Iron-regulated outer membrane proteins and non-siderophore-mediated iron acquisition by Paracoccus denitrificans

Abstract Deprivation of Paracoccus denitrificans of iron in sodium molybdate-containing medium caused a slower rate of growth and lower final cell yield, in contrast to our previous studies in non-sodium molybdate-containing medium, where iron deprivation had little effect on growth rate. Five high M _r outer membrane proteins and catechol production were induced in iron-deprived cultures. The fifth protein, M _r 72 000, was produced later than the others. Growth of iron-deprived cells in medium containing 20 μM ferric citrate repressed siderophore and iron deprivation-induced protein production, and led to production of an M _r 23 000 outer membrane protein (half maximum production after 5 h). Synthesis of the M _r 23 000 and high M _r proteins appeared to be mutally exclusive, and to be regulated by the cell's iron status. Cells inoculated into medium containing 20 μM ferric citrate took up 92% of the iron within 1 h, suggesting the occurrence of a nonsiderophore mediated, 'low affinity' iron uptake pathway.     

Regulation of siderophore production by iron Fe(III) in certain fungi and fluorescent pseudomonads

Regulation of siderophore production in response to iron concentration in the medium was examined. Threshold concentration was recorded for twenty fungi and three rhizobacterial pseudomonads. Organisms showed difference in threshold values at which they stopped siderophore elaboration. In nine fungi (3 aspergilli, 1 penicillium, N. crassa, F. dimerum and 3 mucors) siderophore production was repressed at 3 microM Fe(III). Siderophore production was repressed at 27 microM of Fe (III) in 3 aspergilli, 2 penicillia and 3 pseudomonads. Rest of the fungi had cut off values at 6, 9, 15, 21 microM of Fe(III) concentration.     

Combat of iron-deprivation through a plant growth promoting fluorescent Pseudomonas strain GRP3A in mung bean (Vigna radiata L. Wilzeck)

Microorganisms and plants sustain themselves under iron-deprived conditions by releasing siderophores. Among others, fluorescent pseudomonads are known to exert extensive biocontrol action against soil and root borne phytopathogens through release of antimicrobials and siderophores. In this study, production and regulation of siderophores by fluorescent Pseudomonas strain GRP3A was studied. Among various media tested, standard succinate medium (SSM) promoted maximum siderophore production of 56.59 mg l(-1). There were low levels of siderophore in complex media like King's B medium, trypticase soya medium and nutrient medium (41.27, 29.86 and 27.63 mg l(-1)), respectively. In defferrated SSM, siderophore level was quantified to be 68.74 mg l(-1). Supplementation with iron (FeCl3) resulted in decreased siderophore levels depending on concentration. Siderophore production was promoted by Zn2+ (78.94 mg l(-1)), Cu2+ (68.80 mg l(-1)) whereas Co2+ (57.33 mg l(-1)) and Fe3+ reduced siderophore production (37.44 mg l(-1) as compared to control (55.97 mg l(-1)). Strain GRP3A showed plant growth promotion under iron limited conditions.     

Pyrophosphate increases the efficiency of enterobactin-dependent iron uptake in Escherichia coli.

Exogenous inorganic pyrophosphate increases the biomass yield of Escherichia coli. In this report, we show that the effect of pyrophosphate is related to iron uptake. We have found that addition of pyrophosphate, ammonium iron (III) citrate or iron (III) chloride, in M63 minimal medium containing 1.7 microM of iron, causes an increase in growth yield. In contrast to iron chloride or ammonium iron (III) citrate, exogenous pyrophosphate is deleterious to strains unable to synthesize enterobactin. Thus the positive effect of pyrophosphate is related to the enterobactin uptake system expressed in a low iron content medium. Pyrophosphate in minimal medium has a repressing effect on the expression of Fur-regulated genes. In iron rich medium where enterobactin synthesis is strongly decreased, addition of pyrophosphate increases expression of Fur-regulated genes. Furthermore, this latter regulatory effect of pyrophosphate in iron-rich medium is enhanced in the absence of enterobactin synthesis. It has also been shown that addition of pyrophosphate protects the cell against the oxidative stress caused by the presence of hydrogen peroxide in an iron-rich containing medium. These results indicate that pyrophosphate acts as an iron-chelating agent, could trigger the enterobactin-dependent iron uptake system and could promote an increased binding of iron to enterobactin.     

