Utilization of Iron Sources and Its Possible Roles in the Pathogenesis of Vibrio parahaemolyticus

Vibrio parahaemolyticus is an important enteropathogen in Japan, Taiwan and other coastal regions. The influence of the regulation of iron on the pathogenesis of this pathogen has not been well characterized. The growth of pathogenic and non-pathogenic strains of V. parahaemolyticus on iron-limited agar plates was stimulated by ferritin, lactoferrin and transferrin at 30 μM , and also by hemin, hemoglobin and ferric ammonium citrate at 100 μM . Spontaneous iron-utilizing mutant strains (mutants) were derived from a clinical strain, ST550. Compared with the parent strain, lowered virulence was demonstrated for these mutants, as assayed by adult mouse and suckling mouse models. The in vivo growth and enterotoxigenicity of these mutants were also lower in the suckling mice. Adherence of the mutants to excised mouse intestine was lower as demonstrated by scanning electron microscopy. The iron-regulated outer membrane protein profile also changed in selected mutants. These results indicate that iron-regulated outer membrane proteins and other unknown factors associated with iron utilization may have profound influences, besides iron acquisition, on the pathogenesis of V. parahaemolyticus.     

Factors affecting production of COS and CS2 in Leucaena and Mimosa species

Carbon disulfide (CS₂) and carbonyl sulfide (COS) are colorless, foul-smelling, volatile sulfur compounds with biocidal properties. Some plants produce CS₂ or COS or both. When used as an intercrop or forecrop, these plants may have agronomic potential in protecting other plants. Most of the factors which affect production of these plant-generated organic sulfides are unknown. We determined the effects of sulfate concentration, plant age, nitrogen fixation, drought stress, root injury (through cutting), and undisturbed growth on COS production in Leucaena retusa or Leucaena leucocephala and the effect of some of these factors on CS₂ production in Mimosa pudica. In addition, we determined if organic sulfides were produced in all Leucaena species. When L. retusa and M. pudica seedlings were grown in a plant nutrient medium with different sulfate concentrations (50 to 450 mg SL^-1), COS or CS₂ from crushed roots generally increased with increasing sulfate concentration. COS production was highest (74 ng mg^-1 dry root) for young L. retusa seedlings and declined to low amounts (<5 ng mg^-1 dry root) for older seedlings. Nitrogen fixation reduced the amounts of COS or CS₂ produced in L. leucocephala and M. pudica. Under conditions of undisturbed growth, root cutting, or drought stress, no COS production was detected in 4-to 8-weeks-old L. retusa plants. COS or CS₂ or both was obtained from crushed roots or shoots of all 13 known Leucaena species.     

Study of the role of iron in the anticryptococcal activity of human serum and fluconazole

Anticryptococcal activity of human serum and apotransferrin in RPMI 1640 was studied in vitro. The effects of varying concentrations of FeCl₃ on this activity was investigated. Possible synergy of serum and apotransferrin with fluconazole was also measured. The fungistatic activity of human serum, whether lyophilized, stored at 4 °C, fresh frozen or purchased from commercial sources vs. Cryptococcus neoformans was comparable. There was no significant loss of fungistatic activity after freezing and thawing the serum up to 10 times. The fungistatic activity of human serum was similar when tested in different tissue culture media with the exception of Medium 199. The addition of apotransferrin (2.0 or 0.2 mg/ml) to RPMI 1640 had an inhibitory effect on cryptococcal growth. This effect was reversed by 20 M of FeCl₃ at both apotransferrin concentrations. By contrast, addition of FeCl₃ to human serum and RPMI 1640 did not reverse inhibition of growth. Fluconazole synergized with the human serum preparations described, but not with pooled commercial serum, for fungicidal activity. Synergistic activity of fluconazole and human serum was not affected by the addition of FeCl₃. Apotransferrin did not show any synergistic fungicidal activity with fluconazole.     

