Community analysis of ammonia-oxidising bacteria, in relation to oxygen availability in soils and root-oxygenated sediments, using PCR, DGGE and oligonucleotide probe hybridisation

Abstract

The rhizosphere of oxygen-releasing wetland plants provides a niche for oxygen-consuming microorganisms such as chemolithotrophic ammonia-oxidising bacteria. These bacteria are adapted to oxygen limitation with respect to their affinity for oxygen, ability to survive periods of anoxia, and immediate response to the appearance of oxygen. In this study the techniques of specific amplification of ammonia oxidiser 16S rDNA fragments by PCR, separation of mixed PCR samples by denaturing gradient gel electrophoresis (DGGE), and band identification by specific hybridisation with oligonucleotide probes were combined to allow for the comparison of the community composition of multiple samples over space and time. DGGE bands of interest were also excised for DNA isolation, reamplification, sequence determination and phylogenetic analysis. We compared monthly samples from both the root zone and the bare sediment of a shallow lake inhabited by the emergent macrophyte Glyceria maxima to determine the seasonal effects that the plant roots and the oxygen availability might have on the B-subgroup ammonia-oxidiser populations present. Similarly, five soil or sediment samples, varying in oxygen availability, from different locations in the Netherlands were compared. Although the presence of two previously defined Nitrosospira sequence clusters could be differentially detected in the samples examined, there was no evidence for a particular group which was specific to periodically anoxic environments.