Synechococcus OS-B', a thermophilic unicellular cyanobacterium, recently isolated from the microbial mats in Octopus Spring (Yellowstone National Park), induces a suite of genes, including phosphatases and transporters, in response to phosphorus (P) starvation. Here we describe two different approaches to examine the ability of Synechococcus OS-B' to synthesize and break down polyphosphate (poly P), a key storage compound in many prokaryotes. First, we developed a transformation protocol to create mutants in the polyphosphate kinase (ppk), the major enzyme responsible for the synthesis of poly P. The ppk mutant exhibited a pleiotropic phenotype with defects in poly P accumulation, aberrant levels of Pho regulon transcripts, growth defects, and changes in cell size and exopolysaccharide levels, among others. Second, we measured transcripts of ppk and ppx (encoding the polyphosphatase) directly from mat samples and found that the levels varied dramatically over a diel cycle. We also used Western blot analysis to quantify levels of PPK and PPX and found that these enzymes differentially accumulated during the diel cycle. Levels of polyphosphate kinase peaked at night, while polyphosphatase levels were highest during the early morning hours. We hypothesize that the opposing activities of these two enzymes allow cells to store and utilize poly P to optimize growth over a diel cycle.

Endosymbiosis is a relationship between two organisms wherein one cell resides inside the other. This affiliation, when stable and beneficial for the 'host' cell, can result in massive genetic innovation with the foremost examples being the evolution of eukaryotic organelles, the mitochondria and plastids. Despite its critical evolutionary role, there is limited knowledge about how endosymbiosis is initially established and how host-endosymbiont biology is integrated. Here, we explore this issue, using as our model the rhizarian amoeba Paulinella, which represents an independent case of primary plastid origin that occurred c. 120 million yr ago. We propose the 'chassis and engine' model that provides a theoretical framework for understanding primary plastid endosymbiosis, potentially explaining why it is so rare.

The emergent behaviors of communities of genotypically identical cells cannot be easily predicted from the behaviors of individual cells. In many cases, it is thought that direct cell-cell communication plays a critical role in the transition from individual to community behaviors. In the unicellular photosynthetic cyanobacterium Synechocystis sp. PCC 6803, individual cells exhibit light-directed motility ("phototaxis'') over surfaces, resulting in the emergence of dynamic spatial organization of multicellular communities. To probe this striking community behavior, we carried out time-lapse video microscopy coupled with quantitative analysis of single-cell dynamics under varying light conditions. These analyses suggest that cells secrete an extracellular substance that modifies the physical properties of the substrate, leading to enhanced motility and the ability for groups of cells to passively guide one another. We developed a biophysical model that demonstrates that this form of indirect, surface-based communication is sufficient to create distinct motile groups whose shape, velocity, and dynamics qualitatively match our experimental observations, even in the absence of direct cellular interactions or changes in single-cell behavior. Our computational analysis of the predicted community behavior, across a matrix of cellular concentrations and light biases, demonstrates that spatial patterning follows robust scaling laws and provides a useful resource for the generation of testable hypotheses regarding phototactic behavior. In addition, we predict that degradation of the surface modification may account for the secondary patterns occasionally observed after the initial formation of a community structure. Taken together, our modeling and experiments provide a framework to show that the emergent spatial organization of phototactic communities requires modification of the substrate, and this form of surface-based communication could provide insight into the behavior of a wide array of biological communities.

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the similar to 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

Background: Nucleomorphs are remnants of secondary endosymbiotic events between two eukaryote cells wherein the endosymbiont has retained its eukaryotic nucleus. Nucleomorphs have evolved at least twice independently, in chlorarachniophytes and cryptophytes, yet they have converged on a remarkably similar genomic architecture, characterized by the most extreme compression and miniaturization among all known eukaryotic genomes. Previous computational studies have suggested that nucleomorph chromatin likely exhibits a number of divergent features.

