Crocosphaera watsonii is an ecologically important marine unicellular diazotrophic cyanobacterium. It is often abundant in oligotrophic ocean regions where it provides fixed nitrogen to nutrient-limited phytoplankton communities. Previous genetic studies have observed genetic rearrangements but very little sequence variation among natural populations or cultivated strains of Crocosphaera. Those strains exhibit two phenotypes (large- and small-cell) with characteristics that suggest different ecological roles and niches. Prior to this work, the genetic basis for the phenotypic differences was unknown, and molecular methods for enumerating natural C. watsonii could not differentiate between phenotypes. To address those unanswered scientific questions, these studies compared the genomes of six C. watsonii strains, three of each phenotype, which were isolated over large spatial and temporal distances. A large portion of those genome sequences were shared among all strains with nearly 100% nucleotide identity. However, there were also genes that were specific to each strain, and others were specific to each phenotype, including some which could explain phenotypic differences (e.g. EPS biosynthesis). Relative to small-cell strains, large-cell strains had larger genomes and additional genetic capabilities, including possibly increased adaptations to iron and phosphorus limitation. Clustering based on genome sequences and content showed that strains with a common phenotype were evolutionarily most closely related, regardless of their time and location of isolation. Surprisingly, the genome of the C. watsonii type-strain, WH8501, was quite unusual, even compared to those with the same phenotype, suggesting it may not be appropriately representative of the species. To investigate distributions of Crocosphaera types in the marine environment, molecular assays were developed, based on phenotype-specific genes, and applied to samples from the North and South Pacific. In those samples, small-cells dominated in the upper 75 m where abundance of both types was much greater, while large-cells dominated in samples with lower counts between 100 m and 175 m. There was also more evidence that large-cells form aggregates in the N. Pacific. Future studies will be important to determine which of the initial C. watsonii patterns described here can be generalized, both in genomes, and in natural distributions of the two types.

Chapter 1: The number of known sequences for the nifH gene, commonly used to assess community potential for N2 fixation, has been rapidly growing over the past few decades. Obtaining these sequences from the National Center for Biotechnology Information's GenBank database is problematic because of annotation errors, nomenclature variation, and paralogues; moreover, GenBank's tools are not conducive to searching solely by function. A software retrieval and curation pipeline called ARBitrator was developed that uses a BLAST search followed by a screening phase based on conserved domain similarity to retrieve nifH sequences from Genbank. A total of 34,420 nifH sequences were identified in GenBank. The false-positive rate is 0.033%. The pipeline can be adapted for other genes.

Chapter 2: Marine cyanobacteria capable of fixing molecular nitrogen ("diazotrophs") are key in biogeochemical cycling; the nitrogen fixed is a major external source of nitrogen to the open ocean. Candidatus Atelocyanobacterium thalassa (UCYN-A) is a diazotrophic cyanobacterium known for its widespread geographic distribution, unusually reduced genome, and symbiosis with a single-celled prymnesiophyte alga. Recently a novel strain of this organism, called UCYN-A2, was detected in coastal waters off southern California. We assembled and analyzed the metagenome of this UCYN-A2 population. UCYN-A2 and the open-ocean UCYN-A1 strain share most protein-coding genes with high synteny, yet average amino-acid sequence identity between orthologous genes is only 86%. Our results suggest that UCYN-A1 and UCYN-A2 had a common ancestor and diverged after genome reduction.

Chapter 3: Gene expression in cells fluctuates over time in response to internal and external stimuli. Cyanobacteria, whose metabolism is tightly coupled to the sunlight cycle, have evolved complex patterns of gene expression that may derive from optimizing growth by coordinating photosynthesis and protein synthesis. These patterns can provide much information on how microorganisms grow and respond to the environment. However, analyzing complex information from whole-genome expression studies is difficult, requiring computational approaches that support visual exploration and analysis of data in order to detect related expression signatures.

