Functional gene hybridization patterns of terrestrial ammonia-oxidizing bacteria

The biochemical pathway and genetics of autotrophic ammonia oxidation have been studied almost exclusively in Nitrosomonas europaea. Terrestrial autot rophic ammonia-oxidizing bacteria (AAOs), however, comprise two distinct ph ylogenetic groups in the beta-Proteobacteria, the Nitrosomonas and Nitrosos pira groups. Hybridization patterns were used to assess the potential of fu nctional probes in non-PCR-based molecular analysis of natural AAO populati ons and their activity. The objective of this study was to obtain an overvi ew of functional gene homologies by hybridizing probes derived from N. euro paea gene sequences ranging in size from 0.45 to 4.5 kb, and labeled with P -32 to Southern blots containing genomic DNA from four Nitrosospira represe ntatives. Probes were specific fur genes encoding ammonia monooxygenase (am oA and amoB), hydroxylamine oxidoreductase (hao), and cytochrome c-554 (hcy ). These probes produced hybridization signals, at low stringency (30 degre es C), with DNA from each of the four representatives; signals at higher st ringency (42 degrees C) were greatly reduced or absent. The hybridization s ignals at low stringency ranged from 20 to 76% of the total signal obtained with N. europaea DNA. These results indicate that all four functional gene s in the ammonia oxidation pathway have diverged between the Nitrosomonas a nd Nitrosospira groups. The hao probe produced the most consistent hybridiz ation intensities among the Nitrosospira representatives, suggesting that h ao sequences would provide the best probes for non-PCR-based molecular anal ysis of terrestrial AAOs. Since N. europaea can also denitrify, an addition al objective was to hybridize genomic DNA from AAOs with probes for Pseudom onas genes involved in denitrification. These probes were specific for gene s encoding heme-type dissimilatory nitrite reductase (dNir), Cu-type dNir, and nitrous oxide reductase (nosz). No hybridization signals were observed from probes for the heme-type dNir or nosz, but Nitrosospira sp. NpAV and N itrosolobus sp. 24-C hybridized, under low-stringency conditions, with the Cu-type dNir probe. These results indicate that AAOs may also differ in the ir mechanisms and capacities for denitrification.