, sucrose) deprived of cations produces no morphoregulatory effect. This effect is induced only by the addition of iron. Traces of iron present in the agar are sufficient for the development of globular embryoid into a complete plant. The traces of iron in distilled water and sucrose (p. a.) are insufficient for the morphoregulation. The marked difference between the necessary limiting amount of chelate and necessary limiting amount of iron in non-modified media is explained by the presence of other cations, which saturate and thereby inactivate a greater part of Na₂EDTA. It is recommended to decrease the amount of iron and leave the commonly used amount of chelate in the prepared media. Simultaneously with optimal morphoregulatory effect in androgenesis and somatic embryogenesis, vitality of tissue culture will be lengthened and ageing as well as necrosis of the cultures be decreased.     

Disruption of N-Αcyl Homoserine Lactone-Mediated Cell Signaling and Iron Acquisition in Epiphytic Bacteria by Leaf Surface Compounds

Since N-acyl homoserine lactones (AHLs) are key mediators of cell density-dependent regulation of traits involved in virulence and epiphytic fitness in gram-negative bacteria such as Pseudomonas syringae, a variety of plant species were examined to determine their production of leaf surface compounds that could interact with these signaling systems. Leaf washings of 17 of 52 plant species tested stimulated or inhibited AHL-dependent traits in at least one of the bacterial reporter strains used. The active compounds from most plants could be distinguished from known AHLs due to different patterns of mobility during C₈ and C₁₈ reverse-phase thin-layer chromatography (TLC) and normal-phase TLC compared to the patterns for authentic bacterial AHLs. All plant extracts were also tested to determine their abilities to sequester iron and trigger bacterial siderophore synthesis on a medium containing abundant iron. Leaf washings from 16 of the 52 plant species, as well as tannic acid solutions, stimulated pyoverdine synthesis in P. syringae in a high-iron medium. These preparations also inhibited the growth of a P. syringae mutant unable to produce pyoverdine siderophores but not the growth of the wild-type bacterium. The stimulation of siderophore production and the growth inhibition by plant extracts and purified tannins were both reversed by addition of ferric chloride to culture media, indicating that iron was made unavailable by the compounds released onto the leaf surface.     

Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes.

Glycerol can be oxidized by rat liver microsomes to formaldehyde in a reaction that requires the production of reactive oxygen intermediates. Studies with inhibitors, antibodies, and reconstituted systems with purified cytochrome P4502E1 were carried out to evaluate whether P450 was required for glycerol oxidation. A purified system containing phospholipid, NADPH-cytochrome P450 reductase, P4502E1, and NADPH oxidized glycerol to formaldehyde. Formaldehyde production was dependent on NADPH, reductase, and P450, but not phospholipid. Formaldehyde production was inhibited by substrates and ligands for P4502E1, as well as by anti-pyrazole P4502E1 IgG. The oxidation of glycerol by the reconstituted system was sensitive to catalase, desferrioxamine, and EDTA but not to superoxide dismutase or mannitol, indicating a role for H2O2 plus non-heme iron, but not superoxide or hydroxyl radical in the overall glycerol oxidation pathway. The requirement for reactive oxygen intermediates for glycerol oxidation is in contrast to the oxidation of typical substrates for P450. In microsomes from pyrazole-treated, but not phenobarbital-treated rats, glycerol oxidation was inhibited by anti-pyrazole P450 IgG, anti-hamster ethanol-induced P450 IgG, and monoclonal antibody to ethanol-induced P450, although to a lesser extent than inhibition of dimethylnitrosamine oxidation. Anti-rabbit P4503a IgG did not inhibit glycerol oxidation at concentrations that inhibited oxidation of dimethylnitrosamine. Inhibition of glycerol oxidation by antibodies and by aminotriazole and miconazole was closely associated with inhibition of H2O2 production. These results indicate that P450 is required for glycerol oxidation to formaldehyde; however, glycerol is not a direct substrate for oxidation to formaldehyde by P450 but is a substrate for an oxidant derived from interaction of iron with H2O2 generated by cytochrome P450.     

