High Throughput Cultivation of Microbes

Daniella Nicastro and Dick McIntosh

Univ. of Colorado

Cultivation StrategyCultivation Strategy

Parvularcula bermudensis Parvularcula bermudensis gen. nov., sp. nov.gen. nov., sp. nov.

Fulvimarina pelagi Fulvimarina pelagi gen. nov., sp. nov.gen. nov., sp. nov.

Croceibacter atlanticus Croceibacter atlanticus gen. nov., gen. nov., sp. nov.sp. nov.

Oceanicola granulosus Oceanicola granulosus gen. nov. sp. novgen. nov. sp. nov

Robiginitalea biformata Robiginitalea biformata gen. nov., sp. novgen. nov., sp. nov

Cho & Giovannoni. 2003. IJSEM 53:1853

Cho & Giovannoni. 2003. IJSEM 53: 1031-1036

Cho & Giovannoni. 2004. IJSEM In Press

Cho & Giovannoni. 2004. IJSEM In Press Cho & Giovannoni. 2003. SAM. 26:76

Cho J.-C. et al. 2004. Environ. Microbiol. 6: 611-621

Lentisphaerae: novel bacterial phylumLentisphaerae: novel bacterial phylum

HTC Lab ResultsHTC Lab Results>2000 Strains

> 90% do not grow on agar23 strains in genome sequencing

HTCC1062 Cultivation Scale-upHTCC1062 Cultivation Scale-up

Genome Size Vs. Gene Number for Prokaryotic Genomes

100000

1000000

10000000

100 1000 10000

Number of genes

Genome size

Free living

Host associated

Obligates

Pelagibacter

Prochlorococcus

MMG

Table 1. Metabolic pathways in Pelagibacter . Pathway Prediction* Glycolysis ? TCA cycle + Glyoxylate shunt + Respiration + Pentose phosphate cycle + Fatty acid biosynthesis + Cell wall biosynthesis + Amino acid biosynthesis (20) + Heme biosynthesis + Ubiquinone + Nicotinate and nicotinamide + Folate + Riboflavin + Pantothenate - B6 - Thiamine - Biotin - B12 - * +, present; -, absent; ?, uncertain

Evolution by Gene DuplicationEvolution by Gene Duplication

Median Size of Intergenic Spacers for Prokaryotic GenomesMedian Size of Intergenic Spacers for Prokaryotic Genomes

Genome Streamlining Genome Streamlining HypothesisHypothesis

• Genome streamlining occurs when selection is able to act to directly reduce the 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 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 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 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.eliminated by chance due to a general deletional bias in bacteria cells.

• Kimura described the relationship between population size and selection. Selection Kimura described the relationship between population size and selection. Selection can act on a phenotype when: can act on a phenotype when: s > 1/(2Ne)s > 1/(2Ne), where , where ss is the absolute value of the change is the absolute value of the change in fitness and in fitness and Ne Ne is the effective population size.is the effective population size.

• Because of very large effective population sizes and selection to minimize the amount 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 of N and P needed for cellular replication, selection acts efficiently against "junk" DNA in some marine microbial genomes.some marine microbial genomes.

Kimura, M. Evolutionary Rate at the Molecular Level. Nature 217, 624-626 (1968)

The P. ubiqueThe P. ubique proteorhodopsin is a proton pump proteorhodopsin is a proton pump that is expressed in the dark and in the lightthat is expressed in the dark and in the light

633 nm

488 nm

LightLight

pHpH

DarkDark

MKKLKLFALTAVALMGVSGVANAETTLLASDDFVGISFWLVSMALLASTAFFFIERASVPAGWRVSITVAGLVTGIAFIHYMYMRDVWVMTGESPTVYRYIDWLITVPLLMLEFYFVLAAVNKANSGIFWRLMIGTLVMLIGGYLGEAGYINTTLGFVIGMAGWFYILYEVFSGEAGKNAAKSGNKALVTAFGAMRMIVTVGWAIYPLGYVFGYMTGGMDASSLNVIYNAADFLNKIAFGLIIWAAAMSQPGRAK

MALDI TOF/TOFMALDI TOF/TOF

Sargasso Sea Microbial Observatory

In situ Hybridization Cell Counts: the SAR11 Clade at BATS

Carlson, Morris and Giovannoni, unpublished

P. ubiqueP. ubique growth on seawater in the light and the dark 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 CladeEvolution Within the SAR11 Clade

B

B

B

B

B

B

B

J

J

J

J

J

J

J

H

H

H

H

H

H

H250

200

160

120

80

40

0

0 0.04 0.08 0.12 0.16

B Surface Clade

J Deep Clade

H Surface&Deep Clade

rRNA Hybridization

Depth (Meters)

Field et al., 1997

AA

BB

CC

µM C

Hansell and Carlson

0

50

100

150

200

250

300

12

10

8

6

4

2

091 92 93 94 95 96 97 98 99 00

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

SAR11-IASAR11-II

Spatial

Tem

po

ral

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)Freshwater (IV)

Surface (IA)Surface (IA)

Spring (IB)Spring (IB)

Brackish Brackish (III)(III)

DeepDeep

Evolution Within the SAR11 CladeEvolution Within the SAR11 Clade

SurfaceSurface(II)(II)}

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

SyntigsSyntigs

Proteorhodopsin

(Small Multidrug Resistance

protein)

MOSC Domain Protein

(Unknown Protein)

SAR11 Syntigs From Sargasso Sea in Vicinity of Proteorhodopsin GeneSAR11 Syntigs From Sargasso Sea in Vicinity of Proteorhodopsin Gene

FerrredoxinFerrredoxin

Thioredoxin disulfide reductaseThioredoxin disulfide reductase

Glutathione S-transferaseGlutathione S-transferase

Suppresor ProteinSuppresor Protein

PR Operon ?PR Operon ?

