Effect of Exogenous Reductant on Growth and Iron Mobilization from Ferrihydrite by the Pseudomonas mendocina ymp Strain

Growth of the Pseudomonas mendocina ymp strain on insoluble ferrihydrite is enhanced by exogenous reductants with concurrent increase in soluble iron concentrations. This shows that exogenous reductants play a substantial role in the overall microbial iron bioavailability. The exogenous reductants may work together with the siderophores, Fe-scavenging agents, to facilitate ferrihydrite dissolution.     

Identification, isolation, and analysis of a gene cluster involved in iron acquisition by Pseudomonas mendocina ymp

Microbial acquisition of iron from natural sources in aerobic environments is a little-studied process that may lead to mineral instability and trace metal mobilization. Pseudomonas mendocina ymp was isolated from the Yucca Mountain Site for long-term nuclear waste storage. Its ability to solubilize a variety of Fe-containing minerals under aerobic conditions has been previously investigated but its molecular and genetic potential remained uncharacterized. Here, we have shown that the organism produces a hydroxamate and not a catecholate-based siderophore that is synthesized via non-ribosomal peptide synthetases. Gene clustering patterns observed in other Pseudomonads suggested that hybridizing multiple probes to the same library could allow for the identification of one or more clusters of syntenic siderophore-associated genes. Using this approach, two independent clusters were identified. An unfinished draft genome sequence of P. mendocina ymp indicated that these mapped to two independent contigs. The sequenced clusters were investigated informatically and shown to contain respectively a potentially complete set of genes responsible for siderophore biosynthesis, uptake, and regulation, and an incomplete set of genes with low individual homology to siderophore-associated genes. A mutation in the cluster’s pvdA homolog (pmhA) resulted in a siderophore-null phenotype, which could be reversed by complementation. The organism likely produces one siderophore with possibly different isoforms and a peptide backbone structure containing seven residues (predicted sequence: Acyl-Asp-Dab-Ser-fOHOrn-Ser-fOHorn). A similar approach could be applied for discovery of Fe− and siderophore-associated genes in unsequenced or poorly annotated organisms.     

Reduction of exogenous flavins and mobilization of iron from ferritin by isolated mitochondria

The release mechanism for ferritin iron and the nature of the compound(s) which donate iron to the mitochondria are two important problems of intracellular iron metabolism which still await their solution. We have previously shown that isolated mitochondria reduce exogenously added flavins in a ubiquinol-flavin oxidoreductase reaction at the C-side of the inner membrane and that the resulting dihydroflavins function as reductants in mitochondrial mobilization of iron from ferritin (Ulvik, R. J., and Romslo, I. (1981). Biochim. Biophys. Acta 635, 457-469). In the present study it is shown that the rate at which iron is removed from ferritin depends on the capability of the flavins to penetrate (1) the mitochondrial outer membrane and (2) the intersubunit channels of the ferritin protein shell. Intact mitochondria reduce flavins at rates which decrease in the following order: riboflavin > FAD > FMN. The ferritin iron mobilization rates decrease in the order of riboflavin > FMN > FAD. The results are further support for the operation of a flavin-dependent mitochondrial ferrireductase, and strengthen the suggested role for ferritin as a donor of iron to the mitochondria.     

Kinetic characterization of reductant dependent processes of iron mobilization from endocytic vesicles.

The reductant dependence of iron mobilization from isolated rabbit reticulocyte endosomes containing diferric transferrin is reported. The kinetic effects of acidification by a H(+)-ATPase are eliminated by incubating the endosomes at pH 6.0 in the presence of 15 microM FCCP to acidify the intravesicular milieu and to dissociate 59Fe(III) from transferrin. In the absence of reductants, iron is not released from the vesicles, and iron leakage is negligible. The second-order dependence of rate constants and amounts of 59Fe mobilized from endosomes using ascorbate, ferrocyanide, or NADH are consistent with reversible mechanisms. The estimated apparent first-order rate constant for mobilization by ascorbate is (2.7 +/- 0.4) x 10(-3) s-1 in contrast to (3.2 +/- 0.1) x 10(-4) s-1 for NADH and (3.5 +/- 0.6) x 10(-4) s-1 for ferrocyanide. These results support models where multiple reactions are involved in complex processes leading to iron transfer and membrane translocation. A type II NADH dehydrogenase (diaphorase) is present on the endosome outer membrane. The kinetics of extravesicular ferricyanide reduction indicate a bimolecular-bimolecular steady-state mechanism with substrate inhibition. Ferricyanide inhibition of 59Fe mobilization is not detected. Significant differences between mobilization and ferricyanide reduction kinetics indicate that the diaphorase is not involved in 59Fe(III) reduction. Sequential additions of NADH followed by ascorbate or vice versa indicate a minimum of two sites of 59Fe(III) residence; one site available to reducing equivalents from ascorbate and a different site available to NADH. Sequential additions using ferrocyanide and the other reductants suggest interactions among sites available for reduction. Inhibition of ascorbate-mediated mobilization by DCCD and enhancement of ferrocyanide and NADH-mediated mobilization suggest a role for a moiety with characteristics of a proton pore similar to that of the H(+)-ATPase. These data provide significant constraints on models of iron reduction, translocation, and mobilization by endocytic vesicles.     

