Genetics of hydrogenase from aerobic lithoautotrophic bacteria.

Aerobic facultatively autotrophic hydrogen bacteria are distinguished on the basis of their hydrogen-oxidizing enzyme system (Hox). The major group, represented by Paracoccus denitrificans and Pseudomonas facilis, contains a membrane-bound, electron transport-coupled protein. Species of Nocardia are characterized by the possession of a cytoplasmic NAD-dependent hydrogenase. Both enzymes are present in strains of Alcaligenes. All hydrogenases from lithoautotrophs are H2-consuming nickel-iron-sulfur proteins. Despite these common characteristics, hydrogenases differ in catalytic and molecular properties, in particular in the regulation of enzyme synthesis. Hydrogenase formation is either inducible by H2 (e.g. P. denitrificans strain F1, Alcaligenes hydrogenophilus) or subject to derepression in response to the supply of reductant, temperature, and oxygen (e.g. Alcaligenes eutrophus). The only plasmid-encoded Hox function has been conclusively identified in species of Alcaligenes. Structural and regulatory hox genes reside on megaplasmids, ranging in size between 400 and 500 kilobase pairs (kb). Most of the plasmids are self-transmissible by conjugation. Hox genes of A. eutrophus H16 have been localized by plasmid curing, genetic transfer, molecular cloning and analysis of plasmid deletions and insertions. They seem to be clustered in a DNA sequence of approximately 50 kb, representing several transcriptional units. In addition, a chromosomally encoded regulatory function is required for the expression of plasmid-linked hox genes. Plasmid pHGl of A. eutrophus H16 has been transferred to the non-lithoautotrophic soil bacterium JMP222. Both hydrogenases are expressed in the new host. The current state of hydrogenase genetics in Alcaligenes is discussed in reference to hydrogenase systems of other lithoautotrophic bacteria.

Maturation of membrane-bound hydrogenase of Alcaligenes eutrophus H16.

The formation of the catalytically active membrane-bound hydrogenase (MBH) of Alcaligenes eutrophus H16 requires the genes for the small and large subunits of the enzyme (hoxK and hoxG, respectively) and an accompanying set of accessory genes (C. Kortl ke, K. Horstmann, E. Schwartz, M. Rohde, R. Binsack, and B. Friedrich, J. Bacteriol. 174:6277-6289, 1992). Other genes located in the adjacent pleiotropic region are also required. In the absence of these genes, MBH is synthesized but is catalytically inactive. Immunological analyses revealed that cells containing active MBH produced the small and large subunits of the enzyme in two distinct conformations each; only one of each, presumably the immature form, occurred in cells devoid of MBH activity. The results suggest that the conversion of the two subunits into the catalytically active membrane-associated heterodimer depends on specific maturation processes mediated by hox genes.

Complex I of Rhodobacter capsulatus and its role in reverted electron transport.

The activities of NAD+-photoreduction and NADH/decyl-ubiquinone reductase in membrane preparations of Rhodobacter capsulatus changed to the same extent under different conditions. These results indicated that NADH:ubiquinone oxidoreductase (complex I) catalyzes the electron transport in the downhill direction (respiratory chain) and in the uphill direction (reverted electron flow). This conclusion was confirmed by the characterization of a complex-I-deficient mutant of R. capsulatus. The mutant was not able to reduce NAD+ in the light. Since this mutant was not able to grow photoautotrophically, we concluded that complex I is the enzyme that catalyzes the reverted electron flow to NAD+ to provide reduction equivalents for CO2 fixation. Complex I is not essential for the reverted electron flow to nitrogenase since the mutant grew under nitrogen-fixing conditions. As shown by immunological means, NuoE, a subunit of complex I from R. capsulatus having an extended C-terminus, was modified depending on the nitrogen source present in the growth medium. When the organism used N2 instead of NH4+, a smaller NuoE polypeptide was synthesized. The complex-I-deficient mutant was not able to modify NuoE. The function of the modification is discussed.

Molecular cloning of structural and regulatory hydrogenase (hox) genes of Alcaligenes eutrophus H16.

A gene bank of the 450-kilobase (kb) megaplasmid pHG1 from the hydrogen-oxidizing bacterium Alcaligenes eutrophus H16 was constructed in the broad-host-range mobilizable vector pSUP202 and maintained in Escherichia coli. hox DNA was identified by screening the E. coli gene bank for restoration of hydrogenase activity in A. eutrophus Hox mutants. Hybrid plasmids that contained an 11.6-kb EcoRI fragment restored soluble NAD-dependent hydrogenase activity when transferred by conjugation into one class of Hos- mutants. An insertion mutant impaired in particulate hydrogenase was partially restored in Hop activity by an 11-kb EcoRI fragment. A contiguous sequence of two EcoRI fragments of 8.6 and 2.0 kb generated Hox+ recombinants from mutants that were devoid of both hydrogenase proteins. hox DNA was subcloned into the vector pVK101. The resulting recombinant plasmids were used in complementation studies. The results indicate that we have cloned parts of the structural genes coding for Hos and Hop activity and a complete regulatory hox DNA sequence which encodes the thermosensitive, energy-dependent derepression signal of hydrogenase synthesis in A. eutrophus H16.

A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16.

One of the key enzymes in the chemolithoautotrophic metabolism of Alcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase. The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G. Eberz, C. Hogrefe, C. Kortlüke, A. Kamienski, and B. Friedrich, J. Bacteriol. 168:636-641, 1986). Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA. We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG). The nucleotide sequence contains nine additional closely spaced open reading frames. Immunoelectron microscopy showed that the gene product of one of these open reading frames (hoxM) is involved in the process leading to the attachment of hydrogenase to the membrane. Other open reading frames may encode additional processing functions and components of a hydrogenase-linked electron transport chain. Analysis of Tn5-B21-mediated transcriptional fusions provided evidence that the structural genes and accessory functions belong to at least three coordinately regulated transcriptional units.

Structure of the puf operon of the obligately aerobic, bacteriochlorophyll alpha-containing bacterium Roseobacter denitrificans OCh114 and its expression in a Rhodobacter capsulatus puf puc deletion mutant.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.