Genetics of hydrogenase from aerobic lithoautotrophic bacteria

Friedrich, Bärbel; Kortlüke, C; Hogrefe, Christine; Eberz, Günther; Silber, B.; Warrelmann, Jürgen

doi:10.1016/s0300-9084(86)81078-1

Cited by 25 publications

(6 citation statements)

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“…The key enzymes for autotrophic growth (hydrogen activation) are the NAD ϩ -reducing soluble hydrogenase (SH) and the membrane-bound hydrogenase (MBH) that feeds electrons to the respiratory chain (26,28). Genetic analyses (4,8,14,18,19) have revealed that the gene locus for MBH (hoxP) located on megaplasmid pHG1 is about 100 kb long and contains at least two structural genes and at least eight accessory genes. The gene hoxG codes for the large subunit, and hoxK codes for the small subunit (SSU) of the enzyme.…”

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Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm

et al. 1995

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Electron microscopic immunogold labeling experiments were performed with ultrathin sections of plasmolyzed cells of Alcaligenes eutrophus and ''whole-mount'' samples of spheroplasts and protoplasts. They demonstrated that antigenic determinants of the membrane-bound hydrogenase are exposed, at the outside of the cytoplasmic membrane, to the periplasm.Alcaligenes eutrophus is a hydrogen-oxidizing, obligate aerobic, facultatively chemolithoautotrophic eubacterium. The key enzymes for autotrophic growth (hydrogen activation) are the NAD ϩ -reducing soluble hydrogenase (SH) and the membrane-bound hydrogenase (MBH) that feeds electrons to the respiratory chain (26,28). Genetic analyses (4,8,14,18,19) have revealed that the gene locus for MBH (hoxP) located on megaplasmid pHG1 is about 100 kb long and contains at least two structural genes and at least eight accessory genes. The gene hoxG codes for the large subunit, and hoxK codes for the small subunit (SSU) of the enzyme. The gene product HoxK exhibits a leader peptide sequence 43 amino acids long. Both enzyme subunits undergo maturation (17) to be rendered catalytically active. When A. eutrophus is grown heterotrophically, SH and MBH may be more or less completely derepressed, depending on the carbon source (9, 10).The macromolecular organization of SH (15) and MBH (12) was examined by electron microscopy. In immunocytochemical localization studies, SH was found in the cytoplasm (23). For the MBH (11), a preferentially peripheral location of the enzyme was observed when cells were harvested in the exponential phase of growth. The enzyme-specific label was distributed along the cell periphery and over the cytoplasm in cells harvested in the stationary phase of growth.The gene product HoxM seems to be important for membrane association and activity of MBH (19). Sequence data (19) and biochemical findings (2,6,11,17) indicate that at least part of the active MBH complex may be exposed toward the periplasm. However, this notion has not been verified. Therefore, in the present investigation an approach suitable for this kind of analysis, immunoelectron microscopy performed on plasmolyzed cells, spheroplasts, and protoplasts, was used to further support this assumption. A polyhydroxybutyrate-negative mutant, A. eutrophus H16 PHB Ϫ 4 (DSM 541), was chosen to simplify electron microscopic examination. The cells were grown either autotrophically as previously described (27) on 80% H 2 -10% O 2 -10% CO 2 or heterotrophically (7, 11) on mineral medium containing fructose. Cells were harvested for electron microscopy during the exponential phase of growth by centrifugation. They were washed twice with phosphate-buffered saline (PBS) prior to further treatment.For plasmolysis, 0.5 g (wet weight) of cells was resuspended in 1.0 M potassium phosphate buffer, pH 7.0, and incubated for 30 min at room temperature with continuous stirring. For preparation of spheroplasts and protoplasts, the cells were washed twice in 10 mM Tris-HCl buffer, pH 8.0. One gram (wet weight) of cells was ...

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Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm

et al. 1995

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“…H2-dependent hydrogenase expression has been observed in other bacteria as well. In the hydrogen bacteria Alcaligenes hydrogenophilus (11), Paracoccus denitrificans (31), and Alcaligenes latus (9) and in the methanotroph Methylosinus trichosporium (7), incubation of cultures with high H2 levels resulted in high hydrogenase activities. Also, enhancement of hydrogenase activities in Azotobacter vinelandii has been observed by incubation of the cells with H2 (29).…”

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Common cis-acting region responsible for transcriptional regulation of Bradyrhizobium japonicum hydrogenase by nickel, oxygen, and hydrogen

