Spatial and Temporal Distribution of Two Diazotrophic Bacteria in the Chesapeake Bay

The abundance and biogeochemical importance of marine bacteria has been realized for decades (19). Facilitated by advances in molecular biology techniques, in recent years the taxonomic and phylogenetic identities of marine bacteria have been investigated. Using rRNA genes as phylogenetic markers (18), Giovannoni and coworkers identified previously unknown bacterial phylogenetic lineages in the Sargasso Sea (12), indicating that natural communities of bacteria were not well represented in culture collections. Since that time, studies of the diversity of marine microbial communities have revealed that the majority of bacteria in the ocean are uncultivated and many belong to unexpected taxa (8, 11, 14, 20). With similar molecular methods but aimed at genes encoding enzymes involved in key biogeochemical transformations (sometimes termed functional genes), studies have investigated the diversity and roles of microorganisms involved in specific processes, such as sulfate reduction (9) and nitrogen cycling (31). Biological nitrogen fixation, the reduction of atmospheric dinitrogen to ammonium, is an important process in maintaining the availability of fixed inorganic nitrogen in the biosphere, balancing the opposing process of denitrification. A recent modeling study illustrated the importance of nitrogen fixers to oceanic primary production and argued that nitrogen fixation is the main reason nitrogen availability limits primary production only on short time scales, while phosphorus limits oceanic production over longer time scales (26). Nitrogenase is the multisubunit enzyme that catalyzes nitrogen fixation and is encoded by the nifHDK genes. In the late 1980s, studies directed at the Fe protein subunit (nifH) of the nitrogenase protein complex were initiated by designing PCR primers for the cyanobacterial genus Trichodesmium (29). Since that time, information gathered on the diversity of nitrogenase genes has revealed that nifH is encoded by a wide range of autotrophic and heterotrophic prokaryotes, including Archaea, Firmicutes, spirochetes, and α-, β-, γ-, and δ-Proteobacteria (28). Diverse nitrogen-fixing microorganisms have been detected in aquatic habitats ranging from microbial mats (22, 30) to lakes (15), salt marshes (2), estuaries (1, 6), and hypersaline lakes (23). Although patterns of distribution of nitrogen-fixing microorganisms across habitats and ecosystems are appearing (28), the factors that drive the distribution and diversity of nitrogen-fixing microorganisms in aquatic environments are not understood. Recently, Burns and coworkers (6) discovered that the phylogenetic composition of diazotrophs in Chesapeake Bay and Neuse River sediments varied and concluded that environmental conditions selected for different diazotroph communities. Heidelberg and coworkers examined the seasonality of Chesapeake Bay bacterioplankton (13) and concluded that the distribution of specific taxa along the salinity gradient was patchy but relatively homogeneous. On the other hand, changes in the bacterial response to nutrient amendments (21) and changes in the composition of free-living and particle-associated bacterial communities (5, 7) have been associated with environmental changes along the estuarine gradient. To investigate how the environment affects the distribution of nitrogen-fixing microorganisms and initiate autecological studies on uncultivated natural populations of diazotrophs, we developed a quantitative PCR approach to study the abundance and distribution of specific nifH phylotypes. Ultimately, the goal of this study was to determine if specific bacteria encoding nifH are randomly distributed throughout the Chesapeake Bay.