Siderophore production by the magnetic bacterium Magnetospirillum magneticum AMB-1

Siderophore production by the magnetic bacterium Magnetospirillum magneticum AMB-1 is elicited by sufficient iron rather than by iron starvation. In order to clarify this unusual pattern, siderophore production was monitored in parallel to iron assimilation using the chrome azurol sulfonate assay and the ferrozine method respectively. Iron concentration lowered approximately five times less than its initial concentration only within 4 h post-inoculation, rendering the medium iron deficient. A concentration of at least 6 microM Fe(3+) is required to initiate siderophore production. The propensity of M. magneticum AMB-1 for the assimilation of large amounts of iron accounts for the rapid depletion of iron in the medium, thereby triggering siderophore excretion. M. magneticum AMB-1 produces both hydroxamate and catechol siderophores.     

Effect of Ferric Iron on Siderophore Production and Pyrene Degradation by Pseudomonas fluorescens 29L

The effect of ferric iron [Fe(III)] on pyrene degradation and siderophore production was studied in Pseudomonas fluorescens 29L. In the presence of 0.5 muM of Fe(III) and 50 mg of pyrene per liter of medium as a carbon source, 2.2 mg of pyrene was degraded per liter of medium per day and 25.3 muM of 2,3-DHBA (2,3-dihydroxybenzoic acid) equivalent of siderophores was produced per day. However, the pyrene degradation rate was 1.3 times higher and no siderophores were produced with the addition of 1 muM of Fe(III). Similar trends were seen with 50 mg of succinate per liter of medium as a carbon source, although the growth of strain 29L and the succinate degradation rate were higher. In the absence of siderophore production, pyrene and succinate continued to be biodegraded. This indicates that Fe(III) and not siderophore production affects the hydrocarbon degradation rate. Only 18% of strain 29L mutants capable of growth on pyrene produced siderophores, while among the mutants capable of growth on succinate, only 10% produced siderophores. This indicates that siderophores are not required for pyrene biodegradation. Fe(III) enhances pyrene degradation in Pseudomonas fluorescens 29L but it may be utilized by mechanisms other than siderophores.     

A Catecholic Siderophore Produced by the Thermoresistant Bacillus licheniformis VK21 Strain

Thermophilic and thermoresistant strains of bacilli were screened on a medium containing Chrome Azurol S for the producers of siderophores. It was found that the Bacillus licheniformis VK21 strain dramatically increases secretion of the metabolite, a chelator of Fe³⁺, in response to addition of manganese(II) salts. The growth of the producer on a minimal medium containing MnSO₄ under the conditions of iron deficiency is accompanied by the accumulation of a catecholic product, the content of which reaches maximum at the beginning of the stationary growth phase of culture. In the presence of FeCl₃, the amount of the catecholic product in the medium considerably decreases. The siderophore, called S_VK21, was isolated from the cultural medium and purified by reversed phase HPLC, and its siderophore function was confirmed by the test for the restoration of growth of producer cells in a medium containing EDTA. The UV spectrum of the siderophore has absorption maxima at 248 and 315 nm. According to the amino acid analysis and NMR spectrometry, the metabolite S_VK21 is 2,3-dihydroxybenzoyl-glycyl-threonine.     

Physiological factors affecting carbon tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain KC.

Pseudomonas sp. strain KC was grown on a medium with a low content of transition metals in order to examine the conditions for carbon tetrachloride (CT) transformation. Several carbon sources, including acetate, glucose, glycerol, and glutamate, were able to support CT transformation. The chelators 2,2'-dipyridyl and 1,10-phenanthroline stimulated CT transformation in a rich medium that otherwise did not support this activity. Low (< 10 microM) additions of dissolved iron(II), iron(III), and cobalt(II), as well as an insoluble iron(III) compound, ferric oxyhydroxide, inhibited CT transformation. The addition of 50 microM iron to actively growing cultures resulted in delayed inhibition of CT transformation. CT transformation was seen in aerobic cultures of KC, but with reduced efficiency compared with denitrifying cultures. Inhibition of CT transformation by iron was also seen in aerobically grown cultures. Optimal conditions were used in searching for effective CT transformation activity among denitrifying enrichments grown from samples of aquifer material. No activity comparable to that of Pseudomonas sp. strain KC was found among 16 samples tested.     

