A hydrogen-sensing system in transcriptional regulation of hydrogenase gene expression in Alcaligenes species.

Heterologous complementation studies using Alcaligenes eutrophus H16 as a recipient identified a hydrogenase-specific regulatory DNA region on megaplasmid pHG21-a of the related species Alcaligenes hydrogenophilus. Nucleotide sequence analysis revealed four open reading frames on the subcloned DNA, designated hoxA, hoxB, hoxC, and hoxJ. The product of hoxA is homologous to a transcriptional activator of the family of two-component regulatory systems present in a number of H2-oxidizing bacteria. hoxB and hoxC predict polypeptides of 34.5 and 52.5 kDa, respectively, which resemble the small and the large subunits of [NiFe] hydrogenases and correlate with putative regulatory proteins of Bradyrhizobium japonicum (HupU and HupV) and Rhodobacter capsulatus (HupU). hoxJ encodes a protein with typical consensus motifs of histidine protein kinases. Introduction of the complete set of genes on a broad-host-range plasmid into A. eutrophus H16 caused severe repression of soluble and membrane-bound hydrogenase (SH and MBH, respectively) synthesis in the absence of H2. This repression was released by truncation of hoxJ. H2-dependent hydrogenase gene transcription is a typical feature of A. hydrogenophilus and differs from the energy and carbon source-responding, H2-independent mode of control characteristic of A. eutrophus H16. Disruption of the A. hydrogenophilus hoxJ gene by an in-frame deletion on megaplasmid pHG21-a led to conversion of the regulatory phenotype: SH and MBH of the mutant were expressed in the absence of H2 in response to the availability of the carbon and energy source. RNA dot blot analysis showed that HoxJ functions on the transcriptional level. These results suggest that the putative histidine protein kinase HoxJ is involved in sensing molecular hydrogen, possibly in conjunction with the hydrogenase-like polypeptides HoxB and HoxC.

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 in the facultatively lithoautotrophic bacterium Alcaligenes eutrophus is dependent on the transcriptional activator HoxA and the alternative sigma factor sigma 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 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 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 degrees C because of a mutation in the regulatory gene 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 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.

Cloning and nucleotide sequences of the genes for the subunits of NAD-reducing hydrogenase of Alcaligenes eutrophus H16.

The genes hoxF, -U, -Y, and -H which encode the four subunit polypeptides alpha, gamma, delta, and beta of the NAD-reducing hydrogenase (HoxS) of Alcaligenes eutrophus H16, were cloned, expressed in Pseudomonas facilis, and sequenced. On the basis of the nucleotide sequence, the predicted amino acid sequences, and the N-terminal amino acid sequences, it was concluded that the structural genes are tightly linked and presumably organized as an operon, denoted hoxS. Two pairs of -24 and -12 consensus sequences resembling RpoN-activatable promoters lie upstream of hoxF, the first of the four genes. Primer extension experiments indicate that the second promoter is responsible for hoxS transcription. hoxF and hoxU code for the flavin-containing dimer (alpha and gamma subunits) of HoxS which exhibits NADH:oxidoreductase activity. A putative flavin-binding region is discussed. The 26.0-kilodalton (kDa) gamma subunit contains two cysteine clusters which may participate in the coordination of two [4F3-4S]centers. The genes hoxY and hoxH code for the small 22.9-kDa delta subunit and the nickel-containing 54.8-kDa beta subunit, respectively, of the hydrogenase dimer of HoxS. The latter dimer exhibits several conserved regions found in all nickel-containing hydrogenases. The roles of these regions in coordinating iron and nickel are discussed. Although the deduced amino acid sequences of the delta and beta subunits share some conserved regions with the corresponding polypeptides of other [NiFe] hydrogenases, the overall amino acid homology is marginal. Nevertheless, significant sequence homology (35%) to the corresponding polypeptides of the soluble methylviologen-reducing hydrogenase of Methanobacterium thermoautotrophicum was found. Unlike the small subunits of the membrane-bound and soluble periplasmic hydrogenases, the HoxS protein does not appear to be synthesized with an N-terminal leader peptide.

DNA methyltransferase genes of Bacillus subtilis phages: comparison of their nucleotide sequences.

The phi 3T DNA methyltransferase (Mtase) and most of the SP beta Mtase genes have been sequenced. With the exception of their promoters, no difference was found between the phi 3T and SP beta Mtase genes which code for an enzyme with a Mr of 50 507, consisting of 443 amino acids (aa). Comparison of the deduced aa sequence of the phi 3T/SP beta type Mtase (target specificity: GGCC and GCNGC) with that of the previously established sequence of the SPR Mtase (Buhk et al., 1984) which has the target specificity GGCC and CCGG, reveals strong similarities between these two types of enzymes. There is, however, one striking difference: both the phi 3T/SP beta and the SPR enzymes contain at different positions inserts of 33 aa, which have no homology to each other. We suggest that the methylation specificity unique to each of the two types of Mtases (GCNGC in phi 3T/SP beta; CCGG in SPR) depends on these inserts, while the GGCC-specific modification potential common to all Mtases is determined by structures conserved in both types of enzymes. A DNA fragment of non-modifying phage Z, which shows homology to both flanks of the SPR Mtase gene, was also sequenced. This segment can be described as a derivative of SPR DNA, in which the Mtase gene and sequences at its 5' end have been deleted, with the deletion extending between two direct repeats of 25 bp.