SAR11: Pelagibacter ubique, et al.

Transcription

A genome sequence paper
REPORTS
Genome Streamlining in a
Cosmopolitan Oceanic Bacterium
the cytoplasm to process substrates will be
matched to steady-state membrane transport
rates.
Surprisingly, this genome appears to encode nearly all of the basic functions of aproteobacterial cells (Table 1). The small
genome size is attributable to the nearly complete absence of nonfunctional or redundant
DNA and the paring down of all but the most
fundamental metabolic and regulatory functions. For example, P. ubique falls at the extreme end of the range for intergenic DNA
regions, with a median spacer size of only three
bases (Fig. 2). Intergenic DNA regions vary
considerably among bacteria and archaea, even
including parasites that have small genomes (5).
No pseudogenes, phage genes, or recent gene
duplications were found in P. ubique.
To further explore this trend, we investigated paralogous gene families by means of
BLAST clustering with variable threshold
limits. The genome had the smallest number
of paralogous genes observed in any freeliving cell (Fig. 1) (fig. S1). A steep slope in
Stephen J. Giovannoni,1* H. James Tripp,1 Scott Givan,2
Mircea Podar,3 Kevin L. Vergin,1 Damon Baptista,3 Lisa Bibbs,3
Jonathan Eads,3 Toby H. Richardson,3 Michiel Noordewier,3
Michael S. Rappé,4 Jay M. Short,3 James C. Carrington,2
Eric J. Mathur3
The SAR11 clade consists of very small, heterotrophic marine a-proteobacteria
that are found throughout the oceans, where they account for about 25% of
all microbial cells. Pelagibacter ubique, the first cultured member of this clade,
has the smallest genome and encodes the smallest number of predicted open
reading frames known for a free-living microorganism. In contrast to parasitic
bacteria and archaea with small genomes, P. ubique has complete biosynthetic
pathways for all 20 amino acids and all but a few cofactors. P. ubique has no
pseudogenes, introns, transposons, extrachromosomal elements, or inteins; few
paralogs; and the shortest intergenic spacers yet observed for any cell.
Pelagibacter ubique, strain HTCC1062, belongs to one of the most successful clades of
organisms on the planet (1), but it has the
smallest genome (1,308,759 base pairs) of any
cell known to replicate independently in nature
(Fig. 1). In situ hybridization studies show
that these organisms occur as unattached cells
suspended in the water column (1). They grow
by assimilating organic compounds from the
ocean_s dissolved organic carbon (DOC) reservoir, and can generate metabolic energy either
by a light-driven proteorhodopsin proton pump
1
Department of Microbiology, 2Center for Gene Research and Biotechnology, Oregon State University,
Corvallis, OR 97331, USA. 3Diversa Corporation, 4955
Directors Place, San Diego, CA 92121, USA. 4Hawaii
Institute of Marine Biology, School of Ocean and Earth
Science and Technology, University of Hawaii, Post
Office Box 1346, Kaneohe, HI 96744, USA.
*To whom correspondence should be addressed.
E-mail: [email protected]
10.0
Fig. 1. Number of predicted protein-encoding
genes versus genome
size for 244 complete
published genomes from
bacteria and archaea. P.
ubique has the smallest
number of genes (1354
open reading frames) for
any free-living organism.
Streptomyces coelicolor
Rhodopirellula
baltica
5.0
Silicibacter pomeroyi
Genome size (Mbp)
What is SAR11?
(2) or by respiration (3). The marine planktonic environment is poor in nutrients, and the
availability of N, P, and organic carbon typically limits the productivity of microbial communities. P. ubique is arguably the smallest
free-living cell that has been studied in a laboratory, and even its small genome occupies a
substantial fraction (È30%) of the cell volume.
The small size of the SAR11 clade cells fits a
model proposed by Button (4) for natural selection acting to optimize surface-to-volume ratios
in oligotrophic cells, such that the capacity of
Coxiella burnetii
Bartonella henselae
Thermoplasma acidophilum
Bartonella quintana
Ehrlichia ruminantium
Synechococcus sp.WH8102
Prochlorococcus marinus MIT9313
Prochlorococcus marinus SS120
Prochlorococcus marinus MED4
Pelagibacter ubique
1.0
Rickettsia conorii
Mesoplasma florum
Wigglesworthia glossinidia
0.5
Cultivating the uncultivatable.
Mycoplasma genitalium
Nanoarchaeum equitans
Free-living
Host-associated
Obligate symbionts/parasites
Pelagibacter ubique
0.1
100
What does the genome tell us?
1242
500
1000
Number of protein encoding genes
19 AUGUST 2005 VOL 309
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5000
10000
REPORTS
rates.
Eric J. Mathur3
1
10.0
genes versus genome
Rhodopirellula
baltica
5.0
Genome size (Mbp)
What is SAR11?
Coxiella burnetii
Bartonella henselae
Bartonella quintana
Pelagibacter ubique
1.0
Rickettsia conorii
Mesoplasma florum
0.5
Free-living
Host-associated
Pelagibacter ubique
0.1
100
1242
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1000
SCIENCE
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10000
SAR11 was an “environmental” rRNA sequence
Steve Giovannoni, et al., 1990 Genetic diversity in Sargasso Sea bacterioplankton. Nature 345:60
16S rRNA genes amplified by PCR with “universal” primers from DNA extracted from bacterioplankton
filtered from Sargasso Sea near-surface water.
