Temperature tolerance of hydrogenase expression in Alcaligenes eutrophus is conferred by a single amino acid exchange in the transcriptional activator HoxA

Abstract

Expression of the soluble (SH) and membrane-bound (MBH) hydrogenases in the facultatively lithoautotrophic bacterium Alcaligenes eutrophus is dependent on the transcriptional activator HoxA and the alternative sigma factor 54 . Deletion analysis revealed that a region 170 bp upstream of the transcriptional start of the SH operon is necessary for high-level promoter activity. Mobility shift assays with DNA fragments containing the SH upstream region and purified ␤-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 for HoxA by heparin-Sepharose chromatography and ammonium sulfate fractionation produced a weak but discrete shift in the mobility of the target DNA. This effect was not observed with comparable extracts prepared from hoxA mutants. A similar experiment using antibodies against HoxA 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 temperatures above 33؇C because of a mutation in the regulatory gene hoxA. In this paper, we show that a single amino acid substitution (Gly-4683Val) in the C-terminal part of HoxA is responsible for temperature tolerance. The SH upstream region also contains sequence motifs resembling the E. coli integration host factor (IHF) binding site, and purified E. coli IHF protein shifted the corresponding indicator fragment.Alcaligenes eutrophus, a member of the ␤ subgroup of the class Proteobacteria, can grow lithoautotrophically with hydrogen as the sole energy source and carbon dioxide as the carbon source (reviewed in reference 3). The bacterium harbors a large plasmid on which the genes required for hydrogen oxidation are located. The hox region encodes two hydrogenases: a cytoplasmic NAD-reducing enzyme (SH) and a membranebound electron transport-coupled protein (MBH). Both hydrogenases belong to the family of nickel-iron-containing proteins. The formation of these metalloproteins is an extremely complex and apparently highly conserved process requiring sets of ancillary functions in addition to the hydrogenase enzymes (reviewed in reference 17). The genes coding for these proteins are part of the hox gene complex in A. eutrophus. Their products are involved in nickel uptake (14), metal processing (9), and hydrogenase maturation (24).Physiological experiments have demonstrated that the synthesis of the two hydrogenases of A. eutrophus is coordinately regulated. Tight repression is observed during growth of the cells on preferentially utilized carbon sources, whereas derepression occurs on poor substrates even in the absence of hydrogen (18). Moreover, hydrogenase synthesis in A. eutrophus is temperature sensitive and abolished above 33ЊC (19). Mutant analysis has shown that the expression ...

HoxA is a transcriptional regulator for expression of the hup structural genes in free‐living Bradyrhizobium japonicum

“…In A. eutrophus, HoxA binds at the promoter region of the genes encoding the soluble hydrogenase (Zimmer et al, 1995). Expression of the genes encoding the soluble hydrogenase is dependent on RpoN, and purified E. coli IHF protein was also shown to bind at the promoter region (Zimmer et al, 1995). Also, in R. capsulatus, IHF is required for expression of the hydrogenase structural genes (Toussaint et al, 1991;1993).…”

“…In this regard, the regulatory cascade that governs hupSL expression in free-living B. japonicum cells resembles hydrogenase regulation in the free-living chemolithotroph Alcaligenes eutrophus and in the photosynthetic bacterium Rhodobacter capsulatus. In A. eutrophus, HoxA binds at the promoter region of the genes encoding the soluble hydrogenase (Zimmer et al, 1995). Expression of the genes encoding the soluble hydrogenase is dependent on RpoN, and purified E. coli IHF protein was also shown to bind at the promoter region (Zimmer et al, 1995).…”

The H 2 Sensor of Ralstonia eutropha Is a Member of the Subclass of Regulatory [NiFe] Hydrogenases

“…H 2 -independent O 2 consumption was monitored in N 2 -saturated potassium phosphate buffer. ␤-Galactosidase assays were performed as described previously (54), and the activities (in units) were calculated according to the Miller method (33) except that cell density was measured at 436 nm. The level of protein in extracts was determined by the method of Lowry et al (28).…”

“…HoxA binds specifically at the upstream regions of the MBH and SH operons and activates transcription in concert with the 54 -containing RNA polymerase (42,54). Transcription activation by HoxA is stimulated by at least two environmental signals: the presence of molecular hydrogen and/or limitation of organic carbon and energy sources (25,42).…”

Expression of Genes Encoding Membrane-bound Hydrogenase inPseudomonas hydrogenovoraunder Autotrophic Condition Is Dependent on Two Different Promoters

“…In A. eutrophus, it has been shown that HoxA, the NtrC-homologous product encoded in the hydrogenase gene cluster, activated the expression of hydrogenase genes under conditions of energy limitation. 16 ) Igarashi et al 17 ) reported that P. hydrogenovora did not fix nitrogen but did reduce nitrate, however, the transcriptional activity of P hup region was not affected by the presence or absence of a nitrogen source (Table). Since the structure of the P. hydrogenovora hydrogenase gene cluster is much more similar to that of A. eutrophus than those of other hydrogenase-containing bacteria, P. hydrogenovora is likely to have a transcriptional regulator operating specifically for an expression of hydrogenase genes.…”