High Throughput Cultivation of Microbes Daniella Nicastro and Dick McIntosh Univ. of Colorado

Cultivation Strategy

Parvularcula bermudensis gen. nov., sp. nov. Fulvimarina pelagi gen. nov., sp. nov. Croceibacter atlanticus gen. nov., sp. nov. Oceanicola granulosus gen. nov. sp. nov Robiginitalea biformata gen. nov., sp. nov Cho & Giovannoni IJSEM 53:1853 Cho & Giovannoni IJSEM 53: Cho & Giovannoni IJSEM In Press Cho & Giovannoni SAM. 26:76 Cho J.-C. et al Environ. Microbiol. 6: Lentisphaerae: novel bacterial phylum HTC Lab Results >2000 Strains > 90% do not grow on agar 23 strains in genome sequencing

HTCC1062 Cultivation Scale-up

Evolution by Gene Duplication

Median Size of Intergenic Spacers for Prokaryotic Genomes

Genome Streamlining Hypothesis Genome streamlining occurs when selection is able to act to directly reduce the amount of DNA which serves no useful function for the cell. Introns, inteins, transposons and pesudogenes are examples of "selfish DNA", which persist because their impact on cellular replication efficiency is too small for selection to act directly. This DNA may be eliminated by chance due to a general deletional bias in bacteria cells. Kimura described the relationship between population size and selection. Selection can act on a phenotype when: s > 1/(2Ne), where s is the absolute value of the change in fitness and Ne is the effective population size. Kimura described the relationship between population size and selection. Selection can act on a phenotype when: s > 1/(2Ne), where s is the absolute value of the change in fitness and Ne is the effective population size. Because of very large effective population sizes and selection to minimize the amount of N and P needed for cellular replication, selection acts efficiently against "junk" DNA in some marine microbial genomes. Because of very large effective population sizes and selection to minimize the amount of N and P needed for cellular replication, selection acts efficiently against "junk" DNA in some marine microbial genomes. Kimura, M. Evolutionary Rate at the Molecular Level. Nature 217, (1968)

The P. ubique proteorhodopsin is a proton pump that is expressed in the dark and in the light 633 nm 488 nm Light pH Dark

MKKLKLFALTAVALMGVSGVANAETTLLASDDFVGISFWLVSMALLASTAFFFIERASVP AGWRVSITVAGLVTGIAFIHYMYMRDVWVMTGESPTVYRYIDWLITVPLLMLEFYFVLAA VNKANSGIFWRLMIGTLVMLIGGYLGEAGYINTTLGFVIGMAGWFYILYEVFSGEAGKNA AKSGNKALVTAFGAMRMIVTVGWAIYPLGYVFGYMTGGMDASSLNVIYNAADFLNKIAF GLIIWAAAMSQPGRAK MALDI TOF/TOF

Sargasso Sea Microbial Observatory In situ Hybridization Cell Counts: the SAR11 Clade at BATS Carlson, Morris and Giovannoni, unpublished

P. ubique growth on seawater in the light and the dark Diel light cycle (open symbol) or in darkness (closed symbol) under high-range light intensity (680 µmol m -2 sec -1, circles) or middle-range light intensity (250 µmol m -2 sec -1, squares). Error bars, standard deviation for triplicates. No difference was observed in replicates with and without added retinal (data not shown).

Evolution Within the SAR11 Clade rRNA Hybridization Depth (Meters) Field et al., 1997 A B C

µM C Hansell and Carlson

Prokaryotic Cell Abundance (cells E8 l -1 ) Depth (m)

SAR11-IA SAR11-II Spatial Temporal Spatial and Temporal Structure of Microbial Populations at BATS: Non-metric Multidimensional Scaling of 16S tRFLPs SAR11-IB Morris et al. 2005, L&O

Freshwater (IV) Surface (IA) Spring (IB) Brackish (III) Deep Evolution Within the SAR11 Clade Surface (II) }

Distribution of 16S Genes from the Sargasso Sea WGS Data, by Clade

 Of 725,677 Sargasso sea fragments, ~264,000 have homologues to P. ubique genes (1e -20 ), and of these ~58,000 show conserved gene order.  Of these 58,000 syntigs, 95% passed the second criterion of containing only orfs with best hits to P. ubique.  Synteny is conserved: 96% of the Sargasso Sea SAR11 fragments matched the gene order of the HTCC1062 genome.Syntigs

Proteorhodopsin (Small Multidrug Resistance protein) MOSC Domain Protein (Unknown Protein) SAR11 Syntigs From Sargasso Sea in Vicinity of Proteorhodopsin Gene Ferrredoxin Thioredoxin disulfide reductase Glutathione S-transferase Suppresor Protein PR Operon ?

Rearrangements in the order of SAR11 genes in the Sargasso Sea metagenome

Comparison of the genomes of strains HTCC1062 and HTCC base pair different in 16S differ by 62 gene indels in core regions. 97.4% similarity for the genomes overall The genome of HTCC1002 is 12,298 nucleotides larger than the genome of HTCC1062. Most of the length difference is due to 31 genes inserted in HVR3 of HTCC1002, supporting the conclusion that this hypervariable region is a hotspot for the acquisition of foreign DNA by HGT % similar in nucleotide sequence in HVR2. In addition to few point mutations, the two HVR2 sequences differed by a 13 base deletion that removed one from a set of four tandem repeats within ORFan gene.

Conclusions from Analysis of the SAR11 Metagenome  The Sargasso Sea SAR11 metagenome was substantially similar to the genomes from the two coastal isolates in conserved, core regions of the genome, but differed markedly in islands of genomic variability, and at the sites of gene indels.  The largest variable genomic island was inserted between the 23S and 5S rRNA genes, and encoded genes for cell surface properties.  The variable regions contain gene duplications and deletions, are highly divergent, but show little direct evidence of origins from phage or integrons.  Random gene insertions in core regions of the genomes are common, but apparently are eliminated by selection.  Extraordinarily high allelic variation and rearrangements at operon boundaries appear to mask the conservation of many genome properties in native SAR11 populations, leading to overestimates of species diversity.

SAR11 Genome Proteomics The HTC Lab Jang Cho Kevin Vergin Mike Rappe Craig Carlson RussDesiderio Doug Barofsky Sarah Sowell Bob Morris Mick Noordeweir Noordeweir LisaBibbs Eric Mathur Martha Staples Scott Givan Jim Tripp MirceaPodar DaniellaNicastro DickMcintosh Electron Tomography Stephanie Connon Connon Rachel Parsons Craig Carlson Sargasso Sea Microbial Observatory Crew and Technicians of the RV Weatherbird II & Bermuda Atlantic Time Series Study

Our thanks to: For Supporting our Research Microbial Observatories Program

Marine Bacterioplankton SSU rRNA Gene Cluster Sequence Diversity SAR SAR11 w/freshwater clones SAR11 w/freshwater clones SAR86 w/SAR156 subcluster SAR86 w/SAR156 subcluster SAR406/Group A Marine Picophytoplankton Marine Actinobacteria Roseobacter SAR SAR SAR11Identity (conserved) b Identity (all overlapping) a Cluster/clade a Included all alignment positions for which both sequences possessed nucleotides in the individual pairwise sequence comparisons individual pairwise sequence comparisons b Included the “Lane mask” to omit ambiguous alignment positions and hypervariable regions of the SSU rRNA gene regions of the SSU rRNA gene

Evolutionary distances in the SAR11 clade are much greater than in the marine picophytoplanton clade

Prochlorococcus Venter, 2004 Conserved Properties of the SAR11 Metagenome

Comparison of HVR1 of HTCC 1002 and HTCC1062