Characterisation of Two Novel COG0523-Family Members of the Agrobacterium tumefaciens Zur Regulon

Objective: In Agrobacterium tumefaciens, zinc allocation occurs through zinc chaperones, which are regulated by the Zur (Zinc uptake regulator) repressor. The putative COG0523-family chaperones atu4502 and atu3633 contain consensus Zur-binding sequences upstream of their start codons. The purpose of this study was to investigate their regulation and possible functions.Materials and Methods: Quantitative real-time PCR (qRT-PCR), translational promoter-fusion assays and DNase I footprinting were employed to determine the regulation. All phenotypic assays were carried out in AB minimal media.Results: qRT-PCR analyses revealed that atu4502 and atu3633, encoding novel members of the COG0523-family, are negatively regulated by Zur. DNase I footprinting indicated that Zur binds to consensus Zur box sequences overlapping −35 and −10 promoter sequences upstream of each gene. Translational promoter-fusion assays indicated that Zur boxes are necessary for transcriptional repression. Phenotypic assays showed that an atu4502 inactivation mutant is sensitive to the cell impermeable metal chelator, EDTA, whereas an atu3633 inactivation mutant is sensitive to the cell permeable metal chelator, TPEN, suggesting that the gene products may occupy different subcellular locations. Zinc supplementation was able to rescue both the EDTA sensitivity of atu4502::KM and the TPEN sensitivity of atu3633::GM, consistent with their proposed role as Zinc chaperones. Biofilm and EPS assays showed that in atu4502 and atu3633 inactivation mutants, biofilm and EPS levels were increased relative to wild-type.Conclusion: It was concluded that both atu4502 and atu3633 are regulated by Zur, and likely participate in zinc trafficking.