Cloning and Functional Characterization of Ammonium Transporters From the Marine Diatom Cylindrotheca Fusiformis (Bacillariophyceae)1

The general preference of phytoplankton for ammonium over nitrate as a nitrogen source has long been established, but the underlying molecular mechanisms controlling the uptake and utilization of these nutrients, and the preference for one over the other, are poorly understood. Insights are to be gained by cloning genes involved in nitrogen uptake and metabolism and characterizing the expression of these genes and their encoded proteins. I report the first cloning and characterization of ammonium transporter genes (AMT) from a marine diatom, Cylindrotheca fusiformis Reimann and Lewin. Two distinct types of AMT were isolated, which differed in their encoded amino acid sequences by 5%. A cDNA and genomic copy of AMT1 and three genomic copies of AMT2 were isolated. Analysis of mRNA levels in C. fusiformis cultures grown with different nitrogen sources revealed that AMT1 levels were much higher than AMT2 under all conditions. Highest mRNA levels were detected in nitrogen‐starved cells, followed by nitrate‐grown and then ammonium‐grown cells. A substantial amount of mRNA degradation was observed, suggesting that transcripts for the AMTs are turned over rapidly, which may be a regulatory mechanism over their expression. Transporters AMT1 and 2 were cloned into a Saccharomyces cerevisiae expression vector, and each was able to complement a yeast mutant that lacked all three of its native ammonium transporters. In addition to showing that the AMTs were functional, the data indicated that AMT1 was a much more efficient transporter than AMT2.