FlowCam 8400 and FlowCam Cyano Phytoplankton Classification and Viability Staining by Imaging Flow Cytometry.

This chapter provides a protocol for a detailed evaluation of phytoplankton and nuisance cyanobacteria with the FlowCam 8400 and the FlowCam Cyano. The chapter includes (i) detailed description of the quality control of fluorescent mode of the FlowCam, (ii) detailing methods for discriminating nuisance cyanobacteria using the FlowCam Cyano, how to set up libraries and classification routines for commonly used classification reports, and (iii) detailing methods for viability staining to quantify LIVE versus DEAD phytoplankton using the FlowCam 8400.

Physiology and evolution of nitrate acquisition in Prochlorococcus.

Prochlorococcus is the numerically dominant phototroph in the oligotrophic subtropical ocean and carries out a significant fraction of marine primary productivity. Although field studies have provided evidence for nitrate uptake by Prochlorococcus, little is known about this trait because axenic cultures capable of growth on nitrate have not been available. Additionally, all previously sequenced genomes lacked the genes necessary for nitrate assimilation. Here we introduce three Prochlorococcus strains capable of growth on nitrate and analyze their physiology and genome architecture. We show that the growth of high-light (HL) adapted strains on nitrate is ∼17% slower than their growth on ammonium. By analyzing 41 Prochlorococcus genomes, we find that genes for nitrate assimilation have been gained multiple times during the evolution of this group, and can be found in at least three lineages. In low-light adapted strains, nitrate assimilation genes are located in the same genomic context as in marine Synechococcus. These genes are located elsewhere in HL adapted strains and may often exist as a stable genetic acquisition as suggested by the striking degree of similarity in the order, phylogeny and location of these genes in one HL adapted strain and a consensus assembly of environmental Prochlorococcus metagenome sequences. In another HL adapted strain, nitrate utilization genes may have been independently acquired as indicated by adjacent phage mobility elements; these genes are also duplicated with each copy detected in separate genomic islands. These results provide direct evidence for nitrate utilization by Prochlorococcus and illuminate the complex evolutionary history of this trait.

Genomes of diverse isolates of the marine cyanobacterium Prochlorococcus.

The marine cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism in the oligotrophic oceans, and a model system in marine microbial ecology. Here we report 27 new whole genome sequences (2 complete and closed; 25 of draft quality) of cultured isolates, representing five major phylogenetic clades of Prochlorococcus. The sequenced strains were isolated from diverse regions of the oceans, facilitating studies of the drivers of microbial diversity-both in the lab and in the field. To improve the utility of these genomes for comparative genomics, we also define pre-computed clusters of orthologous groups of proteins (COGs), indicating how genes are distributed among these and other publicly available Prochlorococcus genomes. These data represent a significant expansion of Prochlorococcus reference genomes that are useful for numerous applications in microbial ecology, evolution and oceanography.

Effects of phosphorus starvation versus limitation on the marine cyanobacterium Prochlorococcus†MED4 II: gene expression.

Phosphorus (P) availability drives niche differentiation in the most abundant phytoplankter in the oceans, the marine cyanobacterium Prochlorococcus. We compared the molecular response of Prochlorococcus strain MED4 to P starvation in batch culture to P-limited growth in chemostat culture. We also identified an outer membrane porin, PMM0709, which may allow transport of organic phosphorous compounds, rather than phosphate as previously suggested. The expression of three P uptake genes, pstS, the high-affinity phosphate-binding component of the phosphate transporter, phoA, an alkaline phosphatase, and porin PMM0709, were strongly upregulated (between 10- and 700-fold) under both P starvation and limitation. pstS exhibits high basal expression under P-replete conditions and is likely necessary for P uptake regardless of P availability. A P-stress regulatory gene, ptrA, was upregulated in response to both P starvation and limitation although a second regulatory gene, phoB, was not. Elevated expression levels (> 10-fold) of phoR, a P-sensing histidine kinase, were only observed under conditions of P limitation. We suggest Prochlorococcus in P-limited systems are physiologically distinct from cells subjected to abrupt P depletion. Detection of expression of both pstS and phoR in field populations will enable discernment of the present P status of Prochlorococcus in the oligotrophic oceans.

Effects of phosphorus starvation versus limitation on the marine cyanobacterium Prochlorococcus†MED4 I: uptake physiology.

Recent measurements of natural populations of the marine cyanobacterium Prochlorococcus indicate this numerically dominant phototroph assimilates phosphorus (P) at significant rates in P-limited oceanic regions. To better understand uptake capabilities of Prochlorococcus under different P stress conditions, uptake kinetic experiments were performed on Prochlorococcus MED4 grown in P-limited chemostats and batch cultures. Our results indicate that MED4 has a small cell-specific Vmax but a high specific affinity (αP ) for P, making it competitive with other marine cyanobacteria at low P concentrations. Additionally, MED4 regulates its uptake kinetics in response to P stress by significantly increasing Vmax and αP for both inorganic and organic P (PO4 and ATP). The Michaelis-Menten constant, KM , for PO4 remained constant under different P stress conditions, whereas the KM for ATP was higher when cells were stressed for PO4 , pointing to additional processes involved in uptake of ATP. MED4 cleaves the PO4 moieties from ATP, likely with a 5'-nucleotidase-like enzyme rather than alkaline phosphatase. MED4 exhibited distinct physiological differences between cells under steady-state P limitation versus those transitioning from P-replete to P-starved conditions. Thus, MED4 employs a variety of strategies to deal with changing P sources in the oceans and displays complexity in P stress acclimation and regulatory mechanisms.