12 clones sequenced; 4 cyanobacteria (SAR7 cluster), 8 α-proteobacteria (SAR11 cluster
Dotblot hybridization suggests that SAR11 makes up
a large fraction of the Sargasso bacterioplankton
SAR11 is a group of “uncultivatable” α-proteobacteria
KG Field (Giovannoni) 1997 AEM 63:63
VOL. 63, 1997
DEPTH-SPECIFIC DIVERSITY IN SAR11 CLUSTER rRNA GENES
67
TABLE 3. Phylogenetic subgroups among SAR11 cluster 16S rRNA genesa
Name
(reference)
Accession
no.c
Origin
Depth
(m)
SAR1 (16)
SAR95 (16)
SAR407 (16)
SAR425b
BDA1-25 (15)
OM242b
OM188b
OCS-12b
FL11 (10)
FL1 (10)
ALO21 (41)
ALO38 (41)
ALO39 (41)
NH16-1 (15)
NH25-10 (15)
X52280
M63812
U75253
U75261
L11942
U70689
U70687
U75252
L10935
L10934
M64525
M64532
M64533
L11949
L11967
Atlantic, Hydrostation S
Atlantic, BATS
Atlantic, BATS
Atlantic, near Bermuda
Atlantic, Cape Hatteras
Pacific, Oregon Coast
Pacific, Santa Barbara Channel
Pacific, Santa Barbara Channel
Pacific, ALOHA Station
Northeast Pacific
Northeast Pacific
Surface
Surface
80
80
10
10
10
10
10
10
Surface
Surface
Surface
100
100
20–1191
49–1406
8–1541
1161–1540
537–815
56–467
56–464
9–1005
14–1448
8–1114
307–499
307–499
307–499
537–761
537–817
SAR11 (16)
SAR193b
BDA1-1 (15)
BDA1-20 (15)
BDA1-15 (15)
NH16-2A (15)
NH16-11 (15)
NH25-4 (15)
X52172
U75649
L11934
L11941
L11939
L11961
L11951
L11974
Atlantic, BATS
Northeast Pacific
Northeast Pacific
Northeast Pacific
Surface
250
250
10
10
10
100
100
22–1191
54–613
537–815
537–765
537–752
537–817
537–763
537–857
SAR211b
SAR464b
SAR466b
SAR440b
SAR414b
SAR490b
SAR418b
SAR492b
BDA1-27 (15)
OM239b
OM136b
OM258b
OCS143b
NH49-1 (15)
U75256
U75254
U75263
U75262
U75259
U75264
U75260
U75265
L11943
U70688
U70684
U70691
U75266
L11987
Atlantic, BATS
Atlantic, BATS
Atlantic, BATS
Atlantic, BATS
Atlantic, BATS
Atlantic, BATS
Atlantic, BATS
Atlantic, BATS
Pacific, Oregon Coast
Northeast Pacific
250
80
80
80
80
80
80
80
10
10
10
10
10
500
8–1542
8–1541
8–357
8–357
8–355, 1161–1540
8–357, 1161–1540
8–350, 1180–1540
54–516
537–741
49–319
49–494
60–551
106–411
537–756
SAR220b
SAR241b
BDA1-17 (15)
U75257
U75258
L11940
Atlantic, BATS
Atlantic, BATS
250
250
10
8–1541
8–1542
537–790
SAR203b
NH29-3 (15)
U75255
L11982
Atlantic, BATS
Northeast Pacific
250
100
537–772
537–756
a
b
c
Position
(E. coli numbering)
Genes in boldface type are also shown in the tree in Fig. 1. Line spaces separate phylogenetic subgroups.
This gene was first reported in this paper.
GenBank.
the hybridization to the universal 338R probe providing the
denominator.
depends on whether all templates amplify with equal efficiency.
We tested the hybridization results independently by hybridiz-
VOL. 63, 1997
DEPTH-SPECIFIC DIVE
FIG. 1. Phylogenetic relationships among SAR11 cluster 16S rRNA genes,
inferred by neighbor joining (38) from E. coli positions 9 through 1005 (the
sequence positions found in the shortest gene, OCS12). The gene sequence from
Agrobacterium tumefaciens was used to root the tree. The same branching order
was recovered by the method of Wagner parsimony (44). The numbers above the
internal segments are the percentages of bootstrap replicates which supported
the branching order for the neighbor-joining tree; bootstrap values for the
parsimony analysis are shown below the segments. Bootstrap values below 60%
are not shown. SAR1, -11, and -95, surface; SAR193, -203, -211, -220, and -241,
250 m; SAR407 and -464, 80 m. SAR, Sargasso Sea; OCS12, Oregon coast; FL11,
California coast (10).
neighbor joining (38). We inferred parsimony trees with the heuristic search
option of PAUP (44). The bootstrap (12) with 100 replicates was used to estimate the robustness of branches in both neighbor-joining and parsimony trees.
We edited phylogenetic trees with the program Treetool, provided by Mike
Maciukenas, Ribosomal Database Project (RDP [22, 24]).
Identification of chimeric genes. We obtained secondary structure models for
rDNAs with the program gRNAID, version 1.4 (46a), and examined them for
To assess
SAR11 clust
SSU rRNA g
nucleic acid
coastal and
lyzed the se
group-specifi
probes to nu
depth-specifi
did not use
purpose, bec
tected by th
growing slow
To select
examined p
quences obt
not shown)
ability reve
quenced eigh
SAR203, SA
250 m in t
SAR464 from
from a depth
also analyze
Parsimony
tially the sam
structed from
and nearly f
We used the
branches for
nd five Cy3-labelled oligonucleotide probes (n ¼ 72) within and 1.2 £ 10 cells l (n ¼ 21) below the euphotic
6S rRNA were used in separate hybridization zone. To ensure that cells were not being removed in the hybridizaocessing of raw images was used to restrict tion process, direct cell counts determined from hybridization
wing fluorescence from both the Cy3 probe and preparations were compared with independent preparations that
4 0 ,6-diamidino-2-phenylindole dihydrochlor- used standard DAPI staining procedures for counting cells20. Agreehis approach, there was very little ambiguity in ment between the values was 99.6% in pair-wise comparisons,
nts.
showing that most cells remained on the filters throughout the
ng, fluorescence in situ hybridization (FISH) is hybridization procedure.
for determining the exact abundance of cells in
Morris,
et al.their
(Giovannoni) 2002 Nature 420:806
provided that RM
the probes
circumscribe
nd the fluorescence signals from cells are high
t scoring. Because some microbial cells, such as
bacterioplankton, are difficult to detect by this
tegies have been used to increase signal intenccuracy. Some of these studies have focused
design13,14, whereas others have explored stratfluorescent signal per cell15–17. Our strategy was
that target different regions of the 16S rRNA to
e effect on signal intensity, coupled with a
D (charge-coupled device) camera for detecting
SAR11 make up 20-50% of oceanic bacterioplankton
nce in situ hybridization image composite. Dual image
cells stained with DAPI (blue) and the Cy3 probe (red). Cells
API and the Cy3 probe are both blue and red, and cells
e set of SAR11 probes are blue. The identical fields of view
ed images show the characteristic size and curved rod
SAR11 cell (white box). Scale bar, 1 mm.
