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Directed Isolation of Prochlorococcus

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Directed Isolation of Prochlorococcus Directed isolation of Prochlorococcus Selected tools for the Prochlorococcus model system Berube PM*, Becker JW*, Biller SJ, Coe AC, Cubillos-Ruiz A, Ding H, Kelly L, Thompson JW, Chisholm SW Sampling within the euphotic * co-presenters Massachusetts Institute of Technology, Cambridge, MA zone of oligotrophic ocean regions • Depth specific targeting of Prochlorococcus ecotypes Isolation and characterization of vesicles from marine bacteria • Utilization of ecotype abundance data from time-series Density measurements when available 0.2 µm TFF Pellet by Obtain final gradient Tips for optimal vesicle preparations filter concentration ultracentrifugation sample purification Keep TFF feed pressure < 10 psi at all times Low-light adapted Prochlorococcus populations when concentrating samples. are underrepresented in culture collections Total Prochlorococcus (Flow Cytometry) Ecotype Counts / Flow Cytometry Counts Vesicle pellets are usually colorless, but don’t 0 6 0 6 5 4 worry, it’ll be there! 50 50 100 4 100 2 3 0 150 150 Depth [m] 2 Depth [m] -2 Resuspension of vesicles by gentle manual 200 log cells/mL 200 1 -4 Prochlorococcus Vesicle Other media components 250 250 pipetting works best; keep resuspension 0 -6 log2 ratio (ecotype/FCM) volumes to an absolute minimum. 100 W 90 W 80 W 70W 100 W 90 W 80 W 70W Pre-filtration of Tangential Flow Filtration (TFF) TEM of purified Prochlorococcus Culture Prochlorococcus vesicles Field samples require careful collection of Targeting of low-light adapted Prochlorococcus smaller density gradient fractions to separate phage from vesicles. • Sampling at ≥100m Sampling protocols are designed to capture populations of low-light DNA isolation from vesicles requires 2x TURBO • Large pore-size pre-filtration adapted Prochlorococcus due to DNase (Life Technologies) treatments to ensure their underrepresentation in removal of contaminating DNA; Turbo DNase is • Pro 2 medium additions culture collections and interest in (nitrite,ROS scavengers: much less salt sensitive than traditional DNase I particular traits characteristic of pyruvate,thiosulfate) and more suitable for seawater samples. these populations, including the production of lanthipeptides. • Growth conditions Estimating size and abundance using nanoparticle (low light: 1µE, temperature) tracking analysis (NanoSight, Malvern Instruments). Size Range of Prochlorococcus Vesicles Stability in Seawater at 25oC 0.014 108 Monitoring enrichment progress via flow cytometry 0.012 ~ 100 nm average 0.010 Low percentage of Increasing relative Prochlorococcus 0.008 chl-containing cells; abundance of cells constitute 7 abundance 10 0.006 dominated by Prochlorococcus cells >50% of the 0.004 heterotrophic bacteria enrichment 0.002 Number per mL NanoSight Reference Prochlorococcus Relative 0.000 106 Beads Vesicles 0 50 100 150 200 250 300 0 2 4 6 8 10 12 14 16 18 Biller, S.J., Schubotz, F., Roggensack, S.E., Thompson, A.W., et al. Particle diameter (nm) Time (days) 2014. Bacterial vesicles in marine ecosystems. Science 343:183-186. Integrating diverse data from the Prochlorococcus Expression patterns for COGs of interest can be examined for the diurnal cycle of Prochlorococcus and for Chlorophyll Fluorescence the response of Prochlorococcus to a variety of stressors. model system: An example using the pstS gene Forward Angle Scatter pstS expression over a daily light-dark ProPortal is an interactive data resource Here we present a case study utilizing ProPortal to cycle in Prochlorococcus MED4 10 connecting genomic and transcriptomic examine the pstS gene (encoding a high affinity Isolating cells and maintenance of axenic strains datasets. Our goal is to allow the user to PO 3- binding protein) and a cyanophage specific 4 Dilution Targeting Prochlorococcus Tips for optimizing enrichment & explore questions related to the ecology and COG (PCOG173) of unknown function. isolation of Prochlorococcus evolution of Prochlorococcus and its phage by 1 Frequently monitor enrichments via making connections between multiple types of Correlations between gene abundance and environmental Serial variables can identify COGs that are potentially under fluorescence and flow cytometry. data. In the future, we plan to add improved Dilution functionality for automated phylogenies and selective pressure. Normalized Expression Monitor light levels closely to avoid light mining of metagenome and oceanographic Cyanophage pstS Gene Abundance in 0 shock during transfers and incubation. 