Molecular and Ecological Analysis of Cellular Attachment and Induction of Transparent Exopolymeric Particle Formation in Diatom-Bacteria Interactions

Molecular and ecological analysis of cellular attachment and induction of transparent exopolymeric particle formation in diatom-bacteria interactions

Shalin Seebah

A thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Marine Microbiology

Approved Thesis Committee

Prof. Dr. Matthias Ullrich Jacobs University Bremen

Prof. Dr. Laurenz Thomsen Jacobs University Bremen

Dr. habil. Uta Passow Marine Science Institute University of California Santa Barbara

Date of defense: 23 March 2012

Jacobs University Bremen School of Engineering and Science

ACKNOWLEDGEMENTS ...... I ABSTRACT ...... III LIST OF ABBREVIATIONS ...... IV INTRODUCTION ...... 1 1.1 THE GLOBAL CARBON CYCLE ...... 1 1.1.1 Pre-industrial global carbon cycle ...... 1 1.1.2 Present-day global carbon cycle ...... 2 1.2 OCEANIC CARBON CYCLE ...... 4 1.3 BIOLOGICAL PROCESSES OF THE OCEAN ...... 8 1.3.1 The biological pump ...... 8 1.3.2 Dissolved organic carbon ...... 9 1.3.3 Particulate organic carbon...... 9 1.4 MARINE SNOW...... 12 1.4.1 Marine snow formation ...... 12 1.4.2 Marine gel particles...... 13 1.5 TRANSPARENT EXOPOLYMERIC PARTICLES ...... 15 1.6 MICROSCALE INTERACTIONS ...... 17 1.6.1 Bacterial chemotaxis and motility ...... 18 1.7 BILATERAL MODEL SYSTEM ...... 22 1.7.1 The diatom Thalassiosira weissflogii ...... 22 1.7.2 The marine bacterium Marinobacter adhaerens sp. nov. HP15 ...... 24 AIMS OF THIS STUDY ...... 26 SUMMARY OF RESULTS ...... 27 CHAPTER 1 ...... 29 3.1.1 Marinobacter adhaerens sp. nov., prominent in aggregate formation with the diatom Thalassiosira weissflogii...... 30 3.1.2 Complete genome sequence of Marinobacter adhaerens type strain (HP15), a diatom-interacting marine microorganism ...... 49 3.1.3 Development of a genetic system for Marinobacter adhaerens HP15 involved in marine aggregate formation by interacting with diatom cells ...... 68 CHAPTER 2 ...... 97 3.1.4 Attachment of Marinobacter adhaerens HP15 to Thalassiosira weissflogii is not essential for the induction of transparent exopolymeric particle formation .... 98 CHAPTER 3 ...... 123 3.1.5 Combined effects of lowered pH and elevated temperature on diatom- bacteria interactions ...... 124 DISCUSSION ...... 156 BIBLIOGRAPHY ...... 164 DECLARATION ...... 174

Acknowledgements

This work was carried out from April 2009 to February 2012 as part of the Helmholtz Graduate School for Polar and Marine Research. The work presented in this thesis was carried out in the laboratories of Prof. Dr. Matthias Ullrich, at the Jacobs University Bremen and that of Dr. habil. Uta Passow at the University of California Santa Barbara. Prof. Dr. Laurenz Thomsen was an additional committee supervisor and helped guide the project. This work was performed in close collaboration with Dr. Astrid Gaerdes and Dr. Eva Sonnenschein from April 2009 until January 2011. They both vastly contributed to the project as a whole.

This work was funded by Jacobs University Bremen, the Helmholtz association and the Marine Science Institute, University of California Santa Barbara.

I would firstly like to thank Prof. Dr. Matthias Ullrich for the opportunity to learn and develop in his laboratory, for his unwavering support and for constantly prioritizing his busy schedule to closely supervise and interact with me. Dr. habil. Uta Passow is thanked for giving me the unique opportunity to work and learn from her during a three-month exchange at the Marine Science Institute, University of California Santa Barbara. It has been a humbling experience to work under her wings with constant optimism, motivation and good vibes. Prof. Laurenz Thomsen is thanked for accepting to review my work and for providing to the point criticism, which often prompted me to take a step back and re-focus on the aims of the project. For all the caricatures of scientists as creative loners, science is a richly social endeavor. And for that, I would like to express my heartfelt gratitude to my colleagues for a joyful laboratory atmosphere and for intellectual sustenance. To all the former and current members of the laboratory thank you: Dr. Astrid Gaerdes, Dr. Eva Sonnenschein, Ingrid Torres Monroy, Daniel Pletzer, Gabriela Alfaro,

Shaunak Khandekar, Amna Mehmood, Maria Johansson, Antje Stahl, Desalegne Abebew, Dr. Helge Weingart, Dr. Yannic Ramaye, Dr. Daria Zhurina, Dr. Nehaya Al-Karablieh and Dr. Abhishek Srivastava. My students, Sumana Sharma, Zheyna Kircheva, Yesim Yurtdas and Katharina Flenke are also thanked for giving me the pleasure of supervising them and for their help in the advancement of my project. Sabine Meier and Peter Tsvetkov are thanked for easing the administration and technical matters. Likewise, I extend my warmest thanks to the UCSB team for graciously offering me their help and sharing their equipment whenever I needed them. I would like to especially thank Caitlin Fairfield, the invaluable technician of the laboratory, who contributed on a daily basis both with technical help and experience. Dr. Craig Nelson and Dr. Mary Raven are thanked for their assistance with the epifluorescence microscope. Dr. Konrad Kulacki, Dr. Steve Sadro and Prof. Alice Alldredge are thanked for making feel part of Building 408. I would also like to extend my appreciation to the Bio workshop personnel who promptly repaired the roller tanks which I inadvertly cracked. I am grateful to the POLMAR graduate school and thank everyone who has in a way or the other contributed to the setting up and maintenance of the program. I thank the POLMAR coordinators: Prof. Jelle Bijma, Dr. Claudia Hanfland, Dr. Claudia Sprengel, Dörte Burhop and Dr. Tania Michler-Cieluch who have offered me much more support - moral, educational and financial – than I imagined. I am proud to have been a part of this prestigious program. I would also like to thank Claudia Daniel and Andreas Wagner for providing us with North Sea Water whenever we were in need. In the world beyond my experimental work, I have been extremely lucky for the solid foundation and support of my parents and sisters, even during the times when it was difficult for them that I left home to pursue my aspirations. Thank you for always encouraging me and being a phone call away whenever I needed to vent. My dear friends, Petra Pop Ristova, Tim Kalvelage, Abdul Rahiman Sheik, Renzo Kottmann, Marianne Jacob, Pier-Luigi Buttigieg and Pelin Yilmaz are thanked for making life in Bremen abound with happy memories. Finally, I thank Maté for being my pillar of love, support and fun and without whom none of this would have been possible.

