Metatranscriptomic and Physiological Analyses of Proteorhodopsin-Containing Marine Flavobacteria

Metatranscriptomic and physiological analyses of proteorhodopsin-containing marine flavobacteria

by sA4ETs INSWiJ Hana Kim H OFTECHNOLOGY

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

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 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 all my pursuits.

4 Table of Contents

Abstract...... 2 Acknowledgements ...... 4 List of Figures...... 7 List of Tables...... 10 CHAPTER 1: MICROBIAL OPSINS, AND INTRODUCTION 1. The important role of proteorhodopsin (PR) in marine ecosystem 1.1 Rhodopsins...... 11 1.2 Proteorhodopsins (PRs)...... 12 1.3. Abundance and diversity of PR-containing marine bacteria...... 17 CHAPTER 2. THE INFLUENCE OF LIGHT AND CARBON ON PR-CONTAINING FLAVOBACTERIA 2.1 Introduction...... 18 2.2. Methods and Materials Bacterial strains and culture conditions...... 19 Preparation of starter cultures for growth experiments...... 23 Culture conditions...... 23 Direct microscopic counting methods...... 26 Culture experiments with specific inhibitor ...... 27 2.3 Results and Discussions Responses of various carbon concentrations on light-dependent growth of PR- containing marine flavobacterial strains...... 29 Specific growth rate of proteorhodopsin-containing marine flavobacterial strain ...... 4 1 Culture experiments with an inhibitor of retinal biosynthetic pathway, M P TA ...... 44 Growth characteristics of representative flavobacterial strains on MPTA containing media...... 47 CHAPTER 3. TRNASCRIPTOMIC ANALYSIS OF LOW AND HIGH CARBON CONCENTRATIONS ON LIGHT-DEPENDENT GROWTH OF Dokdonia sp. MED134 3.1 Introduction...... 51 3.2. Materials and methods

5 Cultivation for transcriptomic analyses...... 57 Total RNA extraction...... 58 ribosomal RNA (rRNA) subtraction...... 58 RNA amplification, complementary DNA (cDNA) synthesis and Illumina sequencing...... 59 3.3 Results and Discussions Cultivation for transcriptomic analyses...... 61 Transcriptom ic analyses...... 63 Transcriptomic survey for High carbon conditions...... 64 Conclusion...... 74 References...... 76

6 List of Figures

Fig. 1 Scheme of the carotenoid biosynthesis pathways from farnesyl pyrophosphate

(FPP) to retinal...... 15

Fig. 2 Scheme of the all trans retinal change to 13-cis retinal...... 16

Fig. 3 Geological maps showing location of the sampling sites of strains tested in this

study...... 20

Fig. 4.Phylogenetic tree based on PR amino acid sequences including strains that we

studied in this paper...... 22

Fig. 5. Specific inhibitor of Lycopene cyclization, 2-(4-Methylphenoxy) Triethylamine

(MPTA) (a) Chemical formula of MPTA. (b) The bioregulatory action of MPTA in

-carotene synthesis ...... 28

Fig.6. Growth curves of Dokdonia sp. MED134 (a) and Persicivirgasp. S 1-08 (b)

incubated in the light (o) or in the dark (e)...... 32

Fig.7. Growth curves of Winogradskyella sp. PC-19 incubated in the light (o) or in dark

(0 )...... 33

Fig.8. Growth curves of Winogradskyella sp. PG-2 incubated in the light (o) or in dark

(0 )...... 34

Fig.9. Growth curves of Gilvibactersp. SZ-19 incubated in the light (o) or in dark

(0 )...... 35

Fig.10. Growth curves of Tenacibaculum sp. SZ-18 incubated in the light (o) or in dark

(0 )...... 36

Fig.11. Growth curves of Dokdonia sp. MED134 incubated in the light (o) or in dark

(0 )...... 37

7 Fig. 12. Specific growth rate (p) determined by regression analysis based on the growth

experiment of strain Dokdonia sp. MED134...... 40

Fig. 13. Summary of specific growth rates (p) of Dokdonia sp. MED 134 (a) and

Winogradskyella sp. PG-2 (b)...... 42

Fig. 14. Summary of specific growth rates (p) of Winogradskyella sp. PC-19 (a),

Gilvibactersp. SZ-19(b) and Tenacibaculum sp. SZ-18 (c)...... 43

Fig. 15. Colony images of Dokdonia sp. MED134 in culture experiment with a

M PTA ...... 45

Fig. 16. Colony images of Gilvibactersp. SZ-19 in culture experiment with a

M PTA ...... 46

Fig. 17. Growth curves of Dokdonia sp. MED134 in culture experiments with a MPTA

inhibitor...... 49

Fig. 18. Growth curves of Gilvibactersp. SZ- 19 in culture experiments with a MPTA

inhibitor...... 50

Fig. 19 Scheme showing how the integration of results from different technological levels

of functional genomics leads to construction of a virtual

m icroorganism ...... 55

Fig. 20 Scheme of sample processing pipeline for the metatranscriptomic analysis;

adapted from Stewart et al (2010)...... 56

Fig. 21 Determining optimal sampling time points for transcriptomic experiments, based

on growth curves of Dokdonia sp. MED134...... 62

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

Fig. 23. Plot of normalized mean versus log2 fold change for high carbon TI light sample

8 versus high carbon T1 dark sample (a) and for high carbon T2 light sample versus high carbon T2 dark sample (b)...... 71 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 sam ple (b)...... 72 Fig. 24. Plot of normalized mean versus log2 fold change for T1 light low carbon versus TI light high carbon (a) and for low carbon TI dark sample versus high carbon T I dark sam ple (b)...... 73

9 List of Tables

Table 1. PR-containing Flavobacterial strains used in this work...... 21

Table 2. Composition of agar media used for cultivation studies...... 25

Table 3. Maximum cell density (106 cells ml-1) of PR-containing marine flavobacterial

strain s ...... 3 8

Table 4. Specific growth rate (day-) of PR-containing marine flavobacterial

strain s...... 39

Table 5. Number of reads remaining after quality control step in the pipeline for quality

control...... 65

Table 6. Preliminary analysis of functional enrichment of differential gene expression in

low carbon condition at TI...... 66

Table 7. Read number and significant upregulated genes in the light in low carbon

condition at T2...... 67

Table 8. Read number and significant upregulated genes in the light in high carbon

condition at T I...... 68

Table 9. Preliminary analysis of functional enrichment of differential gene expression in

high carbon condition at TI...... 69

10 CHAPTER 1: MICROBIAL OPSINS, AN INTRODUCTION

1. The important role of proteorhodopsin (PR) in marine ecosystem

1.1 Rhodopsins

Rhodopsins are light harvesting protein that contain retinal (Vitamin A aldehyde) as a

chromophore and have seven transmembrane alpha helices (Spudich 2006). These light

sensitive proteins absorb light and convert it into photonic energy to transport ions across

the cell membrane. More specifically, upon light illumination, the photocycle begins when

the retinal (Vitamin A aldehyde) in the protein undergoes photoisomerization causing

conformational changes of the protein and the creation of the electrochemical gradient that

facilitates ion transportation. Rhodopsins are basically classified into two protein families:

type I and type II rhodopsins. The big difference between type I and type II rhodopsin is

the photoisomerization mechanism of chromophore. In rhodopsin I, the

photoisomerization causes retinal changes from all-trans to 13-cis whereas in rhodopsin II,

changes occur from 11 -cis to all trans retinal (Jung 2007). Type II rhodopsins function as

photosensitive receptor proteins in animal eyes that are human rod and cone visual

pigments. Type I rhodopsin is an archaeal-type rhodopsin and well studied in

Halobacterium salinarium, halophilic archaea. The first discovered rhodopsins are

bacteriorhodopsin (BR), halorhodopsin (HR), sensory rhodopsin I and II (SRI and SRII) in

H. salinarium (Spudich et al. 2000). A number of other rhodopsins have been identified as playing a role in proton-motive force, although their mechanisms differ. Proteorhodopsins have been found in marine bacteria, archaea, and eukarya, Xanthorhodopsins (XRs) use two chromophores, the carotenoids salinixanthin and retinal, to broaden the spectral range for light harvesting (Balashov et al. 2005). Actinorhodopsins (ActRs) have been found in

11 abundance in freshwater, including actinobacteria from a hypersaline lagoon, an estuary,

and a freshwater lake (Sharma et al. 2008). Bacteriorhodopsin (BR) and halorhodopsin

(HR) are light-driven pumps that use light as a catalyst to translocate protons and chloride,

respectively. Rhodopsins may be involved in regulating ionic content and the osmotic state

(Mongodin et al. 2005). Sensory rhodopsins (SRs) function like photoreceptors rather than

ion transporters, mediating phototaxis or signal transduction (Spudich et al. 2000 ).

Recently, type I rhodopsins have been found also in other lineages such as, Eubacteria,

and unicellular Eukarya (Bieszke et al. 1999; Sineshchekov et al. 2002; Jung et al. 2003).

That is the reason why type I rhodopsin is also called microbial rhodopsin.

Archaeal-type rhodopsins, type I rhodopsins, serve as a model for proteorhodopsin

photosystems (PRPS) (Kawanabe et al. 2006). In reaction to a light signal, they establish a

chemical gradient from light energy and change their conformation to facilitate the

translocation of protons. In this study, I am focused on proteorhodopsin (PR), which belongs to the type I rhodopsin family.

1.2 Proteorhodopsins (PRs)

Proteorhodopsins (PRs), a member of the microbrial rhodopsin superfamily of proteins, are retinal-binding transmembrane proteins that play a key role in light-activated proton efflux (Beja et al. 2000; Beja et al. 2001; Martinez et al. 2007).

Earlier studies showed microbial rhodopsins to be associated with Archaea but later studies found PRs in an uncultured marine gammaproteobacterial SAR86 group (Morris et al. 2002; Boichenko et al. 2006). PR-containing bacteria are found in a minimum of 13% of all microorganisms in the photic zone, the layer of the ocean that is exposed of sunlight

12 (Stingl et al. 2007).

