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The synthesis of Rhodobacter capsulatus HupSL hydrogenase is regulated by the two‐component HupT/HupR system
Author(s) -
Dischert Wanda,
Vignais Paulette M.,
Colbeau Annette
Publication year - 1999
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1999.01660.x
Subject(s) - rhodobacter , biology , response regulator , enhancer , two component regulatory system , biochemistry , transcription factor , rpon , transcription (linguistics) , sigma factor , histidine kinase , binding site , microbiology and biotechnology , rna polymerase , mutant , promoter , gene , escherichia coli , gene expression , linguistics , philosophy
The synthesis of the membrane‐bound [NiFe]hydrogenase of Rhodobacter capsulatus (HupSL) is regulated negatively by the protein histidine kinase, HupT, and positively by the response regulator, HupR. It is demonstrated in this work that HupT and HupR are partners in a two‐component signal transduction system. The binding of HupR protein to the hupS promoter regulatory region ( phupS ) was studied using gel retardation and footprinting assays. HupR protected a 50 bp region localized upstream from the binding site of the histone‐like integration host factor (IHF) regulator. HupR, which belongs to the NtrC subfamily, binds to an enhancer site (TTG‐N 5 ‐CAA) localized at −162/−152 nt. However, the enhancer‐binding HupR protein does not require the RpoN sigma factor for transcriptional activation, as is the case for NtrC from enteric bacteria, but functions with σ 70 ‐RNA polymerase, as is the case for R. capsulatus NtrC. Besides, unlike NtrC from Escherichia coli , HupR activates transcription in the unphosphorylated form and becomes inactive by phosphorylation. This was demonstrated by replacing the putative phosphorylation site (D 54 ) of the HupR protein with various amino acids or by deleting it using site‐directed mutagenesis. Strains expressing mutated hupR genes showed high hydrogenase activities even in the absence of H 2 , indicating that hupSL transcription is activated by the binding of unphosphorylated HupR protein. Strains producing mutated HupRD 54 proteins were derepressed for hupSL expression as were HupT − mutants. It is shown that the phosphorylated form of HupT was able to transfer phosphate to wild‐type HupR protein but not to mutated D 54 HupR proteins. Thus, it is concluded that HupT and HupR are the partners of a two‐component regulatory system that regulates hupSL gene transcription.