Metatranscriptomic and physiological analyses of proteorhodopsin-containing marine flavobacteria by sA4ETs INSWiJ H OF TECHNOLOGY Hana Kim B.S. Biology, Second Major in Oceanography Inha University (2006) U BAR IES Submitted to the Department of Civil and Environmental Engineering in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in Civil and Environmental Engineering at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY September 2013 02013 Massachusetts Institute of Technology. All rights reserved. Signature of Author: Hana Kim Department of Civil and Environmental Engineering August 5, 2013 Certified by: . dward F. DeLong Mort-d C ' oulder Professor of Civil and Environmental Engineering and Biological Engineering Thes upervisor Accepted by: Heidi epf Chair, Departmental Committee for Graduate Students
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Metatranscriptomic and physiological analyses of
proteorhodopsin-containing marine flavobacteria
by sA4ETs INSWiJH OF TECHNOLOGYHana Kim
B.S. Biology, Second Major in OceanographyInha University (2006) U BAR IES
Submitted to the Department of Civil and Environmental Engineering
in Partial Fulfillment of the Requirements for the Degree of
MASTER OF SCIENCE
in Civil and Environmental Engineering
at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY
September 201302013 Massachusetts Institute of Technology. All rights reserved.
Signature of Author:Hana Kim
Department of Civil and Environmental Engineering
August 5, 2013
Certified by: .dward F. DeLong
Mort-d C ' oulder Professor ofCivil and Environmental Engineering and Biological Engineering
Thes upervisor
Accepted by:Heidi epf
Chair, Departmental Committee for Graduate Students
Metatranscriptomic and physiological analyses of
proteorhodopsin-containing marine flavobacteria
By
Hana Kim
Submitted to the Department of Civil and Environmental Engineering
on August 5, 2013 in partial fulfillment of the requirements for the degree of Master of
Science in Civil and Environmental Engineering
Abstract
Proteorhodopsin (PR) is a seven-helix integral membrane protein that uses retinal as a
chromophore. PRs transport protons from the cytoplasmic (CP) to the extracellular (EC)
side of the cell membrane utilizing the energy from light. Since PR was first discovered in
marine Gammaproteobacteria, similar types of rhodopsins have been found in all three
domains of life (archaea, bacteria, and eukaryotes). Recent studies have suggested that
some flavobacteria showed a light-dependent increase in cell yield and growth rate of
cultures grown in low carbon media. Although their function as proton pumps with
energy-yielding potential has been suggested in some strains, the photophysiological role
of proteorhodopsins remains largely unexplored. This thesis describes the functional
characterization of PR-containing flavobacteria previously identified from a (Gomez-
Consarnau et al. 2007; Yoshizawa et al. 2012). We describe here experiments performed to
help understand how PR-containing marine flavobacteria respond to varied DOC
concentrations during light-dependent growth, using growth curve observations, inhibitor
experiments and transcriptomic analyses.
The light-dependent growth effects demonstrated a dependence on carbon concentration,
decreasing at increasing carbon concentration in all PR-harboring strains examined in this
study. Interestingly however, the inverse results were observed at high carbon
concentration (48.5 mM C) which resulted in higher cell yields when grown in the dark
than in the light. Growth experiments using 2-(4-methylphenoxy)triethylamine (MPTA) as
an inhibitor of f-carotene synthesis were performed for the representative isolates,
Dokdonia sp. MED134 and Gilvibacter sp. SZ-19, at low and high concentrations of DOC.
These experiments showed that inhibition of retinal biosynthesis abolished the light-
stimulated growth response at low DOC concentrations. Transcriptomic experiments
were designed to determine the effect of DOC concentration on gene expression of PR-
2
containing MED134 under light and darkness. The results show the both PR and retinal
biosynthetic enzymes exhibit significant upregulation in the low carbon condition when
they exposed to the light. Among protein-coding transcripts of high carbon concentration,
beta-oxidation-associated proteins were expressed at significantly higher levels in the dark.
This work furthers our understanding of the details of light-enhanced growth rates and
cell yields in diverse marine flavobacterial isolates, and demonstrate proteorhodopsin-
associated light-dependent growth effects at various carbon concentrations in several
different flavobacterial proteorhodopsin photosystems.