Influence of chelating ligands on bioavailability and mobility of iron in plant growth media and their effect on radish growth

In this study, the effects of chelating ligands on iron movement in growth medium, iron bioavailability, and growth of radish sprouts (Raphanus sativus) were investigated. Iron is an important nutrient for plant growth, yet the insoluble state of iron hydroxides in alkaline conditions decreases its bioavailability. Iron chelates increase iron uptake and have been used in agriculture to correct iron chlorosis. While previous studies have reported the effects of chelating ligands on iron solubility and bioavailability, the present study elucidates the pattern of iron movement by chelating ligands in plant growth medium. The apparent mobility of iron in growth medium was calculated using a ‘4-box’ model. Ethylenediaminedisuccinic acid (EDDS) and hydroxy-iminodisuccinic acid (HIDS) produced the highest apparent mobility of iron from the bottom layer of the medium (initially 10⁻⁴ M Fe(III)) to the upper layer (no iron), followed by glutamatediacetic acid (GLDA), ethylenediaminetetraacetic acid (EDTA), methylglycinediacetic acid (MGDA), and iminodisuccinic acid (IDS). Iron movement in the growth medium was influenced by the chelating ligand species, pH, and ligand exposure time. The iron uptake and growth of radish sprouts were related to the iron mobility produced by the chelating ligands. These results suggest that, in alkaline media, chelating ligands dissolve the hardly soluble iron hydroxide species, thus increasing iron mobility, iron uptake, and plant growth. HIDS, which is biodegradable, was one of the most effective ligands studied; therefore, this compound would be a good alternative to other environmentally persistent chelating ligands.     

The influence of ferrous-complexed EDTA as a solubilization agent and its auto-regeneration on the removal of nitric oxide gas through the culture of green alga Scenedesmus sp

The influence of iron-complexed ehylenediaminetetraacetic acid (EDTA) was studied on nitric oxide (NO) removal using photoautotropic cultivation of green alga Scenedesmus. Fe(II)EDTA is an active solubilization agent of NO in water, while the oxidized Fe(III)EDTA is not. When a gas mixture containing 300 ppm NO was treated through the Scenedesmus culture containing 5 mM Fe(II)EDTA, a constant level of 80–85% NO removal was achieved for a prolonged period. A certain fraction of Fe(II)EDTA remained without being oxidized to Fe(III)EDTA because of the existence of reversible oxidation–reduction balance between Fe(II)EDTA and Fe(III)EDTA. When Fe(III)EDTA was added to the culture instead of Fe(II)EDTA, Fe(II) was generated via reduction of Fe(III), resulting in the increase of NO removal and cell density. This was possible because of the generated Fe(II)EDTA which contributed to the dissolution of NO. Therefore, a long-term NO removal was possible with Fe(III)EDTA, as well as with Fe(II)EDTA, in the present microalgal system. The supplementation of free EDTA was necessary to extend the period of NO removal because EDTA is consumed by biodegradation while the decrease of total iron content was not significant.     

Effect of adenine nucleotides on the oxidation of N-methyl groups in liver mitochondria

In phosphorylating mitochondria, isolated in 0.25 M sucrose and suspended in a glycylglycine-KC1 medium at pH 7.4, the N-methyl group of sarcosine is oxidized to formaldehyde, formate, and CO₂. The initial rate of O₂ uptake in this system is only about half as great as with phosphate-washed mitochondria, in which the N-methyl carbon is oxidized only to the level of “active formaldehyde” and can be recovered as serine-β-carbon and/or formaldehyde. In the glycylglycine-KC1 medium, the O₂ uptake with sarcosine occurs in a biphasic manner and the initial slower rate can be extended by the addition of Mg²⁺, and ADP, AMP, or ATP. O₂ uptake is similarly restrained by ADP in mitochondria buffered with imidazole or pyrophosphate. The ADP effect is not observed in the presence of dinitrophenol. The patterns of O₂ uptake obtained with ADP in these various media are not altered when the oxidation of the formaldehyde, derived from the N-methyl group, is suppressed by the addition of either semicarbazide or rotenone. With dimethylglycine, another component of the “1-C cycle”, the initial rate of oxidation in glycylglycine or imidazole is enhanced by ADP rather than being decreased. These results together with appropriate coenzyme analyses suggest that reactions of “one carbon compounds” can provide sensitive markers for assessing compartition of cofactors such as the pyridine nucleotides, flavins, and folates in the mitochondrial matrix.     

