The marine cyanobacterium, Prochlorococcus, is the smallest yet most abundant known phototroph on the planet and contributes significantly to primary production and biogeochemical cycling in the world's open oceans. Since its first description 20 years ago, this organism has become a well-studied marine microbe that is being developed as a model system for cross-scale systems biology. At least 26 isolates have been obtained since 1988, from which two distinct ecotypes were identified based on the relative photophysiological characteristics: high-light (HL) adapted and low-light (LL) adapted. More than 80% of the isolates belong to the HL adapted ecotype, which is composed of at least two distinct phylogenetic clades. The other isolates (6) are considered LL adapted and are spread among several phylogenetic clades, two of which are represented by single cultured isolates. Evidence indicates that the two HL adapted clades likely evolved from differences in temperature optima resulting in distinct horizontal distributions of their populations. What is not clear are the phenotypic and/or ecological underpinnings of the different LL adapted clades and the distributions of these clades in space and time, in large part because there are so few isolates representing each clade. Thus, despite the relatively large number of isolates and data on Prochlorococcus, only half the story, that of the more dominant HL adapted ecotypes, is understood in terms of the phylogenetic relationships and distributions. This project will study the physiological characteristics that contribute to the ecology and evolution of different phylogenetic clades that make up the LL adapted Prochlorococcus ecotype. This characterization will be achieved through high throughput culturing and physiological assessment experiments that utilize multi-well plates for purifying new LL adapted isolates, physiological determinations, and a 96-well sampling system attached to a flow cytometer.
Broader Impacts: This project will isolate new, axenic cultures with accompanying ecophysiological and phylogenetic information that could be used by biological oceanographers, biogeochemical modelers, microbiologists and evolutionary biologists for further studies of natural population distributions, physiological mechanisms, genomics, transcriptomics, and proteomics of the world's most abundant phototroph. For instance, the 16S-23S rRNA sequences can be used to redesign or develop new primers for environmental detection of LL adapted Prochlorococcus populations and specific ecotypes, which in turn will enable researchers to begin to understand their ecological role in the deeper euphotic zone. New Prochlorococcus isolates will be made available to other researchers as well as deposited in a larger culture collection such as the Center for the Culture of Marine Phytoplankton. Gene sequences will be deposited in the public database, GenBank, and the results from this study will provide information to choose specific strains for further genome sequence analysis. In addition, this project will also provide research opportunities and financial support for several undergraduates to carry out research projects, a MS student in Biology, and the first University of Maine System Cooperative PhD student in Microbiology at USM.
The base of the food chain in the oceans is comprised of extremely small (~ 1 micron in diameter) prokaryotic cells. One of the primary photosynthetic prokaryotes belongs to the genus Prochlorococcus, which is a type of marine cyanobacteria. Although it is tiny, it is very abundant (up to 105 cells per ml, which is ~ 500,000 cells in a teaspoon of seawater) and covers large regions of the oceans from 40 ºN to 40 ºS. At least 26 isolates have been obtained since its discovery in 1988, from which two distinct groupings, called ecotypes, were identified based on their different responses to light: high-light (HL) adapted and low-light (LL) adapted. More than 80% of the isolates belong to the HL adapted ecotype, in large part because are the easiest to study because they dominate the surface waters of the oceans and because these seem to be the easiest to isolate for lab-based studies. When this project started, there were only 6 LL adapted isolates, and these did not group together when examined for their phylogenetic (evolutionary) relationships but formed multiple, distinct groupings, currently referred to as LLI, LLII/III and LLIV ecotypes. Why do these LL adapted Prochlorococcus split into different ecotypes? Are there specific physiological characteristics for any of these different LL adapted ecotypes that might help explain the ecological distributions of these clades in space and time? What I proposed to do in this study was to determine physiological characteristics that might contribute to the ecology and evolution of different phylogenetic clades that make up the LL adapted Prochlorococcus ecotype. The characteristics that I chose to examine were temperature range and optimum growth temperature, growth and pigment response to light, and nitrogen utilization for growth. In order to get enough information for characterizing the different LL ecotypes, I also isolated more LL adapted Prochlorococcus that could be included in this study. Five new isolates of LL adapted Prochlorococcus from the North Atlantic, the South Atlantic and the North Pacific oceans in combination with existing isolates provided four isolates for each LL adapted ecotype to be studied. The temperature physiology for LLI ecotype isolates was determined and compared to that found for HL ecotypes. We found that the temperature range differed only slightly (~ 1-3 degrees) between these ecotypes. More measurements of temperature physiology need to be done for the other LL adapted ecotypes to see if temperature might be a characteristic that distinguishes these from the other Prochlorococcus ecotypes. In terms of growth and pigment physiological responses to light levels, distinct differences in light range and optima were found for each of the LL adapted ecotypes, with LLI Prochlorococcus being able to grow at higher light levels than the other two LL ecotypes and LLIV Prochlorococcus having the lowest and narrowest range of light over which they can survive. Additionally, we found that all LL adapted ecotypes exhibit production of the cyanobacterial accessory pigment, phycoerythrin; but the LLIV ecotype contains only the phycourobilin portion, again setting this LL adapted ecotype apart from the others. Finally, we were successful at isolating and studying the nitrogen dependent growth of Prochlorococcus that could utilize nitrate as a nitrogen source. This physiological capability was not found in previous isolates of Prochlorococcus, which was a surprise because nitrate utilization is common to all other cyanobacteria and an important source of nitrogen for other photosynthetic microbes in the oceans. The genome was sequenced for one of these new isolates in collaboration with researchers from the Chisholm laboratory at Massachusetts Institute of Technology in order to better understand how nitrate utilizing Prochlorococcus might differ from Prochlorococcus that lack this trait. From the studies done for this project, we are getting a better idea of how some of the LL adapted Prochlorococcus differ from each other physiologically. This information will be useful in explaining how Prochlorococcus populations that belong to different ecotypes behave differently with respect to temperature, light and nutrient parameters in the oceans and predicting how some Prochlorococcus populations might respond to future changes in these environmental parameters. Finally, it is important to acknowledge the people who worked on this project and the other institutions involved in this project. Culture-based physiology studies on Prochlorococcus are labor-intensive because it is a slow growing microorganism (reaching maximum growth at ~ one doubling per day) that requires very clean culturing conditions and low nutrients. Two MS students and nine undergraduate students contributed to this project. Two of the students have gone on to PhD programs, three are working in biotechnology and industry, and another is pursuing a nursing degree. In addition to these students, researchers at five other universities around the country (MIT, Duke University, Univ. of Washington, Univ of California Irvine, and Texas A&M University) have been helpful in various aspects of this project.
