The Diversity And Ecology Of Nitrogen-Fixing Bacteria
Biological nitrogen fixation, the enzymatic conversion of gaseous nitrogen to ammonium, is carried out by diverse Bacteria and Archaea. Neither the diversity of nitrogen fixers nor the factors which control nitrogen fixation in the environment are well-characterized. The nifH gene encodes a component of the enzyme which carries out nitrogen fixation. I describe a nifH database, its sequence composition, and its inherent limitations. As an application, the database was used in an evolutionary analysis which compared the nifHDK genes and the 16S rRNA gene, finding support for the horizontal transfer of nitrogen fixation between the Bacteria and Archaea. Next, the database was used to evaluate the global diversity of nitrogen fixers. At an OTU0.95 similarity cutoff, 3,358 nifH OTUs were identified. Soil was found to be contain more nifH diversity than marine waters, and cluster III had the highest richness in the nifH phylogeny. The database was next used to evaluate the in silico coverage of more than 50 published, universal nifH primers, to find that 23 primers target <50% of the known nifH diversity while 15 target >90%. The set DVV/IGK3 had the best laboratory performance against genomic and soil DNA. Next, 5 fulllength, nifH gene templates representing the full diversity of nitrogen-fixers were used against degenerate, nifH primer sets to assess the potential for template-specific bias in quantitative real-time PCR. It was found that bias can lead to over 3 orders of magnitude mis-estimation of template copy number, with the degree of bias dependent on the compared templates, the primer set, and the annealing temperature used. The primer set polF/polR exhibited the least template-specific bias. This primer set was used in an ecological survey of 20 old-field and meadow sites from the Finger Lakes and Champlain regions of New York State. Either nitrogen fixation or nitrogen mineralization tended to dominate in a site, and this corresponded to the [delta] 13C signature of the soil, suggesting that the processes may be driven by distinct biomass inputs. Nitrogen fixation was found to be constrained in most sites, and soil moisture was identified to constrain the abundance of nitrogen fixers.
nitrogen fixation; microbial diversity and ecology; nifH gene database
Buckley, Daniel H
Groffman, Peter Mark; Zinder, Stephen H
Ph.D. of Microbiology
Doctor of Philosophy
dissertation or thesis