Shalin Seebah Bremen, March 2012

Abstract

Transparent exopolymeric particles (TEP) and marine snow aggregates are vital components of the oceanic carbon cycle, leading to a substantial fraction of organic carbon sinking to depth or being provided for further recycling. Diatom-associated bacteria have been recently shown to directly impact TEP production and aggregate formation. However, very little is known about the molecular components that govern this interaction or the dynamics of TEP production and marine aggregate formation under changing environmental conditions. By combining molecular techniques and ecological experiments, we use an interdisciplinary approach to unveil hitherto unknown processes of diatom-bacteria interactions. As part of a collaborative and concerted effort, we initially established a genetically accessible bilateral model system consisting of the diatom Thalassiosira weissflogii and the marine gammaproteobacterium Marinobacter adhaerens HP15. Herein, we taxonomically established M. adhaerens HP15 as a novel member of the Marinobacter genus, revealed its genome sequence and established a genetic system to allow for the precise manipulation of this bacterium at the molecular level. In a second part, we used the established genetic toolbox to investigate the role of M. adhaerens HP15’s motility during its interaction with the diatom. By generating M. adhaerens HP15 flagellum- and MSHA type IV pilus-deficient mutants, we demonstrate that a fully- functional flagellum is a pre-requisite for the bacterial attachment to both abiotic and diatom surfaces. We further show that the MSHA type-IV pilus is important for attachment, albeit to a lesser extent. Although both cellular appendages were shown to be crucial for attachment to diatom surfaces, this type of attachment was demonstrated to not be essential for inducing the formation of diatom-borne transparent exopolymeric particles (TEP). In the final part of this work, how TEP production dynamics and aggregate formation might be impacted in putative future oceanic scenarios was investigated. The results of our study suggest that the combined effect of ocean acidification and increased temperature might lead to a significant reduction in aggregate formation and sinking velocities of marine aggregates. We suggest that a combination of ocean acidification and global warming may severely impact the vertical transport of particulate organic matter in a future ocean.

III

List of abbreviations

Amp Ampicillin CLSM Confocal Laser Scanning Microscopy Cm Chloramphenicol

CO2 Carbon dioxide DAPI 4’, 6’-diamidino-2-phenylindole DIC Dissolved Inorganic Carbon DNA Deoxyribonucleic acid DOC Dissolved organic carbon EDTA Ethylenediamine Tetra-acetic acid EPS Exopolymeric substances EtBr Ethidium Bromide GFP Green fluorescent protein Gm Gentamycin Km Kanamycin LB Luria-Bertani MB Marine Broth Neo Neomycin pCO2 Partial pressure of carbon dioxide PCR Polymerase Chain Reaction PgC Petagrams of Carbon POC Particulate Organic Carbon ppm Parts per million SOC Super Optimal Broth with Catabolite repression medium TA Total Alkalinity TAE Tris-acetate-EDTA TEM Transmission Electron Microscopy TEP Transparent Exopolymeric Particles

Introduction

1.1 The global carbon cycle

1.1.1 Pre-industrial global carbon cycle The global carbon cycle describes the exchanges of carbon between and within its four major reservoirs: the atmosphere, oceans, terrestrial ecosystems and reserves of fossil fuels. A combination of models and paleo-data 1 reveals that carbon fluxes during the pre-industrial global carbon cycle were in steady state [Figure 1], with a remarkably stable atmospheric carbon dioxide (CO2) concentration of 280 ± 20 parts per million (ppm) or approximately 600 ± 43 petagrams of carbon (PgC) [Joos and Prentice 2004].

Figure 1

Schematic presentation of the pre-inudstrial global carbon cycle. Arrows indicate the carbon fluxes in PgC/year and the values in boxes indicate the reservoir sizes in PgC. Carbon fluxes between the different reservoirs were in steady-state [Goudie and Cuff 2002].

Carbon storage in the terrestrial ecosystem was distributed between vegetation (610 PgC), detritus and soil (1,560 PgC). The yearly terrestrial-atmospheric exchange was in balance with 100 PgC taken up by plants and the same amount returned to the

1 measurements on marine and lake sediments, tree rings and historical documents 1

atmosphere by respiration and decomposition processes [Goudie and Cuff 2002]. The pre-industrial ocean reservoir stored the largest amount of carbon with approximately 38,000 PgC in the intermediate and deep ocean, 1,000 PgC in the surface ocean, 3 PgC in the marine biota and 150 PgC in the sediments. Although the estimates of the atmospheric-sea carbon exchanges vary between 70 PgC/year [Sabine et al. 2004] and

90 PgC/year [Goudie and Cuff 2002], it is generally agreed that the net CO2 flux was in balance. Not depicted in the above figure are fossil fuel reserves, which also significantly contributed to the pre-industrial carbon sink. The amount of carbon stored in reserves of coal, oil and natural gas has been estimated to range between 5,000 and 10,000 PgC [Houghton 2007].