Oxidation of Manganese and Iron by Leptothrix discophora: Use of N,N,N′,N′-Tetramethyl-p-Phenylenediamine as an Indicator of Metal Oxidation

A new method for the quantification and characterization of manganese-oxidizing activity by spent culture medium of Leptothrix discophora SS-1 was developed. It is based on the formation of the dye Wurster blue from N,N,N′,N′-Tetramethyl-p-phenylenediamine by oxidized manganese generated in the spent medium. The kinetic parameters thus obtained agreed well with data obtained with other methods. It was also possible to demonstrate iron oxidation by spent culture medium. The kinetics of the process and inhibition by enzyme poisons suggest that iron oxidation is enzymatically catalyzed. Probably two different factors are involved in manganese and iron oxidation.     

Formaldehyde as a carbon and electron shuttle between autotroph and heterotroph populations in acidic hydrothermal vents of Norris Geyser Basin,Yellowstone National Park

The Norris Geyser Basin in Yellowstone National Park contains a large number of hydrothermal systems, which host microbial populations supported by primary productivity associated with a suite of chemolithotrophic metabolisms. We demonstrate that Metallosphaera yellowstonensis MK1, a facultative autotrophic archaeon isolated from a hyperthermal acidic hydrous ferric oxide (HFO) spring in Norris Geyser Basin, excretes formaldehyde during autotrophic growth. To determine the fate of formaldehyde in this low organic carbon environment, we incubated native microbial mat (containing M. yellowstonensis) from a HFO spring with ¹³C-formaldehyde. Isotopic analysis of incubation-derived CO₂ and biomass showed that formaldehyde was both oxidized and assimilated by members of the community. Autotrophy, formaldehyde oxidation, and formaldehyde assimilation displayed different sensitivities to chemical inhibitors, suggesting that distinct sub-populations in the mat selectively perform these functions. Our results demonstrate that electrons originally resulting from iron oxidation can energetically fuel autotrophic carbon fixation and associated formaldehyde excretion, and that formaldehyde is both oxidized and assimilated by different organisms within the native microbial community. Thus, formaldehyde can effectively act as a carbon and electron shuttle connecting the autotrophic, iron oxidizing members with associated heterotrophic members in the HFO community.     

Isolation of 2,4-Diacetylphloroglucinol from a Fluorescent Pseudomonad and Investigation of Physiological Parameters Influencing Its Production

Pseudomonas sp. strain F113 was isolated from the rhizosphere of sugar beets and shown to inhibit a range of plant pathogenic fungi by producing an antibioticlike compound. An antibiotic-negative mutant strain, F113G22, was generated by transposon mutagenesis. This mutant has lost the ability to inhibit both bacterial and fungal microorganisms on high-iron medium. The antibioticlike compound was subsequently identified as 2,4-diacetylphloroglucinol (DAPG), and a high-pressure liquid chromatographic assay was developed for to detect it quantitatively in growth culture media and soil. The growth temperature had a direct bearing on DAPG production by strain F113, with maximum production at 12°C. The iron concentration, pH, and oxygen had no influence on DAPG production by strain F113 under the assay conditions used. However, a low ratio of culture volume to surface area available to the microbe in the growth container was critical for optimum DAPG production. Different types of carbon sources influenced DAPG production by strain F113 to various degrees. For example, sucrose, fructose, and mannitol promoted high yields of DAPG by strain F113, whereas glucose and sorbose resulted in very poor DAPG production.     