Planktonic Bacteria, Archaea and Eukarya reside and compete for light in the photic zone of the ocean (McCarren and DeLong 2007). The PR-bearing marine microbes use light energy for the acquisition of adenosine triphosphate (ATP) by proteorhodopsin

(Frigaard et al. 2006). The proton motive force (PMF) is generated by an electrochemical gradient that transports protons across an energy-transducing membrane. Through oxidative phosphorylation, bacteria use the PMF to synthesize ATP, drive chemiosmotic reactions, and power the rotary flagellar motor (Kashket 1985). The light energy absorbed by rhodopsins is used to translocated protons outside the cell, thereby generating the protonmotive force. Proteorhodopsin have been identified in the world's oceans making this light-transducing protein a key source of solar energy absorption in photosystems.

Protoeorhodopsins are composed of the opsin protein and the chromophore trans-retinal

(Bielawski et al. 2004; Giovannoni et al. 2005). Retinal is formed through the conversion of the carotenoid -carotene (Sabehi et al. 2005; Walter et al. 2007). Many but not all marine bacteria contain the genes required for retinal biosynthesis, or genes that enable the conversion of the precursor beta carotene to retinal(Peck et al. 2001).

The 15, 15' C=C bond is located at the central cleavage of p-carotene to form all-trans retinal. The conversion of the isoprenoid precursors into 0-carotene is catalyzed by four ci genes: crtE, crtB, crtI, and crtY (Fig.1) (Martinez et al. 2007; McCarren and DeLong

2007). One molecule of p-carotene produces two retinal molecules. The rhodopsin chromophore retinal produces the oxidative cleavage of 0-carotene.

This enzyme produced by cleaving the beta-carotene into two retinals is called 15, 15'-

0-carotene-dioxgenase. Some proteobacteria can also synthesize f-carotene and convert it to retinal by using the enzyme encoded by the blh gene to produce functionally active PRs

13 (Kim et al. 2008).

Proteorhodopsin is all a photoactive retinylidene protein that functions as a heptahelical proton pump (de la Torre et al. 2003). It is simple heptahelical proton pumps containing a retinal chromophore covalently bound via a lysine Schiff's base to helix G A single molecule of all-trans retinal is binded by key residues lining the inner surface of the channel. Ion pumping is then induced by a corresponding change in protein conformation which causes ion transport accessory effector proteins to interact with the sensory rhodopsin family of proteins (Fig2) (Ottolenghi and Sheves 1989; Birge 1990).

Abundant evidence exists of PRs function as a transmembrane proton pump, including the light-mediated transport of protons in right-side-out PRs vesicles. Also, genetic community surveys suggested that proteorhodoopsin-harboring microorganisms are common in the different phylogenetic groups of the oceans by lateral gene transfer, indicating a fitness advantage (de la Torre et al. 2003; Frigaard et al. 2006; Martinez et al.

2007)

14 IPP 6-isomerase 0 , (idl) 0 o -P-o-P-O. -* -p-O-P-O0 6- O 6- 0- Isopentenyl iphosphate Dimethylallyl diphosphate (IPP) (DMAPP)

FPP synthase Ipp (ispA) I P

OPP FPP

GGPP synthase PP (crtE)

OPP GGPP

Phytoene synthase GGPP (citB)

Phytoene

Phytoenedehydrogenase (ct/) 4

Lycopene

Lycopene cyclase

Q -Carotene

15,15'-p-carotene dioxygenase OH (b/h)

6(-"b OH Retinal

Fig. 1 Scheme of the carotenoid biosynthesis pathways from farnesyl pyrophosphate (FPP) to retinal. (FPP: farnesyl pyrophosphate, IPP: isopentenyl pysophosphte (diphosphte), CrtE: GGPP synthase, GGPP: geranlygeranyl pyrophosphate, CrtB: phytone synthase, CrtI: phytoene desaturase, CrtY: Lycopene cyclase, BIh: Bacteriorhodopsin- related-protein-like-homolog protein)

15 H Retinal all irma isomer H+ Protonated Schiff base Light induced Spontaneous isomnerization H+N isomedzation

H+N

H Retinal 13-cis isomer Protonated Schiff base

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 Pelagibacterubique (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 Number Organism Reference or Source 16S rRNA PR

Dokdonia sp. Strain DQ481462 Gonzilez et al., 2011 MED134

Winogradskyella sp. PC-19 AB557530 AB557551 Yoshizawa et al., 2007

Winogradskyella sp. PG-2 AB557522 AB557552 Yoshizawa et al., 2007

Gilvibactersp. SZ-19 AB557542 AB557573 Yoshizawa et al., 2007

Persicivirgasp. S 1-08 AB602426 Not yet Yoshizawa et al., 2007

Tenacibaculum sp. SZ-18 AB557536 AB557572 Yoshizawa et al., 2007

21 WnogradsA yana sp PC-O (AI35575W) - WInogradsyeft so PG-3 (AB557553) Wifoogradsktyefia110ogodkyelasoPC- so PG-10 IS (AS567555)AB5575) *1 GopW RaGroup Winogradskyete~4n so PG-20rmsk (AS557559) W2 Wnogradskyofia so P2-1 I (AS565650)

waogodskeyla so PC-8 (AR55549) ~ Group W3 Uncultured 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

numM 024-3C(EA 00000000) Uncuturgoo*dona l bacterd=ra onVsae MEM134 (AAM2DOOO(05) POeyRdbtor sp. MED1 52 MED152 (AAA00005000) Paolbrecfer aeriuM234- (AAG04000M 00) 1 arbactero 1o0 SA4-10m p(A557575) a G tbecter sp SA4-47 (AB55753) 7G pP Uncultured bacteium dcone W1-4 (EU(A83550) Uncultured bectedum clone W-50-2 (EU683584) Uncultured bacterium cone WW1-2 (ELMUM49) Uncultured bacterium clone A140-3 (EU683491)

-ordia sp . Winogradk ye s. PG-2 (A9557551) 10 WUncraistu abacePuclneS.-5iS2> Group W1 Wogradsmyea4 PG-16 (A757557)e I so eigonie douq trainmnsis n O6PRHC (F427093) 84romwsp PZ- 3 (AS557561) 50 PSYChAWOMeUS ftfrgds ATCC 700755 A APR00000000) PAthosate bogr adapte bofoed (AAin00s s sd S n atoo Floolacteiou nBspSA4-32S(AB55758I)* PsF''awobactedumso.SA415 (ASS57576) wiay). ctedum sp.SA4-23 (Ao57579) su Rbstitutiduo s. SA4-4 (AB57585) Flawobactehum so, SA4-8 (AS557584) '00 oareu So. SA4-44 (AB557582) sFlembcrehum so. SA4-20 (ASS57578) Flatobactenumop. SA4-10 (ASS57577) at Flemabscewrrurso, SA4-3W (AB557580) 54Uncultured bacterium olonm WI-13 (E~MOMS Uncultured bacterium ARCTIC02-G-04 (EU?95231) Uncultured bacterium dlone A1-0-7(3) (EL*83481) Uncultured bacterium dlone W6.30-8 (ELM683MI) 96Uncultured bacledurn clone WO-0-10 (EULS3M72) -SpirosameMoguerp DSM 74 (CP001769) -P&Wgirbmtertfi e strain HTWc1062 (EF64091 1)

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. 1 substitutions 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

seawater (ASW, 15 practical salinity units, Sigma CAS No: 7647-14-5) supplemented

with a full strength media (FSM, 1.1 mM C; Table 2). The ASW was filtered through 0.2

mm-pore-size bottle-top vacuum filter system (Nalgene, NY, USA) and autoclaved. A colony grown on marine agar plate streaked from frozen stock was inoculated in ASW

amended with 1.1 mM C FSM media. The starter culture was covered with aluminum foil

(Reynolds) and grown in the dark at 21'C - 22*C until near the middle of the exponential phase, which took from 3 to 5 days, depending on the strain, the conditions of growth, and the initial cell density. Cell density of the starter culture was determined during late- exponential phase by the direct cell counting method. Then, the starter culture was stored in a 4C refrigerator and used to inoculate further growth experiments. And then all cultures were inoculated with a known number of cells (1000 cells/ml).

Culture conditions

Experiments to determine the effect of DOC concentration on light-dependent physiological effect of the PR-containing Flavobacterial strains were inoculated from a stationary phase of starter culture grown on 1.1 mM DOC in a dark condition. Inoculation was taken from this stationary phase culture stored in 4 C. The culture density was followed (cells/mL) up to the time of inoculation and was used to determine the dilution of the starter culture into the DOC concentration experiment cultures. And then all cultures were inoculated with a known number of cells (1000 cells/ml). A common culture was

23 inoculated for all experiment conditions in ASW amended with FSM. Thorough mixing

was provided at each step through rigorous vortexing.

Cultures of PR-containing flavobacterial strains were grown in artificial seawater (ASW,

15 practical salinity units, Sigma CAS No: 7647-14-5) supplemented with a dissolved organic carbon (DOC) at the indicated concentration (Table 2; full strength FSM contains

1.1 mM DOC). The ASW was filter-sterilized through 0.2 mm-pore-size bottle-top

vacuum filter system (Nalgene, Rochester, NY, USA) and autoclaved. Then, 250 ml

aliquots of ASW (containing a background concentration of 0.05 mM C of DOC) were partially supplemented with full strength medium (FSM). Concentrations of DOC in the

ASW media tested were: 0.05 mM, 0.14 mM, 0.39 mM, 1.1 mM, and 48.5 mM. To avoid inorganic nitrogen and phosphate limitation, the media were also amended with 225 Im

NH 4Cl (Sigma CAS No: 12125-02-9) and 44.7 pm Na 2HPO4-12H 20 (Sigma CAS No:

10039-32-4). Cultures were grown in 50 mL glass tubes. Tubes for the dark condition were wrapped with aluminum foil and tubes for the light condition were left unwrapped.

Cultures were incubated in a walk-in light room incubator. The temperature of the incubator is maintained at 21 C - 22 C.