Ferritin and ferrihydrite nanoparticles as iron sources for Pseudomonas aeruginosa

Metabolism of iron derived from insoluble and/or scarce sources is essential for pathogenic and environmental microbes. The ability of Pseudomonas aeruginosa to acquire iron from exogenous ferritin was assessed; ferritin is an iron-concentrating and antioxidant protein complex composed of a catalytic protein and caged ferrihydrite nanomineral synthesized from Fe(II) and O₂ or H₂O₂. Ferritin and free ferrihydrite supported growth of P. aeruginosa with indistinguishable kinetics and final culture densities. The P. aeruginosa PAO1 mutant (ΔpvdDΔpchEF), which is incapable of siderophore production, grew as well as the wild type when ferritin was the iron source. Such data suggest that P. aeruginosa can acquire iron by siderophore-independent mechanisms, including secretion of small-molecule reductant(s). Protease inhibitors abolished the growth of the siderophore-free strain on ferritins, with only a small effect on growth of the wild type; predictably, protease inhibitors had no effect on growth with free ferrihydrite as the iron source. Proteolytic activity was higher with the siderophore-free strain, suggesting that the role of proteases in the degradation of ferritin is particularly important for iron acquisition in the absence of siderophores. The combined results demonstrate the importance of both free ferrihydrite, a natural environmental form of iron and a model for an insoluble form of partly denatured ferritin called hemosiderin, and caged ferritin iron minerals as bacterial iron sources. Ferritin is also revealed as a growth promoter of opportunistic, pathogenic bacteria such a P. aeruginosa in diseased tissues such as the cystic fibrotic lung, where ferritin concentrations are abnormally high.     

Enhancement of growth and toxin production of Clostridium argentinense by co-culture with Pseudomonas mendocina

The growth and toxin production of Clostridium argentinense in co-culture with Pseudomonas mendocina was examined in a micro-fermenter without aeration characterizing the association in terms of several growth parameters. The biomass obtained in co-cultures was 4.4 times higher than that in C. argentinense monocultures with total consumption of the carbon source. The pH and Eh attained in co-cultures at later stages of cultivation were suitable for toxin production by C. argentinense. In comparison with C. argentinense monocultures the production of toxin was 17.5 times higher with a specific toxicity of 0.56 LD50 per g of co-culture biomass.     

Biotransformation of N-nitrosodimethylamine by Pseudomonas mendocina KR1

N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been widely studied, but little information is available concerning the microbial transformation of this compound. The objective of this study was to elucidate the pathway(s) of NDMA biotransformation by Pseudomonas mendocina KR1, a strain that possesses toluene-4-monooxygenase (T4MO). P. mendocina KR1 was observed to initially oxidize NDMA to N-nitrodimethylamine (NTDMA), a novel metabolite. The use of 18O2 and H(2)18O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O2. Experiments performed with a pseudomonad expressing cloned T4MO confirmed that T4MO catalyzes this initial reaction. The NTDMA produced by P. mendocina KR1 did not accumulate, but rather it was metabolized further to produce N-nitromethylamine (88 to 94% recovery) and a trace amount of formaldehyde (HCHO). Small quantities of methanol (CH3OH) were also detected when the strain was incubated with NDMA but not during incubation with either NTDMA or HCHO. The formation of methanol is hypothesized to occur via a second, minor pathway mediated by an initial alpha-hydroxylation of the nitrosamine. Strain KR1 did not grow on NDMA or mineralize significant quantities of the compound to carbon dioxide, suggesting that the degradation process is cometabolic.     

Formation of ortho-benzoquinone from sodium benzoate by Pseudomonas mendocina P2d

Pseudomonas mendocina P2d grew in sodium benzoate at as high as 1% concentration and formed a quinonoid compound, identified as ortho-benzoquinone, that rendered the medium orange to wine-red in colour. The quinone was not metabilised further by the organism. Sodium benzoate was converted to catechol, which was a central metabolite forming ortho-benzoquinone and 2- hydroxymuconic semialdehyde (2-HMS) via. meta ring cleavage pathway.     

