Detection in soil of aerobic hydrogen-oxidizing bacteria related to Alcaligenes eutrophus by PCR and hybridization assays targeting the gene of the membrane-bound (NiFe) hydrogenase

Abstract

The presence and abundance of the aerobic, chemolithoautotrophic Alcaligenes eutrophus, and related hydrogen-oxidizing bacteria, in soil samples were determined by using a hydrogenase gene probe and two different PCR primer sets that were derived from the alignment of (NiFe) hydrogenase gene sequences available in the EMBL data bank. The specificity of both the gene probe and the primer sets was tested on the genomic DNAs of 19 hydrogenase-containing, or hydrogenase-lacking, bacteria. The hydrogenase gene probe detected hydrogenase-containing Knallgas bacteria, N2-fixing bacteria, and enterobacteria, belonging to the alpha, beta, and gamma subclasses of the Proteobacteria. Hydrogenase-containing sulfate reducers (delta subclass) were not detected. One primer set was degenerated and resulted in the amplification of gene fragments of Knallgas bacteria and N2-fixing bacteria. The other primer set was specifically designed for the gene of the small subunit of the membrane-bound hydrogenase of A. eutrophus. In addition to the target organism, only Variovorax (Alcaligenes) paradoxus gave a gene product of the expected length. A. eutrophus-related bacteria were detected in soil by using two different strategies. The first strategy required cultivation and isolation of soil bacteria on complex media. Colonies were isolated from six different soils and tested by colony hybridization with the hydrogenase gene probe. Fifteen of the positive colonies were chosen for further characterization with the PCR primers. Two of the isolates gave amplification products with the A. eutrophus-specific PCR primers, which were sequenced and found to be 89–90% identical on a nucleotide level to the hydrogenase gene of A. eutrophus H16. The second strategy avoided a cultivation step. Total DNA was isolated from the rhizosphere of rice plants and tested in PCR amplification experiments with the A. eutrophus-specific PCR primers. Two different types of hydrogenase fragments were cloned and partially sequenced. The gene fragment sequences showed 73 and 75% identity to the A. eutrophus hydrogenase sequence. In quantitative PCR amplification assays, the number (about 2.6×107 g−1 fresh roots) of these two species of hydrogenase-containing bacteria was determined in the rice rhizosphere and was found to be similar to the number (about 7.4×106 g−1 fresh roots) of Knallgas bacteria that were determined by enumeration on selective media. Thus, it was apparently possible to detect and enumerate A. eutrophus-related bacteria in soil samples by molecular techniques.