A Java application called Dexter was developed to facilitate analysis of single or multiple gene expression time-series data sets. The value of the program was demonstrated by using it to improve operon predictions

Nutrient availability affects primary productivity, and nitrogen (N) is the limiting nutrient over large regions of the ocean. Biological N fixation (BNF) is the conversion of atmospheric dinitrogen (N2) to bioavailable forms of N (ammonium and amino acids). A select group of Bacteria and Archaea, known as diazotrophs, are the only organisms capable of this conversion. BNF is a critical source of N in nutrient-depleted (oligotrophic) marine waters. As such, BNF studies are a fundamental component for better understanding primary productivity and carbon cycling, as well as parameterizing predictive future global ocean models. Unicellular diazotrophs are widespread in marine systems and, in some cases, important contributors of BNF. This dissertation investigates unicellular diazotrophs at the single-cell level, including UCYN-A, a cyanobacterial diazotroph that lives in symbiosis with an algal host, non-cyanobacterial diazotrophs (NCDs), which are putative heterotrophs, and a specific NCD known as Gamma A, which is a commonly occurring organism throughout the tropics and subtropics that belongs to the class Gammaproteobacteria. The single-cell investigations used in these studies can provide high-resolution insights that are not possible from community-level analyses of BNF, such as the N2 fixation rate of individual cells and cell morphology. Single-cell nanoscale secondary ion mass spectrometry (nanoSIMS) analyses were used in chapters 2 and 3 to investigate the cell-specific N2 fixation rates of UCYN-A in the cold waters of the Bering and Arctic Seas where N2 fixation was not previously believed to be possible (chapter 2), and NCDs in the North Pacific Subtropical Gyre where we demonstrated the first direct evidence of NCD N2 fixation on open ocean particles (chapter 3). Chapter 4 focused on a specific uncultivated NCD, Gamma A, using geneFISH to visualize single cells in natural seawater samples. We successfully identified Gamma A cells by developing a fluorescent in situ hybridization (FISH) probe targeting the diazotrophic functional marker gene nifH, revealing Gamma A cells are most often attached to particles. Together these studies provide new insights into the distributions, activities, and physiologies of geographically widespread, unicellular diazotrophs, thus improving our understanding of their roles as sources of fixed N in the surface ocean.

The marine cyanobacterium Crocosphaera significantly contributes to nitrogen fixation and primary production in tropical and subtropical oceans. These regions are often characterized by low phosphorus (P) concentrations that may constrain growth and nitrogen fixation in Crocosphaera. Molecular markers are commonly used as diagnostic tools to detect when microorganisms are under P stress and to better identify the conditions influencing P limitation. Prior to this work, diagnostic biomarkers for P stress in Crocosphaera had yet to be characterized. To identify possible biomarkers, select gene transcripts were evaluated under P stress in cultures of the model strain, WH8501, a small-cell ecotype of Crocosphaera. Transcript levels of two genes were significantly higher: pstS, which codes for the high-affinity P-binding protein, and arsB, coding for the arsenite efflux protein. These genes were up-regulated at discrete stages of the P stress response, with pstS being an early indicator of P limitation and arsB transcripts detected through P starvation. Interestingly, pstS transcripts exhibited a diel expression pattern that was detected in P replete conditions. To further evaluate the efficacy of pstS as a biomarker for P stress in Crocosphaera, changes in its transcript levels were assessed in the presence of various phosphorus sources: inorganic (DIP) and organic (DOP), as well as changing light levels. While pstS transcripts were suppressed by all P sources tested, the response was more sensitive to DIP than DOP. Additionally, pstS was induced by high light levels, independent of the P stress response. Although pstS is a commonly used indicator of P stress, these results suggest that it is not a reliable indicator for Crocosphaera. To evaluate P stress in natural assemblages, biomarkers were used to target environmental populations in the North Pacific. pstS and arsB identified P stress in Crocosphaera, and an alkaline phosphatase gene (phoX) targeted another nitrogen-fixing cyanobacterium, Trichodesmium. Detection of these biomarkers suggest that P is an important driver of Crocosphaera abundance, especially large cell ecotypes, while Trichodesmium abundance is not as negatively impacted by low P concentrations. Future studies with biomarkers differentiating between ecotypes of Crocosphaera will help determine the controls on P limitation between strains.

