Ferrioxamine transport mutants and the identification of the ferrioxamine receptor protein (FoxA) inErwinia herbicola (Enterobacter agglomerans)

Summary Iron deprivation ofErwinia herbicola (Enterobacter agglomerans) induces the biosynthesis of six high-M _r outer-membrane proteins and large amounts of ferrioxamine E. Mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine and selection with ferrimycin A yielded mutants ofE. herbicola K4 (wild type), defective in the expression of a 76-kDa outer-membrane protein, as determined by SDS/polyacrylamide gel electrophoresis. While in bioassays wild-type cells showed growth promotion in the presence of ferrioxamines (B, D₁, D₂, E, G), enterobactin, citrate, ferrichrome and coprogen, these mutants failed to respond to ferrioxamines. Moreover, experiments with⁵⁵Fe-labelled siderophores confirmed that iron transport mediated by ferrioxamine E and B in the mutants was completely inhibited, whereas iron transport by other hydroxamate siderophores, such as ferrichrome and coprogen was unaffected. The results are evidence that the 76-kDa protein in the outer membrane represents the receptor protein (FoxA) for ferrioxamines inE. herbicola.     

Characterization of ferrioxamine E as the principal siderophore ofErwinia herbicola (Enterobacter agglomerans)

Summary Several strains of the enterobacterial groupErwinia herbicola (Enterobacter agglomerans) were screened for siderophore production. After 3 days of growth in a low-iron medium, all strains studied produced hydroxamate siderophores. The retention values of the main siderophore during thin-layer chromatography on silica gel plates and on HPLC reversed-phase columns were identical with those of an authentic sample of ferrioxamine E (norcardamine). Gas-chromatographic analysis of the HI hydrolyzate yielded succinic acid and 1,5-diaminopentane in equimolar amounts; fast-atom-bombardment (FAB) mass spectroscopy showed a molecular mass of 653 Da. Iron from⁵⁵Fe-labelled ferrioxamine E was well taken up by iron-starved cells ofE. herbicola (K _m=0.1 M,V _max=8 pmol mg^–1 min^–1). However, besides ferrioxamine E (100%), several exogenous siderophores such as enterobactin (94.5%), ferric citrate (78.5%), coprogen (63.5%) and ferrichrome (17.5%) served as siderophores, suggesting the presence of multiple siderophore receptors in the outer membrane ofE. herbicola.     

The structure of the O-specific polysaccharide of the lipopolysaccharide from Pantoea agglomerans strain FL1

A neutral O-specific polysaccharide consisting of d-rhamnose was obtained by mild acid hydrolysis of the lipopolysaccharide of the plant pathogenic bacterium Pantoea agglomerans strain FL1, a common epiphyte of many plant species, and associated with Pseudomonas savastanoi pv. savastanoi in young and apparently intact olive knots. By means of compositional and methylation analyses, and NMR spectroscopy, the chemical repeating unit of the polymer was identified as a linear tetrasaccharide of the structure:     

Acid tolerance response induced in the biocontrol agent Pantoea agglomerans CPA-2 and effect on its survival ability in acidic environments

The aim of this work was to optimize acid stress conditions for induction of acid tolerance response (ATR) in the biocontrol agent Pantoea agglomerans and study the effect of ATR induced on the ability to survive under acidic conditions. Initially, Pantoea agglomerans was grown in mild acidic conditions (pH 6.0, 5.5, 5.0 and 4.0) in order to induce ATR. The highest ATR was induced at initial pH of 5 using malic or citric acid. A first in vitro experiment was carried out. Thus, basal liquid medium at different pHs (3.0, 3.5, 4.0 and non-acidified) were then inoculated with acid-adapted and non-adapted inocula of P. agglomerans and survivals were examined during incubation at 25 or 4 °C. It was found that acid adaptation enhanced the survivals of Pantoea agglomerans CPA-2 cells at pH levels at which the cells were unable to grow (<3.5 and 4.0, at 25 and 4 °C, respectively). In contrast, in pH levels at which the cells were able to grow (pH 4.0 at 25 °C and non-acidified medium at 25 and 4 °C) no-differences were found between adapted and non-adapted cells. In in vivo tests, adapted and non-adapted cells were inoculated in wounds on mandarins and pome fruits. No differences were found between adapted and non-adapted cells and biocontrol efficacy was maintained. The present study demonstrated that exposure of Pantoea agglomerans to mild acidic conditions could induce acid resistance in this biocontrol agent.     