Thesis Supervisor: Edward F. DeLong
Title: Morton and Claire Goulder Professor of Civil and Environmental Engineering and
Biological Engineering
3
Acknowledgements
This thesis would not have been possible without the guidance and help of several
individuals who in one way or another contributed and extended their valuable assistance
in the preparation and completion of this study.
First and foremost, I would like to offer my sincerest gratitude to my advisor, Dr.
Edward DeLong, who has supported me throughout my thesis with his patience and
knowledge whilst allowing me the room to work in my own way. I appreciate all his
contributions of time, ideas, and funding to make my Master of Science experience
productive and stimulating. The joy and enthusiasm he has for research was motivational
for me.
I am also thankful to have joined a group of caring, fun, and hardworking colleagues in
the DeLong lab and in Parsons. I would like to especially thank Chon Martinez for her all
the advice she gave me; my fellow graduate students, Mike Valliere, and Tsultrim Palden
for their assistance in the metatranscriptomic data analysis and sharing many happy
moments working on projects together; my lab mates Oscar Sosa, Jessica Bryant and post-
doctoral fellow, Dr. Scott Gifford and Dr. Kristina M Fontanez, and John Eppley, for their
insightful discussions and making my lab experience enjoyable.
Last but not least, I owe my deepest gratitude to my parents, Jong Ho Kim and Kyung
Ja Lee, my brother Jeong Su Kim for their love, encouragement and continual support in
Fig. 2 Scheme of the all trans retinal change to 13-cis retinal.
16
1.3. Abundance and diversity of PR-containing marine bacteria
Oceanic picoplankton play a role in driving biogeochemical cycles. The following
bacteria have been found to be the most prevalent in oceanic picoplankton: phyla
Proteobacteria (63%), Bacteroidetes (13%), Cyanobacteria (7.9%), Firmicutes (7.5%), and
Actinobacteria (4.6%) (Venter et al. 2004).
Following the discovery of PR in uncultured marine gamma proteobacterial SAR86
clade (Beja et al. 2001; Sabehi et al. 2004; Rusch et al. 2007), over 4,000 variants have
been identified (Beja et al. 2001; Dioumaev et al. 2002; Spudich 2006) and several
variants have been cloned into Escherichia coli. PR families have been found in
Monterey Bay (Eastern Pacific Ocean), Hawaii Ocean Time (HOT, Central North Pacific
Ocean), Palmer station (Beja et al. 2001), Mediterranean Sea, Red Sea (Sabehi et al. 2003),
Sargasso Sea (Venter et al. 2004) and Pacific Ocean (Rusch et al. 2007). Most of the
variants of PR fall under one of two groups depending on their photochemical properties:
green-absorbing Proteorhodopsin (GPR) and blue-absorbing Proteorhodopsin (BPR)
(Kralj et al. 2008). GPR has higher absorption efficiency. GPR absorbs light with a
absorption maxima of k.x 525 nm (green) whereas BPR has a maxima of kmx 490nm
(blue) (Man et al. 2003). Green-absorbing pigments predominately function in surface
water while 'blue-absorbing' pigments function in deeper waters depending on light
availability (Beja et al. 2001).
Recently, it has been suggested that there exist PR-like genes in CFB (Cytophyga-
Flavobacteria-Bacteroides) subdivision as well as Proteobacteria (Venter et al. 2004). In
the Northwest Atlantic, among metagenome fragments, the Global Ocean Sampling
(GOS) expedition found that the taxa of the PR gene was predominantly present in two
assembled flavobacterial genomes (Rusch et al. 2007; Woyke et al. 2009). Flavobacteria,
17
which belong to the phylum Bacteroidetes previously called by the Cytophaga-
Flavobacterium-Bacteroides. (CFB), and Alpha- and Gammaproteobacteria are major
carriers of photometabolic genes, including the microbial rhodopsin proteorhodopsin in
marine environments (Giovannoni et al. 2005; Rusch et al. 2007). Flavobacteria play a
role in maintaining the earth's energy balance in the biogeochemical cycle of marine
systems (Kirchman 2002; Abell and Bowman 2005; Alonso et al. 2007)
CHAPTER 2. THE INFLUENCE OF LIGHT AND CARBON ON PR-CONTAINING
FLAVOBACTERIA
2.1 Introduction
Research in recent years has revealed new information about the role and diversity of
PR in proteobacteria; however less attention has focused on the function and diversity of
PR in Bacteroidetes (Zhao et al. 2009). Flavobacteria itself was found both free-living and
attached to organic aggregates and are considered as major mineralizers of organic matter.
