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

Abstract

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 twocomponent regulatory systems present in a number of H 2 -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 H 2 . This repression was released by truncation of hoxJ. H 2 -dependent hydrogenase gene transcription is a typical feature of A. hydrogenophilus and differs from the energy and carbon source-responding, H 2 -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 H 2 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.Alcaligenes hydrogenophilus is an aerobic, facultatively lithoautotrophic proteobacterium capable of obtaining energy from the oxidation of molecular hydrogen (5). Hydrogen oxidation in this strain is catalyzed by two hydrogenases (18). The soluble hydrogenase (SH) is closely related to the NAD-reducing hydrogenases present in Alcaligenes eutrophus, in the gram-positive Rhodococcus sp. strain 1b (5), and in the cyanobacterium Anabaena variabilis (42). The second hydrogenase of A. hydrogenophilus is a membrane-bound hydrogenase (MBH) coupled to the respiratory chain and involved in electron transportdependent phosphorylation (18). The large and the small subunits of this enzyme show 75 and 90% identity, respectively, to the corresponding polypeptides of the A. eutrophus MBH (28,53). Moreover, in both Alcaligenes species H 2 -oxidizing ability is genetically linked to a megaplasmid (18) and dependent on the function of an RpoN-like sigma factor of RNA polymerase which is encoded on the chromosome (32, 38).Despite the high degree of similarity, hydrogenase regulation in A. hydrogenophilus appears to be quite different from the mode of control observed in A. eutrophus. Whi...

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

“…The regulatory region of the megaplasmid-borne hydrogenase gene complex in R. eutropha has previously been extended by three additional open reading frames (ORFs), designated hoxB, hoxC, and hoxJ (25,26). The ORFs fill a gap between hoxA, the response regulator gene, and hoxN, the nickel permease gene.…”

“…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). Recognition of molecular hydrogen by cells of R. eutropha is mediated by a complex signal transduction system consisting of the proteins HoxB and HoxC which share features of [NiFe] hydrogenases, and HoxJ, a histidine protein kinase which has autophosphorylation capacity with ATP as the phosphoryl donor (25,26). Deletions in hoxB or hoxC of R. eutropha prevent hydrogenase from being synthesized, whereas a knockout of hoxJ leads to H 2 -independent high-level hydrogenase gene expression.…”

Molecular Biology of Microbial Hydrogenases

“…Elsen et al, 1996. Black et al, 1994. Lenz et al, 1997 Histidine kinase HupT HupT HoxJ R. capsulatus B. japonicum R. eutropha Elsen et al, 1993Elsen et al, , 1997Elsen et al, , 2003Dischert et al 1999.…”

“…Lenz et al, 1997 Histidine kinase HupT HupT HoxJ R. capsulatus B. japonicum R. eutropha Elsen et al, 1993Elsen et al, , 1997Elsen et al, , 2003Dischert et al 1999. Van Soom et al, 1999. Lenz et al, 1997Lenz and Friedrich, 1998. Response regulator HupR HoxA HoxA R. capsulatus B. japonicum R. eutropha Richaud et al, 1991;Toussaint et al, 1997;Dischert et al, 1999.…”

Fotobiohidrogén termelésében szerepet játszó enzimek bioszintézise és metabolikus kontextusa

“…A hidrogenázok egy családja azonban hidrogénszenzorként működik és a hidrogenázok expressziójának szabályozásában van szerepe. Ilyen hidrogén szenzor enzimeket találunk például a Bradyrhizobium japonicum-ban (Black 1994), a Rhodobacter capsulatus-ban (Elsen 1996), a Ralstonia eutropha-ban (Lenz 1997), a Rhodopseudomonas palustris-ban (Rey 2006) és a Thiocapsa roseopersicina-ban (Kovács et al, 2005a (Vignais 2001, Vignais 2007. Mindegyik csoportra jellemző, hogy rendelkezik egy, a csoporton belül konzervált alegységgel, mely az enzim működéséhez nélkülözhetetlen és tartalmazza az aktív centrumot.…”