ECEMBER 2002 | www.nature.com/nature
Figure 3 SAR11 probe counts, bacterial probe counts and direct cell counts (DAPIstaining particles) in the northwestern Sargasso Sea. a–d, SAR11 clade (squares),
Bacteria (circles) and DAPI (diamonds) counts at 328 N, 648 W (CDOM-01; BATS site;
a), 308 N, 648 W (CDOM-03; b), 288 N, 648 W (CDOM-05; c) and 268 N, 648 W
(CDOM-07; d). e, A transect composite shows the mean abundance values by depth
for SAR11 clade and bacterial cell counts as percentages of direct cell counts (DAPI
staining particles); n ¼ 4, except for depths below 250 m, where n ¼ 1. Standard
deviations are given for depths of 1–250 m. One sample point was obtained for depths
below 250 m at 268 N, 648 W (CDOM-07).
© 2002 Nature Publishing Group
807
method, various strategies have been used to increase signal intensity and counting accuracy. Some of these studies have focused
primarily on probe design13,14, whereas others have explored strategies to amplify the fluorescent signal per cell15–17. Our strategy was
SAR11 are
very
small
(<1µm
long)
curved
to usemostly
several probes
that target
different
regions of
the 16S rRNA
to
produce an additive effect on signal intensity, coupled with a
cooled CCD
(charge-coupled
device) camera for detecting
RM Morris, etsensitive
al. (Giovannoni)
2002
Nature 420:806
Figure 2 SAR11 fluorescence in situ hybridization image composite. Dual image
overlay of DNA-containing cells stained with DAPI (blue) and the Cy3 probe (red). Cells
emitting a signal for both DAPI and the Cy3 probe are both blue and red, and cells
that did not hybridize to the set of SAR11 probes are blue. The identical fields of view
in the DAPI- and Cy3-stained images show the characteristic size and curved rod
morphology of a magnified SAR11 cell (white box). Scale bar, 1 mm.
NATURE | VOL 420 | 19/26 DECEMBER 2002 | www.nature.com/nature
rods
Figure 3 SAR11 probe co
staining particles) in the no
Bacteria (circles) and DAPI
a), 308 N, 648 W (CDOM-0
(CDOM-07; d). e, A transe
for SAR11 clade and bacte
staining particles); n ¼ 4,
deviations are given for de
below 250 m at 268 N, 64
REPORTS
rates.
Eric J. Mathur3
1
10.0
genes versus genome
Rhodopirellula
baltica
5.0
Genome size (Mbp)
What is SAR11?
Coxiella burnetii
Bartonella henselae
Bartonella quintana
Pelagibacter ubique
1.0
Rickettsia conorii
Mesoplasma florum
0.5
Free-living
Host-associated
Pelagibacter ubique
0.1
100
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1000
SCIENCE
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5000
10000
Standard methods grow weeds
SA Connon & SJ Giovannoni 2002 AEM 68:3878
VOL. 68, 2002
Problems with standard methods - and the solutions:
• Slow growers are overwhelmed by “weeds”
Inoculate small cultures with very dilute samples
• Typical media is far too rich for oligotrophs
Use buffered, sterilized seawater for media
• Many organisms grow only to very low densities
Concentrate cultures by filtration for examination
FIG. 1. Flow chart of HTC procedures. DMSO, dimethyl sulfoxide.
and subsequent identification (Fig. 1). Slight variations of the method were
performed during the development of these HTC techniques over the course of
3 years, but the overall approach remained constant. Microtiter plates were used
to culture cells, and cell arrays were made to allow efficient screening of the
plates for growth. The cultures acquired were designated with HTC collection
(HTCC) numbers.
Preparation of media. Water for media was collected on the south side of the
southern jetty in Newport, Oreg., at high tide with a bucket on 19 March 1998 8
km (44°39.1N, 124°10.6W) offshore from the mouth of Yaquina Bay, Oreg., with
counts were done by
filter on triplicate filt
traditional methods,
plates of MA2216 (D
(34), and a 1/10 dilu
diluted into the prepa
48-well non-tissue-cu
lin Lakes, N.J.) to a fi
At least one control p
1-ml aliquots of unin
incubated in the dar
weeks, and the agar c
count, about 1 week
Detection of growt
48-well plate to exam
well in the plate was
manifold of custom
Cells were then DAP
pore-diameter white
Whatman Nuclepore
75-by-50-mm slide (C
48-by-60-mm covergl
each sector of the arr
a cell titer as low as
array was then score
estimated by countin
Culturability statis
for estimation of cult
pure cultures was est
by Button and collea
of pure cultures, n is
p is the proportion o
inoculated wells), an
calculate the error, fi
binomial proportion
(SAS Institute Inc.).
equation and pure cu
and upper 95% confi
number of pure cultu
RFLP analysis an
isolates were identifie
rRNA gene sequenc
through two cycles of
in a 10,000-molecula
United Kingdom). S
buffer (5 M guanidin
concentrator. The lys
water (Specialty Me
buffer. The final volu
Two to three negative
run with each set of
16S rRNA genes w
trated sample was ad
Up to 15% of microbes can be grown using this method
3880
CONNON AND GIOVANNONI
APPL. ENVIRON. MICROBIOL.
TABLE 1. Extinction culturability statistics compared to traditional culturability counts
Date (mo-day-yr)
and location of
inoculation
samplea
Inoculum sample
(cells/ml)
Avg no. of
cells/well
Total no. of
wells inoculated
No. of
positive wellsb
Culture
designations
5-21-98, J
6-5-98, J
7-6-98, 8 km
7-6-98, 25 km
6-17-99, J
10-29-99, J
12-21-99, J
1-26-00, J
1.1 % 106
1.5 % 106
3.7 % 106
1.5 % 106
5.6 % 106
1.9 % 106
8.1 % 105
1.1 % 106
1.1
1.5
3.7
1.5
3.0
3.0
5.0
5.0
144
192
192
192
192
192
384
192
7
37
62
37
21
10
10
11
4-5-00, J
7-12-00, J
9.0 % 105
1.9 % 106
5.0
3.0
192
228
20
33
10-9-00, 8 km
1.3 % 106
3.0
384
5
HTCC1–7
HTCC8–44
HTCC45–106
HTCC107–143
HTCC144–164
HTCC165–174
HTCC175–184
HTCC185–191,
193–196
HTCC197–216
HTCC217–233,
236–251
HTCC252–256
% Culturabilityc
% Culturability on
nutrient-rich agard
1/10R2A
R2A
MA2216
4.5 (1.8, 9.3)
14.3 (10.0, 19.7)
10.5 (8.0, 13.5)
14.3 (10.0, 19.7)
3.9 (2.4, 5.9)
1.8 (0.9, 3.3)
0.5 (0.3, 1.0)
1.2 (0.6, 2.1)
—
—
—
—
—
—
—
0.01
—
—
—
—
—
—
—
0.01
—
—
—
—
—
—
—
0.02
2.2 (1.3, 3.4)
5.2 (3.6, 7.3)
—
0.98
0.15
0.15
0.12
0.12
0.4 (0.1, 1.0)
0.29
0.09
0.02
a
Samples were collected on the date indicated from the jetty (J) or 8 or 25 km out from the mouth of Yaquina Bay, Oreg.
