Download Occurrence of phosphate acquisition genes in Prochlorococcus cells

Environmental Microbiology (2009) 11(6), 1340–1347
doi:10.1111/j.1462-2920.2009.01860.x
Occurrence of phosphate acquisition genes in
Prochlorococcus cells from different ocean regions
Adam C. Martiny,1* Ying Huang2 and Weizhong Li2
Department of Earth System Science and Department
of Ecology and Evolutionary Biology, University of
California, Irvine, 92697 CA, USA.
2
California Institute for Telecommunications and
Information Technology, University of California, San
Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
1
Summary
The cyanobacterium Prochlorococcus is the numerically dominant phototroph in oligotrophic parts of
the oceans. Recently, it was shown that the distribution of phosphate acquisition genes did not match the
16S rRNA phylogeny among isolates from this group
but rather appeared related to phosphate availability
where the strains had been isolated. To further understand adaptation to phosphate limitation in Prochlorococcus, the distribution of phosphate acquisition
genes was investigated in different ocean regions and
related to local ortho-phosphate concentration. In
regions characterized by less than 0.1 mM phosphate,
most Prochlorococcus cells contain genes involved
in phosphate uptake, regulation and utilization of
organic phosphates. In contrast, most of these genes
are absent in regions with more than 0.1 mM phosphate with the exception of genes involved in transport of phosphate (phoE and pstABCS) and three
genes of unknown function. This pattern of phosphate acquisition genes showed no significant correspondence to the distribution of rRNA phylotypes. In
addition, it was demonstrated that several genes in a
separate genomic island were commonly present
in low-P sites while absent in high-P sites. Overall,
this study further demonstrates a linkage between
environmental conditions in the ocean and genome
content of Prochlorococcus.
Introduction
Phosphate (P) plays a key role in regulating primary
productivity in several regions of the world’s oceans
Received 21 August, 2008; accepted 9 December, 2008. *For correspondence. E-mail [email protected]; Tel. (+1) 9498249713; Fax
(+1) 9498243874.
emi_1860
1340..1347
(Wu et al., 2000; Sanudo-Wilhelmy et al., 2001; Moore
et al., 2002a; Thingstad et al., 2005). In these regions, the
marine cyanobacterium Prochlorococcus is commonly
found at high abundance. Thus, P availability may exert
strong influence on the abundance and photosynthetic
activity of Prochlorococcus.
Recently, it was shown that the distribution of P acquisition genes among Prochlorococcus strains was not
consistent with their 16S rRNA phylogeny. Instead, the
distribution of these nutrient uptake genes suggested that
Prochlorococcus cells from P-limited regions contained
more P acquisition gene compared with cells from
P-replete regions or deeper in the photic zone (Martiny
et al., 2006). In contrast, light and temperature adaptation
in Prochlorococcus is closely correlated with phylogeny
(Moore et al., 1998; West and Scanlan, 1999; Johnson
et al., 2006).
In the closely related marine cyanobacterium Synechococcus, a comparison between two strains revealed that
an isolate from a nutrient-rich coastal region lacked the
phosphate sensor–response regulator system (phoBR),
whereas an isolate from a P-depleted region contained
these genes (Palenik et al., 2006). This concept of a
differential distribution of P-uptake genes was further
confirmed by field data from the Global Ocean Sampling (GOS) expedition. Rusch and colleagues (2007)
observed that the phosphate uptake genes, pstABCS
were significantly more abundant among cells including
Prochlorococcus and SAR11 in the Caribbean Sea compared with Eastern Pacific Ocean and attributed this to
differences in phosphate concentration. These results
suggest that lateral gene transfer combined with rapid
exclusion of unnecessary genes could be an important
ecological strategy for Prochlorococcus and other ocean
bacteria to adapt to variations in oceanic phosphate concentration. Furthermore, this adaptive process involving
lateral gene transfer may be a general strategy for competition in the ocean environment considering the large
number of gene gains and losses during the evolution
of Prochlorococcus (Rocap et al., 2003; Coleman et al.,
2006; Kettler et al., 2007). These genetic differences can
have a profound impact on the physiology of individual
cells (Moore et al., 2002b; 2005).
To further elucidate the correspondence between
Prochlorococcus genome content and phosphate availability, we analysed for the presence of P acquisition
© 2009 The Authors
Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd
Phosphate acquisition genes in Prochlorococcus cells 1341
genes in Prochlorococcus cells from GOS samples from
several oceanic regions. Then, we compared the distribution of P acquisition genes with data describing the
average monthly P concentration from these locations
and depths taken from the World Ocean Database. The
goal was to determine whether a relationship between
genome content and seawater phosphate concentration
exists. The outcome of this analysis furthers our understanding of how genome content, cell physiology
and environmental variation interact to shape the biogeochemical role of Prochlorococcus.
