LAKE SUPERIOR PHOTOTROPHIC PICOPLANKTON: NITRATE ASSIMILATION MEASURED WITH A CYANOBACTERIAL NITRATE-RESPONSIVE BIOREPORTER AND GENETIC DIVERSITY OF THE NATURAL COMMUNITY Natalia Valeryevna Ivanikova A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2006 Committee: George S. Bullerjahn, Advisor Robert M. McKay Scott O. Rogers Paul F. Morris Robert K. Vincent Graduate College representative ii ABSTRACT George S. Bullerjahn, Advisor 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 iii 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 environmental clone library, the physiological characterization of these strains may reveal adaptations to unique conditions that exist in Lake Superior. iv ACKNOWLEDGEMENTS First of all, I want to thank my advisor Dr. George S. Bullerjahn for his support and guidance during the three and a half years that I spent in Bowling Green. Thank you George for providing me with an opportunity to work in your lab and encouraging me to think independently. I would like to thank Dr. Robert M. McKay, who helped me to learn the basics of the science of limnology, which I had a very vague idea about when I first came to Bowling Green. I also would like to thank my other committee members: Dr. Scott O. Rogers, Dr. Paul F. Morris, and Dr. Robert K. Vincent. Thank you Dr. Rogers for letting me use your equipment. I would like to acknowledge the Captain and crew of the R/V Blue Heron for their assistance in collection of samples and Michael Twiss and Christel Hassler (Clarkson University) and Rob Sherrell and Eleni Anagnostou (Rutgers University) for sharing their dissolved iron and SRP data, respectively, used in Chapter 3 of the thesis. I want to thank people in George and Mike’s labs Maria Baranova, Audrey Cupp, Linda Popels, Ramakrishna Boyanapali, David Porta and Mamoon Al-Raishadat for creating a friendly atmosphere in the lab and Armeria Vicol for teaching me many useful tips. I also would like to acknowledge my friends that I met here in BG and who also worked in George’ lab Nadejda Vintonenko and Kerry Brinkman. Special thanks to my boyfriend Anton V. Kulikov for tolerating me while I was writing this thesis. And, of course, I want to thank my parents Galina Mazgutovna Gataulina and Valerii Vasylevich. Ivanikov for letting me become who I am and my entire family for their everlasting love and support. v TABLE OF CONTENTS Page CHAPTER 1. INTRODUCTION…………………………………………………………………1 Freshwater picocyanobacteria: diversity…………………………………………………..4 Freshwater cyanobacteria: populational dynamics………………………………………..7 Lake Superior as an example of an extremely oligotrophic system……………………..11 Lake Superior and accumulation of nitrate………………………………………………12 Potential factors limiting primary productivity in Lake Superior………………………..14 Use of cyanobacterial bioreporters to measure nutrient bioavailability…………………16 Regulation of cyanobacterial mitrogen assimilation genes……………………………...19 The importance of studying the endemic picoplankton of Lake Superior……………….22 References………………………………………………………………………………..29 CHAPTER 2. CONSTRUCTION AND PHYSIOLOGICAL CHARACTERIZATION OF A CYANOBACTERIAL BIOREPORTER CAPABLE OF ASSESSING NITRATE ASSIMILATORY CAPACITY IN FRESHWATERS…………………………………………..41 Introduction………………………………………………………………………………………41 Materials and methods…………………………………………………………………………...42 Media and growth conditions…………………………………………………………….42 Construction of the PnirA::luxAB promoter fusions…………………………………….43 Characterization of the AND100A and AND100B promoter fusions and the AND100 bioreporter………………………………………………………………………………..45 Water collection from Lake Superior…………………………………………………....46 Monitoring nitrate depletion in bioreporter assays………………………………………46 vi Results……………………………………………………………………………………………47 Nitrate-Dependent Activation of AND100 Bioluminescence…………………………...47 Factors influencing AND100 nitrate-dependent luminescence in BG-11 media………..50 Induction of bioluminescence during nitrate assimilation……………………………….52 Use of the AND100 bioreporter to assess nitrate assimilation in field samples…………53 Discussion………………………………………………………………………………………..55 Utility of the bioreporter assay…………………………………………………………..55 Application of the AND100 reporter to Lake Superior………………………………….56 Comparison to other cyanobacterial N bioreporters……………………………………..57 Concluding remarks – future prospects………………………………………………….58 References………………………………………………………………………………………..60 CHAPTER 3. NITRATE UTILIZATION IN LAKE SUPERIOR IS IMPAIRED BY LOW NUTRIENT (P, Fe) AVAILABILITY AND SEASONAL LIGHT LIMITATION…………….65 Introduction………………………………………………………………………………………65 Materials and methods…………………………………………………………………………...67 Media and growth conditions…………………………………………………………….67 Sample collection………………………………………………………………………...68 Nitrate assimilation in Lake Superior water: nutrient effects……………………………69 Nitrate assimilation in Lake Superior water: light flux………………………………….70 Monitoring nitrate depletion in bioreporter assays………………………………………70 Measurement of alkaline phosphatase activity…………………………………………..71 Results……………………………………………………………………………………………71 vii Physico-chemical characteristics of Lake Superior……………………………………...71 Nitrate assimilation in Lake Superior water: nutrient effects - The AND100…………...74 Nitrate assimilation in Lake Superior water: light flux………………………………….77 Discussion………………………………………………………………………………………..79 References………………………………………………………………………………………..86 CHAPTER 4. THE PHYLOGENETIC DIVERSITY OF LAKE SUPERIOR CYANOBACTERIA…………………………………………………………………………….91 Introduction………………………………………………………………………………………91 Materials and Methods…………………………………………………………………………...93 Sample collection………………………………………………………………………...93 Isolation of cyanobacterial strains from Lake Superior………………………………….94