Wells were scored for growth after 3 weeks of incubation at 16°C.
c
Ninety-five percent confidence intervals are shown in parentheses.
d
Inoculum was the same as that used for the microtiter plates. —, not determined.
b
positive controls with 108, 2,000, 200, and 20 copies of the 16S rRNA gene from
the clone SAR242 were run in each PCR set. All primers used have no mismatches to the SAR242 sequence, except for 1492R, which does not match the
first and third bases on the 5! end (nonpriming end). The concentration of the
positive control DNA was measured in a Shimadzu UV160U spectrophotometer
(Shimadzu Co., Kyoto, Japan). The 20-copy-positive control could be routinely
amplified with a total of 50 to 66 cycles of nested PCR.
RFLP of the PCR product was done with the restriction enzymes MboI and
HaeIII (MBI Fermentas) (38). HTCC isolates were determined to be a mix of
HTCC230 and HTCC234 (SAR92 clade), and HTCC223 and HTC227 (OM60/
OM241 clade), have been successfully transferred from the initial well, propagated, and stored. Cells were stored in 7% dimethyl sulfoxide and/or 10%
glycerol.
DAPI-stained cell images. Images were obtained with a Hamamatsu
ORCA-ER cooled interline charge-coupled device camera (5 Mz) mounted on a
Leica DMRB microscope. IPLab Spectrum 3.5 image analysis software was used
to acquire images.
Nucleotide sequence accession numbers. The sequences of the HTCC isolates
(32). Phylogenetic analyses were performed with ARB and PAUP! (35). Phylogenetic trees were inferred by neighbor joining with the Jukes and Cantor model
to estimate evolutionary distances. Bootstrap values were obtained in PAUP!
from a consensus of 1,000 neighbor-joining trees. Short sequences of HTCC
isolates were added to the tree by using the parsimony insertion tool in ARB.
The percent similarity of sequences was determined with the distance matrix tool
in ARB; ambiguous bases were not included.
Recovery of HTCC isolates from frozen storage. The probability of recovering
HTCC
isolates from frozen
not been systematically investigated, and
SJ
Giovannoni
2002storage
AEM has
68:3878
not all cultures were saved for further study. However, isolates from three of the
four significant phylogenetic clades in this study, HTCC202 (OM43 clade),
include SAR11 (" sub
SAR92 ($ subclass) (8)
Culturability statisti
culture wells were score
screened for 3 years an
range of 0.4 to 14.3% w
collections (Table 1). T
ples collected between
the average culturabilit
early October and earl
turability were made be
plating on nutrient-rich
from 1.4 to 120 times
addition, MA2216 and
first 143 cultures grown
mer of 1998 to determ
these media. Only thre
on R2A; none of these
(data not shown).
Detection of growth
the HTCC cultures rang
Most grow slowly and only to low density
SA Connon &
TABLE 2. Cell densities and inferred doublings attained after 3
weeks of incubation
Final no. of cells/ml
No. of
culturesa
No. of inferred
doublingsb
1.0 % 103–9.9 % 103
1.0 % 104–9.9 % 104
1.0 % 105–9.9 % 105
1.0 % 106–9.9 % 106
66
120
62
5
10.0–13.3
13.3–16.6
16.6–19.9
19.9–23.3
a
Out of 253 cultures.
This inference is based on the assumption that only one inoculated cell in
each well grew.
b
SAR11 isolates are small, curved rods
VOL. 68, 2002
FIG. 2. Fluorescence microscopy images of several of the novel
isolates. The cells were stained with DAPI. Size bars, 1 "m.
ml, with a mean of 1.1 ! 105 cells per ml and a median of 3.0
! 104 cells per ml. The minimum density for a culture to be
detectable was 1.3 ! 103 cells per ml. This range of cell den-
HIGH-THROUGHPUT
HTCC175 were short rods (ca. 0.8 to 0.5
SAR92 clade isolates HTCC148, HTCC
were short rods (ca. 1 to 0.7 "m by 0.7
OM241 clade isolate HTCC160 was an irr
cus that occasionally formed doublets an
of three (ca. 0.7 by 0.7 "m). These meas
to sizeable error, since these small cells ar
resolution of visible light microscopes. T
stained with a DNA staining dye and h
formaldehyde. The images shown are from
tion dilutions that yielded the four previo
undescribed groups (Fig. 2).
Phylogenetic analysis and culture ident
or undescribed groups SAR11, OM43,
OM241 accounted for the majority of cul
tified out of a subset of 56 cultures (Table
from 13 48-well plates (56 cultures) were
5 different sampling months to minimiz
emerge as a result of seasonal variation
abundance. Forty-seven of the 56 culture
the 9 cultures that were not identified,
unknown mixtures of several cell types ba
sis, and 2 did not amplify under the con
were a total of eight mixed cultures; HTCC
a mix of cells from the SAR11 clade and
failure of two cultures to amplify is pro
problems with the DNA extractions and/o
the cultures. A considerable effort was m
these lineages did not fail to amplify beca
amplification primers. The theoretical stat
the number of pure cultures versus mixed
Correspondence
requests carbon
for materials
should be addressed
M.A.v.Z.
mixture ofand
organic
compounds.
The totechnique
of isolating
efforts. Genetic evidence suggests that diverse uncultivated
14. Rosing, M. T. & Rose, N. M. The role of ultramafic rocks in regulating the concentrations of volatile
cambrian Res. (e-mail:
[email protected]).
microbial taxa dominate most natural ecosystems3–5, which has
cells
by dilution
into during
sterilized
natural
watersChem.
or other
dilute
and non-volatile
components
deep crustal
metamorphism.