To avoid DOC contamination in the growth experiment, it was extremely important to clean all glassware used in the analysis of growth characteristics very carefully. All glassware was acid-washed with 10% HC1 (Macron Chemicals CAS No: 7647-01-0) with an overnight contact time and then thoroughly rinsed with distilled water. Acid-washed glassware was autoclaved before use.

24 Table 2. Composition of agar media used for cultivation studies.

Marine Agar 2216 Full Strength Medium

(g/L) (g/L)

Peptone 5 Peptone 5

Yeast extract 1 Yeast extract 1

Artificial sea water (ASW), Ferric citrate 0.1 1000 ml

Sodium chloride 19.45

Magnesium chloride 5.9

Magnesium sulfate 3.24

Calcium chloride 1.8

Potassium chloride 0.55

Sodium bicarbonate 0.16

Potassium bromide 0.08

Strontium chloride 34 mg

Boric acid 22 mg

Sodium silicate 4 mg

Sodium Fluoride 2.4 mg

Ammonium nitrate 1.6 mg

Disodium phosphate 8 mg

Agar 15

DDW, ml 1000

25 Direct microscopic counting methods

Total cell numbers were determined by direct microscopic count according to the

method described previously (Kimura et al, 2011). The appropriate volume of culture was diluted using ASW and stained with 100 pL of lOOX SYBR Green (Molecular

Probes/Invitrogen) for 15 minutes under darkness. Then, the solution was vacuum filtered with a 25 mm diameter, 15-mL funnel filter apparatus (VWR) onto a black 0.22 pm isopore membrane (Millipore, #GTBP02500). Microscope slides were made using a glass slide (Gold Seal Microslides #3010) with immersion oil (Zeiss Immersol 518) and coverslip (Thermoscientific mSeries Lifterslip 22x251-2-4816). Finally, cellular growth was determined using an epifluorescence microscope (Zeiss Axioskop 2 microscope) with a 100 X magnification objective (Zeiss Plan-NEOFLUAR). Bacterial cell densities with the proper dilution were counted at least 10 separate fields of view across the filter. Total cell densities of samples were calculated using the following equation (from Wetzel and

Likens, 1991).

Bacteria ml' = (membrane conversion factor * ND)

Membrane conversion factor = Filtration area/area of micrometer field

N = Total number of bacteria counted/number of micrometer fields counted

D = Dilution factor; volume of sample stained/total volume of sample available

26 Culture experiments with specific inhibitors

Kimura et al. (2011) proposed that retinal-bound PR has a important role in the light-

stimulated growth by culture experiment with MPTA inhibitor. MPTA act as a inhibitor of

lycopene cyclization in retinal biosynthesis pathway (Cunningham et al., 1994; Armstrong,

1999).

First, to confirm the effect of MPTA inhibitor on PR-containing flavobacterial strains,

we cultured strains on Marine Agar 2216 amended with MPTA at a final concentration of

300 mM.

The agar plates were incubated at room temperature for 3 days. When colonies were

clearly visible, we determined the effect of MPTA against flavobacterial strains on the

inhibition of 0-carotene synthesis as indicated by colony pigmentation. Next, to see the

PR-induced light enhancement in growth, Dokdonia sp. MED134 and Gilvibacter sp. SZ-

19 were grown in ASW slightly enriched with FSM (0.14mM C and 48.5mM C) and

amended with MPTA. MPTA was dissolved in methanol and added to ASW at a final

concentration of 100 mM. The same volume of methanol was added to negative control

cultures without MPTA. These cultures were incubated at 21- 22 C under continuous

white light (approximately 150 mmol of photons m-2 s-1) or in the darkness in both carbon

concentrations. The culture experiments with MPTA were performed in triplicate.

Bacterial cell density was measured every day by the direct counting method with epifluorescence microscope described above.

27 (a)I N CH3

CH 3

2-(4-Methylphenoxy)Triethylamine (MPTA)

(b) Lycopene

Lycopene cycla M PTA (cit)

+-Carotene 15,15'- -carotene dioxygenase OH (b/h)

6"%)%COH Retinal

Fig. 5. Specific inhibitor of Lycopene cyclization, 2-(4-Methylphenoxy)Triethylamine (MPTA) (a) Chemical formula of MPTA. (b) The bioregulatory action of MPTA in 0-carotene synthesis.

28 2.3 Results and Discussions

Responses of various carbon concentrations on light-dependent growth of PR- containing marine flavobacterial strains

Several research studies have been shown that the light-stimulated ATP production or growth in PR-containing bacteria could be detected under carbon limiting conditions

(Gomez-Consarnau et al., 2007; Martinez et al., 2007; Gomez-Consarnau et al., 2010;

Steindler et al., 2011). In this study, we used marine flavobacterial strains to measure the sensitivity to light of the PR light-transducing genes as DOC concentration was varied in a culture media. To determine potential interference with very low levels of DOC caused by endogenous carbon or carryover from the starter culture, the effect of the DOC concentration was determined using a stationary phase culture grown on 1.1 mM glucose in the dark. Then, the cells were washed twice using ASW. Cell density was directly counted under the epifluorescence microscopy with SYBR Green (Applied Biosystems) staining.

Before the beginning of the growth experiment with various carbon concentrations, we confirmed the light-enhanced growth at low carbon concentration (0.14 mM C) on

Dokdonia sp. MED 134 and Persicivirgasp. S 1-08 (Fig. 6). In both strains, cell densities were increased significantly, which were consistent with those seen in before. This shows that further growth experiments with different carbon concentrations are needed to investigate the PR-containing marine flavobacterial strains.

Flavobacteria strains (Dokdonia sp. MED134, Winogradskyella sp. PC-19,

Winogradskyella sp. PG-2, Gilvibacter sp. SZ-19, Persicivirga sp. S1-08, Tenacibaculum 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 (a)

60-

an 40- 0

C 201 0

0 U 0 - + Light -4- Dark

Id 2d 3d 4d 5d 6d 7d Time (day)

40 (b)

30 -

8 an 0 20.

C 0 0 i OW0I U

- Light 0. SDark

Id 2d 3d 4d Sd 6d 7d Time (day)

Fig.6. Growth curves of Dokdonia sp. MED 134 (a) and Persicivirgasp. S 1-08 (b) incubated in the light (o) or in the dark (o). Strains were grown in ASW enriched to 0. 14m C. Error bars indicate standard deviation for triplicate cultures.

32 20- (a) (b)

6- 15

10 50

0* 0.

Id 2d 3d 44 5d 68 70 Id 2d 3d 4d 5d 6d 7d Time (Day) Time (Day)

70 60 (C) 60 (d)

S40 40-

.- 30

20 20-

10 0 0- 0 + LONh --W- Dark

Id 2d 3d 4d 5d 6d 7d Id 2d 3d 4'd ;d 6d 74 Time (Day) 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). 20 (a) (b)

15- I

10-

I C.) 0 - 1~~ -0-I DI 02 03 04 Do D7 01 2 03 0400 06 D7 Time(Day) Time (Day)

E 20

0* i 1 02 03 D4 05 06 D7 Dl DI 02 03 04 06 06 07

ime (Day) 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). 6- 14-

(a) (b) 12-

10 - 4- I 3- 0

2- 4- C 1- 2-

Id 2d 3d 4d 5d 6d id 2d 3d 4d 5d 6d

a0 (C) (d) 30- LA 60- E 2 -

20 - 40- 0

15-

20- 10 -

0 5- 0- 0- +Light r~i-~1 - -Dak L~J

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). (a) a* . (b) 30.

-5' 25-

1412.- 20. 10

6- 10-

4- C) 5- 2.

0- 0-

Id 2d 3d 4d 5d Gd 7d Id 2d 3d 4d 5d Od 7d

so- 35 (c) (d) 30

25- 40-

2,0-

20- 5

1d 2d 3d 4d 5d Gd 7d 18 2d 38 4d(d Sd 7y Time(Day) Time (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 0.14 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). (b) 10. 2.0- II

A 4.

-0.5-

DAY I DAY2 DAYS3 DA YS DCAYS DAYS6 DAYI DAY2 AY3 DAY4 DAYS DAYS ime Time

125 M

i S. 3-

2- = 2-

0- j j F-0---- tE

DAYI DAY2 DAY3 DAY4 DAYS DAY6 DAY I DAY2 DAY3 DAY4 DAYS DAYI Mu. TM.

Fig.11. Growth curvesof 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 mMC (d). Error bars denote standard deviation for the triplicates. 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 + 1.1mMC ASW + 48.5mM C Strain L D L D L D L D L D

Dokdonia sp. MED134 0.23 0.06 9.32 6.28 - - 46.7 47.0 442 529

Winogradskyella sp. PC-19 -- 7.30 4.72 17.5 18.3 47.2 59.6 349 659

Winogradskyella sp. PG-2 - - 10.1 7.44 18.2 19.0 59.9 95.3 41.0 288 wj 00 Gilvibacter sp. SZ-19 0.13 0.05 9.27 6.31 - - 47.5 50.4 526 712

Persicivirga sp. S 1-08 3.26 2.46

Tenacibaculum sp. SZ-18 -- 1.97 1.87 2.56 2.84 3.27 3.51 3.53 3.43 Table 4. Specific growth rate (day-1) 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 C Strain L D L D L D L D L D

Dokdonia sp. MED134 1.53 0.62 3.20 2.96 - - 3.94 4.04 4.99 5.14

Winogradskyella sp. PC-19 - - 1.93 1.75 1.94 2.22 2.17 2.60 3.38 3.95

Winogradskyella sp. PG-2 -- 0.74 0.73 0.81 0.82 1.16 1.29 1.04 1.29 wj Gilvibacter sp. SZ-19 0.76 0.49 1.44 1.31 -- 3.43 3.70 4.20 4.31

