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Zinc uptake regulator (Zur) is a transcriptional regulator that represses zinc acquisition genes under high zinc conditions. The aim of this study was to identify and investigate the role of Zur-binding motifs (Zur boxes) in the differential regulation of Zur target genes, including the zinT, znuA, znuCB-zur operon, the troCBA operon, and yciC, in Agrobacterium tumefaciens. DNase I footprinting and gel shift assays were performed, confirming that Zur directly binds to 18-bp inverted repeat motifs found in the promoter of these Zur-regulated genes. Furthermore, promoter-lacZ fusions and mutagenesis of the identified Zur boxes were performed to assess the role of each Zur box. A Zur box found in the zinT promoter was required for zinc-dependent repression by Zur. The intergenic region between the znuA gene and the znuCB-zur operon contains two Zur boxes, named A and C, which immediately precede the genes znuA and znuC, respectively. Zur box A, but not Zur box C, was essential for the repression of the znuA promoter. Both Zur boxes A and C were implicated in the repression of the znuC promoter, in which mutation of either box alone was sufficient for full derepression of the znuC promoter. Three Zur boxes named T, M, and Y were identified in the intergenic region between the troCBA operon and the yciC gene. Zur box Y, which immediately precedes yciC, was shown to be responsible for Zur repression of the yciC promoter. In contrast, two Zur boxes, T and M, were essential for the complete repression of the troCBA operon, and full derepression of the troC promoter was exhibited when both Zur boxes were mutated simultaneously. Sequence analysis of the identified Zur boxes revealed a correlation between deviation from the core recognition sequence of the Zur box and the requirement of two Zur boxes for Zur regulation of distinctive promoters. [ABSTRACT FROM AUTHOR]

The expression of the Agrobacterium tumefaciens emrAB operon, which encodes a membrane fusion protein and an inner membrane protein, is inducible by various flavonoids, including apigenin, genistein, luteolin, naringenin, and quercetin. Among these flavonoids, quercetin is the best inducer, followed by genistein. The emrR gene is divergently transcribed from the emrAB operon. The EmrR protein, which belongs to the TetR transcriptional regulator family, negatively regulates the expression of emrAB and of itself. Electrophoretic mobility shift assays and DNase I footprinting showed that EmrR binds directly at two EmrR-binding sites in the emrR-emrAB intergenic region and that quercetin inhibits the DNA-binding activity of EmrR. Promoter-lacZ fusion analyses and 5′ rapid amplification of cDNA ends were performed to map the emrR and emrAB promoters. Compared with the wild-type strain, the emrA mutant strain exhibited similar levels of resistance to the tested antibiotics. In contrast, disruption of emrR conferred protection against nalidixic acid and novobiocin, but it rendered A. tumefaciens sensitive to tetracycline and erythromycin. The emrR mutation also destabilized the outer membrane of A. tumefaciens, resulting in increased sensitivity to SDS and low pH. These findings demonstrate that proper regulation of emrR-emrAB is required for free-living A. tumefaciens to survive in deleterious environments in which toxic compounds are present. Nonetheless, A. tumefaciens strains that lack emrR or emrA still have the ability to cause tumors when infecting Nicotiana benthamiana plants. [ABSTRACT FROM AUTHOR]