Net Uptake of Sulfate and its Transport to the Shoot in Spinach Plants Fumigated with H2S or SO2: Does Atmospheric Sulfur Affect the “Inter-Organ” Regulation of Sulfur Nutrition?*

Spinach plants (Spinacea oleracea L. cv. Estivato) were grown on nutrient solutions under deficient, normal and excess sulfate supply. In both young and mature plants net uptake of sulfate and its transport to the shoot increased with increasing sulfate supply, but both processes proceeded at a higher rate in young as compared to mature plants. The relative sulfate transport, i.e. the relative amount of the sulfate taken up that is transported to the shoot, decreased with increasing sulfate supply. Apparently, net uptake of sulfate is not strictly controlled by the sulfur demand of the shoot, but xylem loading appears to counteract excess transport of sulfate to the shoot. Fumigation with H₂S or SO₂ reduced net uptake of sulfate by the roots in sulfur-deficient plants and absolute as well as relative sulfate transport to the shoot independent of the three sulfate levels supplied to the plant. At the same time thiol contents of the shoot and the root were enhanced by fumigation with H₂S and SO₂. These findings are consistent with the idea that thiols produced in the leaves can mediate demand-driven control of sulfate uptake by the roots and its transport to the shoot.     

Biogeochemical cycling of sulfur and iron in sediments of a south-east Asian mangrove,Phuket Island,Thailand

Benthic sulfate reduction and sediment pools of sulfur and iron were examined during January 1992 at 3 stations in the Ao Nam Bor mangrove, Phuket, Thailand. Patterns of sulfate reduction rates (0–53 cm) reflected differences in physical and biological conditions at the 3 stations, and highest rates were found at the vegetated site within the mangrove (Rhizophora apiculata) forest. Due to extended oxidation of mangrove sediments, a large portion of the added³⁵S-label was recovered in the chromium reducible pools (FeS₂ and S⁰) (41–91% of the reduced sulfur). Pyrite was the most important inorganic sulfur component, attaining pool sizes 50–100 times higher than acid volatile pools (FeS). HCl-extractable (0.5 M HCl) iron pools, including Fe(II)_HCl and Fe(III)_HCl, were generally low and Fe(III)_HCl was only present in the upper surface layers (0–5 cm). Maximum concentrations of dissolved Fe²⁺ (35–285 M) occurred just about the depth where dissolved H₂S accumulated. Furthermore Fe²⁺ and H₂S coexisted only where concentrations of both were low. There was an accumulation of organic sulfur in the deep sediment at 2 stations in the inner part of the mangrove. The reoxidation of reduced sulfides was rapid, and storage of sulfur was minor in the upper sediment layers, where factors like bioturbation, the presence of roots, or tidal mixing enhance oxidation processes.Author of correspondence.     

Exchange of reduced sulfur gases between lichens and the atmosphere

Fourteen lichens, 10 green algal lichens and four cyanolichens, as well as a cyanobacterium emitted significant quantities of H₂S (0.01–0.04 pmol g dw^–1 s^–1) and DMS (0.005–0.025 pmol g dw^–1 s^–1) but were sinks for COS (0.015–0.14 pmol g dw^–1 s^–1). In contrast, exchange of CH₃SH and CS₂ were sporatic and inconsistent. Although some interspecific variation occurred for the first three gases, exchange rates were relatively uniform and were not influenced by irradiance conditions. In contrast to DMS and H₂S emission, COS uptake was strongly influenced by degree of thallus hydration. Because lichen dominated systems cover extensive terrestrial habitats, COS uptake is potentially important in the world's sulfur budget.     

Diversity of siderophore genes encoding biosynthesis of 2,3-dihydroxybenzoic acid in Aeromonas spp.

Most species of the genus Aeromonas produce the siderophore amonabactin, although two species produce enterobactin, the siderophore of many enteric bacteria. Both siderophores contain 2,3-dihydroxybenzoic acid (2,3-DHB). Siderophore genes (designated aebC, -E, -B and -A, for aeromonad enterobactin biosynthesis) that complemented mutations in the enterobactin genes of the Escherichia coli 2,3-DHB operon, entCEBA(P15), were cloned from an enterobactin-producing isolate of the Aeromonas spp. Mapping of the aeromonad genes suggested a gene order of aebCEBA, identical to that of the E. coli 2,3-DHB operon. Gene probes for the aeromonad aebCE genes and for amoA (the entC-equivalent gene previously cloned from an amonabactin-producing Aeromonas spp.) did not cross-hybridize. Gene probes for the E. coli 2,3-DHB genes entCEBA did not hybridize with Aeromonas spp. DNA. Therefore, in the genus Aeromonas, 2,3-DHB synthesis is encoded by two distinct gene groups; one (amo) is present in the amonabactin-producers, while the other (aeb) occurs in the enterobactin-producers. Each of these systems differs from (but is functionally related to) the E. coli 2,3-DHB operon. These genes may have diverged from an ancestral group of 2,3-DHB genes.     