1.1.2 Present-day global carbon cycle

The redistribution of anthropogenic CO2 emissions among the atmosphere, land and oceans dominates the present-day global carbon cycle [Figure 2]. The increase of

CO2 emissions from the burning of fossil fuels, cement making and land-use has led to a noticeable imbalance in the carbon fluxes and to a substantial increase of carbon stored in all major carbon reservoirs other than fossil fuel reserves. Although estimates on the redistribution of CO2 emissions between the three major carbon

reservoirs are discrepant, the most recently reported estimates are as follows: the net

release of CO2 emissions in 2009 resulting from fossil fuel combustion and cement making is reported to be 8.4 ± 0.5 PgC/year while that for land use change is 1.1 ± 0.7

PgC/year [Friedlingstein et al. 2010]. The net CO2 uptake flux in the carbon sinks for the period between 1989 to 2007 have been reported to be 3.60 ± 0.28, 1.95 ± 0.25 and 1.15 ± 0.56 PgC/year for the atmosphere, oceans and land respectively [Sarmiento et al. 2010]. These estimates portray an imbalance of approximately 3

PgC/year in the present-day global cycle. Of the various CO2 sinks, the atmosphere is the most tractable for monitoring, and the most recent estimates of the global

concentration of CO2 in the atmosphere have been averaged to 387.2 ppm, equating to approximately 830 PgC [Friedlingstein et al. 2010]. The next most tractable reservoir of carbon are the oceans, for which most recent estimates indicate an additional 140 ± 25 Pg of anthropogenic carbon in present-day oceans [Khatiwala et al. 2009]. The terrestrial ecosystem may be a significant sink for anthropogenic carbon but its detection in this system appears to be a challenging task and cannot be accurately estimated [Carlson et al. 2001]. Not illustrated in Figure 2 are processes such as sea

floor spreading and diagenesis, which further contribute to the overall carbon budget. The amounts of carbon exchanged annually through these processes are however small and are generally not considered [Sunquist and Visser 2004]. It is evident that the magnitude of CO2 emissions from the burning of fossil fuels, cement making and land-use has resulted in noticeable changes in carbon fluxes and carbon sinks. For how long and at what rates the carbon reservoirs will continue to take up CO2 as concentrations of atmospheric CO2 continue to rise is only partially known and is the subject of considerable scientific investigations. The focus of this work lies in understanding carbon cycle processes in the oceans, the largest of the carbon sinks.

Figure 2

Schematic presentation of the present-day global carbon cycle. Carbon fluxes are shown within and between the major carbon sources and reservoirs. The black arrows indicate the natural exchange of CO2 between the various reservoirs and the red arrows indicate the anthropogenic fluxes. [Adapted from the Global Carbon Project 2006. Most recent estimates in orange boxes, from Khatiwala et al. 2009 and Friedlingstein et al. 2010].

1.2 Oceanic carbon cycle

Four major interconnected processes control the uptake, distribution and storage of carbon in the ocean: (i) CO2 exchange at the air-sea interface; (ii) seawater carbonate chemistry; (iii) mixing of surface and deep waters; and (iv) ocean biology. The interplay between these four processes has since long been and continues to be responsible for the majority of carbon being carried by the ocean. The present-day oceanic carbon flux is not in steady state. Instead, the ocean is taking up on average 1.95 ± 0.25 PgC/year more than it releases at the air-sea interface [Friedlingstein et al.

2010]. CO2 flux (F) is primarily controlled by the difference in CO2 partial pressure between the ocean and the atmosphere and is described as:

F = ∆pCO2 Kw where ∆pCO2 is the difference in CO2 partial pressure between the two reservoirs and

Kw is the piston velocity. Piston velocity describes the rate of gas exchange across the air-sea interface and depends on many factors, of which CO2 solubility, turbulence at the ocean surface and chemical reactivity of CO2 are among the most important [Liss and Slater 1974; Carlson et al. 2001]. As atmospheric CO2 dissolves in seawater, it reacts to form carbonic acid (H2CO3) which then dissociates within milliseconds to - 2- form bicarbonate (HCO3 ) and carbonate (CO3 ) ions. This conversion effectively reduces the pCO2 in seawater thereby allowing more CO2 uptake by the ocean. Collectively, these carbonate species make up the dissolved inorganic carbon (DIC) pool of the ocean, which is distributed in the amounts of 1,020 and 38,000 PgC in the surface and deep waters respectively [Carlson et al. 2001]. The draw-down of DIC from the surface to the deep ocean is largely the result of what has been described as the solubility pump [Volk and Hoffert 1985]. The solubility pump is mainly driven by ocean circulation patterns and mixing of surface and deep waters [Figure 3]. Wind- driven circulation transports surface waters from low (warm) to high (cold) latitudes.

Cold waters enhance CO2 solubility and consequently allow more atmospheric CO2 to be taken up at the surface layers. At high latitudes, dense water formation2 leads to the rapid sinking of water masses until they reach depths of 2,000-4,000m where they reside for over 1,000 years due to the slow mixing of surface and deep ocean waters. The exchange of surface waters with the deep ocean is limited because of the strong

2 cold water is denser than warm and sinks below the less dense layer 4

density stratification of the water column. Thermohaline circulation eventually pushes the deep waters up to the surface in the upwelling regions. Thermohaline as compared to wind-driven circulation is a slow process and it takes over 1,000 years for the DIC- rich deep waters to reach the surface again. These two processes of the solubility pump both assist in the transfer of carbon to the deep ocean and maintain a steep vertical gradient of carbon in the ocean thereby making the ocean one of the largest carbon sinks.