Persicivirga sp. S1-08 1.55 1.47

Tenacibaculum sp. SZ-18 - - 1.97 1.87 2.56 2.84 3.27 3.51 3.53 3.43 (a) ASW+ 0.05 mM C (b) ASW+0.14mM C 20 20 - Ught: y = 3.2044x + 6.0143 Ught: y = 1.5296x+ 7.6954 3 15 -+-Ught R =0.9997 -U*-Dark ...... -.- 9 10 - S 10 - Dark: y = 0.6242x+8.5632 Dark: y = 2.9639x + 6.181 5 R= 0.996 5 -Light R2= 0.9986 -+Dark 0 0

1 2 3 1 2 3 TIme(Day) TIme(Day)

(C) ASW+1.1 mM C (d) ASW+ 48.5 mM C 20 - 25 Ught:y = 4.9851x+4.6964 R = 0.9739 J~. 15 20 -+-LUght 0 -m-Dark Ught: y =3.9392x + 5.1 49 j5 a 10- R2=0.9982 Dark: y = 5.1442x + 4.3696 10 7 -- Ight Dark: y = 4.036x + 5.0083 R = 0.989 --- Dark R2=0.995 0- 0 1 2 3 1 2 3 Time (Day) TIme(Day)

Fig. 12. Specific growth rate (p) determined by regression analysis based on the growth experiment of strain Dokdonia sp. MED134. (a) ASW + 0.05 mM C, (b) ASW + 0.14mM C, (c) ASW + 1.1 mM C, (d) ASW + 48.5 mM C. Specific growth rate of proteorhodopsin-containing marine flavobacterial strains

The maximum specific growth rates (g) are one good way of expressing the relative ecological success of a species or strain in adapting to its natural environment or the experimental environment imposed upon it.

The maximum specific growth rates (the slope of a plot of the natural log of cell numbers and time yields the specific growth rate, g) at various carbon concentrations were determined for PR-containing flavobacterial strains. The bacterium's growth was monitored during the exponential phase to measure the specific growth rate at each carbon concentration, as shown in Figure 12 and Table 4. The maximum cell density and specific growth rate reached are shown in Table 3 and 4, respectively.

The specific growth rates of the isolates under various growth conditions were summarized in graphs and compared to each other (Fig. 13 and 14). The g values of the strains under light in ASW plus 0.14 mM C and LNHM plus 48.5 mM C ranged from 0.74 to 3.20 day-1 and

1.04 to 4.99 day-1, respectively (Table 2), and these values were lower than p value of cultures in the dark condition at each strain.

All five strains in the Flavobacteria showed maximum g values at low carbon concentrations (0.05 mM C and 0.14 mM C) in light-incubated cultures, while they showed maximum g values at high carbon concentrations (Fig. 13 and 14). Interestingly, growth characteristics of the PR-containing flavobacterial strains were generally switched at media containing high carbon concentration. From this result, DOC concentration could be one of the criteria for differentiating light-dependent growth within the PR-harboring flavobacteria strains.

41 (a) Dokdonia sp. Strain MED134 6-

3 +Light

0 - 0.05mM C 0. 14 nM C 1.1 nM C 48.5 niM C Carbon concentrations added to ASW

(b) Winogradskyellasp. PG-2 6 5 4

0.05 mM C 0.14 nl C 1.1 NIM C 48.5 niMI C Carbon concentrations added to ASW

Fig. 13. Summary of specific growth rates (g) of Dokdonia sp. MED134 (a) and

Winogradskyella sp. PG-2 (b).

42 (a) Winogradskyellasp. PC-19

-+-Light -U-Dark 0 0.14 nM C 0.39 mM C 11 1mM C 48.5 nAM C Carbon concentrations added to ASW

(b) Gulvibactersp. SZ-19

o -_--_ _-__ -_ 0.14 mM C 0.39 IM C 1.1 IIM C 48.5 uM C Carbon concentrations added to ASW

Tenacibaculunt sp. SZ-18

--- Light -U-Dark

0 0.14 mM C 0.39 NM C 1.1 mM C 48.5 mn C Carbon concentrations added to ASW

Fig. 14. Summary of specific growth rates (p) of Winogradskyella sp. PC-19 (a), Gilvibacter sp. SZ-19(b) and Tenacibaculum sp. SZ-18 (c).

43 Culture experiments with an inhibitor of retinal biosynthetic pathway, MPTA

To determine the effect of retinal biosynthesis inhibitor on light-dependent growth, we studied culture experiment with the MPTA in two different strains of PR-containing flavobacteria: Dokdonia sp. MED134 which was broadly studied and Gilvibactersp.

SZ-19.

In previous studies assessing the role of PR in light-induced growth in strain

Dokdonia sp. MED134, it was observed that only light corresponding to the wavelengths absorbed by PR supported growth (Gomez-Consarnau et al. 2007; Kimura et al. 2011). Additionally, culture experiments on MPTA, an inhibitor of lycopene cyclization in the retinal biosynthetic pathway, were conducted to provide further evidence of the role of PR in stimulating growth in Dokdonia sp. MED 134 (Kimura et aL 2011).

To assess the effectiveness of MPTA as an inhibitor of f-carotene synthesis (the direct precursor of retinal), MED134 was grown on Marine agar plates with and without

MPTA. Bacterial cells accumulating p-carotene display yellow or orange colonies. In the presence of MPTA, MED134 produced light pink colonies, indicating the accumulation of lycopene (Fig. 15). In the absence of MPTA, yellow colonies were observed on agar plates (Fig. 16). Our results indicate that MPTA effectively prevented

J-carotene generation, the precursor for retinal, in both Dokdonia sp. MED134 and

Gilvibactersp. SZ- 19.

44 (a)

(b)

(C)

Fig. 15. Colony images of Dokdonia sp. MED134 in culture experiment with a MPTA. (a, b) Dokdonia sp. MED134 was grown on Marine Agar (Difco 2216) plates with MPTA (a) and without MPTA (b). The strain was also grown on agar plate amended with methanol as negative control (c).

45 (a)

(b)

Fig. 16. Colony images of Gilvibactersp. SZ-19 in culture experiment with a MPTA. (a, b) Gilvibacter sp. SZ-19 was grown on Marine Agar (Difco 2216) plates with MPTA (a) and without MPTA (b). The strain was also grown on agar plate amended with methanol as negative control (c).

46 Growth characteristics of representative flavobacterial strains on MPTA

containing media

To determine the role of MPTA as an inhibitor of p-carotene, culture media at both

low and high concentrations of DOC were used. Culture experiment with MPTA was

performed in both Dokdonia sp. MED134 and Gilvibacter sp. SZ-19.

First, strains were grown in ASW enriched to 0.14 mM C and amended with MPTA

for low carbon concentration media. When Dokdonia sp. MED134 and Gilvibacter sp.

SZ-19 were incubated in ASW without MPTA, bacteria grew well to 1.14 x 106cells/ml

and 1.17 x 106 cells/ml in the presence of light, respectively (Fig. 17a and Fig. 18a).

However, 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 (Fig. 17a and Fig. 18a).

Second, Dokdonia sp. MED134 and Gilvibactersp. SZ-19 were grown on the media

of ASW enriched to 48.5 mM C and amended with MPTA for high carbon concentration

(Fig. 17b and 18b). Under culture experiment with MPTA at high carbon concentration

conditions, whether present of light of not, Dokdonia sp. MED 134 were reached up to

5.15 x 107 cells/ml and 5.77 x 10 7 cells/ml in the light or in the darkness, respectively,

which was at least two times higher than the maximum cell density in the MPTA-treated

cultures. Also, the similar results were shown in the Gilvibactersp. SZ-19.

Although the results of MPTA growth experiment at high carbon concentration are hard to explain clearly at this point, we can still suggest that PR bound to retinal has a

47 important role in the light-stimulated growth of the marine flavobacterial strain containing proteorhodopsin.

48 14 (a)

12 -

JE 10 -

Z x

--- LightControl -0- LIghtMPTA 0 -y- Dark Control -a- DarkMPTA

id 2d 3d 4d Sd 6d Time (Day)

(b) 70 60

U 40 0 7' 30

07 20 0 10 U + LightControl -0- Light -MPTA 0 -v-- DwlControl _a -10 id 2d 3d 4d 5d 6d Time (Day)

Fig. 17. Growth curves of Dokdonia sp. MED 134 in culture experiments with a MPTA inhibitor. (a, b) Dokdonia sp. MED 134 was grown in ASW enriched to 0.14 mM C with and without MPTA (a), in ASW enriched to 48.5 mM C with and without MPTA (b). The cultures in enriched ASW amended with MPTA exposed to light (o) and in the dark (A), and in the enriched ASW without MPTA in culture exposed to light (e) and in the dark (V). The epifluorescence microscopy was used to observe and count bacterial cells.

49 (a) 14* 12 -

10 -

Le 6 -

v 4

0 2- - UghtControl 0 -0- Lght MPTA --- DarkContrl -a- Dark MPTA

1d 2d 3d 4d Sd 6d Time (Day)

(b) 70

60 -

50 -

040

.0 30 - 20 -

10 - -e- LightControl 0 -0- LightMPTA -r- DarkControl A Dark.MPTA

Id 2d 3d 4d Sd 6d Tine (Day)

Fig. 18. Growth curves of Gilvibactersp. SZ-19 in culture experiments with a MPTA inhibitor. (a, b) Gilvibacter sp. SZ-19 was grown in ASW enriched to 0.14 mM C with and without MPTA (a), in ASW enriched to 48.5 mM C with and without MPTA (b). The cultures in enriched ASW amended with MPTA exposed to light (o) and in the dark (A), and in the enriched ASW without MPTA in culture exposed to light (e) and in the dark (V). The epifluorescence microscopy was used to observe and count bacterial cells.

