Identification of Hyphomicrobium spp. Using PCR-Amplified Fragments of the mxaF Gene as a Molecular Marker

Abstract

Summary Fragments of the mxa F gene (nucleic acid position: 1001 to 1557) encoding for the α-subunit of the methanol dehydrogenase (MDH) of Hyphomicrobium spp. were amplified by PCR. Southern blot hybridizations of these PCR products from different methylotrophic bacteria indicated that this specific mxa F gene fragment was a suitable molecular marker for the identification of Hyphomicrobium spp. PCR-amplified mxa F gene fragments were analysed by denaturing gradient gel electrophoresis (DGGE). The DGGE analysis pattern showed a substantial separation of these fragments according to their nucleic acid sequences in the range of 46 to 55% denaturant when they were electrophoresed separately or as a mixture. Fragments of the same species or DNA/DNA-hybridization group revealed an identical electrophoretic mobility. It was investigated if this approach could be used for the identification of new unknown isolates of Hyphomicrobium spp. from different habitats (biofilm, groundwater, and lake water). However, the DNA sequence variation of equal-sized mxa F gene fragments with identical electrophoretic mobility was considerable (9 to 107 bp). Therefore, the identification of environmental isolates by DGGE analysis of the mxa F gene fragment was not possible without verification by sequence analysis. This elucidated the problems arising with DGGE analysis of DNA fragments in taxonomic or environmental studies.