Relationship ofAzotobacter chrooccum siderophores with nitrogen fixation

In iron-limited medium, a siderophore producing soil isolate ofAzotobacter chroococcum showed a high level of hydroxamate with relatively low level of nitrogen fixation. Inclusion of iron in the medium resulted in increased nitrogen fixation with decreased hydroxamate production. Under shake culture conditions, the level of both hydroxamate and catechol type of siderophores decreased after 2 d of incubation in iron-deficient medium. However, under iron-sufficient conditions, both siderophore production and nitrogen fixation increased with time although the level of siderophore was quite low. A number of soil isolates and mutants ofA. chrococcum were tested for nitrogen fixation, hydroxamate and catechol type of siderophore production. Wide variation was observed in the siderophore level and nitrogen fixation in the cultures tested. Nitrogen fixation was higher in the iron-sufficient medium than in iron-limited one while hydroxamate yield was higher in iron-limited medium than in the iron-sufficient one in all the cultures. Inclusion of ammonium acetate in the medium induced catechol synthesis in more than 60% of the cultures.     

Studies of iron-uptake mechanisms in two bacterial species of the shewanella genus adapted to middle-range (Shewanella putrefaciens) or antarctic (Shewanella gelidimarina) temperatures

Iron(III)-uptake mechanisms in bacteria indigenous to the Antarctic, which is the most Fe-deficient continent on Earth, have not been extensively studied. The cold-adapted, Antarctic bacterium, Shewanella gelidimarina, does not produce detectable levels of the siderophore, putrebactin, in the supernatant of Fe(III)-deprived cultures. This is distinct from the putrebactin-producing bacterium from the same genus, Shewanella putrefaciens, which is adapted to middle-range temperatures. The production of putrebactin by S. putrefaciens is optimal, when the pH value of the medium is 7.0. According to the strong positive response from whole cells in the Chrome Azurol S (CAS) agar diffusion assay, Shewanella gelidimarina appears to produce cell-associated siderophores. In the RP-HPLC trace of an Fe(III)-loaded extract from the cell-associated components of S. gelidimarina cultured in media with [Fe(III)] ca. 0 microM, a peak appears at [MeCN] ca. 77%, which decreases in intensity in a parallel experiment in which [Fe(III)] ca. 5 microM, and is barely detectable in Fe(III)-replete media ([Fe(III)] ca. 20 microM). The Fe(III)-dependence of this peak suggests that the attendant species, which is significantly more hydrophobic than putrebactin (RP-HPLC elution: [MeCN] ca. 14%), is associated with Fe(III)-management in S. gelidimarina. This study highlights the diversity in Fe(III)-uptake mechanisms in Shewanella species adapted to different environmental and thermal niches.     

Aerobactin-mediated utilization of transferrin iron

Aerobactin and enterobactin, hydroxamate- and catechol-type siderophores, respectively, were found capable of removing iron (III) from transferrin in buffered solution. Although under these conditions aerobactin displaced the iron much more slowly than did enterobactin, the rate for the former could be accelerated by addition of pyrophosphate as mediator. Transfer of iron (III) from transferrin to aerobactin appeared to proceed via a ternary complex. Cells of Escherichia coli BN 3040 NalR iuc containing transport systems for both enterobactin and aerobactin, the genetic determinants for the latter specified on a ColV-type plasmid, took up iron from [55Fe]transferrin in minimal medium. In this case aerobactin was effective at a much lower concentration, although enterobactin still displayed superior ability to transfer the iron. In serum, however, the rate measured with aerobactin exceeded that found with enterobactin. The results indicate that aerobactin, in spite of its relatively unimpressive affinity for iron (III) as a siderophore, is nonetheless equipped with structural features or properties that enhance its ability to remove the metal ion from transferrin, especially when receptor-bearing cells of E. coli are present to act as a thermodynamic sink for the iron. These attributes of the aerobactin system of iron assimilation may account for its status as a virulence determinant in hospital isolates of E. coli.     