50 CHAPTER 3. TRANSCRIPTOMIC ANALYSIS OF LOW AND HIGH CARBON CONCENTRATIONS ON LIGHT-DEPENDENT GROWTH OF Dokdonia sp. MED134

3.1 Introduction

A branch of biology 'genomics' was coined by Thomas Roderick as a name for the

new journal Genomics in 1986, and refers to a new scientific discipline of mapping,

sequencing, and analyzing genomes (McKusick 1997). And, at the beginning of this

century, genomics greatly changed the face of biology with the completion of large-

scale genome projects. Not only has the scale of molecular biology changed, but also its

scope. Genomics is now undergoinga transition or expansion from the mapping and

sequencing of genomes to an emphasis on genome function (Debouck and Metcalf

2000; DeLong et al. 2006; Rusch et al. 2007; Rodrigue et al. 2009). To reflect this shift,

genome analysis may now be divided into "structural genomics" and "functional

genomics". Structural genomics represents an initial phase of genome analysis such as

linkage analysis, molecular cytogenetics, physical mapping, expressed sequence tag

(EST) sequencing, genome sequencing and genome organization (McKusick 1997).

And functional genomics refers to the development and application of global (genome-

wide or system-wide) experimental approaches to assess gene function by making use

of the information and reagents provide by structural genomics (Hieter and Boguski

1997; Tringe and Rubin 2005). It is characterized by high throughput or large-scale experimental methodologies combined with statistical and computational analysis

(bioinformatics) of the results (Fig. 19)

51 Functional genomics studies for the definition of the functions of large sets of genes that are involved in or regulate processes in plant growth and development are called plant functional genomics.

In functional genomics, 'transcriptomics", the study of gene expressions at transcription level, is one of the flourishing areas that will have a great impact in understanding the functional role of genes (Eymann et al. 2002; Ernst et al. 2005;

DeLong et aL. 2006; Toledo-Arana et al. 2009). Since mRNA is the initial and intermediate product of gene expression, most cases studies at the transcriptional level may reflect the function of the respective genes. To analysis transcriptomes in large- scale, several genomic technologies were developed. They include: PCR-based assays, serial analysis of gene expression (SAGE) (Velculescu et al. 1995), rapid analysis of gene expression (RAGE), expressed sequence tags (ESTs) (Poretsky et al. 2009), macroarrays (Steward et al. 2004) and microarray (biochip) technology (Lindell et al.

2007; Zinser et al. 2009). Among these technologies microarray technology was considered as the powerful tool for transcriptomes analysis. The DNA microarray is invented for studying gene expression patterns simultaneously on a global scale. Large numbers of different gene fragments are immobilized in an ordered array on a solid support; it is available for the interrogation of transcriptomes. To do this, an entire population of mRNAs from a tissue can be simultaneously hybridized, in a single experiment, with tens of thousands of known DNA probes that are immobilized at precisely known positions on the array. After the hybridization, the expression levels of each gene can be determined with a high-resolution laser scanner, and computer programs can group genes that are up- or down- regulated in response to the

52 interrogated stress. Therefore, it is possible to detect the differences in relative

abundance of several mRNAs between two sets of conditions run in parallel.

Despite these advantages (Zhou and Thompson 2002), a non-targeted, sequence

approach is increasingly used to profile community transcripts due to the technological

limitations of microarrays. While microarrays employ complex quantification

algorithms, increasingly more complex gene expression solutions are required. Factors

that can affect probe specificity include outputs of signal intensity instead of nucleotide

sequence identities. Furthermore, microarrays depend on massively parallel nucleic acid

hybridization and therefore have a risk of cross-hybridization of highly related

sequences. The scope and size of studies enabled by non-targeted, sequence is evident in

a 2005 study that generated and sequenced a cDNA clone library of -400 clones by

random priming of microbial community RNA collected from a hypersaline lake

(Poretsky et al. 2005).

Sequence-based profiling of community transcriptomes on a large scale has benefited

from technological advances. Next-generation sequencing techniques can perform

millions of sequence reads in a run. These include pyrosequencing (Margulies et al.

2005), Illumina technology (formerly Solexa sequencing), and Ion Torrent technology

(Ion Torrent Systems, Inc., Guilford, CT). Pyrosequencing, in particular, has been

increasingly used in microbial ecology and its use has been peer reviewed many times

since 2007. First used in soil analysis (Leininger et al. 2006), it is conducive to a number of open ocean microbial assemblage experiment procedures, including measuring size-fractionation and collecting bacterioplankton biomass.

A major advantage of next-generation sequencing applying metatranscriptomic analysis is the lack of a need for cloning in the process of processing the extracted RNA

53 (rRNA subtraction, amplication and so forth) and converting it into cDNA (Fig. 20). In terms of analysis, metatranscriptomic technology provides insight into the regulation and expression patterns of the biological processes of genes at the molecular level (Frias

Lopez et al., 2008). Accordingly, it is being more widely applied to understand the unknown and increasingly complex biology of microbes under different and untested environmental conditions. Methodological and technological approaches and protocols continue to evolve.

54 Gene Genomics (Genome)

mRNAMTranscriptomnics niRNAI (Transcriptomie)

Protein - Proteomics BioinformaticsV -- ru (Proteome) Microorganis1

Metabolite --- + bolorli

Phenotpe Phenonics (Phenome)

Fig. 19 Scheme showing how the integration of results from different technological levels of functional genomics leads to construction of a virtual microorganism.

55 sample

DNA and RNA extraction DNA total RNA PCR amplification wI T7-appended primers 16S ar d 23S rDNA in vitro transcription, T7 polymerase biotin-la beled antisense 16 5 and 23S RNA total RNA hybridizatio

biotin-labeled ds 16S and 23S rRNA unlabeled ss RNA

bead binding, magnetic separation 16S and 23S rRNA rRNA-subtracted RNA RNA amplification, Illumina sequencing

Fig. 20. Scheme of sample processing pipeline for the metatranscriptomic analysis; adapted from Stewart et al (2010).

56 3.2. Materials and methods

Cultivation for transcriptomic analyses

Our experiment was designed to determine the effect of DOC concentration on gene expression of PR-containing Dokdonia sp. MED134 under light and dark growth conditions. For transcriptomic analyses, MED134 was grown in ASW amended with

FSM to yield DOC concentrations of low (0.14mM C) and high (48.5mM C), respectively.

To examine the gene expression of Dokdonia sp. MED134 at low carbon concentration under both light and darkness, MED134 was grown in 4L of ASW enriched to 0.14 mM C at 22 C in the darkness for 2 days. At this time, 500 mL of culture was filtered on to a pore-size 0.22 mm Durapore membrane filter (25mm diameter, Millipore), yielding the TO sample. The remaining culture was split in two 1 L flasks that were incubated again at 22 C under the continuous white light

(approximately 150 mmol of photons m-2 s-1) or in darkness. After 2 more days, the cultures were taken from two time points, mid to late exponential phase and stationary phase and filtered onto Durapore membrane filters (Millipore), yielding samples Li

(exponential phase and light conditions) and L2 (stationary phase and light conditions) and Dl (exponential phase and dark conditions) and D2 (stationary phase and dark conditions), respectively. Sampling time points were determined by pilot experiment.

The same procedures were performed in cultures of high carbon concentration (48.5 mM C) condition. All filtered samples were immediately placed into screw-cap tubes containing 1 mL of RNAlater (Ambion, Austin, TX, USA) and stored at -80 C until

RNA extraction. All treatments were performed in triplicate.

57 Total RNA extraction

Total RNA was extracted from the filter samples using a modification of the mirVana miRNA isolation kit (Ambion) as described previously (Shi et al., 2009; McCarren et al.,

2010). Briefly, Sterivex filter cartridges containing samples were taken from the -80 C freezer, thawed on ice and the RNAlater was carefully removed by pushing through with a syringe. Then, the filters were immersed with Lysis/Binding (Ambion) and mixed to lyse attached cells. The yellowish color on the filter due to cells disappeared during vortexing suggesting cells were lysed. Total RNA was extracted from the lysate according to the manufacturer's protocol. Remaining genomic DNA was removed using a TURBO DNA-free kit (Ambion). Finally, extracted total RNAs (pellets) from the filters were purified, concentrated, and resuspended in 50 pl DEPC treated RNase-free water by using the MinElute PCR Purification Kit (Qiagen) and stored at -80 C.

ribosomal RNA (rRNA) subtraction

rRNA frequently represents 53% - over 90% of the total RNA (Frias-Lopez et al.,

2008; Stewart FJ et al., 2010). Thus, rRNA needs to be removed so that the majority of sequencing effort goes to mRNA.

To remove bacterial 16S and 23S rRNA molecules from total RNA samples, we carried out the subtractive hybridization using sample specific biotinylated rRNA probes as previously described by Stewart et al (Stewart et al., 2010). Ribonucleotide probes were generated from the total DNA extracted from the Dokdonia sp. MED 134.

Templates for probe were first prepared by PCR using strain-specific primers flanking

58 nearly the full length of the bacterial 16S gene and 23S rRNA gene and Herculase II

Fusion DNA Polymerase (Stratagene, La Jolla, CA, USA), with reverse primers

modified to contain the T7 RNA polymerase promoter sequence. Biotin-labeled

antisense rRNA probes were generated by in vitro transcription (IVT) using T7

promoter-containing 16S and 23S amplicons as templates based on MEGAscript High

Yield Transcription Kit (Ambion, Austin, TX, USA). Biotinylated rRNA probes were

hybridized to rRNA in the total RNA sample. The labeled double-strand rRNA was then

removed via hybridization to streptavidin-coated magnetic beads (New England Biolabs,

Ipswich, MA, USA), followed by magnetic separation. After subtracted RNA

purification, purified RNA was confirmed the removal of bacterial rRNAs using a 2100

Bioanalyzer (Agilent, Santa Clara, CA, USA).

RNA amplification, complementary DNA (cDNA) synthesis and Illumina

sequencing

The rRNA-subtracted RNA was amplified using the ScriptSeq" v2 RNA-Seq Library

Preparation kit (Epicentre, Madison, WI). cDNA was synthesized according to the

manufacturer's protocol.