Biodegradation of methyl violet by Pseudomonas mendocina MCM B-402

Pseudomonas mendocina MCM B-402 was found to utilize a triphenylmethane dye, methyl violet as the sole source of carbon when incorporated in synthetic medium. Almost complete decolorization of methyl violet by P. mendocina was observed within 48 h of incubation at ambient temperature (28 ± 2 °C) under aerated culture conditions, when the bacteria were inoculated into Davis Mingioli's synthetic medium at a concentration of 100 mg/l medium. Methyl violet was mineralized to CO₂ through three unknown intermediate metabolites and phenol. The decolorization of the dye involved demethylation. Received: 27 November 1998 / Received revision: 2 March 1999 / Accepted: 5 March 1999     

Purification and characterization of two extracellular polyhydroxyalkanoate depolymerases from Pseudomonas mendocina

Two polyhydroxyalkanoate depolymerases, PHAase I and PHAase II, were purified to homogeneity from the culture supernatant of an effective PHA-degrading bacterium, Pseudomonas mendocina DS04-T. The molecular masses of PHAase I and PHAase II were determined by SDS-PAGE as 59.4 and 33.8 kDa, respectively. Their optimum pH values were 8.5 and 8, respectively. Enzymatic activity was optimal at 50 °C. Both purified enzymes could degrade PHB, PHBV, and P(3HB-co-4HB). Addition of Na⁺ and K⁺ slightly increased the rate of PHAase II. EDTA significantly inhibited PHAase II but not PHAase I. Mercaptoethanol and H₂O₂ also inhibited the activities of both enzymes.     

Induction of toluene oxidation activity in Pseudomonas mendocina KR1 and Pseudomonas sp. strain ENVPC5 by chlorinated solvents and alkanes.

Toluene oxidation activity in Pseudomonas mendocina KR1 and Pseudomonas sp. strain ENVPC5 was induced by trichloroethylene (TCE), and induction was followed by the degradation of TCE. Higher levels of toluene oxidation activity were achieved in the presence of a supplemental growth substrate such as glutamate, with levels of activity of up to 86% of that observed with toluene-induced cells. Activity in P. mendocina KR1 was also induced by cis-1,2-dichloroethylene, perchloroethylene, chloroethane, hexane, pentane, and octane, but not by trans-1,2-dichloroethylene. Toluene oxidation was not induced by TCE in Burkholderia (Pseudomonas) cepacia G4, P. putida F1, Pseudomonas sp. strain ENV110, or Pseudomonas sp. strain ENV113.     

门多萨假单胞菌Pseudomonas mendocina NK-01合成褐藻寡糖及其结构鉴定

研究发现门多萨假单胞菌NK-01在菌体内积累中长链聚羟基脂肪酸酯(PHAMCL)的同时也能够合成褐藻寡糖分泌到发酵液中,其产量与培养基的碳氮比有关,高碳氮比有利于褐藻寡糖的合成。本研究利用紫外-可见分光光度法、傅立叶红外光谱分析、1H和13C核磁共振对褐藻寡糖的结构进行了分析鉴定,发现褐藻寡糖的结构是由β-D-甘露糖醛酸、α-L-古洛糖醛酸通过β-(1→4)/α-(1→4)键连接而成的无支化线性多糖,并且在单体的2位或3位羟基上部分乙酰化。凝胶渗透色谱(GPC)对分子量的测定结果为2054。     

Metabolic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxygen-limited conditions in chemostat cultures.

Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxygen-limited conditions in chemostat cultures.

Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the embryonic axis and exogenous growth regulators on sunflower cotyledon storage protein mobilization

Cotyledons of sunflower seedlings ( Helianthus annuus L. cv. Giant gray stripe) expand and their protein content first rises then begins to decrease during the first three days of growth. Storage protein structures, which are visible with scanning electron microscopy, undergo modification that leads to storage protein disappearance by day 4 post-imbibition. Expansion of cotyledons detached from seeds prior to imbibition is greatly reduced, total protein levels remain high, and storage protein structures remain visible in cells of these cotyledons. Incubation of excised cotyledons in 1.0 μM benzyladenine or kinetin increases the rates of cotyledon expansion and storage protein loss to levels higher than in intact seedling cotyledons, Incubation in 10 μM indole-3-acetic acid inhibits cotyledon expansion and protein mobilization. More rapid hydrolysis of storage proteins in cotyledons of intact seedlings or detached cotyledons treated with cytokinin is further indicated in day 2 specimens by SDS-polyacrylamide gel electrophoresis. These results suggest a possible mechanism for regulation of cotyledon development by interactions of the promotive effects of cytokinin and inhibitory effects of auxin.     

Effect of iron concentration and growth rate on the expression of protein G in Pseudomonas aeruginosa

Protein G of Pseudomonas aeruginosa was found to be strongly induced by iron and repressed by iron restriction in chemically defined medium (CDM). Expression in batch culture was influenced by phase of growth and in iron limited continuous culture by the doubling time. We suggest that protein G may constitute a low-affinity iron uptake system.     

Studies on the mobilization of iron from ferritin by isolated rat liver mitochondria

Rat liver mitochondria and rat liver mitoplasts mobilize iron from ferritin by a mechanism which depends on a respiratory substrate (preferentially succinate), a small molecular weight electron mediator (FMN, phenazine methosulphate or methylene blue) and (near) anaerobic conditions.The release process under optimized conditions (approx. 50 μmol/l FMN, 1 mmol/l succinate, 0.35 mmol/l Fe(III) (as ferritin iron), 37°C and pH 7.40) amounts to 0.9–1.2 nmol iron/mg protein per min.The results suggest that ferritin might function as an intermediate in the cytosolic transport of iron to the mitochondria.