Results and discussion
Distribution of P acquisition genes
To determine the relationship between phosphate availability and uptake genes in Prochlorococcus, we analysed
36 samples from the GOS expedition as listed in Table 1.
These samples contained at least an average of 2.5 hits
affiliated with Prochlorococcus per 1000 bp. Most of the
samples originated from four oceanic regions: Sargasso
Sea, Caribbean Sea, Eastern Pacific Ocean and the
Indian Ocean.
Table 1. Location, depth, date and nutrient profile of GOS expedition samples and Prochlorococcus cultures.
Location
Sample
GS000a
GS000b
GS000d
GS001b
GS015
GS016
GS017
GS018
GS019
GS022
GS023
GS025
GS026
GS029
GS047
GS048
GS108
GS109
GS110
GS111
GS112
GS113
GS114
GS115
GS117
GS119
GS120
GS121
GS122
GS123
Sargasso Sea
Sargasso Sea
Sargasso Sea
Sargasso Sea
Caribbean Sea
Caribbean Sea
Caribbean Sea
Caribbean Sea
Caribbean Sea
E. Tropical Pacific
E. Tropical Pacific
E. Tropical Pacific
E. Tropical Pacific
E. Tropical Pacific
W. Tropical Pacific
W. Tropical Pacific
Indian Ocean
Indian Ocean
Indian Ocean
Indian Ocean
Indian Ocean
Indian Ocean
Indian Ocean
Indian Ocean
Indian Ocean
Indian Ocean
Indian Ocean
Indian Ocean
Indian Ocean
Indian Ocean
Strain
MIT9301
MIT9303
MIT9313
MIT9312
NATL1A
NATL2A
MED4b
MIT9211
MIT9215
AS9601c
MIT9515
SS120
Sargasso Sea
Sargasso Sea
Gulf Stream
Gulf Stream
North Atlantic
North Atlantic
Mediterranean Sea
Equatorial Pacific
Equatorial Pacific
Arabian Sea
Equatorial Pacific
Sargasso Sea
Depth (m)
5
5
5
5
1.7
2
2
1.7
1.7
2
2
1.1
2
2
30
1.4
1.8
1.5
1.5
1.8
1.8
1.8
1.5
1.5
1.8
2.0
2.8
1.5
1.9
2.2
90
100
135
135
30
10
5
83
5
50
15
120
Date
Latitude
Longitude
Phosphatea
Nitratea
N/P
2/26/03
2/26/03
2/25/03
5/15/03
1/8/04
1/8/04
1/9/04
1/10/04
1/12/04
1/20/04
1/21/04
1/28/04
2/1/04
2/8/04
3/28/4
5/17/4
8/3/5
8/5/5
8/6/5
8/7/5
8/8/5
8/9/5
8/15/5
8/16/5
9/9/5
9/26/5
9/27/5
9/29/5
9/30/5
10/1/5
31.5
31.6
31.5
32.5
24.5
24.5
20.4
18.3
10.3
6.2
5.2
5.1
1.2
0.06
-10.1
-17.5
-12.1
-10.9
-10.4
-9.6
-8.5
-7.0
-5.0
-4.7
-4.6
-23.2
-26.0
-29.3
-30.9
-32.4
296.4
295.7
296.4
295.5
276.9
275.7
274.6
276.2
279.7
277.1
273.4
272.9
269.7
268.4
224.6
210.2
96.9
92.1
88.3
84.2
80.4
76.3
65.0
60.5
55.5
52.3
50.1
43.2
40.4
36.6
0.06
0.06
0.06
0.06
0.03
0.02
0.11
0.08
0.05
0.35
0.95
0.95
0.32
0.50
0.20
0.20
0.24
0.14
0.11
0.10
0.04
0.13
0.17
0.25
0.21
0.13
0.16
0.14
0.14
0.28
0.29
0.29
0.29
0.29
1.48
0.98
0.55
0.30
0.00
4.84
10.6
10.6
0.94
5.38
0.03
0.03
0.05
0.04
0.05
0.10
0.21
0.20
0.09
0.28
0.32
0.38
0.09
0.35
0.90
0.15
5.1
5.1
5.1
5.1
54.9
51.7
4.9
3.6
0.0
13.8
11.1
11.1
2.9
10.7
0.2
0.2
0.2
0.3
0.5
1.0
5.4
1.5
0.5
1.1
1.5
2.8
0.5
2.5
6.5
0.6
07/10/93
07/15/93
07/17/93
07/17/93
04/16/90
04/01/90
01/01/89
04/10/92
10/03/92
11/01/95
06/02/95
05/30/88
34.2
34.8
37.5
37.5
37.4
39.0
43.2
0.0
0.0
-19.0
-5.7
29.0
293.7
293.8
291.8
291.8
320.0
310.7
6.9
220.0
220.0
67.0
252.9
295.7
0.06
0.11
0.42
0.42
0.08
0.16
0.21
0.76
0.68
0.60
0.63
0.02
0.98
1.45
6.74
6.74
0.74
1.96
2.96
5.06
4.75
0.72
4.19
0.84
15.9
14.7
16.0
16.0
9.5
12.6
14.2
6.7
7.0
1.2
6.7
41.9
a. Monthly average values at these locations and depth are retrieved from World Ocean Database (unit: mM) ( Boyer et al., 2006).