Production of indole-3-acetic acid, aromatic amino acid aminotransferase activities and plant growth promotion by Pantoea agglomerans rhizosphere isolates

The production of auxins, such as indole-3-acetic acid (IAA), by rhizobacteria has been associated with plant growth promotion, especially root initiation and elongation. Six indole-producing bacteria isolated from the rhizosphere of legumes grown in Saskatchewan soils and identified as Pantoea agglomerans spp. were examined for their ability to promote the growth of canola, lentil and pea under gnotobiotic conditions and for tryptophan (Trp)-dependent IAA production. Five of the isolates enhanced root length, root weight or shoot weight by 15–37% in at least one of the plant species, but isolates 3–117 and 5–51 were most consistent in enhancing plant growth across the three species. Indole concentrations in the rhizosphere of plants grown under gnotobiotic conditions increased in the presence of the rhizosphere isolates and when Trp was added 3 days prior to plant harvest. Isolates 3–117, 5–51 and 5–105 were most effective in increasing rhizosphere indole concentrations. Colony hybridization confirmed that all of the isolates possessed the ipdC gene which codes for a key enzyme in the Trp-dependent IAA synthetic pathway. The activity of amino acid aminotransferase (AAT), catalyzing the first step in the Trp-dependent synthetic pathway, was examined in the presence of Trp and other aromatic amino acids. All of the isolates accumulated Trp internally and released different amounts of IAA. The production of IAA from the isolates was greatest in the presence of Trp, ranging from 2.78 to 16.34 μg mg protein⁻¹ in the presence of 250 μg of Trp ml⁻¹. The specific activity of AAT was correlated with the concentration of IAA produced in the presence of Trp but not when tyrosine (Tyr), phenylalanine (Phe) or aspartate (Asp) was used as a sole nitrogen source. Isolate 3–117, which produced significant concentrations of IAA in the presence and absence of Trp, was able to use aromatic amino acids as sole sources of nitrogen and was most consistent in enhancing the growth of canola, lentil and pea may have potential for development as a plant growth-promoting inoculant. Responsible Editor: Peter A. H. Bakker.     

Exopolysaccharides of Pantoea agglomerans have different priming and eliciting activities in suspension-cultured cells of monocots and dicots

Induced disease resistance of plants is often associated with an enhanced capacity to activate cellular defense responses to pathogen attack, named the "primed" state of the plant. Exopolysaccharides of Pantoea agglomerans have recently been reported as the first priming active component of bacterial origin in wheat cells. We now show that Pantoea exopolysaccharides also prime rice cells for better elicitation of a rapid oxidative burst. In contrast, in tobacco and parsley cell cultures Pantoea exopolysaccharides activate the oxidative burst response directly. Our results point to a different recognition and/or mode of action of Pantoea exopolysaccharides in monocot and dicot plants.     

Purification and Properties of a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

A periplasmatic phytate-degrading enzyme from Pantoea agglomerans isolated from soil was purified about 470-fold to apparent homogeneity with a recovery of 16% referred to the phytate-degrading activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 60°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K_M = 0.34 mmol/l and k_cat = 21 s^-1 at pH 4.5 and 37°C. The enzyme exhibited a narrow substrate selectivity. Only phytate and glucose-1-phosphate were identified as good substrates. Since this Pantoea enzyme has a strong preference for glucose-1-phosphate over phytate, under physiological conditions glucose-1-phosphate is its most likely substrate. The maximum amount of phosphate released from phytate by the purified enzyme suggests myo-inositol pentakisphosphate as the final product of enzymatic phytate degradation.     