Geol. 108,
187–200media
(1993).
Hayes,
J. M., Kaplan,
R. & Wedeking,8,9
W.; initEarth’s
Earliest
Biosphere, its Origin
and Evolution
(ed.
prompted widespread efforts to elucidate the geochemical activibeen
used I. previously
takes
advantage
of the
fact that
reenland, and 15.has
Schopf, J. W.) 93–134 (Princeton Univ. Press, New Jersey, 1983).
ties of these organisms without the benefit of cultures for study6,7.
16.
Oehler,
D.
Z.
&
Smith,
J.
W.
Isotopic
composition
of
reduced
and
oxidized
carbon
in
Early
Archaean
y rocks from
Here we report the isolation of representatives of the SAR11
rocks from Isua, Greenland. Precambr. Res. 5, 221–228 (1977).
clade. Eighteen cultures were initially obtained by means of high17.
Perry,
E.
C.
&
Ahmad,
S.
N.
Carbon
isotope
composition
of
graphite
and
carbonate
minerals
from
3.8
variability in
AE
metamorphosed
sediments,
Isuakasia,
Greenland.
Earth
Planet.
Sci.
Lett.
36,
280–284
(1977).
chniques.
throughput procedures for isolating cell cultures through the
18. Ueno, Y., Yurimoto, H., Yoshioka, H., Komiya, T. & Maruyama, S. Ion microprobe analysis of graphite
dilution of natural microbial communities into very low nutrient
from ca. 3.8 Ga metasediments, Isua supracrustal belt, West Greenland: Relationship between
media. Eleven of these cultures have been successfully passaged
metamorphism and carbon isotopic composition. Geochim. Cosmochim. Acta 66, 1257–1268 (2002).
19. Abed, A. M. & Fakhouri, K. in Phosphorite Research and Development (eds Notholt, A. J. G. & Jarvis, I.)
and cryopreserved for future study. The volume of these cells,
193–203 (The Geological Society, London, 1990).
reduced
about 0.01 mm3, places them among the smallest free-living cells
20. Appel, P. W. U. On the early Archaean Isua iron-formation, West Greenland. Precambr. Res. 11, 73–87
cilities for
in culture.
(1980). S. Rappé, Stephanie A. Connon, Kevin L. Vergin
ng d13C of Michael
French, B. M.
relations of siderite (FeCO3) in the system Fe-C-O. Am. J. Sci. 271, 37–78 (1971).
In an effort to isolate some of the ubiquitous uncultivated
nate phases, &21.Stephen
J.Stability
Giovannoni
part of the 22. van Zuilen, M. A. et al. Graphite and associating carbonates in early Archean Isua supracrustal rocks,
Bacteria and Archaea that dominate marine bacterioplankton
southern West Greenland. Precambr. Res. (submitted).
Wallenberg Department
of
Microbiology,
Oregon
State
University,
Corvallis,
Oregon
97331,
communities2, we inoculated fresh Oregon coast seawater samples
23. Chacko, T., Mayeda, T. K., Clayton, R. N. & Goldsmith, J. R. Oxygen and carbon isotope fractionations
thank
USAbetween CO2 and calcite. Geochim. Cosmochim. Acta 55, 2867–2882 (1991).
into microtitre dish wells by dilution, such that on average each well
script.
.............................................................................................................................................................................
Figure 1 Photomicrographs
of a culture
SAR11 cladeofisolate
HTCC1062.
24. Boak, J. L. & Dymek, R. F. Metamorphism of the ca. 3800 Ma supracrustal rocks at Isua, West Greenland:
received
22 microbial cells.
Mediaofconsisted
sterile
Oregon coast
implications for early Archaean crustal evolution. Earth Planet. Sci. Lett. 59, 155–176 (1982).
a,
b,
Fluorescence
images
of
cells
in
an
identical
field
of
view,
stained
sea water supplemented with either phosphate (as KH2with
PO4the
) and
25. Nagy, B. Porosity and permeability of the Early Precambrian Onverwacht chert: Origin of the
DNA-specific dye
and after
hybridization with
four Cy3-labelled
oligonucleotide
ammonium
(asDAPI
NH(a)
Cl),
or
phosphate,
ammonium
and
a
defined
hydrocarbon content. Geochim. Cosmochim. Acta 34, 525–527 (1970).
4
probes targeting
SAR11carbon
cells (b). compounds.
Scale bar (a, b), The
1 mm.technique
c, d, Transmission
electron
26. Lepland, A., Arrhenius, G. & Cornell, D. Apatite in early Archean Isua supracrustal rocks, southern
mixture
of organic
of isolating
West Greenland: its origin, association with graphite and potential as a biomarker. Precambrian Res.
micrographs
of strain
HTCC1062.
c, Shadowed
cells withortheother
typicaldilute
SAR11media
clade
cells
by dilution
into
sterilized
natural waters
1
(in the press).
morphology.
d, Negatively
stained8,9cell.
latexadvantage
beads in c and
have fact
a diameter
has
been used
previously
; it The
takes
ofd the
that of
27. Fedo, C. M. & Whitehouse, M. J. Metasomatic origin of quartz-pyroxene rock, Akilia, Greenland, and
0.514
m
m.
implications for Earth’s earliest life. Science 296, 1448–1452 (2002).
28. Rosing, M. T. 13C-Depleted carbon microparticles in .3700-Ma sea-floor sedimentary rocks from
630West Greenland. Science 283, 674–676 (1999).
NATURE | VOL 418 | 8 AUGUST 2002 | www.nature.com/nature
29. Macpherson, C. G., Hilton, D. R., Newman, S. & Mattey, D. P. CO2, 13C/12C and H2O variability in
natural basaltic glasses: A study comparing stepped heating and FTIR spectroscopic techniques.
Geochim. Cosmochim. Acta 63, 1805–1813 (1999).
Cultivation of SAR11 Pelagibacter ubique
..............................................................
Cultivation of the ubiquitous SAR11
marine
bacterioplankton
clade
MS Rappé
2002 Cultivation of the
ubiquitous SAR11 marine bacterioplankton clade. Nature 418:630
The a-proteobacterial lineage that contains SAR11 and related
ribosomal RNA gene clones was among the first groups of
organisms to be identified when cultivation-independent
approaches based on rRNA gene cloning and sequencing were
applied to survey microbial diversity in natural ecosystems . This
group accounts for 26% of all ribosomal RNA genes that have
........