In brief, the rRNA-depleted sample is fragmented using the RNA Fragmentation

Solution. The fragmented RNA was converted to double-stranded cDNA via random-

sequence primers containing a tagging sequence at their 5' ends. The 5'-tagged cDNA is then tagged at its 3' end by the terminal-tagging reaction to make di-tagged at both its

5'- and 3'-end, single-stranded cDNA. Following purification, the di-tagged cDNA is

amplified by PCR, which completes the addition of the Illumina adaptor sequences,

amplifies the library for subsequent cluster generation and adds an optional Illumina

59 Index. The amplified RNA-seq library is purified using AMPure kit (Beckman Coulter

Genomics, Danvers, MA, USA) and is ready for cluster generation and sequencing. The resulting cDNA libraries were then pyrosequenced on the MiSeq platform (Illumina).

60 3.3 Results and Discussions

Cultivation for transcriptomic analyses

Before the beginning of the transcriptomic analysis with various carbon concentrations, we confirmed the light-enhanced growth on Dokdonia sp. MED134 at both low (0.14 mM C) and high (48.5 mM C) carbon concentration. Sampling time points were also determined by this pilot experiment.

Cell densities were increased significantly in light condition at low DOC concentration whereas MED134 grew well slightly in darkness at high DOC concentration, which were consistent with those seen in before. This shows that further transcriptomic analysis with different carbon concentrations are needed to investigate the PR-containing marine flavobacterial strains. Twelve samples were collected at TO,

TI and T2 of low DOC condition and TO, TI and T2 of high DOC condition under both light and darkness, respectively (Fig. 21).

61 (a) 14 12- TI T2

1lo-

S6 - .a TO C 4 2-1

0- -0-- Ught - Dark

Od 2d 3d 4d 5d 6d Time (Day)

(b) 70

60- T2 TI

S0-

. 30- TO 20-

10 -

0 - -0- Ught *e Dark

Od 2d 3d 4d 5d 6d Mime (Day)

Fig. 21 Determining optimal sampling time points for transcriptomic experiments, based on growth curves of Dokdonia sp. MED 134. Growth curves of Dokdonia sp. MED 134 incubated in the light (o) or in the dark (*). MED 134 was grown in ASW enriched to 0. 14 mM C (a), and in

ASW enriched to 48.5 mM C. Error bars indicate standard deviation for triplicate cultures. Red arrows represent the sampling time points for each condition.

62 Transcriptomic analyses

Illumina sequencing of Dokdonia sp. MED134 transcriptomes (samples TO, TI

triplicates in light, TI triplicates in dark at low carbon concentrations) yielded 3.16E4 to

6.72E5 reads per sample (Table 5). Poor quality reads were removed by demultiplexing,

dereplication, removal of short reads and reads with low fastq quality scores. Quality

control was performed using pipelines for CLC Genomics workbench. Table 5 reflects

that the number of reads that survive quality-control step in read processing via our

scripts across samples.

Transcriptomic analyses revealed that several genes showed significant upregulation

in the presence of light at Ti and T2 low carbon condition (Table 6). In particular, two

predicted Fe assimilation proteins (MED134_06839, MED134_06834,

MED134_22457) were significantly upregulated in the light at TI low carbon condition

(Table 6). In T2 low carbon cultures, genes of a proteorhodopsin (MED134_07119),

retinal biosynthetic enzymes (MED134_13081), putative adhesion protein

(MED134_03969) are exhibited significant upregulation in the light (Table 7). Further, bacterial cryptochrome gene (MED134_10201) also exhibited significant upregulation

in the light at T2 low carbon cultures. These results suggested the importance of the proteorhodopsin for the light-stimulated growth in the light.

Also, MED134 has a gene associated with light sensing contains the BLUF (blue light sensing using FAD) domain, which specifically responds to blue light (Gomelsky and Klug, 2002). The BLUF domain is also found in the genome of Polaribacter sp.

MED152 (Gonzalez et al., 2008). In addition to light sensors, ABC transporter

(MED134_03404) and molecular chaperone DnaK (MED134_02409), which might have important roles for response regulation, were significantly upregulated in the light.

63 Transcriptomic survey for High carbon conditions In transcriptomic experiments of high carbon concentration condition, upregulated genes were similar to the results with low carbon condition at T2 in the light T1 (Table

8). At TI in high carbon condition, genes of a proteorhodopsin (MED134_07119), ABC transporter (MED134_03404), bacterial cryptochrome gene (MED134_10201), retinal biosynthetic enzymes (MED134_13081), putative adhesion protein (MED134_03969) are exhibited significant upregulation in the light (Table 8). However, the pattern of upregulated genes in T2 high carbon condition was changed compared to the results of low carbon condition. The lesser upregulation of a preoterhodopsin (MED134_07119) was observed at T2 higher carbon concentration. Also, P-oxidation pathways involved in ATP/energy production are up-regulated in the high carbon concentration at T2 culture with the former being statistically significant (Table 9). In particular, genes encoding electron transfer flavoprotein alpha-subunit (MED134_12996), electron transfer flavoprotein beta-subunit (MED134_00130) and Enoyl-CoA hydratase

(MED134_06229) exhibited significant upregulation in the dark.

Fatty acid P-oxidation is a multi step process by which fatty acids are broken down by various tissues to produce energy. The long chain acyl-CoA enters the fatty acid p- oxidation pathway, which results in the production of one acetyl-CoA from each cycle of fatty acid P-oxidation. This acetyl-CoA then enters the mitochondrial tricarboxylic acid (TCA) cycle. The NADH and FADH 2 produced by both fatty acid P-oxidation and the TCA cycle are used by the electron transport chain to produce ATP. The proton gradient built up in electron transport chain then allows for ATP synthesis. These results suggested the potential importance of the P-oxidation for growth of PR-containing

MED 134 in the high carbon concentration condition.

64 Table 5. Number of reads remaining after quality control step in the pipeline for quality control.

TO Ti-Light T1-Dark T2-Light T2-Dark

Low 67255 45458 40861 43189 25478 39672 43456 31609 39134 81880 15280 68029 Carbon 1 2 7 3 6 2 9 8

High 38174 38888 40066 24880 36447 24691 10279 24187 363742 35520 39483 35637 43794 Carbon 0 3 6 5 7 9 4 8 8 2 2 7 Table 6. Preliminary analysis of functional enrichment of differential gene expression in low carbon condition at T1.

Read number Locus tag ligh dmr foldChange log2FoldChange pval pval. Genbank annotation light dark FDRadj

MED134_06839 107 27 0.3 -2.0 2.37E-05 0.017 Putative uncharacterized protein

ON~ MED134_06834 1798 463 0.3 -2.0 1.95E-09 5.55E-06 TonB-dependent outer membrane M~ receptor MED134_22457 11827 3598 0.3 -1.7 1.16E-07 1.65E-04 #N/A MED134_01225 109 360 3.3 1.7 5.00E-06 0.005 Secretion protein HlyD

Red character indicates significant upregulation in the light. Black character indicates significant upregulation in the darkness. Table 7. Read number and significant upregulated genes in the light in low carbon condition at T2.

Read Locus tag number foldChange log2FoldChange pval Genbank annotation FDRadj light dark

MED134_07119 156 11 0.1 -3.8 7.39E-08 3.49E-05 Bacteriorhodopsin MED134_07089 25 2 0.1 -3.7 4.94E-09 2.80E-06 Putative uncharacterized protein -3.4 MED134_10201 41 4 0.1 1.29E-10 1.82E-07 Probable bacterial cryptochrome Transcriptional regulator, MerR family MED134_13081 69 11 0.2 -2.7 4.29E-09 2.80E-06 protein MED134_14276 34 6 0.2 -2.6 1.43E-05 0.005 Putative uncharacterized protein MED134_04999 97 18 0.2 -2.4 1.95E-10 1.84E-07 Putative uncharacterized protein 0.2 MED134_01275 456 102 -2.2 3.15E-13 8.92E-10 Putative uncharacterized protein 0.3 MED134_13076 33 9 -1.9 2.36E-04 0.061 Phytoene dehydrogenase 0.4 MED134_05424 66 27 -1.3 0.001 0.140 ThiJ/PfpIfamily protein 0.4 MED134_03409 161 66 -1.3 9.89E-06 0.004 (dnaK) Molecular chaperone DnaK 0.5 MED134_03969 590 268 -1.1 4.27E-04 0.101 Putative adhesion lipoprotein 0.5 MED134_03404 206 99 -1.1 6.54E-05 0.021 ABC-1 0.5 MED134_11080 642 331 -1.0 8.57E-05 0.024 Putative uncharacterized protein Table 8. Read number and significant upregulated genes in the light in high carbon condition at T1.

Locustag Read number foldChange log2FoldChange pval pval FDRadj Genbank annotation light dark MED134_07119 1197 84 0.1 -3.8 3.76E-13 1.07E-09 Bacteriorhodopsin MED134_03969 1368 139 0.1 -3.3 2.80E-10 3.98E-07 Putative adhesion lipoprotein -3.0 MED134_07089 73 9 0.1 4.46E-07 3.18E-04 Putative uncharacterized protein -3.0 MED134_01275 2537 317 0.1 -3.0 4.95E-05 0.023 Putative uncharacterized protein 00 MED134_03404 1194 151 0.1 -3.0 6.38E-05 0.024 ABC-1 MED134_04999 836 107 0.1 6.79E-05 0.024 Putative uncharacterized protein MED134_10201 337 45 0.1 -2.9 6.08E-08 5.77E-05 Probable bacterial cryptochrome MED134_14271 149 25 0.2 -2.6 1.46E-06 0.001 Short chain dehydrogenase MED134_14276 198 40 0.2 -2.3 9.18E-05 0.029 Putative uncharacterized protein MED134_13076 234 59 0.3 -2.0 1.12E-04 0.032 Phytoene dehydrogenase Table 9. Preliminary analysis of functional enrichment of differential gene expression in high carbon condition at T1.

