Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd 1 -containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N 2 -fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N 2 fixation are not genetic traits of most of the uncultured bacteria.

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This paper originates from an address at the 8th International Symposium on Nitrogen Fixation with Non-Legumes, Sydney, NSW, December 2000 Different Azospirillumstrains and some other plant growth-promoting rhizobacteria (PGPR) were screened for the occurrence of genes coding for denitrification and nitrogenase reductase (nifH) using polymerase chain reaction (PCR)-based techniques. All PGPR examined were nitrogenase-positive. Azospirillum strains were remarkably dissimilar with respect to denitrification capabilities, in particular with respect to genes of the dissimilatory nitrite reductase. A. brasilense, A. lipoferum and A. halopraeferens strains possess a cytochrome cd1-containing nitrite reductase with low sequence similarities among them. A. irakense and A. doebereinerae have a Cu-containing nitrite reductase and A. amazonense is unable to denitrify. The molecular data were corroborated by activity measurements. The current results indicate that the inability to perform denitrification is unlikely a selective advantage for Azospirillum spp. and other associative bacteria for forming an association with plant roots.

To assess the diversity of total, denitrifying and N 2 -fixing bacteria in a nitrogen (N)-limited, acid forest soil, isolated DNA was analysed for the genes 16S rRNA, nosZ and nifH. Sequence information for these genes was obtained from clone libraries and from our TReFID computer program, which employs terminal restriction patterns for bacteria using multiple restriction enzymes. Both approaches indicated that Proteobacteria (α-and γ -groups) and Acidobacteria dominated. A comprehensive list of bacteria retrieved from this soil is provided and compared with literature data on the bacterial community compositions from other sites. The study indicated that the current PCR conditions with the primers employed allowed retrieval of only a portion of the bacteria occurring in soils. Massive treatment of a soil plot with NH 4 NO 3 caused an increase in the N content, which was rapidly followed by an enhancement of carbon (C) content. Thus the C/N ratio stayed below 16.0 and the soil remained N-limited. This may explain why the bacterial diversity did not undergo drastic shifts as was tentatively inferred from the available data sets.

Any attempt to characterize a bacterial community and their functional genes coding for enzymes of the nitrogen cycle is faced with its extreme biodiversity. Novel techniques, based on PCR amplification of target genes in DNA from environmental samples, have been developed for characterizing both cultured and as yet uncultured bacteria in the last few years. Computer-based assignment tools have now been developed utilizing terminal restriction fragments obtained from digestions with multiple restriction enzymes. Such programs allow the gross characterization of bacterial life in any complex bacterial community with confidence.

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