H-053. Characterization of Fur in Nitrosomonas europaea

N. Vajrala, X. Wei, L. A. Sayavedra-Soto, D. J. Arp;
Oregon State Univ., Corvallis, OR.

Nitrosomonas europaea is an ammonia-oxidizing beta-proteobacterium with high requirements for Fe. Bioinformatics suggests that this bacterium has ~90 genes dedicated to Fe acquisition. A ferric uptake regulator (fur gene, NE0616), homologous to E. coli fur, was identified in the N.europaea genome. A consensus nucleotide sequence of the Fur binding motifs (Fur boxes) in N.europaea was deduced using HMMER package version 2.3.2. The deduced consensus sequence for the Fur box sites in the genome of N.europaea was shown to be functional by the Fur Titration Assay (FURTA). Positive FURTA results were obtained in Fur boxes of 2 outer membrane receptors (Genes NE1205, NE1540) and of gene NE0616, showing that the Fur box nucleotide sequence consensus is valid. We tested further functionality of the Fur box in gene NE0616, and concomitantly, whether gene NE0616 indeed regulates other Fe acquisition genes. We inserted a Kanamycin cassette in the Fur box upstream of gene NE0616 to produce the mutant strain N. europaea furbox::kan. The mutant did not transcribe NE0616 gene and over-expressed proteins with molecular weights similar to Fe-siderophore outer membrane receptors. This negative regulation of expression of Fe acquisition systems by Fur in response to iron availability is similar to that documented for other species such as E. coli and P. aeruginosa. Analysis of the cellular Fe contents by ICP-AES showed that N. europaea furbox::Kan mutant grown in Fe-replete (10 µM) conditions had 1.5 fold more Fe than wild-type grown in similar conditions. Consistently N. europaea furbox::Kan mutant was more sensitive than the wild-type to increasing concentrations of Fe in the growth media. Growth was inhibited likely to Fe toxicity and consequent oxidative damage to the cells. Together these suggest that NE0616 plays a key role in regulation of iron-repressed N. europaea genes and likely is fur in N. europaea.