Sub-micron picoplankton shape, orientation, and internal structure combined to preferentially amplify the forward scatter.

Compelling evidence is presented that sub-micron picoplankton shape, internal structure and orientation in combination leads to a disproportionate enhancement of differential forward scatter compared with differential side scatter when analyzed with a flow cytometer. Theoretical evidence is provided which results in an order of magnitude amplification in the forward scatter direction, with little or no change in the side scatter: this discounts the possibility of "doublets" caused by multiple particles simultaneously present in the laser beam. Observational evidence from progressively finer filtered seawater samples shows up to three orders of magnitude enhancement in the forward scatter direction and sizes of Prochlorococcus close to that reported in the literature (0.61 ± 0.17 µm). It therefore seems likely that flow cytometrically observed "bi-modal size distributions" of Prochlorococcus are instead the manifestation of intra-population differences in shape (spherical - prolate with preferential alignment) and internal structure (homogenous - heterogenous).

Marine picoplankton size distribution and optical property contrasts throughout the Atlantic Ocean revealed using flow cytometry.

Depth-resolved flow cytometric observations have been used to determine the size distribution and refractive index (RI) of picoplankton throughout the Atlantic Ocean. Prochlorococcus frequently showed double size distribution peaks centered on ${0.75 \pm 0.25}$0.75±0.25 and ${1.75 \pm 0.25}\,\,{\rm \unicode{x00B5}{\rm m}}$1.75±0.25µm; the smallest peak diameters were ${\le}{0.65}\,\,{\rm \unicode{x00B5}{\rm m}}$≤0.65µm in the equatorial upwelling with larger cells (${\sim}{0.95}\,\,{\rm \unicode{x00B5}{\rm m}}$∼0.95µm) in the surface layers of the tropical gyres. Synechococcus was strongly monodispersed: the smallest (${\sim}{1.5}\,\,{\rm \unicode{x00B5}{\rm m}}$∼1.5µm) and largest cells (${\sim}{2.25{-}2.50}\,\,{\rm \unicode{x00B5}{\rm m}}$∼2.25-2.50µm) were encountered in the lowest and highest abundance regions, respectively. Typical RI for Prochlorococcus was found to be ${\sim}{1.06}$∼1.06, whereas for Synechococcus surface RI varied between 1.04-1.08 at high and low abundances, respectively.

Picophytoplankton size and biomass around equatorial eastern Indian Ocean.

The cellular size and biomass of picophytoplankton were studied by flow cytometer during spring monsoon (March-May of 2015) in equatorial eastern Indian Ocean. We established an empirical relationship between forward scatter and cellular size to address the size and biomass of picophytoplankton. Results indicated that mean cell diameter of Prochlorococcus (0.60 µm) was the smallest, and then followed by Synechococcus (0.98 µm) and picoeukaryotic phytoplankton (1.05 µm). Thereafter, the biomass converted by abundance reached 0.64 µg·C·L-1 for Prochlorococcus, 0.34 µg·C·L-1 for Synechococcus, and 0.20 µg·C·L-1 for picoeukaryotic phytoplankton. Additionally, the distinct biomass contribution of picophytoplankton appeared to be affected by abundance, but not changes in cellular size. Vertically, the cellular sizes of picophytoplankton were remarkably small in upper waters, which was predominantly controlled by the nutrient availability. In contrast, they were larger in deeper waters, which was primarily attributed to the combined effects of low temperature and reduced light availability. Spatially, under the influence of high nutrient concentration induced by the different circulations and coastal upwelling, slightly high carbon biomass of picophytoplankton was observed around the coastal zones of Sri Lanka island and Sumatra, as well as the southern Bay of Bengal.

Biophysical clocks face a trade-off between internal and external noise resistance.

Many organisms use free running circadian clocks to anticipate the day night cycle. However, others organisms use simple stimulus-response strategies ('hourglass clocks') and it is not clear when such strategies are sufficient or even preferable to free running clocks. Here, we find that free running clocks, such as those found in the cyanobacterium Synechococcus elongatus and humans, can efficiently project out light intensity fluctuations due to weather patterns ('external noise') by exploiting their limit cycle attractor. However, such limit cycles are necessarily vulnerable to 'internal noise'. Hence, at sufficiently high internal noise, point attractor-based 'hourglass' clocks, such as those found in a smaller cyanobacterium with low protein copy number, Prochlorococcus marinus, can outperform free running clocks. By interpolating between these two regimes in a diverse range of oscillators drawn from across biology, we demonstrate biochemical clock architectures that are best suited to different relative strengths of external and internal noise.

Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles.

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.Single-cell genomics can be used to study uncultured microorganisms. Here, Stepanauskas et al. present a method combining improved multiple displacement amplification and FACS, to obtain genomic sequences and cell size information from uncultivated microbial cells and viral particles in environmental samples.

Prochlorococcus.

Move over plants-make way for tiny Prochlorococcus, the smallest and most abundant photosynthetic cell on earth! Penny Chisholm tells us all about this powerhouse marine bacterium.

The allometry of the smallest: superlinear scaling of microbial metabolic rates in the Atlantic Ocean.

Prokaryotic planktonic organisms are small in size but largely relevant in marine biogeochemical cycles. Due to their reduced size range (0.2 to 1 µm in diameter), the effects of cell size on their metabolism have been hardly considered and are usually not examined in field studies. Here, we show the results of size-fractionated experiments of marine microbial respiration rate along a latitudinal transect in the Atlantic Ocean. The scaling exponents obtained from the power relationship between respiration rate and size were significantly higher than one. This superlinearity was ubiquitous across the latitudinal transect but its value was not universal revealing a strong albeit heterogeneous effect of cell size on microbial metabolism. Our results suggest that the latitudinal differences observed are the combined result of changes in cell size and composition between functional groups within prokaryotes. Communities where the largest size fraction was dominated by prokaryotic cyanobacteria, especially Prochlorococcus, have lower allometric exponents. We hypothesize that these larger, more complex prokaryotes fall close to the evolutionary transition between prokaryotes and protists, in a range where surface area starts to constrain metabolism and, hence, are expected to follow a scaling closer to linearity.

An improved protocol for flow cytometry analysis of phytoplankton cultures and natural samples.

Preservation of cells, choice of fixative, storage, and thawing conditions are recurrent issues for the analysis of phytoplankton by flow cytometry. We examined the effects of addition of the surfactant Pluronic F68 to glutaraldehyde-fixed photosynthetic organisms in cultures and natural samples. In particular, we examined cell losses and modifications of side scatter (a proxy of cell size) and fluorescence of natural pigments. We found that different marine phytoplankton species react differently to the action of Pluronic F68. In particular, photosynthetic prokaryotes are less sensitive than eukaryotes. Observed cell losses may result from cell lysis or from cell adhesion to the walls of plastic tubes that are commonly used for flow cytometry analysis. The addition of the surfactant, Pluronic F68, has a positive effect on cells for long-term storage. We recommend to modify current protocols for preservation of natural marine planktonic samples, by fixing them with glutaraldehyde 0.25% (final concentration) and adding Pluronic F68 at a final concentration of 0.01% in the samples before preservation. Pluronic F68 also appears effective for preserving samples without fixation for subsequent sorting, e.g. for molecular biology analyses. © 2014 International Society for Advancement of Cytometry.

In situ activity of a dominant Prochlorococcus ecotype (eHL-II) from rRNA content and cell size.

In the open ocean genetically diverse clades of the unicellular cyanobacteria Prochlorococcus are biogeographically structured along environmental gradients, yet little is known about their in situ activity. To address this gap, here we use the numerically dominant Prochlorococcus clade eHL-II (eMIT9312) as a model organism to develop and apply a method to examine their in situ activity using rRNA content and cell size as metrics of cellular physiology. For two representative isolates (MIT9312 and MIT9215) rRNA cell(-1) increases linearly with specific growth rate but is anticorrelated with cell size indicated by flow cytometrically measured (SSC). Although each strain has a unique relationship between cellular rRNA (or cell size) and growth rate, both strains have the same strong positive correlation between rRNA cell(-1) SSC(-1) and growth rate. We field test this approach and observe distinct patterns of eHL-II clade specific activity (rRNA cell(-1) SSC(-1)) with depth that are consistent with patterns of photosynthetic rates. This molecular technique provides unique insight into the ecology of Prochlorococcus and could potentially be expanded to include other microbes to unravel the ecological and biogeochemical contributions of genetically distinct marine side scatter microbes.

Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus.