b. Value retrieved from grid cell 42.5 N, 6.5 E as no data were available for 43.5 N, 6.5 E.
c. Value retrieved from JGOFS Arabian Sea cruise TTN53, station N7 (Shalapyonok et al., 2001).
© 2009 The Authors
Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1340–1347
1342 Adam C. Martiny, Y. Huang and W. Li
Fig. 1. Occurrence of phosphate acquisition genes in Prochlorococcus.
A. Genes located in proximity to phoB in strain MED4. Red star denotes upregulated genes during phosphate limitation (Martiny et al., 2006).
Prochlorococcus gene occurrence in DNA libraries from the surface waters of the (B) Sargasso Sea (GS00d), (C) Caribbean Sea (GS17), (D)
Eastern Pacific Ocean (GS23) and (E) Indian Ocean (GS114) (Rusch et al., 2007). Abundance of individual genes is determined by reciprocal
best BLAST hit and normalized against length. The frequency is calculated as the length-normalized occurrence of a specific gene divided by
the length-normalized mean occurrence of single-copy core Prochlorococcus MED4 genes at each site (Kettler et al., 2007). The shaded box
represents the abundance of 95% of the single core genes (fitted to a gamma distribution). The unfilled area of pstS represents copies that
are associated with phages.
Martiny and colleagues (2006) observed that many
genes upregulated during P stress in Prochlorococcus
MED4 were located in a genomic region around the phosphate stress response regulator phoB (also illustrated in
Fig. 1A). These genes are mostly involved in P assimilation (e.g. P uptake, organic-P utilization and regulation).
Here we determined the relative abundance of each of
these P acquisition genes in each GOS sample and compared the values with the mean abundance of core genes
(see Table S1 for abundance data for all genes). This
value indicates the relative frequency of Prochlorococcus
cells that contain a given gene. Figure 1B–E show the
results of selected samples from different regions; other
samples from each of these regions show similar trends.
Most Prochlorococcus cells in the Sargasso and Caribbean Sea samples contain the majority of genes from this
genomic region in MED4 [i.e. the ratio of P acquisition to
core genes are within the 95% confidence interval of core
genes (shaded area)], whereas cells lack many genes
in the eastern Pacific and Indian Ocean samples. Cells
from the eastern Pacific and Indian Ocean appear only
to contain genes involved in the direct uptake of ortho-
© 2009 The Authors
Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1340–1347
Phosphate acquisition genes in Prochlorococcus cells 1343
phosphate (phoE and pstABCS) and three genes of
unknown function (PMM0715, PMM0719 and PMM721).
With the exception of PMM0715 and 721, the genes
detected in the Pacific and Indian Ocean are also present
in all sequenced Prochlorococcus strains. This pattern
suggests that Prochlorococcus cells require these proteins for P assimilation regardless of nutrient concentration. The rest of the genes found in the Sargasso or
Caribbean Sea may represent a sophistication that allows
for regulation (e.g. phoBR and ptrA) and utilization of
alternative P sources (e.g. phoA) in addition to several
unknown components.
Despite the presence of pstS in all known Prochlorococcus genomes, Rusch and coworkers found DNA
fragments containing pstS in higher abundance in the
Caribbean Sea compared with the Eastern Pacific Ocean
(Rusch et al., 2007) (see also Fig. 1C and D for the same
trend). Some cyanophages carry phosphate uptake
genes including pstS (Sullivan et al., 2005), so we
removed any GOS hit if the paired-end sequence mate
had best match to a phage (unfilled area in Fig. 1).
