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.