Settlement of the diazotrophic,phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat: An investigation using ELISA and transmission electron microscopy

The aim of this study was to investigate the ability of Pantoea agglomerans, a plant growth-promoting bacterium, to colonize various regions and tissues of the wheat plant (Triticum aestivum L.) by using different inoculation methods and inoculum concentrations. In addition, the enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) were used to determine: (a) the ability of the bacterial cells to grow and survive both on the surface and within internal tissue of the plant and (b) the response of the plant to bacterial infection. After inoculation, cells of the diazotrophic bacterial strain P. agglomerans were found to be located in roots, stems and leaves. Colony development of bacterial cells was only detected within intercellular spaces of the root and on the root surface. However, single bacterial cells were observed in leaves and stems on the surface of the epidermis, in the vicinity to stomatal cells, within intercellular spaces of the mesophyll and within xylem vessels. Inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relationship between endophyte and host. The density of bacterial settlement in the plant in all experiments was about 10⁶ to 10⁷ cells per mL root or shoot sap. Establishment was confirmed by a low coefficient of variation of ELISA means at these concentrations.     

Transport properties of N-acyl derivatives of the coprogen and ferrichrysin classes of siderophores inNeurospora crassa

Several derivatives of the coprogen and ferrichrysin classes of siderophores were synthesized as potential affinity labels of the iron uptake system inNeurospora crassa. While only one of these compounds has proved useful as an affinity label, all were recognized and transported byNeurospora crassa. One derivative, chloroacetyl-ferrichrysin, proved to be an unexpectedly potent reversible inhibitor (K ₁=0.4 M) of both ferrichrysin and coprogen uptake, similar to the natural siderophore, ferrirubin. The reported results provide further understanding of the steric and electronic requirements of siderophores for the iron uptake system inNeurospora crassa.Abbreviations amu atomic mass units - DMF dimethylformamide - FAB tast atom bombardment - NMR nuclear magnetic resonance - ppm parts per million - tlc thin layer chromatography     

Transport and utilization of rhizoferrin bound iron in Mycobacterium smegmatis

Transport and metabolization of iron bound to the fungal siderophore rhizoferrin was analyzed by transport kinetics, Mössbauer and EPR spectroscopy. Saturation kinetics (v _max=24.4 pmol/(mg min), K _m=64.4M) and energy dependence excluded diffusion and provided evidence for a rhizoferrin transport system in M. smegmatis. Based on the spectroscopic techniques indications for intracellular presence of the ferric rhizoferrin complex were found. This feature could be of practical importance in the search of novel drugs for the treatment of mycobacterial infections. EPR and Mössbauer spectroscopy revealed different ferritin mineral cores depending on the siderophore iron source. This finding was interpreted in terms of different protein shells, i.e. two types of ferritins.     

A study of the growth condition and solubilization of phosphate from hydroxyapatite byPantoea agglomerans

The growth conditions ofPantoea agglomerans, a phosphate solubilizing organism, were studied in our laboratory to determine the optimal conditions.Pantoea agglomerans showed the highest growth rate at 30°C, pH 7.0 and 2 vvm, after 50 h cultivation. A certain relationship between pH and phosphate concentration, was evident when the glucose concentration in the medium was changed. Increasing glucose concentration increased the pH buffer action of the broth. At glucose concentrations higher than the optimum concentration of 0.2 M, the cell growth was retarded.P. agglomerans consumed glucose as a substrate to produce organic acids which caused the pH decrease in the culture medium. The phosphate concentration in the medium was increased by the presence of the organic acids, which solubilized insoluble phosphates such as hydroxyapatite.     

Formation of iron(VI) in ozonalysis of iron(III) in alkaline solution

Here we report the formation of iron in hexavalent state, in ozonalysis of iron(III) in alkaline medium. The formation of tetrahedral ion is confirmed by UV-Visible and Mössbauer spectroscopic techniques. The value of isomer shift, δ, of the tetra-oxy anion is consistent with known δ values for various salts of iron(VI) ion.     

Nicotianamine and the distribution of iron into the apoplasm and symplasm of tomato (Lycopersicon esculentum Mill.)