The Cyanobacteria Prochlorococcus and Synechococcus account for a substantial fraction of marine primary production. Here, we present quantitative niche models for these lineages that assess present and future global abundances and distributions. These niche models are the result of neural network, nonparametric, and parametric analyses, and they rely on >35,000 discrete observations from all major ocean regions. The models assess cell abundance based on temperature and photosynthetically active radiation, but the individual responses to these environmental variables differ for each lineage. The models estimate global biogeographic patterns and seasonal variability of cell abundance, with maxima in the warm oligotrophic gyres of the Indian and the western Pacific Oceans and minima at higher latitudes. The annual mean global abundances of Prochlorococcus and Synechococcus are 2.9 ± 0.1 × 10(27) and 7.0 ± 0.3 × 10(26) cells, respectively. Using projections of sea surface temperature as a result of increased concentration of greenhouse gases at the end of the 21st century, our niche models projected increases in cell numbers of 29% and 14% for Prochlorococcus and Synechococcus, respectively. The changes are geographically uneven but include an increase in area. Thus, our global niche models suggest that oceanic microbial communities will experience complex changes as a result of projected future climate conditions. Because of the high abundances and contributions to primary production of Prochlorococcus and Synechococcus, these changes may have large impacts on ocean ecosystems and biogeochemical cycles.

Ultraviolet stress delays chromosome replication in light/dark synchronized cells of the marine cyanobacterium Prochlorococcus marinus PCC9511.

BACKGROUND: The marine cyanobacterium Prochlorococcus is very abundant in warm, nutrient-poor oceanic areas. The upper mixed layer of oceans is populated by high light-adapted Prochlorococcus ecotypes, which despite their tiny genome (approximately 1.7 Mb) seem to have developed efficient strategies to cope with stressful levels of photosynthetically active and ultraviolet (UV) radiation. At a molecular level, little is known yet about how such minimalist microorganisms manage to sustain high growth rates and avoid potentially detrimental, UV-induced mutations to their DNA. To address this question, we studied the cell cycle dynamics of P. marinus PCC9511 cells grown under high fluxes of visible light in the presence or absence of UV radiation. Near natural light-dark cycles of both light sources were obtained using a custom-designed illumination system (cyclostat). Expression patterns of key DNA synthesis and repair, cell division, and clock genes were analyzed in order to decipher molecular mechanisms of adaptation to UV radiation. RESULTS: The cell cycle of P. marinus PCC9511 was strongly synchronized by the day-night cycle. The most conspicuous response of cells to UV radiation was a delay in chromosome replication, with a peak of DNA synthesis shifted about 2 h into the dark period. This delay was seemingly linked to a strong downregulation of genes governing DNA replication (dnaA) and cell division (ftsZ, sepF), whereas most genes involved in DNA repair (such as recA, phrA, uvrA, ruvC, umuC) were already activated under high visible light and their expression levels were only slightly affected by additional UV exposure. CONCLUSIONS: Prochlorococcus cells modified the timing of the S phase in response to UV exposure, therefore reducing the risk that mutations would occur during this particularly sensitive stage of the cell cycle. We identified several possible explanations for the observed timeshift. Among these, the sharp decrease in transcript levels of the dnaA gene, encoding the DNA replication initiator protein, is sufficient by itself to explain this response, since DNA synthesis starts only when the cellular concentration of DnaA reaches a critical threshold. However, the observed response likely results from a more complex combination of UV-altered biological processes.

Choreography of the transcriptome, photophysiology, and cell cycle of a minimal photoautotroph, prochlorococcus.

The marine cyanobacterium Prochlorococcus MED4 has the smallest genome and cell size of all known photosynthetic organisms. Like all phototrophs at temperate latitudes, it experiences predictable daily variation in available light energy which leads to temporal regulation and partitioning of key cellular processes. To better understand the tempo and choreography of this minimal phototroph, we studied the entire transcriptome of the cell over a simulated daily light-dark cycle, and placed it in the context of diagnostic physiological and cell cycle parameters. All cells in the culture progressed through their cell cycles in synchrony, thus ensuring that our measurements reflected the behavior of individual cells. Ninety percent of the annotated genes were expressed, and 80% had cyclic expression over the diel cycle. For most genes, expression peaked near sunrise or sunset, although more subtle phasing of gene expression was also evident. Periodicities of the transcripts of genes involved in physiological processes such as in cell cycle progression, photosynthesis, and phosphorus metabolism tracked the timing of these activities relative to the light-dark cycle. Furthermore, the transitions between photosynthesis during the day and catabolic consumption of energy reserves at night- metabolic processes that share some of the same enzymes--appear to be tightly choreographed at the level of RNA expression. In-depth investigation of these patterns identified potential regulatory proteins involved in balancing these opposing pathways. Finally, while this analysis has not helped resolve how a cell with so little regulatory capacity, and a 'deficient' circadian mechanism, aligns its cell cycle and metabolism so tightly to a light-dark cycle, it does provide us with a valuable framework upon which to build when the Prochlorococcus proteome and metabolome become available.