Kim

Maier

1991

J Bacteriol

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Bradyrhizobiumjaponicum expresses hydrogenase in microaerophilic free-living conditions in the presence of nickel. Plasmid-borne hup-lacZ transcriptional fusion constructs were used to study the regulation of the hydrogenase gene. The hydrogenase gene was transcriptionally induced under microaerobic conditions (0.1 to 3.0% partial pressure 02). The hydrogenase gene was not transcribed or was poorly transcribed in strictly anaerobic conditions or conditions above 3.0% 02. Hydrogen gas at levels as low as 0.1% partial pressure induced hydrogenase transcription, and a high level of transcription was maintained up to at least 10% H2 concentration. No transcription was observed in the absence of H2. Hydrogenase was regulated by H2, 02, and Ni when the 5'-upstream sequence was pared down to include base number -168. However, when the upstream sequence was pared down to base number -118, the regulatory response to 02, H2, and Ni levels was negated. Thus, a common cis-acting regulatory region localized within 50 bp is critical for the regulation of hydrogenase by hydrogen, oxygen, and nickel. As a control, the B. japonicum hemA gene which codes for 8-aminolevulinic acid synthase was also fused to the promoterless lacZ gene, and its regulation was tested in the presence of various concentrations of 02 and H2. hemA-lacZ transcription was not dependent on levels of Ni, 02, or H2. Two different hup-lacZ fusions were tested in a Hup-background, strain JH47; these hup-lacZ constructs in JH47 demonstrated dependency on nickel, 02, and H2, indicating that the hydrogenase protein itself is not a sensor for regulation by 02, H2, or nickel.In soybean root nodules, the Bradyrhizobium japonicum bacterium undergoes a morphological transformation into N2-fixing bacteroids. Within the nodule, conditions are microaerobic because of an 02 diffusion barrier and 02-binding leghemoglobins; because of this, the 02 concentration at the bacteroid surface is estimated to be 11 nM (1). Many new proteins are expressed in the 02-limited nodule environment. In addition to those encoded by the nitrogen fixation genes, a membrane-bound H2-oxidizing hydrogenase enzyme that utilizes the H2 evolved by nitrogenase is expressed. The Ni-containing hydrogenase (3,32,33) activates H2 and sends the electrons through an energy-generating respiratory electron transport chain (reviewed in reference 27). Therefore, it is thought to be a component responsible for maintaining efficient N2 fixation in the nodules.Studying the regulation of bacteroid hydrogenase in response to environmental stimuli supplied to the root nodules is difficult if not physically impossible. Conditions have been worked out so that B. japonicum hydrogenase activity can be induced in the free-living form in an H2-and C02-containing atmosphere (16)(17)(18). It is within these controlled circumstances that most hydrogenase regulation studies have been done (16-19, 22, 26, 35, 36). (30) and 20 bases downstream of it. The transcription initiation site has previously been mapped (30). pBJ3-1 is ...

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“…However, heterologous expression of the adh gene in A. eutrophus JMP222 and in P. oxalaticus supports the conclusion that the cloned HS10 fragment does contain the complete DNA sequence necessary for the synthesis of active fermentative alcohol dehydrogenase. The heterotrophic strain JMP222 has already served as host for the expression of plasmidencoded hydrogenases of A. eutrophus H16 (8). Similarly, the herbicide-degrading plasmid pJP4 of strain JMP134, which is the parent strain of JMP222, is expressed in, e.g., A. eutrophus H16 (9).…”

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confidence: 99%

Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene

et al. 1988

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Mutants of Akcaligenes eutrophus which are altered with respect to the utilization of 2,3-butanediol and acetoin were isolated after transposon mutagenesis. The suicide vehicle pSUP5011 was used to introduce the drug resistance transposable element TnS into A. eutrophus. Kanamycin-resistant transconjugants of the 2,3-butanediol-utilizing parent strains CF10141 and AS141 were screened for mutants impaired in the utilization of 2,3-butanediol or acetoin. Eleven mutants were negative for 2,3-butanediol but positive for acetoin; they were unable to synthesize active fermentative alcohol dehydrogenase protein (class 1). Forty mutants were negative for 2,3-butanediol and for acetoin (class 2). Tn5-mob was also introduced into a Smr derivative of the 2,3-butanediol-nonutilizing parent strain H16. Of about 35,000 transconjugants, 2 were able to grow on 2,3-butanediol. Both mutants synthesized the fermentative alcohol dehydrogenase constitutively (class 3). The TnS-labeled EcoRI fragments of genomic DNA of four class 1 and two class 3 mutants were cloned from a cosmid library. They were biotinylated and used as probes for the detection of the corresponding wild-type fragments in a AL47 and a cosmid gene bank. The gene which encodes the fermentative alcohol dehydrogenase in A. eutrophus was cloned and localized to a 2.5-kilobase (kb) Sall fragment which is located within a 11.5-kb EcoRI-fragment. The gene was heterologously expressed in A. eutrophus JMP222 and in Pseudomonas oxalaticus. The insertion of TnS-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb Sall fragment.Recently, the ability of strictly respiratory bacteria to form fermentation enzymes under conditions of restricted oxygen supply was demonstrated (2, 28, 37). A whole physiological group of bacteria, the aerobic chemolithoautotrophic bacteria, relies only on respiratory energy generation, and not a single member has been reported to be able to grow on organic compounds anaerobically on the basis of fermentative energy generation. Since fermentation enzymes were not expected to be present in these bacteria, the question of the metabolic role of these enzymes and the significance of these cryptic genes in strict aerobes was raised. The hydrogen-oxidizing bacterium Alcaligenes eutrophus serves as a model for our study on enzyme derepression in strictly respiratory bacteria under conditions of oxygen deficiency.In A. eutrophus an NAD-dependent lactate and an NAD(P)-dependent alcohol dehydrogenase appear to function as a safety valve for the release of excess reducing power in the absence of exogenous hydrogen acceptors such as oxygen or nitrate (38-41). A. eutrophus is even able to evolve molecular hydrogen (43) and to synthesize poly-,-hydroxybutyric acid (29) to dispose of the reductant if a suitable electron acceptor is lacking. The cytoplasmic, NAD-reducing hydrogenase is responsible for the evolution of hydrogen (19).The fermentative alcohol dehydrogenase is a tetramer of relative molecular mass 156,000 an...

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Genetics of hydrogenase from aerobic lithoautotrophic bacteria

Cited by 25 publications

References 58 publications

Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm

Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm

Common cis-acting region responsible for transcriptional regulation of Bradyrhizobium japonicum hydrogenase by nickel, oxygen, and hydrogen

Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene

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