Negative regulation of ectoine uptake and catabolism in Sinorhizobium meliloti

Characterization of the EhuR gene

Qinli Yu, Hanlin Cai, Yanfeng Zhang, Yongzhi He, Lincai Chen, Justin Merritt, Shan Zhang, Zhiyang Dong

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Ectoine has osmoprotective effects on Sinorhizobium meliloti that differ from its effects in other bacteria. Ectoine does not accumulate in S. meliloti cells; instead, it is degraded. The products of the ehuABCD-eutABCDE operon were previously discovered to be responsible for the uptake and catabolism of ectoine in S. meliloti. However, the mechanism by which ectoine is involved in the regulation of the ehuABCD-eutABCDE operon remains unclear. The ehuR gene, which is upstream of and oriented in the same direction as the ehuABCD-eutABCDE operon, encodes a member of the MocR/GntR family of transcriptional regulators. Quantitative reverse transcription-PCR and promoter-lacZ reporter fusion experiments revealed that EhuR represses transcription of the ehuABCD-eutABCDE operon, but this repression is inhibited in the presence of ectoine. Electrophoretic mobility shift assays and DNase I footprinting assays revealed that EhuR bound specifically to the DNA regions overlapping the -35 region of the ehuA promoter and the +1 region of the ehuR promoter. Surface plasmon resonance assays further demonstrated direct interactions between EhuR and the two promoters, although EhuR was found to have higher affinity for the ehuA promoter than for the ehuR promoter. In vitro, DNA binding by EhuR could be directly inhibited by a degradation product of ectoine. Our work demonstrates that EhuR is an important negative transcriptional regulator involved in the regulation of ectoine uptake and catabolism and is likely regulated by one or more end products of ectoine catabolism.

Original languageEnglish (US)
Article numbere00119-16
JournalJournal of Bacteriology
Volume199
Issue number1
DOIs
StatePublished - 2017

Fingerprint

Sinorhizobium meliloti
Operon
Genes
Surface Plasmon Resonance
ectoine
Deoxyribonuclease I
DNA
Electrophoretic Mobility Shift Assay
Genetic Promoter Regions
Reverse Transcription
Bacteria
Polymerase Chain Reaction

Keywords

  • Ectoine
  • EhuR
  • MocR
  • Sinorhizobium meliloti
  • Transcriptional regulator

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Negative regulation of ectoine uptake and catabolism in Sinorhizobium meliloti : Characterization of the EhuR gene. / Yu, Qinli; Cai, Hanlin; Zhang, Yanfeng; He, Yongzhi; Chen, Lincai; Merritt, Justin; Zhang, Shan; Dong, Zhiyang.

In: Journal of Bacteriology, Vol. 199, No. 1, e00119-16, 2017.

Research output: Contribution to journalArticle

Yu, Qinli ; Cai, Hanlin ; Zhang, Yanfeng ; He, Yongzhi ; Chen, Lincai ; Merritt, Justin ; Zhang, Shan ; Dong, Zhiyang. / Negative regulation of ectoine uptake and catabolism in Sinorhizobium meliloti : Characterization of the EhuR gene. In: Journal of Bacteriology. 2017 ; Vol. 199, No. 1.
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abstract = "Ectoine has osmoprotective effects on Sinorhizobium meliloti that differ from its effects in other bacteria. Ectoine does not accumulate in S. meliloti cells; instead, it is degraded. The products of the ehuABCD-eutABCDE operon were previously discovered to be responsible for the uptake and catabolism of ectoine in S. meliloti. However, the mechanism by which ectoine is involved in the regulation of the ehuABCD-eutABCDE operon remains unclear. The ehuR gene, which is upstream of and oriented in the same direction as the ehuABCD-eutABCDE operon, encodes a member of the MocR/GntR family of transcriptional regulators. Quantitative reverse transcription-PCR and promoter-lacZ reporter fusion experiments revealed that EhuR represses transcription of the ehuABCD-eutABCDE operon, but this repression is inhibited in the presence of ectoine. Electrophoretic mobility shift assays and DNase I footprinting assays revealed that EhuR bound specifically to the DNA regions overlapping the -35 region of the ehuA promoter and the +1 region of the ehuR promoter. Surface plasmon resonance assays further demonstrated direct interactions between EhuR and the two promoters, although EhuR was found to have higher affinity for the ehuA promoter than for the ehuR promoter. In vitro, DNA binding by EhuR could be directly inhibited by a degradation product of ectoine. Our work demonstrates that EhuR is an important negative transcriptional regulator involved in the regulation of ectoine uptake and catabolism and is likely regulated by one or more end products of ectoine catabolism.",
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