R11
on 97331,
......................
d related
oups of
pendent
ing were
ms1. This
hat have
Acknowledgements
We thank M. Wahlen and B. L. Deck for providing facilities for extraction of reduced
carbon and subsequent isotopic measurement, D. R. Hilton for providing facilities for
stepped-combustion extraction of reduced carbon, J. L. Teranes for measuring d13C of
carbonate phases, J. Finarelli for determination of cation composition of carbonate phases,
and P. W. U. Appel for providing coordination and facilities for field work as part of the
Isua Multidisciplinary Research Project. Support by the Marianne and Marcus Wallenberg
Foundation (for A.L.) and NASA Exobiology is gratefully acknowledged. We thank
L. P. Knauth, S. Moorbath and J. M. Hayes for their comments on this manuscript.
Figure 1 Photomicrographs of a culture of SAR11 clade isolate HTCC1062.
a, b, Fluorescence images of cells in an identical field of view, stained with the
Competing
interests
DNA-specific
dye DAPIstatement
(a) and after hybridization with four Cy3-labelled oligonucleotide
The
authors
declare
that
they
have(b).
no Scale
competing
financial
probes targeting SAR11 cells
bar (a,
b), 1 minterests.
m. c, d, Transmission electron
micrographs
of
strain
HTCC1062.
c,
Shadowed
cells
with
theM.A.v.Z.
typical SAR11 clade
Correspondence and requests for materials should be addressed to
morphology.
d,
Negatively
stained
cell.
The
latex
beads
in
c
and
d have a diameter of
(e-mail: [email protected]).
0.514 mm.
002 Nature Publishing Group
SAR11 clade cells were identified preliminarily. Cultures were clone library that has been constructed with universal or bacterial
obtained in microtitre plates incubated in the dark or under a polymerase chain reaction (PCR) primers from marine prokaryotic
light/dark cycle, and in both the medium containing only natural plankton samples, including coastal and near-shore waters16,17 and
organic carbon and the medium supplemented with a defined seawater samples from depths up to 3,000 m (ref. 18). Relatives of
mixture of carbon compounds. Eleven isolates were propagated the SAR11 clade have even been detected in freshwater lakes19.
and cryopreserved for future study.
Growth rates for the 11 SAR11 clade isolates replicating at 15 8C
Phylogenetic relationships of the isolates were investigated by a in sterile Oregon coast seawater supplemented with 0.1 mM phoscombination of 16S rRNA gene and 16S–23S rDNA intergenic phate, 1.0 mM ammonium and a defined mixture of organic carbon
spacer sequence analysis. Ribosomal RNA nucleotide sequences of compounds ranged from 0.40 to 0.58 d21. Although this rate of cell
414–608 bases, obtained from the 3 0 end of the 16S rRNA gene of all division is low in comparison to values typical of cultivated bacteria,
11 isolates, were identical. Intergenic spacer sequences (415–417 it is not dissimilar to the measured growth rates of marine
MS bases)
Rappé
2002 however,
Cultivation
ofwere
the
ubiquitous
SAR11bacterioplankton
marine bacterioplankton
clade.
indicated,
that there
three
genetically distinct
communities in nature,
whichNature
vary from418:630
0.05 to
21
0.3 d (ref. 20). All of the isolates produced a logistic growth curve
(Fig. 3). In subsequent experiments with strain HTCC1062, which
was originally isolated in seawater media supplemented with the
defined organic carbon compound and vitamin mixtures, removal
of these amendments did not negatively affect growth rate or
maximum cell abundance (Fig. 3). However, the addition of dilute
proteose peptone (0.001%) inhibited growth (Fig. 3).
Pelagibacter ubique is a robust SAR11
Figure 2 Phylogenetic relationships between strain HTCC1062 and representatives of the
SAR11 clade and a-Proteobacteria inferred from 16S rRNA gene sequence comparisons.
The Gram-positive bacteria Bacillus subtilis and Marinococcus halophilus were used as
outgroups. Bootstrap proportions over 70% that supported the branching order are
shown. Scale bar corresponds to 0.05 substitutions per nucleotide position. Also included
in the analysis were the g-Proteobacteria Alteromonas macleodii and Marinobacter
hydrocarbonoclasticus, and the b-Proteobacteria Methylophilus methylotrophus and
Polynucleobacter necessarius.
Figure 3 Growth of strain HTCC1062 in Oregon coast seawater media. Media consisted of
sterile sea water supplemented with 1.0 mM NH4Cl and 0.1 mM KH2PO4 (filled circles),
1.0 mM NH4Cl, 0.1 mM KH2PO4, and mixed carbon (open circles), 1.0 mM NH4Cl, 0.1 mM
KH2PO4, and Va vitamins (filled triangles), 1.0 mM NH4Cl, 0.1 mM KH2PO4, mixed carbon
and Va vitamins (open triangles), 1.0 mM NH4Cl, 0.1 mM KH2PO4, mixed carbon, Va
vitamins and 0.001% (w/v) proteose peptone (filled squares). For all cultures, cell counts
attempted on days 7 and 12 were below the limit of detection (dotted line, 3,000 cells per
ml), as were counts on day 31 and after day 33 for the culture containing proteose
peptone. The point at day 0 is the inoculum density.
631
Pelagibacter ubique is really, really small
MS Rappé 2002 Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature 418:630
E. coli : 1.3um x 4um average : considered small for a typical bacterium
1.3^2 x 4 = 6.76um^3 (volume)
2(1.3^2)+4(1.3x4) = 24um^2 (surface area)
24/6.76 = 3.6um^2/um^3 (surface/volume ratio)
20,000 ribosomes make up 30% of the cell mass
cell wall & membranes make up 20% of cell mass
DNA makes up 2% of cell mass
P. ubique : 0.15um x 0.6um average
0.15^2 x 0.6 = 0.0135um^3 (1/500th the volume of E. coli)
2(0.15^2)+4(0.15x0.6) = 0.4um^2 (1/100th the SA of E.coli)
0.0135/0.4 = 30um^2/um^3 (8X the SA/V ratio of E.coli)
DNA makes up 30% of the cell volume
REPORTS
rates.