Figure 3

Schematic presentation of the solubility pump of the ocean. The black arrows indicate the movement of carbon and the white arrows indicate the movement of water between the low and high latitudes. [Carlson et al. 2001]

It would appear intuitive from the above-described physical and chemical processes that an increase in atmospheric CO2 would lead to an increase in pCO2 difference at the air-sea interface thereby leading to enhanced CO2 uptake and draw-down into the ocean. The interplay between oceanic processes are however much more complex. 3 Rising atmospheric CO2 concentrations are coupled to increased radiative forcing which result in higher sea-surface temperatures [Houghton et al. 1995]. Since CO2 solubility and the processes of the solubility pump are dependent on temperature it is inevitable that the efficiency of the solubility pump is impacted. Climate models suggest a weakening of the thermohaline circulation at future projected atmospheric

3 the rate of energy change per unit area as measured at the top of the atmosphere 5

CO2 and temperature levels [Joos et al. 1999; Montegro et al. 2007]. The dissociation of dissolved CO2 into DIC is another process likely to be impacted by the increase in atmospheric CO2 concentrations. In fact, a doubling in atmospheric CO2 is not predicted to cause a doubling in DIC but may result in only approximately 10% increase of DIC in the ocean [Zeebe and Wof-Gladrow 2001]. This is due to the intricate nature of the underlying seawater carbonate chemistry. The dissociation of dissolved CO2 into the different carbon species has been equated as:

- + 2- + CO2 (aq) + H20 ↔ H2CO3 ↔ HCO3 + H ↔ CO3 + 2H where the different carbonate species exist in defined proportions maintaining the pH of seawater within a narrow range [Figure 4]. The carbonate species exist in the - 2- approximate ratio of 1 : 90 : 10 [CO2] : [HCO3 ] : [CO3 ] at the average seawater pH of 8.2 ± 0.3 [Tyrrell 2007]. [CO2] encompasses both CO2 (aq) and H2CO3.

Figure 4

The seawater carbonate system represented in a Bjerrum plot. The relative proportions of the carbonate ions control the pH of the ocean [Zeebe and Wolf-Gladrow 2001].

Compared to the pre-industrial ocean, the pH of the current ocean has already decreased by 0.1 units which in fact corresponds to a 30% decrease [Caldeira and Wickett 2003]. The capacity of the carbonate system to continue to buffer the changes due to increasing CO2 concentrations is however finite and together with the potential perturbations in the solubility pump could lead to a severe impact on the overall functioning of the oceanic carbon sink. It is important however to realize that the

ocean is not an abiotic structure and understanding the interplay of oceanic biological processes with the above-described physical and chemical processes is fundamental.

1.3 Biological processes of the ocean

1.3.1 The biological pump Carbon stored in the form of marine biota is approximately 3 PgC/year [Houghton 2007]. Although the standing stock4 is small, the activity associated with the biota is extremely important for the cycling of carbon between the atmosphere and the ocean.

Climate models suggested that pre-industrial atmospheric CO2 ranged between 450 and 530 ppm instead of 280 ppm without a functioning biological pump [Sarmiento and Orr 1991]. The biological pump describes the biologically mediated processes whereby DIC in the upper ocean is transformed and transported to the ocean interior as dissolved organic matter or sinking biogenic particles [Figure 5]. Phytoplankton use light energy in the euphotic zone to convert DIC into organic compounds by the process of photosynthesis. Although phytoplankton encompass less than 1% of the total photosynthetic biomass in the biosphere, they impressively account for roughly half of the total annual primary production [Field et. al 1998].

Figure 5

Schematic presentation of the biological pump depicting oceanic biological processes responsible for the transformation of DIC into organic carbon and its re-distribution in the ocean [OCTET workshop report 2000]

4 the weight or biomass of a stock of organisms 8

The transformation of DIC into organic matter effectively leads to a reduction of pCO2 in the upper ocean, thereby promoting more atmospheric CO2 uptake [Falkowski and Raven 2007]. In addition to converting DIC into organic matter, certain phytoplankton such as the coccolithophores combine calcium with carbonate ions in the upper ocean to form hard calcium carbonate (CaCO3) body parts. CaCO3 is dense and sinks from surface water, thereby also promoting the uptake of additional atmospheric CO2 [Carlson et al. 2001]. The sinking of carbon in the form of organic matter is more efficient than in CaCO3. The ratio of carbon sinking as CaCO3 to organic carbon varies from 1:4 up to 1:17 [Li et al. 1969; Sarmiento et al. 2002].

1.3.2 Dissolved organic carbon Dissolved organic carbon (DOC) is the largest reservoir of organic carbon in the ocean and amounts to approximately 662 PgC [Hansell and Carlson 2001]. Although the mechanisms leading to DOC production have not been fully elucidated, it appears that it is mainly produced as a by-product of primary production [Kepkay et al. 1993; Biddanda and Brenner 1997]. Newly formed DOC is labile and when channeled into the microbial loop 5, vast heterotrophic microbial populations rapidly utilize it as substrate [Azam et al. 1983]. The microbial loop is dynamic, and active processes such as viral cell lysis, sloppy feeding by zooplankton and dissolution of faecal pellets continuously release DOC which then re-enters the oceanic organic carbon pool [Lampert 1978; Middelboe and Lyck 2002]. The remineralized DOC pool is not entirely labile but can exist as semi-labile and recalcitrant DOC. These two types of transformed DOC can persist for months to years in the ocean and account for a significant portion of DOC that is exported from the euphotic zone to the ocean depths [Jiao et al. 2010].

1.3.3 Particulate organic carbon Operationally delineated from DOC by virtue of size and ability to passively sink, particulate organic carbon (POC) forms the second largest oceanic organic carbon pool and amounts for up to 30 PgC [Verdugo et al. 2004; Hansell and Carlson 2001]. A global compilation of POC fluxes shows that oceanic POC concentrations are highest at the surface and exponentially decline with depth [Figure 6]. This suggests

5 a pathway in the aquatic food web whereby DOC is taken up by bacteria and archaea, which are in turn eaten by protists and so on up the food chain 9

that the efficiency of the biological pump is low at exporting POC into the depth of the ocean. The ocean is however not homogenous and there is considerable regional and temporal variability in the fraction of POC that is eventually exported to depth [De La Rocha and Passow 2007]. Primary production is tightly coupled to POC export and during periods of bloom formation. Between 30 and 100% of the net primary production can eventually sink out of the euphotic zone as POC [Buesseler 1998]. The sinking flux of POC below the euphotic zone is dependent on phytoplankton community composition and on the supply of minerals. The presence of large phytoplankton cells (≥ 5 µm) for example has been shown to be positively correlated with an enhanced POC export from the euphotic zone to depth [Smayda 1971; Buesseler 1998; Pommier et al. 2008]. In addition, the higher concentrations of suspended minerals at the continental margins as compared to the open ocean have been suggested as a probable reason for the higher POC export fluxes observed at the continental margin regions [De La Rocha and Passow 2007].