(DeLong et al. 1993; Abell and Bowman 2005; Cottrell and Kirchman 2009; Zhao et al.
2009). Among them, PR in Flavobacteria (Dokdonia sp. MED134) was revealed to
enhance the cell yields in the presence of light compared to in dark so that PR fulfils a
phototrophic function in marine bacteria (Gomez-Consarnau et al. 2007). Also, in a PR-
containing marine flavobacterial suspension, light-driven proton transport activity was
sufficient for ATP generation was demonstrated (Yoshizawa et al. 2012). The effect of
light on cell yields has been little studied in cultivated marine alpha proteobacterium, and
primarily limited to the SAR11 strain HTCC1062 Pelagibacter ubique (Giovannoni et al.
2005) and gamma proteobacterium SAR92 strain HTCC2207. Therefore, it is expected
18
that flavobacteria will give us information about in vivo function of PR as well as the
relationship between light and cell growth (Stingl et aL 2007).
This study will focus on the discovery of the effect of a light-enhanced growth yield in
proteorhodopsin-containing flavobacterial strains. To see the physiological characteristics
of PR-containing Flavobacteria, we performed growth experiment with various carbon
concentrations in the light or in the darkness. Also, we explored the effect of retinal
biosynthesis inhibitor, MPTA, on light-enhancement growth. To our knowledge, this is the
first time a light-dependent growth yield effect under low and high carbon DOC
concentrations has been demonstrated in native cells. The chapter will conclude with
discussion of the significance of the findings and future directions.
2.2. Methods and Materials
Bacterial strains and culture conditions
Dr. Kazuhiro Kogure (The University of Tokyo, Japan) kindly provide us PR-containing
marine Flavobacteria isolates from sea ice collected in Saroma-ko Lagoon and from the
surface seawater collected at Sagami Bay Station P and western North Pacific Station S
(Fig. 3.).
The bacterial strains used are listed in Table 1. The Flavobacteria strains were routinely
grown in Marine Agar 2216 (Difco Laboratories, Detroit, MI, USA) at 22 'C for 48 h.
19
Fig. 3. Geological maps showing location of the sampling sites of strains tested in this
study.
20
Table 1. PR-containing Flavobacterial strains used in this work
GenBank Accession NumberOrganism
16S rRNA PR
Dokdonia sp. Strain
MED134
Winogradskyella sp. PC-19
Winogradskyella sp. PG-2
Gilvibacter sp. SZ-19
Persicivirga sp. S 1-08
Tenacibaculum sp. SZ-18
DQ481462
AB557530
AB557522
AB557542
AB602426
AB557536
AB557551
AB557552
AB557573
Not yet
AB557572
Reference or Source
Gonzilez et al., 2011
Yoshizawa et al., 2007
Yoshizawa et al., 2007
Yoshizawa et al., 2007
Yoshizawa et al., 2007
Yoshizawa et al., 2007
21
WnogradsA yana sp PC-O (AI35575W) -
WInogradsyeft so PG-3 (AB557553)
Wifoogradsktyefia so PG-10 (AS567555)110ogodkyelasoPC- IS AB5575) *1 GopW~4n rmsk RaGroup W2Winogradskyete so PG-20 ( AS557559)Wnogradskyofia so P2-1 I (AS565650)
waogodskeyla so PC-8 (AR55549) ~ Group W3Uncultured bactedum clone A-04-6(3) (EU663483)
Polsittwoor so, PG-S 4ASS57564)100 Pcaa.boctar sp PGA19 (AB57558) Group P2
U fed bacter su PGo2 (A557556) *
w Group T1
Group T2
Uncuturgo l bacter num M 024-3C (EA 00000000)o*dona a d=r onVsae MEM134 (AAM2DOOO(05)
POeyRdbtor sp. MED1 52 MED152 (AAA00005000)Paolbrecfer aeriuM 234- (AAG04000M 00)1o0 1 o arbacter m p SA4-10 (A557575) a Gtbecter sp SA4-47 (AB55753) 7G pP
Fig. 4.Phylogenetic tree based on PR amnino acid sequences including strains that we studied in
this paper. Figure adapted from Yoshizawa et al., 2007. Red boxes indicate strains examined in this
study. Strains are color coded to indicate site of isolation (Blue, Saromako-Lagoon; Orange,western North Pacific, Green, Sagami Bay). Bootstrap values are shown at each node. Bar, 0. 1substitutions per nucleotide position.