Eric J. Mathur3
1
10.0
genes versus genome
Rhodopirellula
baltica
5.0
Genome size (Mbp)
What are SAR11 & P.ubique?
Coxiella burnetii
Bartonella henselae
Bartonella quintana
Pelagibacter ubique
1.0
Rickettsia conorii
Mesoplasma florum
0.5
Free-living
Host-associated
Pelagibacter ubique
0.1
100
1242
500
1000
SCIENCE
www.sciencemag.org
5000
10000
living cell (Fig. 1) (fig. S1). A steep slope in
availability of N, P, and organic carbon typorganisms on the planet (1), but it has the
ically limits
the productivity of microbial comsmallest genome
(1,308,759 base pairs) of any
ically limits the productivity of microbial comsmallest genome (1,308,759 base pairs) of any
munities.
P. ubique is arguably the smallest
known tomunities.
replicateP.independently
in nature
1
ubique is arguably
the smallest
cell known to replicate independentlycell
in nature
Department of Microbiology, 2Center for Gene Refree-living
has beenOregon
studied
in University,
a lab(Fig.show
1). In free-living
situ hybridization
showin a labsearchcell
and that
Biotechnology,
State
cell that hasstudies
been studied
(Fig. 1). In situ hybridization studies
3
Corvallis,
OR
97331,
USA.
Diversa
Corporation,
4955
oratory,
and
even
its
small
genome
occupies
a
that
these
organisms
occur
as
unattached
cells
oratory, and even its small genome occupies a
substantial
fraction (È30%) of the cell volume.
suspended
the waterfraction
column
(1). They
(È30%)
of thegrow
cell volume.
suspended in the water column (1). They
grow insubstantial
The small
sizecompounds
of the SAR11from
cladethe
cells fits
a small
by assimilating organic compoundsby
from
the
The
sizeandofTechnology,
the SAR11
clade ofcells
fits Post
a
assimilating
organic
Science
University
Hawaii,
model
proposed
by
Button
(4)
for
natural
selecocean_s dissolved organic carbon (DOC)
reserOffice
Box
1346,
Kaneohe,
HI
96744,
USA.
model proposed by Button (4) for natural selecocean_s dissolved organic carbon (DOC) resertion
acting
to
optimize
surface-to-volume
ratios
voir, and can generate metabolic energy
either
*To whom
correspondence
should be addressed.
tion acting
to optimize
surface-to-volume
ratios
voir, and can generate metabolic energy either
1
Department of Microbi
search and Biotechnolo
Corvallis, OR 97331, USA
Directors Place, San Die
Institute of Marine Biolog
Science and Technology
Office Box 1346, Kaneoh
P. ubique has the smallest genome, with the fewest genes,
of any free-living organism
*To whom correspond
E-mail: steve.giovannoni
Steve Giovannoni, et al., 2005 Genome streamlining in a cosmopolitan oceanic bacterium. Science 309:1242
10.0
Genome size (Mbp)
5.0
genes versus genome
1.0
0.5
10.0
Streptomyc
Rhodopirellula
baltica
5.0
Coxiella burnetii
Bartonella henselae
Bartonella quintana
Genome size (Mbp)
genes versus genome
S
Prochlorococcus marinus MIT9313Coxiella burnetii
Bartonella
Prochlorococcus marinus
SS120 henselae
Prochlorococcus
marinus MED4
Thermoplasma
acidophilum
Prochlorococcus
Prochlorococcus
1,308,759bp
Pelagibacter ubique
1.0
Rickettsia conorii
Mesoplasma florum
Mesoplasma florum
0.5
Free-living
Host-associated
Free-living
Pelagibacter ubique
0.1
100
Prochlorococcu
Pelagibacter ubiqueBartonella quintana
Rickettsia conorii
Synecho
500
Host-associated
1000
10000
5000
Pelagibacter ubique
0.1
19 AUGUST 2005 VOL 309 SCIENCE www.sciencemag.org
100
500
1000
genes that would confer alternate metabolic
genomes—for example, recombination and
were found in the genome. Autoradiogra
lifestyles, motility, or other complexities of
the propagation of self-replicating DNA (e.g.,
with native populations of SAR11 has d
structure and function were nearly absent. Conintrons, inteins, and transposons)—overwhelm
onstrated high uptake activity for amino a
spicuous exceptions were genes for carotenoid
the simple economics of metabolic costs.
and 3-dimethylsulfoniopropionate (13). He
synthesis, retinal synthesis, and proteorhodopHowever, evolutionary theory predicts that
efficiency is achieved in a low-nutrient sys
sin. P. ubique constitutively expresses a lightthe probability that selection will act to
by reliance on transporters with broad s
dependent retinylidine proton pump and is the
eliminate DNA merely because of the metastrate ranges (14) and a number of special
first cultured bacterium to exhibit the gene
bolic cost of its synthesis will be greatest in
substrate targets, in particular, nitrogen
that encodes it (2). The genome also contained
very large populations of cells that do not excompounds and osmolytes.
perience drastic periodic declines (6).
Steve Giovannoni, et al., 2005 Genome streamlining in a cosmopolitan oceanic bacterium. Science 309:1242
The P.ubique genome is highly streamlined, but
hasn’t discarded any basic metabolism
Table 1. Metabolic pathways in Pelagibacter.
Pathway
Prediction
Glycolysis
TCA cycle
Glyoxylate shunt
Respiration
Pentose phosphate cycle
Fatty acid biosynthesis
Cell wall biosynthesis
Biosynthesis of all 20 amino acids
Heme biosynthesis
Ubiquinone
Nicotinate and nicotinamide
Folate
Riboflavin
Pantothenate
B6
Thiamine
Biotin
B12
Retinal
Uncertain
Present
Present
Present
Present
Present
Present
Present
Present
Present
Present
Present
Present
Absent
Absent
Absent
Absent
Absent
Present
Fig. 2. Median siz
intergenic spacers
bacterial and arch
genomes. Inset sh
expanded view of ra
for organisms w
the smallest interg
spacers.
• Entner-Deuteroff pathway / gluconeogenesis
• Carbon from organics - no C fixation
• Energy from respiration or proteorhodopsin-based phototrophy
• No duplications, pseudogenes, or prophages genes
• No recent lateral transfers - genes for DNA uptake for N & P?
