Influences on napa Genetic Diversity in Freshwater Microbial Populations

Microbial communities in freshwater sediments remove nitrate from the water column via denitrification. The first step in this process is the reduction of nitrate (NO₃^1-) to nitrite (NO₂^1-). Two enzyme complexes, Nar and Nap, independently catalyze this step under  different but overlapping environmental and metabolic conditions. Oxygen concentration and pH have been shown to influence the activity of these enzymes. Other environmental factors such as season have also been shown to influence denitrification rates. How these factors impact the genetic diversity of these genes in freshwater systems has not been well explored. This study examines the genetic diversity of the catalytic subunit in the Nap complex, napa, in the freshwater, non-tidal portion of Beaver Dam Creek, near Salisbury, Maryland. Three sampling sites were selected to potentially examine the impact of land use and residential population density as well as the impact of season and sample depth. Small (~10" x 1") sediment cores were collected bimonthly for over a year. Microbial genomic DNA was extracted from the upper two inches and polymerase chain reaction (PCR) was used to amplify the napa gene. The PCR reactions were digested with the restriction enzyme Alu I and then separated on 2% agarose gels to create a community DNA fingerprint.  Cluster analysis demonstrated that there is a seasonal influence on the napa gene diversity for the uppermost portion of the cores (0-1 inch) with one cluster dominated by winter samples (most representative sample = 1/21/13), a second cluster dominated by summer/fall samples (7/10/12),  and a third cluster dominated by spring samples (5/6/13).  However, at a depth of only 1-2 inches, geographic location appears to have a much stronger influence where samples from the least urban sample site segregate away from the other two sites. Analysis of clone libraries representing the spring communities (6/17/13), indicate that samples from this site had the highest genetic diversity (~18 unique RFLP types)  whereas the other two sites had similar, but lower diversity (11-12 unique RFLP types).  Thus, it appears that both geographic location and season impact the diversity of a gene critical to nitrate removal. Current experiments are examining napa in the winter cluster in order to fully examine the dynamic nature of this gene's diversity.