22
Preparation of starter cultures for growth experiments
Starter cultures for the growth experiment at various carbon concentrations were grown
in acid-washed borosilicate glass culture bottles (1 L, VWR), with 500 mL of artificial
sp. SZ-18; Yoshizawa et al, 2012) were grown under light and dark conditions while the
29
concentrations of DOC were varied. Our results showed that as the concentration of DOC
was increased there was a diminishing advantage of growth in the light over the dark (Fig.
7-11). This result is consistent with those of other studies analyzing the effect of different
carbon concentrations on light-induced increases in the growth yield in PR-containing
flavobacterium. Figure 7-11 shows characteristic growth curves for flavobacterial strains
grown on ASW media enriched with varied concentrations of carbon.
The measure of carbon concentrations showed that smaller amounts of carbon resulted
in a different growth yields between cultures grown in light and dark conditions.
Specifically, DOC concentrations below 1.1 mM showed a significant difference in the
maximum growth yield with increased yields in cultures grown in the light compared to
those in the dark (Fig. 7-11). In contrast, the difference between the light and dark cultures
was not significant at a DOC levels of 1.1 mM. The cell yield ratio between light and dark
showed a decreasing trend with increasing DOC concentration at DOC concentrations
greater than 1.1mM (Figure 7-11). Therefore, it can be concluded that proteorhodopsin has
a favorable impact on yields at low carbon concentrations. However, interestingly, the
inverse was true at high carbon concentrations (48.5mM C) in the dark, which resulted in
higher yields when grown in the dark than in the light. We could not fully explain the
reason at this time, but it has been suggested that PMF generated respiration could interact
with the PR.
In some microbial strains containing light transport-inducing rhodopsins, biomass
yields increased and rates of aerobic respiration decreased when the bacteria were grown
in light conditions (Gomez-Consarnau et al. 2007; Lami et al. 2009; Gomez-Consarnau et
al. 2010; Kimura et al. 2011). Several theories have been presented to explain the
mechanism, largely unknown, that could interfere with the ability of PRs to maintain
30
cellular proton mobility force even during respiratory distress. One theory states that
"backpressure" from the PMF directly interacts between the respiratory proteins and
proteorhodopsin, reducing its effect on proton pumping. As a result, PR proton pumping is
replaced by respiration. With the electrochemical potential decreased, the PRs are unable
to translocate protons and electrons across the cell membrane to a terminal electron
acceptor during cellular respiration.
Another possibility would be regulation of metabolic pathways, such as through a
decrease in available ATP, which results in fewer electron donors of the respiratory chain
being formed. These include NADH and the FADH 2 of the tricarboxylic acid (TCA) cycle.
Anabolic pathways competing for metabolites could result in a decrease in respiratory flux.
The high respiration rate may be caused by the excess nutrients which may, in turn,
interfere with the function of proteorhodopsin.
Studies on marine flavobacterial strains containing proteorhodopsin have similarly
found that the light-stimulated growth effect in PR-harboring flavobacteria only occurs at
low carbon substrate concentrations. However, the mechanism by which an increase in
carbon decreases the light effect was not elucidated in this study. To get a better
understanding, we performed transcriptomic analysis from cultures of Dokdonia sp.
MED134 at various carbon concentration and different incubation time.
31
so
60-
40-
201
0 -
Id 2d 3d 4d 5d 6d 7d
Time (day)
40
30 -
20.
OW0I
0.
Id 2d 3d 4d
Time (day)
Sd 6d 7d
Fig.6. Growth curves of Dokdonia sp. MED 134 (a) and Persicivirga sp. S 1-08 (b)
incubated in the light (o) or in the dark (o). Strains were grown in ASW enriched to 0. 14mC. Error bars indicate standard deviation for triplicate cultures.