• No motility genes
• 30% G+C - lower N requirement
• Transporters are mostly ABC - low Km, high ATP cost
• Only 2 sigma factors - vegetative & heat-shock
• Few regulatory networks - only 3 2-component regulators
Do not respond to pulses of nutrients
• 1 rrn operon - cannot modulate growth rate over a wide range
• No quorum sensing genes - why does it stop growing at 10^6?
The streamlined genome is the result of opposing evolutionary forces - the demand to retain
www.sciencemag.org SCIENCE VOL 309 19 AUGUST 2005
the ability to make what it needs
to independently live is a sparse environment, and the
need to minimize the genome size to lower it’s resource cost and physical size
genes that would confer alternate metabolic
bination and
were found in the genome. Autoradiography
lifestyles, motility, or other complexities of
g DNA (e.g.,
with native populations of SAR11 has demstructure and function were nearly absent. Con—overwhelm
onstrated high uptake activity for amino acids
spicuous exceptions were genes for carotenoid
abolic costs.
and 3-dimethylsulfoniopropionate (13). Hence,
synthesis, retinal synthesis, and proteorhodoppredicts that
efficiency is achieved in a low-nutrient system
sin. P. ubique constitutively expresses a lightwill act to
by reliance on transporters with broad subdependent retinylidine proton pump and is the
of the metastrate ranges (14) and a number of specialized
first cultured bacterium to exhibit the gene
e greatest in
substrate targets, in particular, nitrogenous
that
encodes it (2).
The2005
genome
also streamlining
contained in
at do not ex-Steve
compounds
and osmolytes.
Giovannoni,
et al.,
Genome
a cosmopolitan
oceanic bacterium. Science 309:1242
(6).
P.ubique has the smallest intergenic spacers of any
organism known - only 3bp on average!
Fig. 2. Median size of
intergenic spacers for
bacterial and archaeal
genomes. Inset shows
expanded view of range
for organisms with
the smallest intergenic
spacers.
agibacter.
Prediction
Uncertain
Present
Present
Present
Present
Present
Present
Present
Present
Present
Present
Present
Present
Absent
Absent
Absent
Absent
Absent
Present
www.sciencemag.org
SCIENCE
VOL 309
19 AUGUST 2005
1243
P.ubique has an RNase P RNA gene (rnpB) in one of it’s largest
“intergenic spacers” (not annotated, of course).
A AC
U A
A U
C G
C G
G C A
120
C G A 140
A C G G
A U
U
U
G
A
A
Pelagibacter ubique
RNase P RNA
A
A
A
A
A
A
G
G
GG UG U G U A
A
A
C
C C A C GC G A G
A
G
160
C
GU
C
UC U
C
G
AU
G AA
U A GU
C
G
UG
U
A
U
A A C C
G
AA
C U100
C A A A G G
A A G
A
U
C
G G
C
G G GG
A
G CA
180
A
200
C
G
220
U
C
C
G U A
A
U
A
A
A
G
G
G
CU
80
A
A
G
A
C GU
U
G
G
C
G
G
A
A
A
G
A
C
U
C
240
U
C
G
GA UG
A U
A U
C
AC
A C
A
C
A
C C UC
U UG A
G
A
C U A C
C
A
A
G
U
UG
G
A
G
A
U
U G G GA
U
A
C G
C
G A
60
260 A U
A A
A
G
A
A
G
C
C C G
U 280
G
C
U
G
C
G
U U UC
A GC U UA A G A G
U
U
G
U U C U AG
A
C
G
A
A
C
U
A
A
300
40
A
G
G
A
G
G
A
A
A
A
U U UA A A G
A
U A A U UU C
U
U
G C G
20
U A
A U
U A
U G
320
G 1
C G
U U U A A A U G
C UA GA U GA A C G A
A
G A U C UG UU U G U
C
A A
C
A
340
A
U U C G G C C C A A G
A
C
C
C
G
140 U
A
C
C
G
C
AC
A
A
UU
G
C
A
U
G
G
UA
GA
GG
U
Agrobacterium tumefaciens
RNase P RNA
160
G
A
A
A
G UA
G
GG
A
GGU G
C C G AG
CCAC
G 180
CG
CC
GC
GU
UG
GG
G
G
UG
C
U
A
A
200
C G
A
G
A
G
A
G
A
G
A
C
A
C
G AA
C
C
120 G
A
G
260
A U
G CG
A G A A
AUG
G C
AG
C
C
A
G GU
C
A
G CCCC GG C
G
220
AU
U
A
A 240
C G G GG
CGG U
CA
G
G
G
A
C
CC
CA A
C
G GGAG
G G A U GA C
GACC
G
100
G
A
G C U GG
C
C
C
C C U A CU G
UUGG
CCUC
C
80
A
A
G
C
C G
G 300 G
A
G
G
U
G
C
G
U
AAAG
G
C
C C
U
GC G
A
G
G
G C
G
A
U
G
G
C
AA
C
G
GC C U U U G
60
A
C
U
G G C G G C A U GC
G
A
C U G C C G U A A A 320
A
C
A
C
G
A
G
40
G
A
A
U
A
A GA C GGU A AGGUG
G
A C U G C C A U U C C GC
A
G
A
UCA
G
U A
U
20
A
G
A 360
C G 340
G
A
C G
C
G
G C
C
C
A U
G
C
1
C G
U G
G C
U G
C C A G U U GG C C G G C A C G G
C C A UA
GGU C A A CC GG C
C
A A
C
A
G
G 400
UUCGGCCCAA
A
380
A
Pelagibacter ubique is a member of the SAR11 “unculturable” group
of alpha-proteobacteria that predominate the oceanic pelagic
ecosystem. This organism, like most SAR11 species, is a free-living,
planktonic oligotrophic facultative photochemotroph. It is very
small, 0.15 x 0.6um, 1/500th the volume of E.coli, providing a large
surface/volume ratio for absorbing trace nutrients and light.
The 1.3Mbp genome of Pelagibacter
ubique is extremely streamlined, with no
repeated sequences, prophage, &c, and
has the smallest known intergenic spacers.
However, the genome retains all of the
usual metabolic capabilities of alphaproteobacteria, and is specialized for slow
growth, extracting trace dissolved
organics, nitrogen, and phosphorous from
the open ocean water.

SAR11: Pelagibacter ubique, et al.

Transcription

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