Biology Ph.D. Dissertations

Lake Superior Phototrophic Picoplankton: Nitrate Assimilation Measured with a Cyanobacterial Nitrate-responsive Bioreporter and Genetic Diversity of the Natural Community

Date of Award

2006

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

George Bullerjahn

Abstract

Cyanobacteria of the picoplankton size range (picocyanobacteria) Synechococcus and Prochlorococcus contribute significantly to total phytoplankton biomass and primary production in marine and freshwater oligotrophic environments. Despite their importance, little is known about the biodiversity and physiology of freshwater picocyanobacteria. Lake Superior is an ultra-oligotrophic system with light and temperature conditions unfavorable for photosynthesis. Synechococcus-like picocyanobacteria are an important component of phytoplankton in Lake Superior. The concentration of nitrate, the major form of combined nitrogen in the lake, has been increasing continuously in these waters over the last 100 years, while other nutrients remained largely unchanged. Decreased biological demand for nitrate caused by low availabilities of phosphorus and iron, as well as low light and temperature was hypothesized to be one of the reasons for the nitrate build-up. One way to get insight into the microbiological processes that contribute to the accumulation of nitrate in this ecosystem is to employ a cyanobacterial bioreporter capable of assessing the nitrate assimilation capacity of phytoplankton. In this study, a nitrate-responsive biorepoter AND100 was constructed by fusing the promoter of the Synechocystis PCC 6803 nitrate responsive gene nirA, encoding nitrite reductase to the Vibrio fischeri luxAB genes, which encode the bacterial luciferase, and genetically transforming the resulting construct into Synechocystis. The transcription of luciferase in the transformant is regulated by the availability of nitrate in the sample. Therefore, the bioluminescent signal produced by the bioreporter reflects the nitrate assimilation capacity of the cell. The dynamic range of the bioreporter response was found to be between 1 and 100 µM nitrate. The results of a series of bioreporter assays conducted on preserved water samples collected from several stations in Lake Superior in May and September 2004 suggest that low availability of phosphorus is the major factor that constrains nitrate depletion in the lake with low seasonal or spatial variability. In addition, iron was found to be a secondary limiting factor, whose effect is evident only of phosphorus is added to the sample. During the period of isothermal mixing, light was shown to significantly reduce nitrate depletion in the lake. Overall, the bioreporter AND100 is a suitable model for elucidating the factors that regulate nitrate depletion by phytoplankton in natural waters. However, understanding the physiology of the natural cyanobacterial assemblages in the lake helps to prove the validity of the bioreporter approach. Since the information on the endemic Lake Superior phytoplankton is very scarce, an initial characterization of the genetic diversity of cyanobacteria in the lake was conducted. High throughput sequencing of a library of cyanobacterial 16S ribosomal DNA clones amplified by PCR from DNA isolated from the lake water resulted in 368 successful reactions. In a neighbor-joining tree the majority of the 16S rDNA sequences clustered within the “picocyanobacterial clade” that consists of both freshwater and marine Synechococcus and Prochlorococcus picocyanobacteria. Two new groups of picocyanobacteria LSI and II that do not cluster within any of the known freshwater picocyanobacterial clusters were the most abundant (> 50% of the sequences) in the samples collected from pelagic Lake Superior stations. Conversely, at station KW located in a nearshore urban area, only 4% of the sequences belonged to these clusters, and the remaining of the sequences reflected the freshwater biodiversity described previously. In addition, several picocyanobacterial strains were isolated from Lake Superior between years 2004 and 2005. Despite their low representation in the ...

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