Studies on some factors affecting solasodine contents in tissue cultures of Solanum nigrum

Tissue cultures of Solanum nigrum L. were initiated from leaf explants on a solid medium containing inorganic salts [Murashige and Skoog (1962), Physiol. Plant. 15: 473–497], vitamins [Gamborg et al. (1968) Exp. Cell Res. 50:151–158], 3% sucrose and combinations of indoleacetic acid and benzyladenine. Solasodine content was determined in differentiated and undifferentiated (callus) tissues by a colorimetric technique and thin layer chromatography. Indoleacetic acid and sucrose in the medium markedly stimulated the production of solasodine in the tissue cultures. In the cultures grown in darkness the differentiated tissues produced significantly more (anywhere from 1.5 to 10 times) solasodine than the callus in several media. When sucrose concentration was increased to 4, 6 and 10% level in the medium which contained 10 μ M benzyladenine as the sole growth regulator, a significant increase of solasodine production in cultures was found.     

Iron mediated regulation of growth and siderophore production in a diazotrophic cyanobacterium Anabaena cylindrica

Iron mediated regulation of growth and siderophore production has been studied in a diazotrophic cyanobacterium Anabaena cylindrica. Iron-starved cells of A. cylindrica exhibited reduced growth (30%) when the cells were growing under N2-fixing conditions. In contrast, N03-, NO2-, NH4' and urea grown cells exhibited almost 50% reduction in their growth in the absence of iron as compared to their respective counterparts cultured in the presence of iron. However, at 60 microM of iron, A. cylindrica cells exhibited almost equal growth regardless of the nitrogen source available. Siderophore production in A. cylindrica was started after day 2nd of the cell growth and attained its optimal level on day 5th when the cells were at their mid-log phase. No siderophore production was, however, recorded on day 2nd at all the concentrations of iron tested. The production of siderophore in A. cylindrica further increased with increase in iron concentration and attained its optimum level on day 5th at 60 microM iron. A. cylindrica cells took at least 3 days for initiation of siderophore production and produced about 60% siderophore on day 5th even under iron-starved condition. A. cylindrica produced dihydroxamate type of siderophore.     

Hydroxamate siderophore synthesis by Phialocephala fortinii, a typical dark septate fungal root endophyte

The siderophore production of various isolates of Phialocephala fortinii was assessed quantitatively as well as qualitatively in batch assays under pure culture conditions at different pH values and iron(III) concentrations. We found a distinct effect of both of these parameters on siderophore synthesis and as well as on fungal growth. In comparative analyses of two of the isolates, maximum siderophore production was found at a pH in the range of pH 4.0 to 4.5 while, under the experimental conditions employed, the optimal concentration of ferric iron was determined to be between 20–40 g iron (III) l^–1 (0.36–0.72 M, respectively). HPLC analysis of the culture filtrate of most of the isolates of P. fortinii revealed the excretion of ferricrocin as main hydroxamate siderophore, followed by ferrirubin and ferrichrome C. The pattern of release of these three substances proved to be dependent on pH and iron(III) concentration of the culture medium, and to be specific for each isolate under investigation.     

Directing the Biosynthesis of Putrebactin or Desferrioxamine B in Shewanella putrefaciens through the Upstream Inhibition of Ornithine Decarboxylase

To manage iron acquisition in an oxic environment, Shewanella putrefaciens produces the macrocyclic dihydroxamic acid putrebactin (PB) as its native siderophore. In this work, we have established the siderophore profile of S. putrefaciens in cultures augmented with the native PB precursor putrescine and in putrescine-depleted cultures. Compared to base medium, PB increased by two-fold in cultures of S. putrefaciens with 10?mM NaCl and 20?mM exogenous putrescine. In cultures augmented with 1,4-diaminobutan-2-one (DAB), PB decreased with only 0.02-fold PB detectable at 10?mM DAB. As an ornithine decarboxylase (ODC) inhibitor, DAB depleted levels of endogenous putrescine which attenuated downstream PB assembly. Under putrescine-depleted conditions, S. putrefaciens produced as its replacement siderophore the cadaverine-based desferrioxamine B (DFO-B), as characterised by ESI-MS of the Fe(III) -loaded form (m/z(obs) 614.13; m/z(calc) 614.27). A third siderophore, independent of DAB, was observed in low levels. LC/MS Analysis of the Fe(III) -loaded extract gave m/z(obs) 440.93, which, formulated as a 1?:?1 Fe(III) complex with a macrocyclic dihydroxamic acid, comprising one putrescine- and one cadaverine-based precursor (m/z(calc) 440.14). These results show that the production of native PB or non-native DFO-B by S. putrefaciens can be directed though upstream inhibition of ODC. This approach could be used to increase the molecular diversity of siderophores produced by S. putrefaciens and to map alternative diamine-dependent metabolites.     

