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

  • Puttamas Nuonming
  • Sasimaporn Khemthong
  • Rojana SukchawalitEmail author
  • Skorn Mongkolsuk
Applied genetics and molecular biotechnology
  • 69 Downloads

Abstract

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.

Keywords

Zur-DNA interaction Mutagenesis of Zur boxes 

Notes

Funding information

This study was funded by the Chulabhorn Research Institute and the Thailand Research Fund (RSA5880010).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2020_10346_MOESM1_ESM.pdf (92 kb)
ESM1 (PDF 92 kb).

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Authors and Affiliations

  • Puttamas Nuonming
    • 1
  • Sasimaporn Khemthong
    • 1
  • Rojana Sukchawalit
    • 1
    • 2
    • 3
    Email author
  • Skorn Mongkolsuk
    • 1
    • 3
  1. 1.Laboratory of BiotechnologyChulabhorn Research InstituteBangkokThailand
  2. 2.Applied Biological SciencesChulabhorn Graduate InstituteBangkokThailand
  3. 3.Center of Excellence on Environmental Health and Toxicology (EHT)Ministry of EducationBangkokThailand

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