Figure 6

Mean annual global POC flux at different depths in the ocean. Figure modified from Lutz et al. (2002) by C. De La Rocha [marum.de/en/Page9564.html]

In situ observations reveal that the majority of POC sinks out of the euphotic zone in the form of large aggregated particles collectively termed as marine snow [Fowler and Knauer 1986]. The fact that large aggregates enhance POC export can be correlated to

the enhanced sinking rates of the large particles. For example, the sinking rates of solitary phytoplankton cells have been determined to be less than 2 m d-1 [Fowler and Knauer 1986]. Assuming an average oceanic depth of 4000 m and no degradation of the particles during their vertical flux, these cells would reach the bottom of the ocean after more than 5 years. Realistically though, within this time most of the organic carbon would have been degraded before reaching the seabed. With settling speeds ranging between 6 and 368 m d-1, the sinking of marine snow significantly reduces the transit time of organic carbon from surface waters thereby leading to more POC to be exported from the euphotic zone [Turner 2002]. The formation and eventual sinking of marine snow is a complex process and depends both on the formation and degradation rates of the marine aggregates [Kiorboe 2001]. Understanding the processes of marine snow formation and their eventual vertical flux is consequently of utmost importance to further our knowledge of vertical fluxes of organic carbon in the ocean.

1.4 Marine snow

Marine snow is term collectively used to describe macroscopic marine aggregates which are greater than 500 µm in length and are variably composed of planktonic cells, detritus material, faecal pellets as well as inorganic materials such as clay and sediment particles [Alldredge and Silver 1988].

1.4.1 Marine snow formation Marine snow can either be produced de novo by marine plankton or by the physical coagulation of smaller particles. The latter process is often biologically enhanced [Figure 7]. Additionally, members of the zooplankton community such as pteropods, larvaceans and salps secrete mucopolysaccharide feeding webs and houses, which together with their faecal pellets contribute to the de novo production of marine snow particles [Alldredge and Silver 1988; Hansen et al. 1996]. The gelatinous houses of appendicularians are particularly significant in the formation of marine snow due to the rapid turnover between the formation and abandonment of their mucus feeding structures [Lombard and Kiorboe 2010].

Figure 7

Schematic depiction of the major processes leading to marine snow formation. Marine plankton produce marine snow aggregates de novo as mucus webs, houses, sheaths, and flocculent fecal pellets. Small particles including phytoplankton, fecal pellets, microaggregates, bacteria and inorganic particles collide together via physical processes and adhere together by biological processes [Alldredge and Silver 1988].

Many species of phytoplankton especially of the diatom genus Thalassiosira and the prymnesiophyte genus Phaeocystis produce fibrils and mucus sheaths around their colonies and contribute to the de novo formation of marine snow [Fryxell et al. 1984; Alldredge and Silver 1988]. The collision and coagulation of phytoplanktonic cells, faecal pellets, microaggregates, microorganisms and inorganic particles further contribute to the formation of marine snow. The collisions of the above-mentioned particles are facilitated by physical processes such as differential settlement mediated by differences in the sinking velocities of the particles, fluid shear, which is mediated by differences in fluid movements and brownian motion, which describes the collision of particles due to random walk [Kiorboe 2001]. Upon collision, the subsequent coagulation into larger aggregates depends on a multitude of factors that are often biologically enhanced. The cell surfaces of many species of phytoplankton are sticky by nature and immediately form aggregates upon collision [Kiorboe et al. 1990]. The sticking efficiency of these species can vary depending on their physiological state. Under nutrient-rich conditions, for example, the cell surface stickiness of the diatom Thalassiosira pseudonana is very low but then increases by more than two orders of magnitude as cell growth ceases and nutrient become limiting [Kiorboe et al. 1990]. Therefore it could be speculated that in regions where these diatoms occur in abundance, enhanced diatom flocculation and sinking would be observed, especially at the end of diatom blooms. The stickiness of other phytoplankton species on the other hand can be less variable. Skeletonema costatum cells for example are sticky under nutrient replete conditions but can reach their highest sticking efficiency during the transition from exponential to stationary phase of growth [Kiorboe et al. 1990].

1.4.2 Marine gel particles The successful coagulation of particles to form larger marine aggregates is also enhanced by the presence and abundance of extracellular polymer gel particles [Alldredge and Silver 1988; Verdugo et al. 2004]. These gel particles are mostly exudates from living or lysed cells and have been defined as three-dimensional networks of polymers imbedded in seawater. These particles range from single macromolecules to complexed colloidal networks and large assembled polymer networks several hundreds of microns or larger in size [Alldredge and Silver 1988; Verdugo et al. 2004]. Extracellular polymer gel particles are significant in the

formation of larger aggregates and their sedimentation. Due to their gelatinous and sticky nature, these gels are both able to scavenge further particles as they sink and to self-coagulate. This provides an abiotic mechanism to increase sizes and sinking rates [Verdugo et al. 2004]. Marine gels can form within minutes to hours from the self- assembly of free DOC polymers [Figure 8]. The self-assembly initially forms fibrils and thereafter nanogels of 100-150 nm and microscopic gels reaching 4-5 µm in size [Chin et al. 1998; Verdugo 2012].

Figure 8

Schematic presentation of size distributions and abiotic processes leading to the aggregation of marine gel particles in the ocean [Verdugo et al. 2004].

Classified as POC by virtue of their size, macrogels such as transparent exopolymeric particles (TEP) are predominantly formed by the aggregation of nano- and microgels [Verdugo et al. 2004]. TEP have been shown to be critical in marine snow formation and aggregation of diatom blooms as they provide a stable underlying matrix for aggregate formation [Alldredge et al. 1993; Passow and Alldredge 1994].

