Identification of Zur boxes and determination of their roles in the differential regulation of the Zur regulon in Agrobacterium tumefaciens C58.

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.

Regulation and function of the flavonoid-inducible efflux system, emrR-emrAB, in Agrobacterium tumefaciens C58.

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.

The Agrobacterium tumefaciens atu3184 gene, a member of the COG0523 family of GTPases, is regulated by the transcriptional repressor Zur.

Analysis of the Agrobacterium tumefaciens C58 genome revealed a potential Zur (zinc uptake regulator) binding site (5'-GATATGTTATTACATTAC-3', the underlined letters are the center of symmetry of the inverted palindrome) located in the upstream region of atu3184, whose gene product is a member of the COG0523 subfamily of G3E GTPases. The specific interaction of the Zur protein with the 18-bp inverted repeat operator motif in the presence of zinc was demonstrated in vitro by a DNA band shift assay and a DNase I footprinting assay. A LacZ reporter fusion assay further confirmed that Zur negatively regulates atu3184 promoter activity in vivo. The expression of atu3184 was upregulated in response to zinc limitation in the wild-type strain, but the zur mutant strain exhibited high-level constitutive expression of atu3184 under all conditions, irrespective of the zinc levels. It is likely that A. tumefaciens Zur senses zinc and directly regulates the atu3184 promoter by a molecular mechanism similar to that of Escherichia coli Zur, where the operator DNA is surrounded by four Zur monomers forming two dimers bound on the opposite sides of the DNA duplex. Disruption of atu3184 did not affect cell growth under metal-limited conditions and had no effect on the total cellular zinc content. Furthermore, an A. tumefaciens strain lacking atu3184 caused a tumor disease in a host plant.

Characterization and regulation of AcrABR, a RND-type multidrug efflux system, in Agrobacterium tumefaciens C58.

Agrobacterium tumefaciens AcrR is the transcriptional repressor of the acrABR operon. The AcrAB efflux pump confers resistance to various toxic compounds, including antibiotics [ciprofloxacin (CIP), nalidixic acid (NAL), novobiocin (NOV) and tetracycline (TET)], a detergent [sodium dodecyl sulfate (SDS)] and a biocide [triclosan (TRI)]. The sequence to which AcrR specifically binds in the acrA promoter region was determined by EMSA and DNase I footprinting. The AcrR-DNA interaction was abolished by adding NAL, SDS and TRI. Quantitative real time-PCR analysis showed that induction of the acrA transcript occurred when wild-type cells were exposed to NAL, SDS and TRI. Indole is a signaling molecule that increases the antibiotic resistance of bacteria, at least in part, through activation of efflux pumps. Expression of the A. tumefaciens acrA transcript was also inducible by indole in a dose-dependent manner. Indole induced protection against CIP, NAL and SDS but enhanced susceptibility to NOV and TRI. Additionally, the TET resistance of A. tumefaciens was not apparently modulated by indole. A. tumefaciens AcrAB played a dominant role and was required for tolerance to high levels of the toxic compounds. Understanding the regulation of multidrug efflux pumps and bacterial adaptive responses to intracellular and extracellular signaling molecules for antibiotic resistance is essential. This information will be useful for the rational design of effective treatments for bacterial infection to overcome possible multidrug-resistant pathogens.