The Functionality of the three-sited Ferroxidase center of E. coli Bacterial Ferritin (EcFtnA)

At least three ferritins are found in the bacterium Escherichia coli, the heme-containing bacterioferritin (EcBFR) and two non-heme bacterial ferritins (EcFtnA and EcFtnB). In addition to the conserved A- and B-sites of the diiron ferroxidase center, EcFtnA has a third iron-binding site (the C-site) of unknown function that is nearby the diiron site. In the present work, the complex chemistry of iron oxidation and deposition in EcFtnA has been further defined through a combination of oximetry, pH stat, stopped-flow and conventional kinetics, UV-visible, fluorescence and EPR spectroscopic measurements on the wildtype protein and site-directed variants of the A-, B- and C-sites. The data reveal that, while H2O2 is a product of dioxygen reduction in EcFtnA and oxidation occurs with a stoichiometry of Fe(II)/O2 ~ 3:1, most of the H2O2 produced is consumed in subsequent reactions with a 2:1 Fe(II)/H2O2 stoichiometry, thus suppressing hydroxyl radical formation. While the A- and B-sites are essential for rapid iron oxidation, the C-site slows oxidation and suppresses iron turnover at the ferroxidase center. A tyrosyl radical, assigned to Tyr24 near the ferroxidase center, is formed during iron oxidation and its possible significance to the function of the protein is discussed. Taken as a whole, the data indicate that there are multiple iron-oxidation pathways in EcFtnA with O2 and H2O2 as oxidants. Furthermore, the data are inconsistent with the C-site being a transit site, providing iron to the A- and B-sites, and does not support a universal mechanism for iron oxidation in all ferritins as recently proposed

Soil enzyme activities in the rhizosphere of field-grown sugar beet inoculated with the biocontrol agent Pseudomonas fluorescens F113

The original publication is available at www.springerlink.com . Copyright Springer DOI : 10.1007/s003740050397Pseudomonas fluorescens F113, which produces the antimicrobial compound 2,4-diacetylphloroglucinol is a prospective biocontrol agent. Soil enzyme activities were used to investigate the ecological impact of strain F113 in the rhizosphere of field-grown sugar beet. There were distinct trends in rhizosphere enzyme activities in relation to soil chemistry (studied by electro-ultrafiltration). The activities of enzymes from the phosphorus cycle (acid phosphatase, alkaline phosphatase and phosphodiesterase) and of arylsulphatase were negatively correlated with the amount of readily available P, whereas urease activity was positively correlated with the latter. Significant correlations between electro-ultrafiltration nutrient levels and enzyme activity in the rhizosphere were obtained, highlighting the usefulness of enzyme assays to document variations in soil nutrient cycling. Contrary to previous microcosm studies, which did not investigate plants grown to maturity, the biocontrol inoculant had no effect on enzyme activity or on soil chemistry in the rhizosphere. The results show the importance of homogenous soil microcosm systems, used in previous work, in risk assessment studies, where inherent soil variability is minimised, and where an effect of the pseudomonad on soil enzymology could be detected.Peer reviewe

Predominance of Nonculturable Cells of the Biocontrol Strain Pseudomonas fluorescens CHA0 in the Surface Horizon of Large Outdoor Lysimeters

The persistence of the biocontrol agent Pseudomonas fluorescens CHA0 in the surface horizon of 12 large outdoor lysimeters planted with winter wheat, Phacelia tanacetifolia followed by spring wheat, or maize was monitored for 1 year. Soil was inoculated with a spontaneous rifampin-resistant mutant (CHA0-Rif) of CHA0, and the strain was studied by using colony counts, Kogure's direct viable counts, and total counts (immunofluorescence). The number of culturable cells of the inoculant decreased progressively from 8 to 2 log CFU/g of soil or lower. However, culturable cells of CHA0-Rif accounted for less than 1% of the total cells of the inoculant 8 months after release in autumn. Since viable but nonculturable cells represented less than a quarter of the latter, most cells of CHA0-Rif in soil were thus inactive-dormant or dead at that time. Nonculturable cells of the inoculant were predominant also in the surface horizon of the lysimeters inoculated in the spring, and a significant fraction of them were viable. Results suggest that the occurrence of nonculturable cells of CHA0-Rif was influenced by climatic factors (water availability and soil temperature) and the abundance of roots in soil. The fact that the inoculant persisted as mixed populations of cells of different physiological states, in which nonculturable cells were predominant, needs to be taken into account when assessing the autecology of wild-type or genetically modified pseudomonads released into the soil ecosystem

Applicability of the 16S-23S rDNA internal spacer for PCR detection of the phytostimulatory PGPR inoculant Azospirillum lipoferum CRT1 in field soil

Aims: To assess the applicability of the 16S-23S rDNA internal spacer regions (ISR) as targets for PCR detection of Azospirillum ssp. and the phytostimulatory plant growth-promoting rhizobacteria seed inoculant Azospirillum lipoferum CRT1 in soil. Methods and Results: Primer sets were designed after sequence analysis of the ISR of A. lipoferum CRT1 and Azospirillum brasilense Sp245. The primers fAZO/rAZO targeting the Azospirillum genus successfully yielded PCR amplicons (400-550 bp) from Azospirillum strains but also from certain non-Azospirillum strains in vitro, therefore they were not appropriate to monitor indigenous Azospirillum soil populations. The primers fCRT1/rCRT1 targeting A. lipoferum CRT1 generated a single 249-bp PCR product but could also amplify other strains from the same species. However, with DNA extracts from the rhizosphere of field-grown maize, both fAZO/rAZO and fCRT1/rCRT1 primer sets could be used to evidence strain CRT1 in inoculated plants by nested PCR, after a first ISR amplification with universal ribosomal primers. In soil, a 7-log dynamic range of detection (102-108 CFU g-1 soil) was obtained. Conclusions: The PCR primers targeting 16S-23S rDNA ISR sequences enabled detection of the inoculant A. lipoferum CRT1 in field soil. Significance and Impact of the Study: Convenient methods to monitor Azospirillum phytostimulators in the soil are lacking. The PCR protocols designed based on ISR sequences will be useful for detection of the crop inoculant A. lipoferum CRT1 under field conditions

Biological control of phytopathogens by phloroglucinol and hydrolytic enzyme producing bacterial inoculants

Envionmental and consumer concerns have focused interest on the development of biological control as an alternative, environmentally-friendly strategy for the protection of agricultural and horticultural crops against phytopathogens. Biological control agents. producing a variety of secondary metabolites and hydrolytic enzymes, have been identified among fungi, actinomyceles and bacteria. Pseudomonas fluorescens FII3 and Stenotrophomonas maltophilia W81 inhibit growth of the phytopathogenic fungus Pythium ultimum in vitro and are capable of protecting sugar beet against the effects of damping-off under soil conditions. Transposon mutagenesis of strains FIl3 and W81 has demonstrated that the biocontrol abilities of these strains are mediated by 2,4-diacetylphloroglucinol (pm...) or lytic enzyme production, respectively. Globodera roslochiensis is a phytopathogenic cyst neml1.tode of major agronomic importance. Purified PHL, lytic enzymes, and chitinolytic or phloroglucinol-producing bacterial inoculants negatively influence hatch of G. rostochiensis eggs and decrease subsequent viability of juvenile cyst nematodes in vitro. Similar results were obtained under soil conditions