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Infections with extraintestinal avian pathogenic Escherichia coli (APEC) cause significant economic losses in the poultry industry worldwide. In a previous study we applied signature-tagged transposon mutagenesis and identified 28 virulence-associated genes in APEC strain IMT5155 (O2 : H5 : K1). One of them, yjjQ, encodes a putative transcriptional regulator whose function and role in pathogenesis are still unknown. In the present study, this mutant has been characterized. The yjjQ-defective mutant of IMT5155 (M18E10) was out-competed by the wild-type strain in vivo, and infection of chickens with this yjjQ mutant led to strongly reduced bacterial loads in several organs. Expression studies showed that transcription of yjjQ was significantly upregulated in M9 minimal medium. Correspondingly, the yjjQ mutant showed significantly reduced growth in M9 medium. Although the mutation could not be complemented, a yjjQ deletion mutant showed phenotypes similar to the transposon-generated mutant M18E10, whereas deletion and overexpression of the downstream gene bglJ did not cause a growth defect in M9. To identify virulence genes regulated by YjjQ, one- and two-dimensional protein gel electrophoresis was performed. The proteomic analysis revealed that in the yjjQ mutant M18E10 the expression of several genes involved in iron uptake was downregulated and some other genes were upregulated. The regulation of genes involved in iron uptake was shown to occur at the transcription level using real-time RT-PCR. Taking the results together, this functional analysis strongly suggests that YjjQ is a regulator involved in virulence of APEC by affecting iron uptake.