Induction of siderophore activity in Anabaena spp. and its moderation of copper toxicity.

Growth of Anabaena sp. strain 7120 (in the absence of chelators or added iron) was inhibited by the addition of 2.1 to 6.5 microM copper and was abolished by copper concentration of 10 microM or higher. When the copper was chelated to schizokinen (the siderophore produced by this organism in response to iron starvation), the toxic effects were eliminated. Analysis of culture filtrates showed that the cupric schizokinen remains in the medium, thereby lowering the amount of copper taken up by the cells. Although this organism actively transports ferric schizokinen, it apparently does not recognize the cupric complex. Thus, Anabaena sp. is protected from copper toxicity under conditions in which siderophore is being produced. For cells grown in low iron, the accumulation of extracellular schizokinen was observed to parallel cell growth and continue well into stationary phase. The actual iron status of the organism was monitored by using iron uptake velocity as an assay. Cultures grown on 0.1 microM added iron were found to be severely iron limited upon reaching stationary phase, thus explaining the continued production of schizokinen. These data show that the siderophore system in Anabaena spp. has developed primarily as a response to iron starvation and that additional functions such as alleviation of copper toxicity or allelopathic inhibition of other algal species are merely secondary benefits.     

Induction of siderophore activity in Anabaena spp. and its moderation of copper toxicity

Growth of Anabaena sp. strain 7120 (in the absence of chelators or added iron) was inhibited by the addition of 2.1 to 6.5 microM copper and was abolished by copper concentration of 10 microM or higher. When the copper was chelated to schizokinen (the siderophore produced by this organism in response to iron starvation), the toxic effects were eliminated. Analysis of culture filtrates showed that the cupric schizokinen remains in the medium, thereby lowering the amount of copper taken up by the cells. Although this organism actively transports ferric schizokinen, it apparently does not recognize the cupric complex. Thus, Anabaena sp. is protected from copper toxicity under conditions in which siderophore is being produced. For cells grown in low iron, the accumulation of extracellular schizokinen was observed to parallel cell growth and continue well into stationary phase. The actual iron status of the organism was monitored by using iron uptake velocity as an assay. Cultures grown on 0.1 microM added iron were found to be severely iron limited upon reaching stationary phase, thus explaining the continued production of schizokinen. These data show that the siderophore system in Anabaena spp. has developed primarily as a response to iron starvation and that additional functions such as alleviation of copper toxicity or allelopathic inhibition of other algal species are merely secondary benefits.     

Effects of iron level on the anatagonistic action of siderophores from non-pathogenicStaphylococcus spp

The production, detection and the effects of iron concentration on the siderophore production ofStaphylococcus strains used as meat starter cultures were studied. Non-pathogenicStaphylococcus strains produce extracellular low molecular weight compounds which exhibited positive reactivity when measured by a universal detection method for siderophores. The production of siderophores was very closely associated with the iron concentration in the medium, and very low additions considerably reduced siderophore production. Although the production of siderophores was highly iron-dependent, the antimicrobial activity of spent medium fromStaphylococcus cultures against selected yeasts and moulds remained considerable under high iron concentrations.     

Polyamine-mediated inhibition of in-vitro cell proliferation is not due to acrolein

The influence of acrolein or spermine on the viability and growth of phytohaemagglutinin-stimulated rat thymic lymphocytes in cultures supplemented with foetal calf serum have been investigated. Acrolein (greater than 20 microM) was cytotoxic; spermine had little effect on viability, but inhibited [3H]TdR incorporation at low concentrations (approximately 10 microM). Cells treated with greater than 8 microM acrolein 3 hr before stimulation exhibited irreversible inhibition of protein synthesis, whereas 50 microM spermine had no effect, even when cells were treated for 24 hr before stimulation. However, addition of 25 microM spermine after stimulation did inhibit both [3H]-uridine incorporation and protein synthesis: this was reversible if cells were freed of polyamine within 4 hr, but not if washed after 24 hr. These results show that, contrary to several previous reports, in-vitro inhibition of cell proliferation by spermine is not due to the formation and action of acrolein.