The apoplasmic and symplasmic iron pools were determined in roots and leaves of Lycopersicon esculentum Mill. cv. Bonner Beste and its mutant chloronerva. The mutant is auxotrophic for the ubiquitous plant constituent nicotianamine (NA) and exhibits an impaired iron metabolism. Formation of apoplasmic iron pools in roots was dependent on the iron source in the nutrient solution. With Fe-ethylenediaminedi-(2-hydroxyphenylacetate) (FeEDDHA) only a very small apoplasmic iron pool was formed in the roots of both genotypes. Plants grown with FeEDTA increased their apoplasmic iron pool with increasing exogenous iron concentrations in the nutrient solution. The size of the apoplasmic pools in roots did not differ between the wild-type and the mutant (about 85 mol Fe · g^–1 DW). By contrast, the symplasmic iron concentrations in roots and leaves of the mutant were significantly higher when compared to the wild-type. An exogenous NA supply to the leaves of the mutant reduced the high symplasmic iron concentrations to the level of the wild-type. Mutant leaves exhibited a gradient of symplasmic iron concentrations depending on the developmental age of the leaves. The oldest leaves contained considerably more symplasmic iron than the youngest. The results demonstrate that the apparent iron deficiency of the mutant is not the consequence of an impaired iron transport from the apoplasm to the symplasm. Therefore, it is concluded that NA is not required for the transport of Fe(II) through the plasmalemma into the cell.Abbreviations BPDS bathophenanthroline disulfonic acid, Na₂ salt - FeEDDHA ferric N-N-ethylenediaminedi-(2-hydroxy-phenylacetate) - NA nicotianamine Part 40 in the series The normalizing factor for the tomato mutant chloronerva. For part 39 see Pich et al. (1991)The valuable technical assistance of Mrs. Christa Kallas and Mr. Günter Faupel is gratefully acknowledged.     

Expression of klebsiella his and nif genes in serratia marcescens,Erwinia herbicola and Proteus mirabilis

Plasmid pRD1, an R plasmid of the P incompatibility group which carries his and nif genes from Klebsiella pneumoniae in addition to drug resistance markers derived from RP4, was transferred to His^- mutants of Serratia marcescens, Erwinia herbicola and Proteus mirabilis. His⁺ transconjugants were obtained at low but different frequencies according to recipient genus. Transconjugants all acquired the drug resistance, and were Nif⁺ in S. marcescens and E. herbicola, having acetylene-reducing activities of the same order of magnitude as the parent K. pneumoniae and fixing ¹⁵N₂. No evidence for nif expression in P. mirabilis transconjugants was obtained though the nif genes were present.     

Biotechnological application of homo- and heterotrinuclear iron(III) furoates for cultivation of iron-enriched Spirulina

New trinuclear iron(III) furoates with the general formula [Fe₃O(α-fur)₆(R-OH)₃]X, where α-fur C₄H₃OCOO, R = CH₃ (1), C₂H₅ (2), n-C₃H₇ (3), n-C₄H₉ (4), X = NO₃⁻ (1-4); [Fe₃O(α-Fur)₆(DMF)(CH₃OH)₂]NO₃ (5); [Fe₃O(α-Fur)₆(H₂O)(CH₃OH)₂]Cl (6); [Fe₂MO(α-Fur)₆(L)(H₂O)₂], where L = THF (7-9), DMF (10-12), M = Mn²⁺ (7, 10), Co²⁺ (8, 11), Ni²⁺ (9, 12) and [Fe₂MO(α-Fur)₆(3Cl-Py)₃], where M = Mn²⁺ (13), Co²⁺ (14), Ni²⁺ (15); have been prepared and investigated by Mössbauer and IR spectroscopy. The X-ray crystal structure for the 1·2CH₃OH complex indicates that it crystallizes in the monoclinic crystal system (P2₁/n) and has a structure typical of μ₃-O-bridged trinuclear iron(III) compounds. Coordination compounds 1, 4, 7, 8 can be used as regulators of the biochemical composition of cyanobacterium Spirulina platensis biomass. The supplementation of these compounds, in concentrations exceeding 5-10 mg/l, increases the content of iron, amino acids, peptides and carbohydrates in Spirulina.     

