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High Throughput Cultivation of
Microbes
Daniella Nicastro and
Dick McIntosh
Univ. of Colorado
Cultivation Strategy
HTC Lab Results
>2000 Strains
> 90% do not grow on agar
23 strains in genome sequencing
Lentisphaerae: novel bacterial phylum
Oceanicola granulosus gen. nov. sp. nov
Cho J.-C. et al. 2004. Environ. Microbiol. 6: 611-621
Parvularcula bermudensis gen. nov., sp. nov.
Cho & Giovannoni. 2004. IJSEM In Press
Fulvimarina pelagi
gen. nov., sp. nov.
Cho & Giovannoni. 2003. IJSEM 53: 1031-1036
Robiginitalea biformata gen. nov., sp. nov
Cho & Giovannoni. 2003.
IJSEM 53:1853
Croceibacter atlanticus gen. nov.,
sp. nov.
Cho & Giovannoni. 2004. IJSEM In Press
Cho & Giovannoni. 2003. SAM. 26:76
HTCC1062 Cultivation Scale-up
Genome Size Vs. Gene Number for Prokaryotic Genomes
10000000
Genome size
Free living
Host associated
1000000
Obligates
Pelagibacter
Prochlorococcus
MMG
100000
100
1000
Number of genes
10000
Table 1. Metabo lic pat hways 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
+
Ubiquinon e
+
Nicotinate and nicotinamide
+
Folate
+
Riboflavin
+
Pantothenate
B6
Thiamine
Biotin
B12
* +, present; -, absent; ?, uncertain
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.
• 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, 624-626 (1968)
The P. ubique proteorhodopsin is a proton pump
that is expressed in the dark and in the light
Light
633 nm
488 nm
pH
Dark
MALDI TOF/TOF
MKKLKLFALTAVALMGVSGVANAETTLLASDDFVGISFWLVSMALLASTAFFFIERASVPAGWRVS
ITVAGLVTGIAFIHYMYMRDVWVMTGESPTVYRYIDWLITVPLLMLEFYFVLAAVNKANSGIFWRL
MIGTLVMLIGGYLGEAGYINTTLGFVIGMAGWFYILYEVFSGEAGKNAAKSGNKALVTAFGAMRMI
VTVGWAIYPLGYVFGYMTGGMDASSLNVIYNAADFLNKIAFGLIIWAAAMSQPGRAK
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
A
Depth (Meters)
0
HJ
40
H
J
80
H
120
H
160
0
J
B
B
B
J
BH
H
B
J
H
B
J
H
200
250
B
B
J
B
0.04
J
0.08
0.12
0.16
rRNA Hybridization
Field et al., 1997
C
Surface Clade
Deep Clade
Surface&Deep Cla
µM C
Hansell and Carlson
Prokaryotic Cell Abundance (cells E8 l-1)
Depth (m)
0
12
50
10
100
8
150
6
200
4
250
2
300
0
91
92
93
94
95
96
97
98
99
00
Spatial and Temporal Structure of Microbial Populations at BATS:
Non-metric Multidimensional Scaling of 16S tRFLPs
Temporal
SAR11-IA
SAR11-II
SAR11-IB
Spatial
Morris et al. 2005, L&O
Evolution Within the SAR11 Clade
Surface (IA)
Spring (IB)
Surface
Deep
Brackish
(III)
Freshwater (IV)
}
(II)
Distribution of 16S Genes from the Sargasso Sea WGS Data, by Clade
Syntigs



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.
SAR11 Syntigs From Sargasso Sea in Vicinity of Proteorhodopsin Gene
Proteorhodopsin
Ferrredoxin
Thioredoxin disulfide reductase
Glutathione S-transferase
MOSC Domain Protein
Suppresor Protein
(Small Multidrug
Resistance
protein)
(Unknown Protein)
PR Operon ?
Rearrangements in the order of SAR11 genes in the Sargasso Sea metagenom
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 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

Proteomics
SAR11
Genome
Doug Barofsky
The HTC Lab
Jang Cho
Mick
Jim Tripp Noordeweir
Craig Carlson
Russ
Desiderio
Sargasso Sea
Microbial Observatory
Rachel Parsons
Scott Givan
Kevin Vergin
Martha Staples
Sarah Sowell
Mike Rappe
Bob Morris
Electron
Tomography
Eric Mathur
Craig Carlson
Dick
Daniella
Stephanie Nicastro Mcintosh
Connon
Crew and Technicians of the RV Weatherbird II &
Bermuda Atlantic Time Series Study
Lisa
Mircea Bibbs
Podar
Our thanks to:
Microbial Observatories Program
For Supporting our Research
Marine Bacterioplankton SSU rRNA Gene Cluster
Sequence Diversity
Cluster/clade
SAR11
SAR11 w/freshwater clones
SAR86
SAR86 w/SAR156 subcluster
SAR116
Roseobacter
SAR324
Marine Actinobacteria
Marine Picophytoplankton
SAR406/Group A
aIncluded
Identity
(all overlapping)a
0.889
Identity
(conserved)b
0.900
0.872
0.887
0.935
0.945
0.845
0.875
0.897
0.884
0.897
0.940
0.962
0.946
0.908
0.904
0.922
0.966
0.969
0.968
all alignment positions for which both sequences possessed nucleotides in the
individual pairwise sequence comparisons
bIncluded the “Lane mask” to omit ambiguous alignment positions and hypervariable
regions of the SSU rRNA gene
Evolutionary distances in the SAR11 clade are much greater than in the
marine picophytoplanton clade
Conserved Properties of the SAR11 Metagenome
Prochlorococcus
Venter, 2004
Comparison of HVR1 of HTCC 1002 and HTCC1062