Nitrogen (N2) fixation is a major source of nitrogen that supports primary production in the vast oligotrophic areas of the world’s oceans. The Southwestern Pacific Ocean has been identified as a potential hotspot for N2 fixation based on empirical and modeled data. In the southwest Lagoon of New Caledonia (SLNC), high abundances of the unicellular N2-fixing cyanobacteria group A (UCYN-A), coupled with daytime N2 fixation rates associated with the < 10 µm size fraction, suggest UCYN-A may be an important diazotroph (N2-fixer) in this region. However, little is known about the seasonal variability and diversity of UCYN-A in this region. To assess this, surface waters from a 12 km transect from the mouth of the Dumbéa River to the Dumbéa Pass were sampled monthly between July, 2012 and March, 2014. UCYN-A abundances for two of the defined sublineages, UCYN-A1 and UCYN-A2, were quantified using qPCR targeting the nifH gene, and the nifH-based diversity of UCYN-A was characterized using oligotyping. Total UCYN-A abundances were dominated by the UCYN-A1 sublineage, peaked in September and October and could be predicted by a suite of nine environmental parameters. At the sublineage level, UCYN-A1 abundances could be predicted based on lower temperatures (<23C), nitrate concentrations, precipitation, and wind speed, while UCYN-A2 abundances could be predicted based on silicate, chlorophyll a concentrations, wind direction, precipitation and wind speed. Using UCYN-A nifH oligotyping, new UCYN-A sublineages were discovered, and similar environmental variables explained the relative abundances of sublineages and their associated oligotypes, with the notable exception of a UCYN-A2 oligotype (oligo43) which had relative abundance patterns distinct from the dominant UCYN-A2 oligotype (oligo3). The results support the emerging picture that UCYN-A is comprised of a diverse group of strains, with sublineages that likely have different ecological niches. By identifying environmental factors that influence the composition and abundance of UCYN-A sublineages, this study helps to explain global UCYN-A abundance patterns, and is important for understanding the significance of N2 fixation at local and global scales.

Nitrogen (N2) fixation is an important component of the nitrogen cycle, supports primary production by supplying biologically available nitrogen, particularly in oligotrophic regions of the world’s oceans where nitrogen availability often limits growth. This critical process is mediated by diazotrophs, a diverse group of organisms including both Bacteria and Archaea.

This dissertation focuses on the distribution, activity, and physiology of diazotrophs. It places a particular emphasis on the globally distributed UCYN-A/haptophyte symbiosis, which has been observed in a wide range of environments from the oligotrophic open ocean to coastal regions and colder high-latitude waters. The UCYN-A symbiosis is comprised of the N2-fixing cyanobacterial symbiont, UCYN-A, and a photosynthetic alga (haptophyte) host, Braarudosphaera bigelowii. In the first chapter, the autotrophic and phagotrophic modes of nutrition of the UCYN-A/B. bigelowii symbiosis were investigated. The second chapter investigates the range of temperatures in which the UCYN-A/B. bigelowii symbiosis can grow and fix carbon and N, with a particular focus on lower temperatures representative of high-latitude waters. Chapter three reports on a study conducted during a winter cruise through the undersampled North Pacific subtropical gyre (NPSG), specifically in the often-overlooked NPSG, which analyzes the environmental determinants influencing diazotroph distributions and abundances along a transect from Guam to the west coast of California.

In summary, this dissertation illuminates aspects of the physiology of the UCYN-A/B. bigelowii symbiosis, specifically its mixotrophic survival strategy and temperature responses. It also provides valuable insights into the biogeographic distributions of a variety of diazotrophs across an undersampled region of the vast Pacific Ocean. Taken together, this dissertation provides new information on the function and role of diazotrophs in open ocean N cycling.