1.5 Transparent exopolymeric particles

TEP are defined as macrogels retained on 0.4 µm polycarbonate filters and stainable with the cationic dye alcian blue [Figure 9]. These particles are both ubiquitous and abundant in the ocean and have been found in all aggregates investigated to date [Alldredge et al. 1993; Passow and Alldredge 1994; Passow 2002]. Although TEP production is predominantly associated with phytoplankton, organisms such as bacteria, macroalgae and more recently the jellyfish Aurelia aurita have also been shown to be TEP-producers [Stoderegger and Herndl 1999; Passow 2000; Ramaiah et al. 2001; Dicker 2011]. TEP composition varies depending on the species producing the primary DOC polymers. Therefore, attributing a precise chemical composition to TEP in general is challenging. However, it has been shown that TEP primarily comprise acidic polysaccharides, dominated by the simple sugars fucose and rhamnose, and their acidity is derived from the presence of sulfate half ester groups [Mopper et al. 1995; Zhou et al. 1998].

Figure 9

Microscopic view of alcian-blue stained TEP produced by the diatom Thalassiosira weissflogii. [Gaerdes 2010]. Scale bar = 100 µm

The abiotic self-assembly of DOC polymers released by phytoplankton and other organisms is the major pathway of TEP production [Passow 2000; Passow 2002a]. The release of these polymers in turn depends on the species releasing them as well as their individual physiological state and the prevailing growth conditions. For example, the production of TEP by the coccolithophore Emiliana huxleyi is significantly lower than that produced by the prymnesiophyte Phaeocystis antarctica

[Passow 2002; Hong et al. 1997]. The physiological state of the organism can influence TEP production; exponentially growing Chaetoceros sp. produce significantly less TEP than senescent cells [Schuster and Herndl 1995]. The induction of diatom-borne TEP production as a direct result of bacterial activity has recently gained attention. In their study, Gaerdes et al. 2011 demonstrate that certain specific bacterial strains enhance TEP production and aggregate formation of an axenic culture of T. weissflogii [Figure 10].

Figure 10

Total aggregated volume of the diatom Thalassiosira weissflogii under the influence of different bacterial strains [Gaerdes et al. 2011].

The direct impact of bacteria on phytoplanktonic TEP production offers a new and potentially important pathway for TEP production. Understanding the micro-scale interactions between phytoplankton and those bacteria that enhance TEP production could therefore provide important insights into TEP production and the dynamics of marine snow formation.

1.6 Microscale interactions

The heterogeneous spatial distribution of organic matter in the ocean creates microscale gradients to which microbial interactions are restricted. As a pelagic analogy to the rhizosphere 6 of terrestrial plant-associated ecosystems, the phycosphere describes the region surrounding an algal cell that favors the growth of certain microbial taxa [Bell and Mitchell 1972; Cole 1982]. In fact, several studies have documented species-specific bacterial interactions with phytoplankton. The microbial diversity found in the phycosphere of the dinoflagellate Alexandrium fundyense for example is distinctly different from that of the bacterial community in the surrounding seawater. The majority of bacterial taxa recovered in association with the dinoflagellate belonged to the Gammaproteobacteria class of bacteria [Hasegawa et al. 2007]. Other phylogenetic groups known to be associated with dinoflagellates included members of the families Alteromonadaceae, Pseudoalteromonadaceae, Rhodobacteraceae and Flavobacteraceae [Hasegawa et al. 2007]. Diatom-associated bacteria on the other hand have been mainly reported to belong to the Flavobacteria– Sphingobacteria group of the Bacteroidetes phylum, whereas free-living bacteria identified in the same study comprised mainly of members from the Roseobacter group of Alphaproteobacteria [Grossart et al. 2005]. Predominantly studied in vitro, four modes of interactions between bacteria and phytoplanktonic cells have been described. Mutualism describes the interaction where both the bacteria and phytoplankton benefit from each other. Marinobacter spp for example has been shown to promote the algal assimilation of iron by facilitating photochemical redox cycling and the algal cells in turn release organic molecules that are used by the bacteria for growth [Amin et al. 2009]. Parasitism describes the interaction where the presence of certain bacteria has detrimental effects on phytoplankton. The presence of the algicidal bacterium Kordia algicida for example significantly inhibits the growth of certain species of diatoms [Paul and Pohnert 2011]. Commensalism describes the interaction whereby bacteria benefit from phytoplankton cells without having any detrimental or beneficial effects on the algal cell. However, this type of interaction tends to be rather transient and often reverts back to either mutualism or parasitism. Loose associations between bacteria and phytoplankton have also been recognized

6 region of soil that is directly influenced by root secretions and associated soil microorganisms 17

and this latter type of interaction highly depends on the prevailing environmental conditions [Rhee 1972].

1.6.1 Bacterial chemotaxis and motility In the context of organic matter transformations, bacteria have evolved various adaptive strategies among which chemotaxis, motility and production of hydrolytic enzymes are thought to be the most significant [Figure 11]. Marine bacteria cluster around living and lysed marine plankton to take advantage of the organic exudates released by these cells [Azam and Ammerman 1984; Blackburn et al. 1998]. This active clustering requires that the bacterial cells sense gradients of the chemicals released by plankton and respond to them by movement. Chemotaxis describes the process by which bacteria sense the concentration changes of chemical stimuli within their immediate microenvironment. Chemotaxis further comprises bacterial motility, which describes their active movement towards or away from attractants and repellents, respectively and is common among marine bacteria. Chemical attractants in the ocean include dissolved low-molecular-weight organic matter, photosynthetic products, zooplankton excretion products, marine plankton lysates and organic matter leaking from particles [Bell and Mitchell 1972; Blackburn et al. 1998; Malmcrona- Friberg et al. 1990, Fenchel 2002; Barbara and Mitchell 2003a; Seymour et al. 2010]. As a consequence of chemotaxis and motility, heterotrophic bacteria are often found in close spatial associations with phytoplanktonic cells.