32
(a)
.2
an0
C0
0U
(b)
8an0
C00iU
+ Light-4- Dark
- LightSDark
(a) (b)20-
15
50
0.
Id 2d 3d 44 5d 68 70
Time (Day)
Id 2d 3d 4d 5d 6d 7d
Time (Day)
0
40-
20-
0-
Id 2d 3d 4d 5d 6d 7d
Time (Day)
+ LONh--W- Dark
Id 2d 3d 4'd ;d 6d 74
Time (Day)
Fig.7. Growth curves of Winogradskyella sp. PC-19 incubated in the light (o) or in dark (e). Winogradskyella sp. PC-19 was grown in ASW enriched to
0.14 mM C (a), in ASW enriched to 0.39 mM C (b), in ASW enriched to 1.1 mM C (c), and , in ASW enriched to 48.5 mM C (d).
60(d)
10
6-
4.
0*
70
(C) 60
S40
.- 30
20
10
0
(a)20
(b)
15 -
10-
0 -
DI 02 03 04 Do D7Time (Day)
1~~ -0-I01 2 03 04 00 06 D7
Time (Day)
Dl 02 03 D4 05 06 D7
ime (Day)
30
E 20
20
15
0
0* i 1DI 02 03 04 06 06 07
Time (Day)
Fig.8. Growth curves of Winogradskyella sp. PG-2 incubated in the light (o) or in dark (9). Winogradskyella sp. PG-2 was grown in ASW enriched to 0.14
mM C (a), in ASW enriched to 0.39 mM C (b), in ASW enriched to 1.1 mM C (c), and), in ASW enriched to 48.5 mM C (d).
(d)
I
IC.)
(c)
6-
(a)
4-
3-
2-
1-
(C)
LA
E
0
0
30-
2 -
20 -
15-
10 -
5-
0-
(b)
I0
C
14-
12-
10 -
4-
2-
0-
id 2d 3d 4d 5d 6d
a0
(d)60-
40-
20-
0-
Id 2d 3d 4d Sd Od 1d 2d 3d 4d 5d Od
Time (Day) Time (Day)
Fig.9. Growth curves of Gilvibacter sp. SZ-19 incubated in the light (o) or in dark (e). Gilvibacter sp. SZ-19 was grown in ASW enriched to 0.14 mM C
(a), in ASW enriched to 0.39 mM C (b), in ASW enriched to 1.1 mM C (c), and ), in ASW enriched to 48.5 mM C (d).
Id 2d 3d 4d 5d 6d
+Light- -Dak
r~i-~1L~J
12.14 -
10
a.
6-
4-
2.
0-
so-
(b)30.
25-
20.
10-
5-
0-
Id 2d 3d 4d 5d Gd 7d
1d 2d 3d 4d 5d Gd 7d
Time (Day)
Id 2d 3d 4d 5d Od 7d
18 2d 38 4d Sd (d 7yTime (Day)
Fig.10. Growth curves of Tenacibaculum sp. SZ-18 incubated in the light (o) or in dark (9). Tenacibaculum sp. SZ-18 was grown in ASW enriched to 0.14mM C (a), in ASW enriched to 0.39 mM C (b), in ASW enriched to 1.1 mM C (c), and), in ASW enriched to 48.5 mM C (d).
(a)
-5'
C)
(c)35
(d)30
25-
5
40-
2, 0-
20-
a* .
2.0-
II
-0.5-
(b)
A
10.
4.
0.
DAY I DAY 2 DAYS3 DA YS DCA YS DAYS6
ime
DAY I DAY 2 DAY 3 DAY 4 DAY S DAY 6Mu.
DAY I DAY 2 AY3 DAY4 DAYS DAYS
Time
DAY I DAY 2 DAY 3 DAY 4
TM.
DAY S DAY I
Fig.11. Growth curves of Dokdonia sp. MED134 incubated in the light (o) or in dark(*). Dokdonia sp. MED134 was grown in unenriched ASW (0.05
mM C) (a), in ASW enriched to 0.14 mM C (b), in ASW enriched to 1.1 mM C (c), and ), in ASW enriched to 48.5 mM C (d). Error bars denote standard
deviation for the triplicates.
3-
2-
0-
M 125
i S.
4.