However, we still observe a higher abundance of pstS in
the Caribbean Sea compared with other regions. In some
Prochlorococcus strains, pstS is present in two or three
copies (MIT9301, MIT9303, MIT9313 and SS120). This
trend is also observed among marine Synechococcus
(Moore et al., 2005) and may explain the difference in the
frequency of pstS between individual oceanic sites. In
other words, most Prochlorococcus cells in the Sargasso
Sea, Pacific and Indian Ocean only contain one pstS copy
whereas cells in the Caribbean Sea contain two or more.
In MIT9313, only one copy of pstS responded to P stress
indicating that individual copies may be under different
regulatory control (Martiny et al., 2006).
To compare the gene stoichiometries of the GOS
samples to the local phosphate concentration, we
retrieved monthly average values for phosphate and
nitrate for each location and depth from the World Ocean
Database (Table 1) (Boyer et al., 2006). A caveat is that
this approach may introduce additional variance in the
analysis of correspondence between genome content and
phosphate availability, as these values do not represent
the exact nutrient concentration at the time of sampling.
At the month of sampling, the Sargasso Sea and
Caribbean Sea regions are characterized by a low P
concentration (average = 0.06 mM). Samples from the
Indian Ocean generally contained between 0.08 and
0.24 mM phosphate, whereas the P concentration in the
Pacific Ocean sites was above 0.2 mM. This concentration
difference is clearly reflected in the average occurrence of
P acquisition genes in Prochlorococcus (Fig. 2). A detailed
analysis of each gene shows that that cells proliferating
in ocean water with less than approximately 0.1 mM phosphate contain almost all P acquisition genes, cells found in
Fig. 2. Relationship between phosphate concentration and average
occurrence of Prochlorococcus P acquisition genes in GOS
samples and isolated strains. The average occurrence of P genes
is the logarithmic mean of relative abundance of genes from the
phoB genomic region (as defined in MED4) in Prochlorococcus in
a water sample or isolated strain. Thus, MED4 has a value of one.
Phosphate concentration values for each site are retrieved from
the World Ocean Database (Boyer et al., 2006) and are listed in
Table 1.
areas with approximately 0.1–0.3 mM P have some
assimilation genes (including mfs and gap1), whereas
cells from P-rich waters have few of these genes.
Figure 2 also includes 12 Prochlorococcus strains
representing all known major phylogenetic clades. Most
strains isolated from ocean waters with low phosphate
have many P acquisition genes, whereas strains from
P-rich regions have few genes with two notable exceptions. Strain MIT9312 and MIT9313 contain many genes
involved in P uptake but were isolated from the relative
nutrient-rich Gulf Stream at 135 m depth. Both strains
contain partly degraded P acquisition genes (phoA in
MIT9312 and phoR in MIT9313) (Rocap et al., 2003;
Moore et al., 2005; Martiny et al., 2006), so these
lineages could have been in the process of adapting to a
more phosphate-rich environment. Also, the metagenomic analysis measures the average genome content
of Prochlorococcus at a given site whereas the genome
of a single strain represents the history of the cell and not
necessarily the gene content of the population. Thus,
MIT9312 and MIT9313 may have been adapted to a more
nutrient-poor environment but recently transported by the
ocean current to the position where they were isolated.
In contrast, there was no clear relationship between the
nitrogen to phosphate (N/P) ratio and the distribution of P
acquisition genes (Table 1). This result may partly be due
to difficulties of measuring P at very low concentrations
(Karl and Tien, 1992), leading to an overestimation of P in
© 2009 The Authors
Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1340–1347
1344 Adam C. Martiny, Y. Huang and W. Li
the Sargasso Sea (as represented in the World Ocean
Database). However, many samples and strains do not
originate from such extremely low-P environments, but
still do not show any relationship between genome
content and N/P ratio. This suggests that the concentration of phosphate per se controls the distribution of P
acquisition genes rather than the role of P as a limiting
nutrient.
important role in the response to P stress in Prochlorococcus in natural populations. Furthermore, it provides
additional evidence that genes located in genomic
islands may regulate the physiology of individual
Prochlorococcus cells in response to variation in specific
environmental factors as proposed earlier (Coleman
et al., 2006).