Signal transduction in bacteria is complex, ranging across scales from molecular signal detectors and effectors to cellular and community responses to stimuli. The unicellular, photosynthetic cyanobacterium Synechocystis sp. PCC6803 transduces a light stimulus into directional movement known as phototaxis. This response occurs via a biased random walk toward or away from a directional light source, which is sensed by intracellular photoreceptors and mediated by Type IV pili. It is unknown how quickly cells can respond to changes in the presence or directionality of light, or how photoreceptors affect single-cell motility behavior. In this study, we use time-lapse microscopy coupled with quantitative single-cell tracking to investigate the timescale of the cellular response to various light conditions and to characterize the contribution of the photoreceptor TaxD1 (PixJ1) to phototaxis. We first demonstrate that a community of cells exhibits both spatial and population heterogeneity in its phototactic response. We then show that individual cells respond within minutes to changes in light conditions, and that movement directionality is conferred only by the current light directionality, rather than by a long-term memory of previous conditions. Our measurements indicate that motility bias likely results from the polarization of pilus activity, yielding variable levels of movement in different directions. Experiments with a photoreceptor (taxD1) mutant suggest a supplementary role of TaxD1 in enhancing movement directionality, in addition to its previously identified role in promoting positive phototaxis. Motivated by the behavior of the taxD1 mutant, we demonstrate using a reaction-diffusion model that diffusion anisotropy is sufficient to produce the observed changes in the pattern of collective motility. Taken together, our results establish that single-cell tracking can be used to determine the factors that affect motility bias, which can then be coupled with biophysical simulations to connect changes in motility behaviors at the cellular scale with group dynamics.

Advances in sequencing technology have allowed for the study of complex and previously unexplored microbial and viral populations; however, linking host-phage partners using in silico techniques has been challenging. Here, we describe the flow-through for creation of a virome, and its subsequent analysis with the viral assembly and analysis module "Viritas," which we have recently developed. This module allows for binning of contigs based on tetranucleotide frequencies, putative phage-host partner identification by CRISPR spacer matching, and identification of ORFs.

Organelle positioning in cells is associated with various metabolic functions and signaling in unicellular organisms. Specifically, the microalga Chlamydomonas reinhardtii repositions its mitochondria, depending on the levels of inorganic carbon. Mitochondria are typically randomly distributed in the Chlamydomonas cytoplasm, but relocate toward the cell periphery at low inorganic carbon levels. This mitochondrial relocation is linked with the carbon-concentrating mechanism, but its significance is not yet thoroughly understood. A genotypic understanding of this relocation would require a high-throughput method to isolate rare mutant cells not exhibiting this relocation. However, this task is technically challenging due to the complex intracellular morphological difference between mutant and wild-type cells, rendering conventional non-image-based high-event-rate methods unsuitable. Here, we report our demonstration of intelligent image-activated cell sorting by mitochondrial localization. Specifically, we applied an intelligent image-activated cell sorting system to sort for C. reinhardtii cells displaying no mitochondrial relocation. We trained a convolutional neural network (CNN) to distinguish the cell types based on the complex morphology of their mitochondria. The CNN was employed to perform image-activated sorting for the mutant cell type at 180 events per second, which is 1-2 orders of magnitude faster than automated microscopy with robotic pipetting, resulting in an enhancement of the concentration from 5% to 56.5% corresponding to an enrichment factor of 11.3. These results show the potential of image-activated cell sorting for connecting genotype-phenotype relations for rare-cell populations, which require a high throughput and could lead to a better understanding of metabolic functions in cells.

Extensive fine-scale genetic diversity is found in many microbial species across varied environments, but for most, the evolutionary scenarios that generate the observed variation remain unclear. Deep sequencing of a thermophilic cyanobacterial population and analysis of the statistics of synonymous single-nucleotide polymorphisms revealed a high rate of homologous recombination and departures from neutral drift consistent with the effects of genetic hitchhiking. A sequenced isolate genome resembled an unlinked random mixture of the allelic diversity at the sampled loci. These observations suggested a quasisexual microbial population that occupies a broad ecological niche, with selection driving frequencies of alleles rather than whole genomes.