Read number pval Locus tag foldChange log2FoldChange pval Genbank annotation light dark FDRadj

MED134_07119 27 8 0.3 -1.7 1.60E-04 0.051 Bacteriorhodopsin MED134_00750 85 46 0.5 -0.9 2.90E-04 0.075 Purine nucleoside phosphorylase (EC 2.4.2.1) MED134_22457 6752 4431 0.7 -0.6 1.27E-04 0.049 #N/A MED134_12996 333 545 1.6 0.7 4.78E-05 0.023 Putative recognition particle-docking protein MED134_00125 407 694 1.7 0.8 8.84E-06 0.005 Electron transfer flavoprotein alpha-subunit

M. MED134_05174 2604 4669 1.8 0.8 1.11E-07 1.11E-04 Putative heat shock ClpB protein MED134_13401 737 1344 1.8 0.9 1.17E-07 1.11E-04 GrpE protein (Hsp-70 cofactor) MED134_00130 311 574 1.8 0.9 4.66E-07 3.32E-04 Electron transfer flavoprotein(Beta subunit) MED134_06234 136 257 1.9 0.9 3.79E-04 0.090 Thiolase family protein MED134_06229 409 779 1.9 0.9 1.38E-04 0.049 Enoyl-CoA hydratase 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. C- (a)

-si

I I I 1 10 100 1000 10000 mean of normalized counts

(b)

0 W.~' ~

CYI

I?. II I 1 10-01 10+00 10+01 10+02 1e+03 mean of normalized counts

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 CM - (a)

t~J

10-01 10+00 10+01 16+02 le+03

mean of nomalized counts

en (b)

0 -L

l-01 1+00 1o+01 1o+02 10+03 mean of normelized counts

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 (a)

(~1

10 100 1000 10000 mean of normalized counts

(b)

0 "I.Av

10-01 10+01 le+03 mean of normalized counts

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 (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 Abell, G C. and J. P. Bowman (2005). "Ecological and biogeographic

relationships of class Flavobacteria in the Southern Ocean." FEMS Microbiol Ecol

51(2): 265-77.

Alonso, C., F. Warnecke, R. Amann and J. Pernthaler (2007). "High local and global diversity of Flavobacteria in marine plankton." Environ Microbiol 9(5): 1253-66.

Balashov, S. P., E. S. Imasheva, V. A. Boichenko, J. Anton, J. M. Wang and J. K. Lanyi (2005). "Xanthorhodopsin: a proton pump with a light-harvesting carotenoid antenna." Science 309(5743): 2061-4.

Beja, 0., L. Aravind, E. V. Koonin, M. T. Suzuki, A. Hadd, L. P. Nguyen, S. B. Jovanovich, C. M. Gates, R. A. Feldman, J. L. Spudich, E. N. Spudich and E. F. DeLong (2000). "Bacterial rhodopsin: evidence for a new type of phototrophy in the sea." Science 289(5486): 1902-6.

Beja, 0., E. N. Spudich, J. L. Spudich, M. Leclerc and E. F. DeLong (2001). "Proteorhodopsin phototrophy in the ocean." Nature 411(6839): 786-9.

Bielawski, J. P., K. A. Dunn, G. Sabehi and 0. Beja (2004). "Darwinian adaptation of proteorhodopsin to different light intensities in the marine environment." Proc Natl Acad Sci U S A 101(41): 14824-9.

Bieszke, J. A., E. L. Braun, L. E. Bean, S. Kang, D. 0. Natvig and K. A. Borkovich (1999). "The nop-1 gene of Neurospora crassa encodes a seven transmembrane helix retinal-binding protein homologous to archaeal rhodopsins." Proc Natl Acad Sci U S A 96(14): 8034-9.

Birge, R. R. (1990). "Nature of the primary photochemical events in rhodopsin and bacteriorhodopsin." Biochim Biophys Acta 1016(3): 293-327.

Boichenko, V. A., J. M. Wang, J. Anton, J. K. Lanyi and S. P. Balashov (2006). "Functions of carotenoids in xanthorhodopsin and archaerhodopsin, from action spectra of photoinhibition of cell respiration." Biochim Biophys Acta 1757(12): 1649-56.

Cottrell, M. T. and D. L. Kirchman (2009). "Photoheterotrophic microbes in the Arctic Ocean in summer and winter." Appl Environ Microbiol 75(15): 4958-66.

de la Torre, J. R., L. M. Christianson, 0. Beja, M. T. Suzuki, D. M. Karl, J. Heidelberg and E. F. DeLong (2003). "Proteorhodopsin genes are distributed among

76 divergent marine bacterial taxa." Proc Nati Acad Sci U S A 100(22): 12830-5.

Debouck, C. and B. Metcalf (2000). "The impact of genomics on drug discovery." Annu Rev Pharmacol Toxicol 40: 193-207.

DeLong, E. F, D. G. Franks and A. L. Alldredge (1993). "Phylogenetic diversity of aggregate-attached vs. free-living marine bacterial assemblages." Limnologh and Oceanography 38(5): 924-934.

DeLong, E. F., C. M. Preston, T. Mincer, V. Rich, S. J. Hallam, N. U. Frigaard, A. Martinez, M. B. Sullivan, R. Edwards, B. R. Brito, S. W. Chisholm and D. M. Karl (2006). "Community genomics among stratified microbial assemblages in the ocean's interior." Science 311(5760): 496-503.

Dioumaev, A. K., L. S. Brown, J. Shih, E. N. Spudich, J. L. Spudich and J. K. Lanyi (2002). "Proton transfers in the photochemical reaction cycle of proteorhodopsin." Biochemistry 41(17): 5348-58.

Ernst, F. D., S. Bereswill, B. Waidner, J. Stoof, U. Mader, J. G Kusters, E. J. Kuipers, M. Kist, A. H. van Vliet and G Homuth (2005). "Transcriptional profiling of Helicobacter pylori Fur- and iron-regulated gene expression." Microbiology 151(Pt 2): 533-46.

Eymann, C., G. Homuth, C. Scharf and M. Hecker (2002). "Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis." J Bacteriol 184(9): 2500-20.

Frigaard, N. U., A. Martinez, T. J. Mincer and E. E DeLong (2006). "Proteorhodopsin lateral gene transfer between marine planktonic Bacteria and Archaea." Nature 439(7078): 847-50.

Fuhrman, J. A., M. S. Schwalbach and U. Stingl (2008). "Proteorhodopsins: an array of physiological roles?" Nat Rev Microbiol 6(6): 488-94.

Giovannoni, S. J., L. Bibbs, J. C. Cho, M. D. Stapels, R. Desiderio, K. L. Vergin, M. S. Rappe, S. Laney, L. J. Wilhelm, H. J. Tripp, E. J. Mathur and D. F. Barofsky (2005). "Proteorhodopsin in the ubiquitous marine bacterium SARIl." Nature 438(7064): 82-5.

Gomez-Consarnau, L., N. Akram, K. Lindell, A. Pedersen, R. Neutze, D. L. Milton, J. M. Gonzalez and J. Pinhassi (2010). "Proteorhodopsin phototrophy promotes survival of marine bacteria during starvation." PLoS Biol 8(4): e1000358.

77 Gomez-Consarnau, L., J. M. Gonzalez, M. Coll-Llado, P. Gourdon, T. Pascher, R. Neutze, C. Pedros-Alio and J. Pinhassi (2007). "Light stimulates growth of proteorhodopsin-containing marine Flavobacteria." Nature 445(7124): 210-3.

Jung, K. H. (2007). "The distinct signaling mechanisms of microbial sensory rhodopsins in Archaea, Eubacteria and Eukarya." Photochem Photobiol 83(1): 63-9.

Jung, K. H., V. D. Trivedi and J. L. Spudich (2003). "Demonstration of a sensory rhodopsin in eubacteria." Mol Microbiol 47(6): 1513-22.

Kashket, E. R. (1985). "The proton motive force in bacteria: a critical assessment of methods." Annu Rev Microbiol 39: 219-42.

Kawanabe, A., Y. Furutani, K. H. Jung and H. Kandori (2006). "FTIR study of the photoisomerization processes in the 13-cis and all-trans forms of Anabaena sensory rhodopsin at 77 K." Biochemistry 45(14): 4362-70.

Kim, S. Y., S. A. Waschuk, L. S. Brown and K. H. Jung (2008). "Screening and characterization of proteorhodopsin color-tuning mutations in Escherichia coli with endogenous retinal synthesis." Biochim Biophys Acta 1777(6): 504-13.

Kimura, H., C. R. Young, A. Martinez and E. F. Delong (2011). "Light-induced transcriptional responses associated with proteorhodopsin-enhanced growth in a marine flavobacterium." Isme J 5(10): 1641-5 1.

Kirchman, D. L. (2002). "The ecology of Cytophaga-Flavobacteria in aquatic environments." FEMS Microbiol Ecol 39(2): 91-100.

Kralj, J. M., V. B. Bergo, J. J. Amsden, E. N. Spudich, J. L. Spudich and K. J. Rothschild (2008). "Protonation state of Glu142 differs in the green- and blue-absorbing variants of proteorhodopsin." Biochemistry 47(11): 3447-53.

Lami, R., M. T. Cottrell, B. J. Campbell and D. L. Kirchman (2009). "Light- dependent growth and proteorhodopsin expression by Flavobacteria and SAR11 in experiments with Delaware coastal waters." Environ Microbiol 11(12): 3201-9. Hieter, P. and M. Boguski (1997). "Functional genomics: it's all how you read it." Science 278(5338): 601-2.

Leininger, S., T. Urich, M. Schloter, L. Schwark, J. Qi, G. W. Nicol, J. I. Prosser, S. C. Schuster and C. Schleper (2006). "Archaea predominate among ammonia- oxidizing prokaryotes in soils." Nature 442(7104): 806-9.

78 Lindell, D., J. D. Jaffe, M. L. Coleman, M. E. Futschik, I. M. Axmann, T. Rector, G Kettler, M. B. Sullivan, R. Steen, W. R. Hess, G. M. Church and S. W. Chisholm (2007). "Genome-wide expression dynamics of a marine virus and host reveal features of co-evolution." Nature 449(7158): 83-6.

Man, D., W. Wang, G Sabehi, L. Aravind, A. F. Post, R. Massana, E. N. Spudich, J. L. Spudich and 0. Beja (2003). "Diversification and spectral tuning in marine proteorhodopsins." Embo J 22(8): 1725-31.

