TEMPERATURE TOLERANCE OF HYDROGENASE EXPRESSION IN ALCALIGENES-EUTROPHUS IS CONFERRED BY A SINGLE AMINO-ACID EXCHANGE IN THE TRANSCRIPTIONAL ACTIVATOR HOXA

Expression of the soluble (SH) and membrane-bound (MBH) hydrogenases i n the facultatively lithoautotrophic bacterium Alcaligenes eutrophus i s dependent on the transcriptional activator HoxA and the alternative sigma factor sigma(54). Deletion analysis revealed that a region 170 b p upstream of the transcriptional start of the SH operon is necessary for high-level promoter activity. Mobility shift assays with DNA fragm ents containing the SH upstream region and purified beta-galactosidase -HoxA fusion protein isolated from Escherichia coli or authentic HoxA isolated by immunoaffinity chromatography from A. eutrophus failed to detect specific binding. In contrast, A. eutrophus extracts enriched f or HoxA by heparin-Sepharose chromatography and ammonium sulfate fract ionation produced a weak but discrete shift in the mobility of the tar get DNA. This effect was not observed with comparable extracts prepare d from hoxA mutants. A similar experiment using antibodies against Hox A confirmed that HoxA was responsible for the observed mobility shift. Extracts prepared from a temperature-tolerant mutant of A. eutrophus gave a stronger retardation than did those from the wild type. Unlike the wild type, the hox(Tr) mutant is able to grow with hydrogen at tem peratures above 33 degrees C because of a mutation in the regulatory g ene hoxA, In this paper, we show that a single amino acid substitution (Gly-468-->Val) in the C-terminal part of HoxA is responsible for tem perature tolerance. The SH upstream region also contains sequence moti fs resembling the E. coli integration host factor (IHF) binding site, and purified E. coli IHF protein shifted the corresponding indicator f ragment.