Hyphomicrobium spp. were counted and isolated for 12 months in a sewage treatment plant with a combination of simultaneous and intermittent nitrification and denitrification using Most-Probable-Number methods. Genomic DNA of these hyphomicrobia was investigated by Southern or dot blot hybridizations with gene probes specific for genes of dissimilatory nitrate reduction (nitrate reductase, narG; cytochrome c,d-containing nitrite reductase, nirS; Cu-containing nitrite reductase, nirK; nitrous oxide reductase, nosZ), nitrification (ammonia monooxygenase, amoA) and N2-fixation (nitrogenase, nifH). In particular, the Hyphomicrobium DNA/DNA-hybridization group HG 27 constituted one of the dominant denitrifying Hyphomicrobium populations in the activated sludge of this sewage treatment plant. A species-specific gene probe (Hvu-1) for HG 27 was generated from a transposon Tn5-132 insertion mutant defective in methanol oxidation using the inverse polymerase chain reaction. With this probe the abundance of this group in activated sludge of the sewage treatment plant and its receiving lake was determined as a subfraction of the total cultivable hyphomicrobia. Fragments of the mxaF gene encoding for the α-subunit of the methanol dehydrogenase of Hyphomicrobium spp. were amplified by PCR and analysed by denaturing gradient gel electrophoresis (DGGE). The DGGE analysis pattern showed a substantial separation of these fragments according to their nucleic acid sequences.

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