Role of a Fur homolog in iron metabolism in Nitrosomonas europaea

Abstract

BackgroundIn response to environmental iron concentrations, many bacteria coordinately regulate transcription of genes involved in iron acquisition via the ferric uptake regulation (Fur) system. The genome of Nitrosomonas europaea, an ammonia-oxidizing bacterium, carries three genes (NE0616, NE0730 and NE1722) encoding proteins belonging to Fur family.ResultsOf the three N. europaea fur homologs, only the Fur homolog encoded by gene NE0616 complemented the Escherichia coli H1780 fur mutant. A N. europaea fur:kanP mutant strain was created by insertion of kanamycin-resistance cassette in the promoter region of NE0616 fur homolog. The total cellular iron contents of the fur:kanP mutant strain increased by 1.5-fold compared to wild type when grown in Fe-replete media. Relative to the wild type, the fur:kanP mutant exhibited increased sensitivity to iron at or above 500 μM concentrations. Unlike the wild type, the fur:kanP mutant was capable of utilizing iron-bound ferrioxamine without any lag phase and showed over expression of several outer membrane TonB-dependent receptor proteins irrespective of Fe availability.ConclusionsOur studies have clearly indicated a role in Fe regulation by the Fur protein encoded by N. europaea NE0616 gene. Additional studies are required to fully delineate role of this fur homolog.

Global analysis of the Nitrosomonas europaea iron starvation stimulon

“…Nitrosomonas europaea genome encodes 3 fur homologs (Chain et al 2003;Vajrala et al 2011). Of these 3 fur homologs, transcript of the NE0616 fur homolog that encodes all conserved residues of iron-sensing Fur proteins from other organisms did not show any signiWcant changes in our microarrays; however, transcript level of NE0730, the second Fe-sensing fur homolog was 15-fold higher in iron limitation compared to iron-replete conditions ( Table S2).…”

“…The glassware, iron-replete, iron-limited, and irondepleted media used in this study are prepared as described (Wei et al 2006(Wei et al , 2007Vajrala et al 2010Vajrala et al , 2011. Lyophilized hemoglobin from bovine blood was purchased from Sigma.…”

“…The Fe-limited cells have depleted internal iron pools and hence are pale-yellowish in color when compared to reddish-brown-colored cells grown in iron-replete medium. Cellular Fe contents in thoroughly washed N. europaea cells were determined by the ferrozine assay following HNO 3 (5%) digestion of cells at 100°C (Vajrala et al 2011). Nitrosomonas europaea cells from three independent experiments (three iron-replete [300 ml culture at 0.04 OD 600nm ] and three iron-limited cultures [500 ml culture at 0.025 OD 600nm ]) were harvested at mid-exponential phase by centrifugation.…”

Ab Initio Structural Modeling of and Experimental Validation for Chlamydia trachomatis Protein CT296 Reveal Structural Similarity to Fe(II) 2-Oxoglutarate-Dependent Enzymes

“…This may have resulted from their system of comparing ␤-galactosidase activity levels between induced and noninduced conditions. Other groups researching Fur homologs have allowed for constitutive expression of protein and have compared ␤-galactosidase activity levels between irondepleted and iron-supplemented conditions (40,52,54). Utilizing this approach, we were able to assay the repression activities of Fur and CT296 with a very low level of background repression.…”

Cycloheximide prevents thede novopolypeptide synthesis required to recover from acetylene inhibition inNitrosopumilus maritimus

“…The concentrated cells were resuspended in water and lysed with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer. Proteins (~100 lg per lane) were loaded for each sample, and the protein composition of cell membranes was analyzed using SDS-PAGE with 12% (w/v) acrylamide in the resolving gel (Vajrala et al, 2011). Autoradiograms were prepared and analyzed by phosphorimaging and IMAGEQUANT softwares as described by the manufacturer (Molecular Dynamics, Piscataway, NJ).…”