Lateral gene transfer
Environmental profiling
Having established that Prochlorococcus cells proliferating in environments with less than approximately 0.1 mM
phosphate have many P acquisition genes whereas cells
from P-rich regions contain only a few of these genes,
we searched for other genes with a similar profile in the
GOS samples. Figure 3 shows the ratio of occurrence
of genes in low- to high-P regions (i.e. samples from
Sargasso plus Caribbean Sea versus Pacific and
Indian Ocean) of each gene in Prochlorococcus MED4.
This analysis underscores the findings described above
whereby P acquisition genes surrounding phoB are
more prevalent in the Sargasso and Caribbean Sea
compared with the Indian and Pacific Ocean. In addition,
we found that a region spanning PMM1403 to PMM1419
is commonly found among cells in low-P areas but not
in high-P areas. Interestingly, these genes belong to a
genomic island (ISL5) unique to MED4 (Coleman et al.,
2006) and in a microarray experiment these genes were
shown to respond to P limitation (Martiny et al., 2006).
This result suggests that this genomic island plays an
Fig. 3. Ratio of occurrence of Prochlorococcus MED4 genes in
low- versus high-P sites. The ratio is determined as the average
frequency of genes in samples from Sargasso and Caribbean Sea
divided by average frequency of genes from Pacific and Indian
Ocean (frequency of genes in Prochlorococcus is calculated as
described for Fig. 1).
From the analysis of P acquisition genes in Prochlorococcus strains, it was clear that the presence or absence
of these genes did not correspond with major phylogenetic groupings (Martiny et al., 2006). However, the
distribution of phosphate acquisition genes in Prochlorococcus could still be explained by the existence of a
number of monophyletic subgroups within major phylogenetic clades that dominate the surface water of the
analysed sites (i.e. eMED4 and eMIT9312). Thus, some
groups within these major clades might be adapted
to low and others to high P concentration. This would
suggest that lateral gene transfer is a relatively rare
event. Alternatively, if phosphate acquisition genes are
commonly transferred laterally between Prochlorococcus
cells, there should be no linkage between the phylotype
(and thereby the core genome of Prochlorococcus) and
the presence or absence of these P genes. To test this,
we compared the 16S/23S rRNA intergenic transcribed
spacer (ITS) sequence similarity from two groups – high(Pacific and Indian Ocean) and low-P environments
(Sargasso and Caribbean Sea) – and observed that
sequences from one environment were not more closely
related to one another than those across environments
(ANOSIM, P = 0.9, n = 126). This result indicates a lack
of phylogenetic clustering related to P physiology and
is consistent with a recent study of the biogeography of
Prochlorococcus where no correspondence between
diversity and P concentration was detected (Martiny
et al., 2009). Furthermore, it suggests that lateral transfer
of P acquisition genes commonly occur between cells
and is an important mechanism for adaptation to differences in P concentration.
Some cyanophages carry host genes including the
phosphate uptake gene pstS, so they may facilitate lateral
gene transfer between Prochlorococcus cells (Sullivan
et al., 2005). To further investigate this, we compared
the phylogeny of pstS in host and phage (Fig. 4), and
observed that pstS associated with phages formed a
distinct cluster. Although cyanophages in all likelihood
gained this gene from Prochlorococcus, this phylogenetic
pattern indicates that the gene has not been transferred
back to the host. Thus, phages may not be the vector
for lateral transfer of P acquisition genes between
Prochlorococcus cells.
© 2009 The Authors
Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1340–1347
Phosphate acquisition genes in Prochlorococcus cells 1345
Fig. 4. Phylogenetic relationship of pstS in
cyanobacteria and cyanophages. The
reciprocal best match of the paired-end
sequence mate of GOS reads was
determined by BLASTX. Numbers on the
tree represents bootstrap values (100 total)
estimated in PHYLIP by neighbor-joining,
maximum parsimony and maximum
likelihood of the protein alignment.
© 2009 The Authors
Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1340–1347
1346 Adam C. Martiny, Y. Huang and W. Li
Role of genome adaptation to P starvation
The analysis presented here suggests a phosphate concentration threshold at approximately 0.1 mM for different
genomic variants in relation to P acquisition genes. Below
this threshold, Prochlorococcus cells contain a variety
of genes involved in orthophosphate uptake, organic P
utilization and regulation as well as many genes of yet
unknown function. Above this threshold many genes are
absent. This ecological trade-off may be driven by the
value of increased P uptake versus the cost of carrying
and expressing additional genes. The P half-saturation
constant (K) – which reflects the ability to take up P at low
concentrations – could be affected by the presence or
absence of some of these genes. This could include the
ability to change the cell protein content when starved
by phosphate using the regulatory system phoBR. The
K-value for P is unknown in Prochlorococcus, but several
marine Synechococcus strains have K-values of 0.014
[measured based on growth, Km (Timmermans et al.,
2005)] to 0.04 mM [measured based on phosphate
uptake, Ks (Ikeya et al., 1997)]. Considering that Prochlorococcus likely has a lower K-value due to the larger
surface-to-volume ratio compared with Synechococcus,
phosphate uptake is probably maximized independently
of extra genes at a phosphate concentration of 0.1 mM
or more. Thus, above this nutrient level it appears advantageous to have a smaller genome compared with an
improved P uptake system and this may drive the gene
content of Prochlorococcus.