12 24 12 24 • 1 to 5 Prochlorococcus cells/well data. A version soon to be released will South Pacific Metagenome Libraries Clock Hour • Pyruvate or thiosulfate as ROS Diversify enrichments into multiple include a total of 41 Prochlorococcus, 15 Coastal Transition Gyre conditions (light, temperature, medium) to pstS expression during phosphate starvation of Prochlorococcus MED4 scavengers Synechococcus, and 11 single-cell genomes. Zone enrich for distinct populations. 0.3 • Organic carbon mix to easily Clusters of orthologous groups of proteins 0.2 identify heterotrophic contamination Accurately quantify cells prior to serial (COGs) derived from closely related 0.1 • Monitor visually for green wells dilution. cyanobacterial and cyanophage genomes are Normalized Abundance 0.0 Do not attempt isolations in outer wells: the foundation of ProPortal and form the fill with sterile media to slow evaporation. framework for linking diverse sets of data. Phosphate Concentration The Future ProPortal Will... Upon detection, immediately transfer green (non-cloudy) wells at multiple PCOG173 is a conserved cyanophage gene The distribution of pstS among genomes shows that it is a Automate the addition of new genomes, assignment of genes to dilutions to increase viability. cluster with unknown function. Network core gene and sometimes multicopy in host genomes; in COGs, and functional annotation of COGs and pathways. analysis of GOS metagenome reads contrast, pstS is found in many T4-like cyanophage Link RNAseq data, from both the lab and field, to COGs in order Conduct backup transfers whenever showed that PCOG173 is genomes but not other phage morphotypes. to facilitate comparisons of gene regulation among strains. possible to guard against loss. Check for purity in commonly found adjacent to Link metagenome data to COGs to examine the distribution of Frequently monitor new isolates via Cyanophage Genomes multiple test broths pstS and a phoA-like gene functional potential in different environments. fluorescence and time all transfers within in wild cyanophage Develop flexible frameworks to describe functional networks of log phase growth. populations. COGs that govern cellular functions and functional modules (e.g. photosynthesis and immune reaction to phage infection). Cyanobacterial Host Genomes LLII-III SS120 With these additions, the future ProPortal could enable users to: 0.0090 MIT9211 1 pstS copy NATL2A 1) Select a pathway of interest and view the expression of LLI 2 pstS copies NATL1A host genes in this pathway under stress conditions; BATS_4A1C3 2) Select a set of COGs of interest and examine their Clean Contaminated MIT9303 7E6 distribution across pathways and transcription under different 4C5 The 23S-16S ITS phylogeny Examination of the PCOG173 gene cluster in sequenced environmental conditions; Scale up pure isolates to larger of 11 newly isolated strains volumes and ID via sequencing of 3E4 phage genomes revealed potential regulation by PhoBR. LLIV 10D5 reveal they belong to the 3) Select host and phage COGs and examine potential effects of transcriptional regulation on metabolic pathways of the 23S-16S ITS region 8C5 low-light adapted IV (LLIV) pho 3F8 pstS interest; clade of Prochlorococcus. box 4E3 8 of these new isolates Nodes: Genes 4) Cross-reference environmental profiles (e.g. phosphate MIT9313 S-SM1 appear to produce Edges: Adjacent Gene on Read PCOG173 phoA concentration) with COG distribution. 8E2 2E3 lanthipeptides. 14G2 Henn, M.R., Sullivan, M.B., Stange-Thomann, N., Osburne, M.S., et al. 2010. Analysis of Kelly, L., Ding, H., Huang, K.H., Osburne, M.S. & Chisholm, S.W. 2013. Genetic diversity in cultured and wild Sullivan, M.B., Krastins, B., Hughes, J.L., Kelly, L., et al. 2009. The genome and structural proteome of an ocean 4C3 high-throughput sequencing and annotation strategies for phage genomes. PLoS One 5:e9083. marine cyanomyoviruses reveals phosphorus stress as a strong selective agent. ISME J. 7:1827-1841. siphovirus: a new window into the cyanobacterial 'mobilome'. Environ. Microbiol. 11:2935-2951. WH8102 Marine Kelly, L., Huang, K.H., Ding, H. & Chisholm, S.W. 2011. ProPortal: a resource for integrated systems Kettler, G.C., Martiny, A.C., Huang, K., Zucker, J., et al. 2007. Patterns and implications of gene gain and loss Sullivan, M.B., Huang, K.H., Ignacio-Espinoza, J.C., Berlin, A.M., et al. Genomic analysis of oceanic cyanobacterial 7803 biology of Prochlorococcus and its phage. Nucl. Acids Res. 40:D632-D640. in the evolution of Prochlorococcus. PLoS Genet. 3:e231. myoviruses compared with T4-like myoviruses from diverse hosts and environments. Environ. Microbiol. 12:3035-3056. Synechococcus.
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