Regulation of androgenesis inNicotiana tabacum L. cv. White Burley andDatura innoxia Mill. Effect of bivalent and trivalent iron and chelating substances

The effect of FeSO₄.7H₂O, Fe₂(SO₄)₃.9H₂O, disodium salt of ethylene-diaminotetraacetic acid, dihydrate (EDTA) and N-(2-acetamido) iminodiacetic acid (ADA) and their combinations on the androgenesis was studiedin vitro in tobacco (cv. White Burley) and datura (Datura innoxia Mill.). Simultaneously the reversibility and irreversibility of the morphogenic process leading to the conversion of the pollen embryoid into complete plant was followed. Complete plants developed in anthers on media with trivalent iron, chelated trivalent iron, chelated bivalent iron, bivalent iron in the presence of ADA and of media with EDTA. The number of androgenic plants in anthers increased in the following order: Fe₃₊ < Fe₃₊ EDTA ≦ ≦ EDTA < Fe²⁺ EDTA. The marked brown colour of cultured anthers was due to the presence of trivalent iron in the medium. The androgenic development was most rapid on the medium containing only trivalent iron, slower on media with chelated iron and slowest on medium with EDTA. The viability of cultures with complete plants decreased in the reverse order. No complete plants grew on media without trivalent iron and without EDTA and on media containing only bivalent iron whereas globular embryoids arose and developed continuously on these media. The anthers reacted in the same way on both complete and minimal media. Isolated embryoids formed complete plants in corresponding variants on complete media only. The development of pollen embryoids into complete plants was stopped by the transfer of globular and torpedo-shaped embryoids from medium with EDTA to the medium without EDTA. Isolated greenish cotyledonar embryoids continued to grow even on the medium without EDTA.     

Influence of cultural and physiochemical factors on ascorbate stability in plant tissue culture media

Ascorbic acid rapidly decays in plant tissue culture media. Within 50 min to 3 h after preparing 100 mM solutions, ascorbic acid was destroyed. Autoclaving, shaking flasks, high light intensity and increasing pH over a range from 4.5–7 accelerated decay. Ascorbic acid was oxidized to dehydroascorbic acid which also underwent decay. Within 11 h and 15 min after adding ascorbic acid both ascorbic acid and its oxidation product, dehydroascorbic acid, disappeared from medium. Since ascorbic acid is rapidly destroyed in plant tissue culture media it may not exert its effect as an intact molecule. Instead its antioxidant/antibrowning role in plant cell, tissue and organ cultures may be mediated by some product of further oxidation.     

Effects of medium composition, calcium, iron and oxygen on haemolysin production by Plesiomonas shigelloides isolated from water

AIMS: The effects of medium composition, calcium, iron and oxygen tension on the haemolytic activity of Plesiomonas shigelloides were investigated. METHODS AND RESULTS: The haemolytic activity of seven strains of Ple. shigelloides was tested on the surface of Luria Agar (LA), Brain Heart Infusion Agar (BHIA) and Trypitic Soy Agar (TSA) containing 5% (v/v) sheep blood, and in the Agar Overlay (AO) assay. All strains produced beta-haemolysis in the AO assay in three media, and on the surface of LA. The kinetics of growth and haemolytic activity of Ple. shigelloides 9P3-1 were evaluated in six different media, and the highest production of haemolysin occurred in Luria Broth (LB). The haemolytic activity of 9P3-1 was stimulated by Ca2+ and inhibited by EDTA. Addition of iron to the culture medium did not affect bacterial growth, although it reduced bacterial haemolytic activity. In the presence of an iron chelator, growth of the 9P3-1 was inhibited, but its haemolytic activity was enhanced. CONCLUSION: The haemolytic activity of Ple. shigelloides depends on medium composition, and that it is regulated by iron and is calcium-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: These results show the importance of optimization of media composition and oxygen tension for detection of Ple. shigelloides haemolytic activity.     

Biodegradation of Metal-EDTA Complexes by an Enriched Microbial Population

A mixed culture utilizing EDTA as the sole carbon source was isolated from a mixed inoculum of water from the River Mersey (United Kingdom) and sludge from an industrial effluent treatment plant. Fourteen component organisms were isolated from the culture, including representatives of the genera Methylobacterium, Variovorax, Enterobacter, Aureobacterium, and Bacillus. The mixed culture biodegraded metal-EDTA complexes slowly; the biodegradability was in the order Fe>Cu>Co>Ni>Cd. By incorporation of inorganic phosphate into the medium as a precipitant ligand, heavy metals were removed in parallel to EDTA degradation. The mixed culture also utilized a number of possible EDTA degradation intermediates as carbon sources.     

Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by microsomes, a minor pathway of alcohol metabolism, involved generation of hydroxyl radical (.OH). Microsomes oxidized chemical .OH scavengers (KMB, DMSO, t-butyl alcohol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal .OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal .OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize .OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal .OH production, the oxidation of ethanol by .OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 and NADPH-P-450 reductase indicated that the reductase is the critical enzyme locus for interacting with iron and catalyzing production of reactive oxygen species. Microsomes isolated from rats chronically fed ethanol catalyzed oxidation of .OH scavengers, light emission, and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a .OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.     

Iron phosphate precipitation in Murashige and Skoog media

Murashige and Skoog revised medium, a standard plant tissue culture medium, precipitated on keeping because iron was ineffectively chelated. Most of the precipitate formed after two days and analysis indicated that it was predominantly iron phosphate. It was found that, after precipitation, the supernatant medium had lost c. 45% of its original iron, 20% zinc and 13% phosphate. The following modifications prevented precipitation: lowering the pH to below 3.2, increasing to 3 the molar ratio of EDTA:Fe or preparing the stock solution of FeEDTA with inadequate heating. Most of these modifications had a deleterious effect on the growth and greening of Ocimum basilicum suspension cultures. However, when an increase in the molar ratio of EDTA:Fe was achieved by reducing the iron concentration to one-third, there was no inhibitory effect on growth or greening of these suspension cultures.     

IRON-MEDIATED CHANGES IN THE GROWTH OF LAKE ERIE PHYTOPLANKTON AND AXENIC ALGAL CULTURES1

The effect of iron enrichment on algal growth and photosynthesis was investigated using natural assemblages of Lake Erie phytoplankton and axenic cultures of Anabaena, Scenedesmus and Selenastrum. Cell yield and photosynthesis were frequently inhibited in the presence of unchelated iron over the range of 3.6 to 53.7 μM iron as FeCl₃. In lake water and in a defined medium with low nutrient concentrations, the degree of inhibition by iron could be reduced by chelating the iron with EDTA or by enriching the cultures with phosphorus. Chemical analyses revealed that the EDTA efectively reduced the ability of the ferric iron to remove soluble phosphorus from the media. EDTA was also observed to reduce rather than enhance iron uptake by axenic cultures of A. flos-aquae. These data support the hypothesis that additions of EDTA to low-nutrient media may serve to stimulate algal growth in the presence of iron by preventing the iron from altering extracellular concentrations of soluble ions essential for algal metabolism. In medium with high nutrient concentrations, the soluble phosphorus concentration was not appreciably altered by either EDTA-chelated or unchelated iron enrichment (0.9 to 53.7 μM). Instead, the observed enhancement of cell yield by EDTA-chelated iron in nutrient-rich media appeared to be due to the direct effect of iron on intracellular metabolic processes.     

Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae

We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1–100 nM were rapidly iron-depleted when ferric citrate—but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.     

Iron limitation by transferrin promotes simultaneous cheating of pyoverdine and exoprotease in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a primary bacterial model to study cooperative behaviors because it yields exoproducts such as siderophores and exoproteases that act as public goods and can be exploited by selfish nonproducers behaving as social cheaters. Iron-limited growth medium, mainly casamino acids medium supplemented with transferrin, is typically used to isolate and study nonproducer mutants of the siderophore pyoverdine. However, using a protein as the iron chelator could inadvertently select mutants unable to produce exoproteases, since these enzymes can degrade the transferrin to facilitate iron release. Here we investigated the evolutionary dynamics of pyoverdine and exoprotease production in media in which iron was limited by using either transferrin or a cation chelating resin. We show that concomitant loss of pyoverdine and exoprotease production readily develops in media containing transferrin, whereas only pyoverdine loss emerges in medium treated with the resin. Characterization of exoprotease- and pyoverdine-less mutants revealed loss in motility, different mutations, and large genome deletions (13–33 kb) including Quorum Sensing (lasR, rsal, and lasI) and flagellar genes. Our work shows that using transferrin as an iron chelator imposes simultaneous selective pressure for the loss of pyoverdine and exoprotease production. The unintended effect of transferrin uncovered by our experiments can help to inform the design of similar studies.Subject terms: Bacteriology, Microbial ecology