Figure 11

Schematic depiction of the adaptive strategies of bacteria coupling to organic matter. Bacterial motility and chemotaxis as well as hydrolytic enzymes play important roles in the coupling of bacteria to organic matter [Azam and Malfatti 2007].

Although bacterial motility is a common phenotype in the ocean, the fraction of bacteria that are motile vary anywhere between 5 and 70% depending on the study

and used analysis method [Grossart et al. 2001]. It was suggested that most microorganisms are motile only in part of their life cycle [Fenchel 2002]. One probable reason for this could be that since the expression of chemotaxis and motility genes are energetically costly, bacteria possessing these adaptations preferentially use them under certain defined environmental conditions. In fact, motility in the ocean has been described as being highly intermittent, with bacterial swimming speeds reaching up to 407 µm s-1 during bursts of nutrients [Mitchell et al. 1995]. The most predominant strategy of bacterial motility is movement mediated by their flagella. By the usage of their flagella, motile bacteria can swim or swarm, depending on the viscosity of the medium they encounter [Jarell 2009]. Twitching motility is another type of bacterial motility that is mediated by the Type IV pilus. This mode of motility is especially relevant when bacteria move on solid surfaces [Jarell 2009].

Flagellum-mediated motility The significance of flagellum-mediated motility and attachment of bacteria to both abiotic and biotic surfaces has been evidenced in various studies. In the formation of biofilms for example, the initial attachment of Pseudomonas aeruginosa to both abiotic and biotic surfaces has been shown to be mediated by the bacterial flagellum [O' Toole and Kolter 1998]. The symbiotic interaction between Vibrio fischeri and the squid Euprymna scolopes also depends on the flagellum-mediated attachment of the bacterium to its host and bacteria deficient in flagellum are unable to cause bioluminescence of the squid organ, an important survival strategy for this bacterial species [Nvholm and McFall-Ngai 2004]. The bacterial flagellum is a complex multi- component structure that spans both cell membranes to the outside of the cell. The flagellum self-assembles to form a helical propeller that enables prokaryotic cells to either swim or swarm in their environments. The flagellum comprises three major subunits: (a) the basal body which is embedded in the inner cell membrane and contains the motor, the switch complex and the rod, (b) the hook, which is a short, highly curved structure made by the polymerization of about 100 copies of the FlgE protein and serves as the junction between the basal body and the filament of the flagellum, and, (c) the filament, which is made by the polymerization of tens of thousands of copies of the flagellin protein FliC and which acts as a propeller when rotated at its base [Figure 12]. The formation and assembly of the proteins involved

in creating the flagellum structure is orchestrated by the interplay between more than 50 gene products [Jarrell 2009].

Figure 12

Schematic depiction of the structure of the bacterial flagellum [Adapted from Liu and Ochman 2007]

Type IV pilus-mediated motility Although different Type IV pili in bacteria have been shown to have diverse functions that provide bacteria with selective advantages to perform their functions, the prevalence of the MSHA Type IV pilus in bacteria from marine environments indicate an advantageous role of this type of cell appendage for microbes in the ocean. The significance of Type IV-mediated motility and attachment of bacteria to both abiotic and biotic surfaces has been evidenced in several diverse studies. The attachment of the marine bacterium Pseudoalteromonas tunicata to both abiotic substrata and cellulose-containing surfaces of the green alga Ulva australis has been shown to be mediated by the MSHA pilus [Dalisay et al. 2006]. Analysis of the genome of V. parahaemolyticus, the causative agent of seafood-associated gastroenteritis, revealed that this bacterium contains two sets of Type IV pili: a chitin- regulated pilus (ChiRP) and the MSHA pilus [Shime-Hattori et al. 2006]. Although both pili have been reported to be crucial in biofilm formation, each pilus has a

defined function during the formation of the biofilm. While the initial attachment of the bacterium to surfaces is mediated by the MSHA pilus, ChiRP plays a role in bacterial agglutination during the later steps of biofilm formation [Shime-Hattori et al. 2006]. The MSHA pili are produced by a wide variety of V. cholerae strains [Albert et al. 1997] and similarly to the biogenesis of the Type IV pilus in P. aeruginosa, which is well characterized. It appears that the MSHA biogenesis and structural genes are organized as an operon.

Figure 13

Schematic representation of the predicted MSHA gene locus in P. tunicata (top) and V. cholera El Tor (bottom). The entire locus is 17,525 bp in length and consists of 17 continuous ORFs. The scale bar represents approximately 2 kb. Black shading >45% identity; dark grey 35-45% identity, pale grey 25-35%, white <25% identity [Figure and legend from Dalisay et al. 2006]

Although there is ample evidence that bacterial motility promotes bacterial-eukaryotic interactions, it remains to be investigated whether bacterial motility and the attachment to phytoplanktonic cells, such as diatoms, induce the production of TEP, thereby impacting marine snow formation and the vertical flux of carbon. In this context, establishing a bilateral model system to mechanistically investigate the interactions at a molecular level is therefore imperative.

1.7 Bilateral model system

Diatom aggregation significantly contributes to the formation of marine snow and is especially relevant during diatom blooms. TEP have been shown to be critical in marine snow formation and aggregation of diatom blooms as they provide a stable underlying matrix for aggregate formation [Alldredge et al. 1993; Passow and Alldredge 1994]. Although the main pathway for TEP production is the abiotic coagulation of smaller gel particles, the presence of certain specific bacteria has been shown to be important for the induction of TEP production in diatoms (Gaerdes et al. 2011). This interaction represents a new and potentially significant pathway for TEP production. In an attempt to investigate the underlying molecular mechanisms of diatom-bacteria interactions with relevance to bacterial attachment, TEP production and marine snow formation, an in vitro bilateral model system consisting of the diatom Thalassiosira weissflogii and the marine gammaproteobacterium Marinobacter adhaerens sp. nov. HP15 was previously established [Figure 14].

Figure 14

Scannning electron micrograph showing the close interaction between the diatom T. weissflogii and the marine bacterium M. adhaerens [Gaerdes et al. 2011].