Margulies, M., M. Egholm, W. E. Altman, S. Attiya, J. S. Bader, L. A. Bemben, J. Berka, M. S. Braverman, Y. J. Chen, Z. Chen, S. B. Dewell, L. Du, J. M. Fierro, X. V. Gomes, B. C. Godwin, W. He, S. Helgesen, C. H. Ho, G P. Irzyk, S. C. Jando, M. L. Alenquer, T. P. Jarvie, K. B. Jirage, J. B. Kim, J. R. Knight, J. R. Lanza, J. H. Leamon, S. M. Lefkowitz, M. Lei, J. Li, K. L. Lohman, H. Lu, V. B. Makhijani, K. E. McDade, M. P. McKenna, E. W. Myers, E. Nickerson, J. R. Nobile, R. Plant, B. P. Puc, M. T. Ronan, C T. Roth, G J. Sarkis, J. F. Simons, J. W. Simpson, M. Srinivasan, K. R. Tartaro, A. Tomasz, K. A. Vogt, C A. Volkmer, S. H. Wang, Y. Wang, M. P. Weiner, P. Yu, R. F. Begley and J. M. Rothberg (2005). "Genome sequencing in microfabricated high- density picolitre reactors." Nature 437(7057): 376-80.

Martinez, A., A. S. Bradley, J. R. Waldbauer, R. E. Summons and E. F. DeLong (2007). "Proteorhodopsin photosystem gene expression enables photophosphorylation in a heterologous host." Proc Natl Acad Sci U S A 104(13): 5590-5.

McCarren, J. and E. F. DeLong (2007). "Proteorhodopsin photosystem gene clusters exhibit co-evolutionary trends and shared ancestry among diverse marine microbial phyla." Environ Microbiol 9(4): 846-58.

McKusick, V. A. (1997). "Genomics: structural and functional studies of genomes." Genomics 45(2): 244-9.

Mongodin, E. F., K. E. Nelson, S. Daugherty, R. T. Deboy, J. Wister, H. Khouri, J. Weidman, D. A. Walsh, R. T. Papke, C Sanchez Perez, A. K. Sharma, C. L. Nesbo, D. MacLeod, E. Bapteste, W. F Doolittle, R. L. Charlebois, B. Legault and F. Rodriguez- Valera (2005). "The genome of Salinibacter ruber: convergence and gene exchange among hyperhalophilic bacteria and archaea." Proc Natl Acad Sci U S A 102(50): 18147-52.

Morris, R. M., M. S. Rappe, S. A. Connon, K. L. Vergin, W. A. Siebold, C. A. Carlson and S. J. Giovannoni (2002). "SAR11 clade dominates ocean surface bacterioplankton communities." Nature 420(6917): 806-10.

79 Ottolenghi, M. and M. Sheves (1989). "Synthetic retinals as probes for the binding site and photoreactions in rhodopsins." J Membr Biol 112(3): 193-212.

Peck, R. F., C. Echavarri-Erasun, E. A. Johnson, W. V. Ng, S. P. Kennedy, L. Hood, S. DasSarma and M. P. Krebs (2001). "brp and blh are required for synthesis of the retinal cofactor of bacteriorhodopsin in Halobacterium salinarum." J Biol Chem 276(8): 5739-44.

Poretsky, R. S., N. Bano, A. Buchan, G LeCleir, J. Kleikemper, M. Pickering, W. M. Pate, M. A. Moran and J. T. Hollibaugh (2005). "Analysis of microbial gene transcripts in environmental samples." Appl Environ Microbiol 71(7): 4121-6.

Poretsky, R. S., 1. Hewson, S. Sun, A. E. Allen, J. P. Zehr and M. A. Moran (2009). "Comparative day/night metatranscriptomic analysis of microbial communities in the North Pacific subtropical gyre." Environ Microbiol 11(6): 1358-75.

Rodrigue, S., R. R. Malmstrom, A. M. Berlin, B. W. Birren, M. R. Henn and S. W. Chisholm (2009). "Whole genome amplification and de novo assembly of single bacterial cells." PLoS One 4(9): e6864.

Rusch, D. B., A. L. Halpern, G Sutton, K. B. Heidelberg, S. Williamson, S. Yooseph, D. Wu, J. A. Eisen, J. M. Hoffman, K. Remington, K. Beeson, B. Tran, H. Smith, H. Baden-Tillson, C. Stewart, J. Thorpe, J. Freeman, C. Andrews-Pfannkoch, J. E. Venter, K. Li, S. Kravitz, J. F. Heidelberg, T. Utterback, Y H. Rogers, L. I. Falcon, V. Souza, G. Bonilla-Rosso, L. E. Eguiarte, D. M. Karl, S. Sathyendranath, T. Platt, E. Bermingham, V. Gallardo, G. Tamayo-Castillo, M. R. Ferrari, R. L. Strausberg, K. Nealson, R. Friedman, M. Frazier and J. C. Venter (2007). "The Sorcerer II Global Ocean Sampling expedition: northwest Atlantic through eastern tropical Pacific." PLoS Biol 5(3): e77.

Sabehi, G, 0. Beja, M. T. Suzuki, C. M. Preston and E. F. DeLong (2004). "Different SAR86 subgroups harbour divergent proteorhodopsins." Environ Microbiol 6(9): 903-10.

Sabehi, G, A. Loy, K. H. Jung, R. Partha, J. L. Spudich, T. Isaacson, J. Hirschberg, M. Wagner and 0. Beja (2005). "New insights into metabolic properties of marine bacteria encoding proteorhodopsins." PLoS Biol 3(8): e273.

Sabehi, G, R. Massana, J. P. Bielawski, M. Rosenberg, E. F. Delong and 0. Beja (2003). "Novel Proteorhodopsin variants from the Mediterranean and Red Seas." Environ Microbiol 5(10): 842-9.

80 Sharma, A. K., 0. Zhaxybayeva, R. T. Papke and W. F. Doolittle (2008). "Actinorhodopsins: proteorhodopsin-like gene sequences found predominantly in non- marine environments." Environ Microbiol 10(4): 1039-56.

Sineshchekov, 0. A., K. H. Jung and J. L. Spudich (2002). "Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii." Proc Natl Acad Sci U S A 99(13): 8689-94.

Spudich, J. L. (2006). "The multitalented microbial sensory rhodopsins." Trends Microbiol 14(11): 480-7.

Spudich, J. L., C. S. Yang, K. H. Jung and E. N. Spudich (2000). "Retinylidene proteins: structures and functions from archaea to humans." Annu Rev Cell Dev Biol 16: 365-92.

Steward, G F., B. D. Jenkins, B. B. Ward and J. P. Zehr (2004). "Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity." Appl Environ Microbiol 70(3): 1455-65.

Stingl, U., R. A. Desiderio, J. C. Cho, K. L. Vergin and S. J. Giovannoni (2007). "The SAR92 clade: an abundant coastal clade of culturable marine bacteria possessing proteorhodopsin." Ayp Environ Microbiol 73(7): 2290-6.

Toledo-Arana, A., 0. Dussurget, G. Nikitas, N. Sesto, H. Guet-Revillet, D. Balestrino, E. Loh, J. Gripenland, T. Tiensuu, K. Vaitkevicius, M. Barthelemy, M. Vergassola, M. A. Nahori, G Soubigou, B. Regnault, J. Y Coppee, M. Lecuit, J. Johansson and P. Cossart (2009). "The Listeria transcriptional landscape from saprophytism to virulence." Nature 459(7249): 950-6.

Tringe, S. G. and E. M. Rubin (2005). "Metagenomics: DNA sequencing of environmental samples." Nat Rev Genet 6(11): 805-14.

Velculescu, V. E., L. Zhang, B. Vogelstein and K. W. Kinzler (1995). "Serial analysis of gene expression." Science 270(5235): 484-7.

Venter, J. C., K. Remington, J. F. Heidelberg, A. L. Halpern, D. Rusch, J. A. Eisen, D. Wu, 1. Paulsen, K. E. Nelson, W. Nelson, D. E. Fouts, S. Levy, A. H. Knap, M. W. Lomas, K. Nealson, 0. White, J. Peterson, J. Hoffman, R. Parsons, H. Baden-Tillson, C. Pfannkoch, Y H. Rogers and H. 0. Smith (2004). "Environmental genome shotgun sequencing of the Sargasso Sea." Science 304(5667): 66-74.

81 Walter, J. M., D. Greenfield, C. Bustamante and J. Liphardt (2007). "Light- powering Escherichia coli with proteorhodopsin." Proc Natl Acad Sci U S A 104(7): 2408-12.

Woyke, T., G Xie, A. Copeland, J. M. Gonzalez, C. Han, H. Kiss, J. H. Saw, P. Senin, C. Yang, S. Chatterji, J. F. Cheng, J. A. Eisen, M. E. Sieracki and R. Stepanauskas (2009). "Assembling the marine metagenome, one cell at a time." PLoS One 4(4): e5299.

Yoshizawa, S., A. Kawanabe, H. Ito, H. Kandori and K. Kogure (2012). "Diversity and functional analysis of proteorhodopsin in marine Flavobacteria." Environ Microbiol 14(5): 1240-8.

Zhao, M., F. Chen and N. Jiao (2009). "Genetic diversity and abundance of flavobacterial proteorhodopsin in China seas." Appl Environ Microbiol 75(2): 529-33.

Zhou, J. and D. K. Thompson (2002). "Challenges in applying microarrays to environmental studies." Curr Opin Biotechnol 13(3): 204-7.

Zinser, E. R., D. Lindell, Z. I. Johnson, M. E. Futschik, C. Steglich, M. L. Coleman, M. A. Wright, T. Rector, R. Steen, N. McNulty, L. R. Thompson and S. W. Chisholm (2009). "Choreography of the transcriptome, photophysiology, and cell cycle of a minimal photoautotroph, prochlorococcus." PLoS One 4(4): e5135.