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

Comparison of the genomes of strains HTCC1062 and HTCC1002

• 1 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.

• 99.96% 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 Conclusions from Analysis of the SAR11 MetagenomeMetagenome

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 LabJang ChoJang Cho Kevin VerginKevin Vergin

Mike RappeMike Rappe

Craig CarlsonCraig Carlson

RussRussDesiderioDesiderio

Doug BarofskyDoug Barofsky

Sarah SowellSarah Sowell

Bob MorrisBob Morris

MickMick NoordeweirNoordeweir

Lisa Lisa BibbsBibbs

Eric MathurEric Mathur

Martha StaplesMartha Staples

Scott GivanScott Givan

Jim TrippJim Tripp

MirceaMirceaPodarPodar

DaniellaDaniellaNicastroNicastro

DickDickMcintoshMcintosh

Electron Electron TomographyTomography

StephanieStephanie ConnonConnon

Rachel ParsonsRachel Parsons

Craig CarlsonCraig Carlson

Sargasso SeaSargasso SeaMicrobial ObservatoryMicrobial Observatory

Crew and Technicians of the RV Weatherbird II & Crew and Technicians of the RV Weatherbird II & Bermuda Atlantic Time Series StudyBermuda Atlantic Time Series Study

Our thanks to:Our thanks to:

For Supporting our ResearchFor Supporting our Research

Microbial Observatories ProgramMicrobial Observatories Program

Marine Bacterioplankton SSU rRNA Gene Marine Bacterioplankton SSU rRNA Gene Cluster Sequence DiversityCluster Sequence Diversity

0.9220.9220.8970.897SAR324SAR324

0.8870.8870.8720.872 SAR11 w/freshwater clonesSAR11 w/freshwater clones

0.8750.8750.8450.845 SAR86 w/SAR156 subclusterSAR86 w/SAR156 subcluster

0.9680.9680.9460.946SAR406/Group ASAR406/Group A0.9690.9690.9620.962Marine PicophytoplanktonMarine Picophytoplankton0.9660.9660.9400.940Marine ActinobacteriaMarine Actinobacteria

0.9040.9040.8840.884RoseobacterRoseobacter0.9080.9080.8970.897SAR116SAR116

0.9450.9450.9350.935SAR86SAR86

0.9000.9000.8890.889SAR11SAR11

IdentityIdentity

(conserved)(conserved)bb

IdentityIdentity

(all overlapping)(all overlapping)aaCluster/cladeCluster/clade

aaIncluded all alignment positions for which both sequences Included all alignment positions for which both sequences possessed nucleotides in thepossessed nucleotides in the individual pairwise sequence comparisonsindividual pairwise sequence comparisonsbbIncluded the “Lane mask” to omit ambiguous alignment positions Included the “Lane mask” to omit ambiguous alignment positions and hypervariableand hypervariable regions of the SSU rRNA generegions of the SSU rRNA gene

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

Table 3. Regulatory Proteins Gene Protein Class Function C134_0606 32 sigma factor heat shock transcrip tion

factor C134_0037 70 sigma factor vegetative transcription factor C134_0089/C134_0088 envZ/OmpR* sensor/regulator osmolarity C134_0198/C134_0199 Unidentified* sensor unknown C134_0946/C134_0948 ntrY/ntrX sensor/regulator N regulation C134_0447 RegB sensor redox C134_0203 RegA regulator redox C134_0363 Unidentified regulator unknown C134_1180/C134_1174 PhoR/PhoB sensor/regulator Activates high-affinity

phosphate uptake C134_0382 Fur Negative regulator Iron uptake

C134_0516 Unidentified ? regulation of amino-acid metabolism

C134_0741 sufD ? regulation of nitrogen and sulphur utilization

C134_0738 Unidentified ? regulation of nitrogen and sulphur utilization

C134_0297 PhoE ? regulation of phosphate utilization

C134_1135 Unidentified ? regulation of C-compound and carbohydrate utilization

C134_0824 recX ? recombination and DNA repair

C134_0423 Unidentified ? transcriptional control C134_0138 MarR family ? transcriptional control C134_0064 Unidentified ? transcriptional control C134_0087 petP ? transcriptional control C134_0047 Unidentified ? transcriptional control C134_0273 Unidentified ? transcriptional control C134_0860 Unidentified ? transcriptional control C134_0974 Unidentified ? transcriptional control C134_0964 Unidentified ? transcriptional control C134_1242 Unidentified ? transcriptional control C134_1034 MerR family ? transcriptional control C134_0958 Unidentified ? transcriptional control C134_0768 NAGC-like ? transcriptional control C134_1243 Unidentified ? transcriptional control C134_1175 PhoU ? transcriptional control C134_1248 DNA-binding ? transcriptional control

C134_0881 clpX ? protein targeting, sorting and translocation

*sensor and regulatory element adjacent on same strand. ** sensor and regulatory element on opposite strands.

ProchlorococcusProchlorococcus

Venter, 2004Venter, 2004

Conserved Properties of the SAR11 MetagenomeConserved Properties of the SAR11 Metagenome

Comparison of HVR1 of HTCC 1002 and HTCC1062Comparison of HVR1 of HTCC 1002 and HTCC1062