Diazotrophic diversity, nifH gene expression and nitrogenase activity in a rice paddy field in Fujian, China

Biological nitrogen fixation is an important source of fixed nitrogen for the biosphere. Microorganisms catalyse biological nitrogen fixation with the enzyme nitrogenase, which has been highly conserved through evolution. Cloning and sequencing of one of the nitrogenase structural genes, nifH, has provided a large, rapidly expanding database of sequences from diverse terrestrial and aquatic environments. Comparison of nifH phylogenies to ribosomal RNA phylogenies from cultivated microorganisms shows little conclusive evidence of lateral gene transfer. Sequence diversity far outstrips representation by cultivated representatives. The phylogeny of nitrogenase includes branches that represent phylotypic groupings based on ribosomal RNA phylogeny, but also includes paralogous clades including the alternative, non-molybdenum, non-vanadium containing nitrogenases. Only a few alternative or archaeal nitrogenase sequences have as yet been obtained from the environment. Extensive analysis of the distribution of nifH phylotypes among habitats indicates that there are characteristic patterns of nitrogen fixing microorganisms in termite guts, sediment and soil environments, estuaries and salt marshes, and oligotrophic oceans. The distribution of nitrogen-fixing microorganisms, although not entirely dictated by the nitrogen availability in the environment, is non-random and can be predicted on the basis of habitat characteristics. The ability to assay for gene expression and investigate genome arrangements provides the promise of new tools for interrogating natural populations of diazotrophs. The broad analysis of nitrogenase genes provides a basis for developing molecular assays and bioinformatics approaches for the study of nitrogen fixation in the environment.

There is a paucity of knowledge on microbial community diversity and naturally occurring seasonal variations in agricultural soil. For this purpose the soil microbial community of a wheat field on an experimental farm in The Netherlands was studied by using both cultivation-based and molecule-based methods. Samples were taken in the different seasons over a 1-year period. Fatty acid-based typing of bacterial isolates obtained via plating revealed a diverse community of mainly gram-positive bacteria, and only a few isolates appeared to belong to the Proteobacteria and green sulfur bacteria. Some genera, such as Micrococcus, Arthrobacter, and Corynebacterium were detected throughout the year, while Bacillus was found only in July. Isolate diversity was lowest in July, and the most abundant species, Arthrobacter oxydans, and members of the genus Pseudomonas were found in reduced numbers in July. Analysis by molecular techniques showed that diversity of cloned 16S ribosomal DNA (rDNA) sequences was greater than the diversity among cultured isolates. Moreover, based on analysis of 16S rDNA sequences, there was a more even distribution among five main divisions, Acidobacterium, Proteobacteria, Nitrospira, cyanobacteria, and green sulfur bacteria. No clones were found belonging to the gram-positive bacteria, which dominated the cultured isolates. Seasonal fluctuations were assessed by denaturing gradient gel electrophoresis. Statistical analysis of the banding patterns revealed significant differences between samples taken in different seasons. Cluster analysis of the patterns revealed that the bacterial community in July clearly differed from those in the other months. Although the molecule- and cultivation-based methods allowed the detection of different parts of the bacterial community, results from both methods indicated that the community present in July showed the largest difference from the communities of the other months. Efforts were made to use the sequence data for providing insight into more general ecological relationships. Based on the distribution of 16S rDNA sequences among the bacterial divisions found in this work and in literature, it is suggested that the ratio between the number of Proteobacteria and Acidobacterium organisms might be indicative of the trophic level of the soil.