Nitrogenase gene expression in the North Pacific Subtropical Gyre

J. P. Zehr¹, J. P. Montoya², C. Short¹, A. Hansen³, B. D. Jenkins¹, M. J. Church³, D. M. Karl³

¹University of California, Department of Ocean Sciences, E&MS 438, Santa Cruz, CA 95064

²Georgia Institute of Technology, School of Biology, Atlanta, GA 30332

³Department of Oceanography, University of Hawaii, 1000 Pope Road, Honolulu, HI 96822

Abstract

We performed phosphorus addition experiments with natural assemblages of oceanic phytoplankton in the Pacific Ocean to determine the effect of nutrient enrichment on diazotrophic population dynamics and nitrogenase gene expression. Cloning and sequencing of PCR and RT-PCR nifH amplification products from experiments with water collected from Station ALOHA and from Kaneohe Bay, Hawaii, yielded the same groups of nifH phylotypes previously reported: cyanobacteria belonging to the unicellular Groups A and B, the genus Trichodesmium, heterocystous groups as well as gamma Proteobacteria. QPCR and QRT-PCR showed that the abundance of the bacteria, including cyanobacteria, did not change significantly during experimental incubations, but all had distinct diel patterns of gene expression. The Group B cyanobacterial nifH gene abundance was correlated with the presence of 3-8 micrometer diameter phycoerythrin-containing cells, whereas the Group A cyanobacterial abundance was not, suggesting that Group A may be smaller cells or do not contain phycoerythrin. The Group A cyanobacteria, which have not yet been successfully maintained in culture, express nitrogenase primarily during the day, which is unusual for a unicellular cyanobacterium. Patterns of gene expression in the experiments were similar to those documented for natural populations, and abundances of individual populations were stable over the time period of the experiment (36 hours), indicating that the experimental design simulated the natural environment. Phosphorus additions had no short-term (36 hr) effect on either abundance or nifH gene expression. The QPCR and QRT-PCR methods, coupled with experimental manipulations, provide a powerful approach for examining the factors controlling the distribution and activity of diazotrophs in the sea.