Spin-crossover iron(II) complexes [Fe(Medpq)(py)2(NCS)2] and [Fe(Medpq)(py)2(NCSe)2]: syntheses, characterization and magnetic properties

Two new spin-crossover complexes, [Fe(Medpq)(py)₂(NCS)₂] · py · 0.5H₂O (1) and [Fe(Medpq)(py)₂(NCSe)₂] · py (2) (Medpq = 2-methyldipyrido[3,2-f:2′,3′-h]-quinoxaline, py = pyridine), have been synthesized. The crystal structures were determined at both room temperature (298 K) and low temperature (110 K). Complexes 1 and 2 crystallize in the orthorhombic space group Pbca and monoclinic space group P2₁/n, respectively. In both complexes, the distorted [FeN₆] octahedron is formed by six nitrogen atoms from Medpq, the trans pyridine molecules and the cis NCX⁻ groups. The thermal spin transition is accompanied by the shortening of the mean Fe–N distances by 0.194 Å for 2. The mononuclear [Fe(Medpq)(py)₂(NCS)₂] and [Fe(Medpq)(py)₂(NCSe)₂] neutral species interact each other via π-stacking, resulting in a one-dimensional extended structure for both 1 and 2. There exist C–HX (X = S, Se) hydrogen bonds for both complexes. Variable-temperature magnetic susceptibility measurements and Mössbauer spectroscopy reveal the occurrence of a gradual spin transition. The transitions are centered at T_1/2 = 120 K for 1 and T_1/2 = 180 K for 2, respectively.     

Surface iron polymers and hydroxy acids. A model of iron supply in sideramine-free fungi

Biochemical and electron microscopic evidence is presented that sideramine-free fungi form iron hydroxide polymer layers on the cell surface when grown in an iron containing medium.Iron hydroxide polymer formation on the cell surface is completely prevented in sideramine producing strains of Neurospora crassa. After feeding a sideramine-free mutant of Neurospora crassa with ornithine in order to restore the sideramine synthesis the iron hydroxide coat is gradually dissolved.The addition of excess citrate and malate to the incubation medium also prevents iron polymer adsorption, suggesting that hydroxy acids may be involved in iron supply, when sideramine-free organisms are grown in iron containing media.In order to study the interaction between iron hydroxide polymer deposition upon the cell surface and iron chelating acids in Neurospora crassa, the amount and the proportion of excreted acids was studied under various experimental conditions. Gas chromatographic analysis of the acids produced under iron deficient conditions revealed that succinate, malate and citrate were present within the cells in the early growth phase. The acids were sequentially excreted into the medium in the order succinate, malate and citrate. The amount of succinate decreased after 2 days of cultivation, whereas the amount of malate and citrate continually increased. Although citrate was present within the cells from the 1st day, excretion occurred very late, generally after the 3rd day.It is suggested that sideramine-free fungi first adsorb iron as a hydroxide polymer on the cell surface, and that it is gradually solubilized by excreted hydroxy acids such as citrate or malate. Thus high local concentrations of iron chelated by hydroxy acids provide sideramine-free fungi with a continuous iron supply.Abbreviations BSTFA N,O-Bis(trimethylsilyl)-trifluoracetamide - GC Gaschromatography - EGTA Ethylenglykol-bis(2-aminoethylether) N,N-tetraacetic acid - TMS Trimethylsilyl     

Pyoverdine-mediated iron transport Fate of iron and ligand inPseudomonas aeruginosa

Summary Incubated in the presence of [⁵⁵Fe]ferri[¹⁴C]pyoverdine, iron-poorPseudomonas aeruginosa accumulated more⁵⁵Fe than¹⁴C over a 60-min period. Distribution studies showed (a) more¹⁴C than⁵⁵Fe in the soluble fraction during the first 20 min, (b) approximately 60% of the⁵⁵Fe associated with the membranes at 60 min, and (c) approximately 85% of the¹⁴C in the soluble fraction at 60 min. Cells osmotically shocked after incubating with [⁵⁵Fe]ferri[¹⁴C]pyoverdine for 60 min released⁵⁵Fe but not¹⁴C, suggesting separation of metal and ligand in the periplasmic space. Whereas the mechanism of dissociation of iron and ligand is not known, the decrease in transport observed in the presence of dipyridyl suggests involvement of reduction in this process. Transport of iron was energized by the proton motive force instead of by intracellular levels of ATP. The hydrogen ion gradient was the major driving force of transport. Cyanide-poisoned cells accumulated more¹⁴C than⁵⁵Fe over 60 min. Here, iron accumulated in the soluble fraction instead of on the membranes.