Effects of chelators on iron uptake and release by the brain in the rat

The iron chelators desferrioxamine (DFO), pyridoxal isonicotinoyl hydrazone (PIH), 2,2-bipyridine, diethylenetriamine penta-acetic acid (DTPA) and 1,2 dimethyl-3-hydroxy pyrid-4-one (CP20) were analysed for their ability to change⁵⁹Fe uptake and release from the brain of 15- and 63-day rats either during or after intravenous injection of⁵⁹Fe-¹²⁵I-transferrin. DTPA was the only chelator unable to significantly reduce iron uptake into the brain of 15-day rats. This indicates that iron is not released from transferrin at the luminal surface of brain capillary endothelial cells. CP20 was able to reduce iron uptake in the brain by 85% compared to 28% with DFO. Only CP20 was able to significantly reduce brain iron uptake in 63 day rats. Once⁵⁹Fe had entered the brain no chelator used was able to mediate its release. All of the chelators except CP20 had similar effects on femur iron uptake as they did on brain uptake, suggesting similar iron uptake mechanisms. It is concluded that during the passage of transferrin-bound iron into the brain the iron is released from transferrin within endothelial cells after endocytosis of transferrin.     

Diffusion of Fe(II) from an iron propagation cage and its effect on tissue iron and pigments of macroalgae on the cage

Iron propagation cages were settled on sand and/or rock beds in coastal areas of Hokkaido. The cage was oxidized by dissolved oxygen and the released Fe(II) diffused into the seawater around the cage. Fe(II) concentrations in the range of 10–50 nM were detected within a 20-m distance around the cage. For comparison, in the Japan Sea, the total iron concentration is less than 2 nM.Laminaria japonica was grown in an indoor semi-continuous culture system. The critical Fe level for maintaining maximum growth, and the subsistence Fe level for survival were measured. The concentrations obtained were 14–21 and 8 g Fe g^–1 tissue, respectively. Iron found inL. japonica growing on rocks and/or rock beds in the Japan Sea was close to the subsistence level. However, the Fe level inL. japonica on the cage in the Japan Sea was considerably higher. The concentrations of chlorophyll-a and fucoxanthin collected from the cage were significantly higher for sporophytes, demonstrating that iron is a very important element for the growth of seaweeds.     

Ethylene production in rice bronzing leaves induced by ferrous iron

Bronzing, a nutritional disorder of rice plants which is widely distributed in tropical lowlands, was induced by dipping the cut end of rice leaves into FeSO₄ solution (pH 3.5). Ethylene production; the activities of peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase; and the effects of Co²⁺, aminoethoxyvinylglycine, Ag⁺, cycloheximide, and 1-aminocyclopropane-1-carboxylate, were investigated in the course of bronzing development. It was found that ethylene production could be stimulated up to about 20 times that of the control by Fe²⁺, and a peak could be reached at about 24 h after incubation. The Fe²⁺-treated leaves also had 10-fold higher peroxidase activity than the control, whereas in vitro enzyme activity was inhibited by Fe²⁺. Cycloheximide retarded in vivo stimulation of peroxidase, indicating that in vivo stimulation resulted from inducing de novo synthesis of the enzyme. No changes in the activities of phenylalanine ammonia-lyase and polyphenol oxidase were observed. The results, obtained from the incubation of leaves with Co²⁺, aminoethoxyvinylglycine, Ag⁺, cycloheximide, or 1-aminocyclopropane-1-carboxylate, showed that ethylene production was the effect of Fe²⁺ stress and that it was not involved in the process of bronzing development, which is probably an acclimation process to enable plants to cope with stress. The accelerated peroxidase activity may be associated with bronzing development.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - EFE ethylene forming enzyme - PAL phenylalanine ammonia-lyase - POD peroxidase - PPO polyphenol oxidase - SE standard error