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Identification of an Interaction Between a Histidine Kinase Response Regulator Hybrid Protein and 2-keto-3-deoxy-D-manno-octulosonate-8- Phosphate Synthase in Gluconobacter oxydans

[ Vol. 24 , Issue. 11 ]

Author(s):

Cuijuan Yuan, Yangchen Lin, Yushu Ma* and Dongzhi Wei   Pages 990 - 995 ( 6 )

Abstract:


Background: Two-component signaling systems are wildly distributed in most bacteria, which allow bacteria to sense and respond to the stimuli for survival in fluctuating environmental conditions. Histidine kinase response regulator hybrid protein (HK) from G. oxydans, a polypeptide with a response regulator attached to the histidine protein kinase, was identified as a member of two-component signaling system in the bacterium. However, the role of the protein in the bacterium remained unknown.

Objectives: The main objective of this study was to screen and identify the proteins in G. oxydans, potentially interactive with HK, to reveal the roles played by HK in the bacterium.

Methods: Potential HK interactive proteins were screened by yeast two-hybrid assay. Among those candidate proteins, a specific interaction between HK and a 2-keto-3-deoxy-D-manno-octulosonate- 8-phosphate synthase (KDO8PS) was established by GST pull-down assay in vitro and BiFC assay in vivo.

Results: 60 positive clones, representing 27 different prey proteins in G. oxydans, were screened by yeast two-hybrid assay with HK as the bait. Among these interactive proteins, KDO8PS can catalyze the condensation reaction between phosphoenolpyruvate (PEP) and arabinose 5-phosphate (A5P) to produce a precursor essential for LPS biosynthesis in Gram-negative bacteria. After expression and purification, the interaction between HK and KDO8PS was further verified by GST pull-down assay. Subsequent BiFC assay revealed the interaction between these two proteins in vivo.

Conclusion: Our results revealed an interaction between a two-component system protein HK with KDO8PS in G. oxydans. As KDO8PS plays a vital role in lipopolysaccharide biosynthesis required for growth in Gram-negative bacteria, and its activity strongly depends on several conserved histidine residues, the interaction described here implied that HK may play certain roles in bacterial growth under stress.

Keywords:

Gluconobacter oxydans, two-component system, 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate synthase, yeast two-hybrid assay, GST pull-down, bimolecular fluorescence complementation.

Affiliation:

State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237

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