= 2-
j j F-0- --- tE
Table 3. Maximum cell density (106 cells ml-) of PR-containing marine flavobacterial strains
ASW +0.05mM C ASW +0.14mM C ASW + 0.39mM C ASW + 1.1mM C ASW + 48.5mM CStrain
MED134_03499 189 364 1.9 0.9 2.23E-04 0.063 Putative uncharacterized protein
MED134_04044 1158 2381 2.1 1.0 1.99E-10 0.000 Putative heat-shock related protein
Red character indicates significant upregulation in the light. Black character indicates significant upregulation in the darkness.
M.
C-
II I10-01 10+00 10+01 10+02
mean of normalized counts
11e+03
Fig. 22. Plot of normalized mean versus log2 fold change for low carbon TI light
sample versus low carbon TI dark sample (a) and for low carbon T2 light sample versus
low carbon T2 dark sample (b).
70
(a)
-si
I I I
1 10 100 1000 10000
mean of normalized counts
W.~' ~
a
(b)
C4
I0
CYI
I?.
CM -
a
t~J
en
(b)
q
C
0
9
0
(a)
l-01 1+00 1o+01 1o+02
mean of normelized counts
10+03
Fig. 23. Plot of normalized mean versus log2 fold change for high carbon TI light
sample versus high carbon TI dark sample (a) and for high carbon T2 light sample
versus high carbon T2 dark sample (b).
71
10-01 10+00 10+01 16+02 le+03
mean of nomalized counts
-L
(a)
10+01
mean of normalized counts
le+03
Fig. 24. Plot of normalized mean versus log2 fold change for TI light low carbon versus
TI light high carbon (a) and for low carbon TI dark sample versus high carbon TI dark
sample (b). Red dots indicate differentially expressed genes.
72
N
0
(~1
10 100 1000 10000
mean of normalized counts
"I. Av
'9
(b)
0
10-01
(a) -- - -
H ; -
10-01 10+00 10+01 le+02 10+03
mean of normalized counts
(b) - --
I V
10-01 1o+01 10+03
mean of nonnalized counts
Fig. 25. Plot of normalized mean versus log2 fold change for T2 light low carbon versus
T2 light high carbon (a) and for low carbon T2 dark sample versus high carbon T2 dark
sample (b). Red dots indicate differentially expressed genes.
73
Conclusions
The light-enhanced growth rates and cell yields in marine flavobacterial strains
demonstrate the proteorhodopsin-associated light-dependent growth effect at various
carbon concentrations for the first time in a native cell in a proteorhodopsin
photosystem (PRPS). In this study, we found that increasing concentrations of DOC in
the culture media decreased the effect of the PR-stimulated growth. Also, interestingly,
we found that cell densities of dark-incubated cultures with PR-containing
flavobacterial strains were higher than light-incubated cultures at high carbon
concentration. Together, the results show strong evidence that PR is beneficial in carbon
and respiration limited conditions. The results also show that this effect is seen in a
variety of different PR-containing flavobacteria, suggesting that this phenomena is
widespread and not just limited to a few strains.
To verify that proteorhodopsins are responsible for the observed effect, we conducted
a growth experiment in which we inhibited the synthesis of proteorhodopsin with an
inhibitor of MPTA. Cultures incubated in ASW with MPTA in the light showed
significantly lower yields than those in ASW without MPTA. In culture experiments in
the dark with or without MPTA, cell density of both strains similar to the results with
MPTA-treated condition in the light. This result demonstrated the critical role of PR in
light-enhanced growth.
Additionally, to better characterize the photophysiology of PR-containing marine
flavobacteria, metatranscriptomics studies were performed to see the gene expression
patterns in strain MED134 in this study. Metatranscriptomic analysis will lead to a
better understanding of functional capacity under different carbon conditions different
staged of growth. We still need to work more on metatranscriptomics analyses, but this
study has addressed several questions including: "What are the active genes at
74
different DOC conditions in light?", "When are they active?", and "How do
flavobacteria to different DOC conditions in the dark compared to light" which provides
new insight into the photophysiological characteristics of PR-containing marine
flavobacteria. In the future, it will be useful to examine the gene expression patterns
associated with the higher growth rates and cell yields in flavobacterium strain
MED134 in both low-carbon and high-carbon media in even greater detail.
75
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