Experimental procedures
Abundance of Prochlorococcus genes
The abundance of each Prochlorococcus MED4 gene was
determined in two steps. First, we searched all GOS samples
(Table 1) with TBLASTN (e-value 1E-6 and minimum length of
25 letters) to find hits matching any Prochlorococcus strain
(4.6 million hits). The translated GOS sequences of these hits
were compared with the reference database of all sequenced
genomes (GenBank as of 01/28/08) using fast CD-HIT-2D
program (accurate mode) to determine whether a read had
reciprocal best match to Prochlorococcus (Li and Godzik,
2006). For sequences with no match using CD-HIT-2D, we
also applied BLASTX to find top matches at e-value ⱕ1e-10.
Noteworthy, we used the GenBank annotation for MED4 and
not the newer annotation presented in Kettler and colleagues
(2007), as the GenBank annotation is widely available.
However, this will not influence our results as both annotation
contain all core and phosphate assimilation genes in MED4.
Second, we assigned each Prochlorococcus hit to a specific protein in MED4. We used this genome as we have gene
expression data under P stress and it have the most expansive gene cluster involved in P acquisition (Martiny et al.,
2006). A GOS hit was considered an orthologue if: (i) it was
assigned to Prochlorococcus and (ii) the reciprocal hit
matched the original query protein when only the MED4
genome was searched. We only assigned one hit per GOS
read for each Prochlorococcus query protein to accommodate errors such as frame shifting of the GOS reads. The
motivation for this two-step approach was to avoid problems
with assigning orthologues within Prochlorococcus.
The relative abundance of each gene was calculated as
the number of hits of a specific gene divided by the mean
number of hits of single-copy core genes (as identified in
Kettler et al. 2007). All hits were normalized for gene length
(see Table S1 for raw and normalized values). Using Matlab
(Mathworks, MA), a maximum likelihood estimate of the
mean abundance of Prochlorococcus genes and variance for
each sample is calculated by fitting the number of hits of
single-copy core genes (n = 1209) to a gamma distribution
(Table S1).
We also tested if GOS fragments matching pstS were
located in cyanophages or Prochlorococcus by comparing
the paired end sequence mates against all sequenced
genomes of prokaryotes and cyanophages using BLASTX
(e-value = 1E-6).
Phylogenetic analysis of ITS sequences
We aligned all 16S/23S rRNA ITS sequences affiliated
to Prochlorococcus from the analysed GOS sites (Table 1,
n = 126) using ARB (Ludwig et al., 2004). Next, a distance
matrix of these sequences using maximum likelihood correction was calculated in Phylip v3.66 (Felsenstein, 1989) and
imported into Primer v6 (PrimerE, UK) (see also Table S2
for the distance matrix). We divided the sequences into two
groups – high- (Pacific and Indian Ocean) and low-P environments (Sargasso and Caribbean Sea) – and tested if
sequences from one environment were not more closely
related to one another than those across environments using
ANOSIM.
Acknowledgements
We would like to thank Paul Gilna and John Wooley at
CAMERA (http://camera.calit2.net) for providing computational
resources, Zackary Johnson and Jennifer Martiny for many
helpful comments on the manuscript, and the J. Craig Venter
Institute and Gordon and Betty Moore Foundation for allowing early access to the Global Ocean Survey samples from
the Indian Ocean. The work was supported by the University
of California, Irvine (A.C.M.) and the Gordon and Betty Moore
Foundation (Y.H. and W.L.).
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Supporting information
Additional Supporting Information may be found in the online
version of this article:
Table S1. Raw number of hits to GOS samples, number of
hits normalized to gene length, number of hits normalized to
mean abundance core genes.
Table S2. Phylip distance matrix for GOS reads encoding
Prochlorococcus ITS sequences based on an alignment of
452 positions.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
© 2009 The Authors
Journal compilation © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 1340–1347