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In vitro characterization of constitutive CysB proteins from Salmonella typhimurium
Author(s) -
Colyer Tracey E.,
Kredich Nicholas M.
Publication year - 1996
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.1365-2958.1996.6301347.x
Subject(s) - biology , inducer , promoter , serine , mutant , biochemistry , repressor , transcription (linguistics) , cysteine , regulon , microbiology and biotechnology , escherichia coli , in vitro , transcription factor , gene expression , gene , phosphorylation , enzyme , linguistics , philosophy
Expression of the cysteine regulon in Salmonella typhimurium and Escherichia coli is controlled by the LysR‐type transcriptional activator CysB and by the inducer N ‐acetyl‐ l ‐serine. Sulphide and thiosulphate are anti‐inducers. Two highly purified constitutive CysB proteins, CysB(T149M) and CysB(T149P), were found to bind to the cysJIH cysK and cysP promoters, to activate transcription from the cysJIH and cysK promoters in the absence of N ‐acetyl‐ l ‐serine, and to be insensitive to the effects of anti‐inducers. At 10mM MgCl 2 , the in vitro transcription activity of CysB(T149M) was maximal without N ‐acetyl‐ l ‐serine, but that of CysB(T149P) was increased by inducer. At 2mM MgCl 2 , both proteins were fully active without inducer. A third mutant protein, CysB(W166R), was totally inactive at 10mM MgCl 2 , but gave constitutive expression of the cysK and cysJIH promoters at 2 mM MgCl 2 . Surprisingly, wild‐type CysB was also constitutive for the cysK promoter at 2mM MgCl 2 but not at 10mM MgCl 2 ; it required inducer for cysJIH promoter activation at both concentrations. Mutagenic studies indicated that this difference between promoters is due to the distance between activation site half‐sites, which are separated by 1 bp in the cysJIH promoter and by 2 bp in the cysK promoter. We speculate that inducer acts to decrease the distance between the binding domains of two CysB subunits that interact with an activation site. In vitro activities of wild‐type and mutant CysB proteins correlated much better with in vivo behaviour at 2mM than at 10mM MgCl 2 , suggesting that the former is the more physiological concentration.