1.7.1 The diatom Thalassiosira weissflogii

Taxonomically affiliated to the phylum Bacillariophyta, the class Coscinodiscophycea, order Thalassiosirales, family Thalassiosiraceae and genus

Thalassiosira, the ubiquitously distributed diatom T. weissflogii belongs to a diverse and ecologically successful group of microalgae found in most aquatic environments [Fryxell and Hasle 1977; Fryxell 1981; Hallegraeff 1992]. The Thalassiosira genus encompasses approximately 180 marine and 12 freshwater species [Round et al. 1990; Silva and Hasle 1994]. Due to their predominance in diverse regions of the ocean they are reasonably considered as a representative model for marine diatoms. Characteristic of diatoms, T.weissflogii possesses an ornamented siliceous cell wall with a frustule composed of two overlapping thecae: an epitheca and a hypotheca [Figure 15]. Each theca consists of a silica valve, one or more girdle bands that possess a distinctive micro-architecture of pores, and slits displayed in a highly organized arrangement [Fryxell and Hasle 1977]. These structures facilitate cell growth and provide avenues for nutrient and gas exchange as well as for exopolysaccharide secretion [Molino and Wetherbee 2008].

Figure 15

Micrograph of the marine diatom T. weissflogii showing the typical structure of diatoms with the silica cell wall and frustules. [Courtesy of F. Hinz, Alfred-Wegener Institute, Bremerhaven, Germany].

T. weissflogii typically occur as solitary cells ranging between 12 - 22 m in length and 10 - 12 µm in width [Provasoli-Guillard National Center for Marine Algae and Microbiota]. With a doubling time of approximately 1.1 days and ease of cultivation and maintenance under laboratory conditions, T. weissflogii has become an attractive diatom model to work with. Diverse studies including diatom physiology [Armbrust

et al. 2004], copepod grazing experiments [Koski et al. 2008; Ceballos and Ianora 2003], gene expression analysis [Armsbrust 1999] and toxicity studies [Casotti et al. 2005; Windust et al. 1997] have made use of this model. T. pseudonana is the only species of Thalassiosira that has its genome sequence determined thus far, which allows for more in-depth molecular investigations of the diatom [Armbrust et al. 2004].

1.7.2 The marine bacterium Marinobacter adhaerens sp. nov. HP15

M. adhaerens sp. nov. HP15 is a gram-negative, heterotrophic marine bacterium, taxonomically affiliated to the phylum Proteobacteria, the class of Gammaproteobacteria, order Alteromonadales, family Alteromonadaceae and the genus Marinobacter. The marine bacterium M. adhaerens sp. nov. HP15 was isolated from a pool of particle-associated bacteria from the surface waters of the German Wadden Sea [Grossart et al. 2004]. The Marinobacter genus encompasses approximately 30 species and has attracted increasing interest because together with the genera Alcanivorax, Thallassolituus, Cycloclasticus and Oleispira, they form the hydrocarbonoclastic group of bacteria recognized to play a significant role in the biological removal of petroleum hydrocarbons from polluted marine waters [Gauthier et al. in 1992; Yakimov et al. 2007]. Marinobacter are ubiquitously distributed and have been isolated from a variety of marine environments ranging from oil- contaminated environments to surface waters and polar regions [Huu et al. 1999; Yoon et al. 2003; Grossart et al. 2004; Green et al. 2006; Montes et al. 2008]. Marinobacter species have also been isolated from living cultures of dinoflagellate, bryzooan and from the marine sponge Xestospongia testudinaria [Romanenko et al. 2005; Green et al. 2006; Lee et al. 2011]. From a pool of 82 bacterial strains, M. adhaerens sp. nov. HP15 was selected as the bacterial counterpart of the bilateral model system based on the observation that it showed optimal attachment to diatom cells and significantly enhanced TEP production and aggregate formation during its interaction with the axenic T. weissflogii cultures [Gaerdes et al. 2011]. M. adhaerens sp. nov. HP15 forms brownish mucoid colonies when grown on marine broth agar plates and appears rod-shaped with a single polar flagellum when observed under the transmission electron microscope [Figure 16]. 16S rRNA gene sequence analysis in the Ribosomal Database Project [Cole et al. 2003] revealed that the closest relative of

M. adhaerens sp. nov. HP15 is M. flavimaris, with a similarity score of 0.985. At the start of this thesis work, the genome sequences of three Marinobacter species had been determined: M. aquaeolei VT8 (similarity score 0.811), M. algicola DG893 (similarity score 0.913) and Marinobacter sp. ELB17 (no open access data available).

Figure 16

Upper left panel: M. adhaerens HP15 grown on MB agar plates; Upper right panel: M. adhaerens HP15 as observed under the light microscope; Lower panel: M. adhaerens HP15 as observed under transmission electron microscope. [TEM micrograph courtesy of Y. Ramaye, Jacobs University Bremen].

Aims of this study

TEP and marine snow aggregates are vital components of the oceanic carbon cycle, leading to a substantial fraction of organic carbon sinking to depth or being provided for further recycling. Diatom-associated bacteria have been recently shown to directly impact TEP production and aggregate formation. However, very little is known about the molecular components that govern this interaction or the dynamics of TEP production and marine aggregate formation under changing environmental conditions. Through a combination of molecular and ecological experiments, this work aimed to clarify the molecular basis of diatom-bacteria interactions and to uncover hitherto unknown dynamics of TEP production and aggregate formation under future ocean scenarios. The initial aim of this work was the characterization and establishment of a genetic system that allows the precise manipulation of the bacterial counterpart of the bilateral model system. Once established, the second aim of this work was the elucidation of the roles of M. adhaerens HP15 motility appendages during its interaction with the diatom T. weissflogii. The final aim of this work was the characterization of TEP production and aggregate formation dynamics under changed carbonate chemistry and temperature regimes. Taken together, the ultimate aim of the study was to gain a better mechanistic understanding of the molecular components governing diatom-bacteria interactions, and understand how the conditions of a future ocean will impact TEP production and aggregate formation dynamics.

Summary of Results

Chapter 1

Taxonomic affiliation and establishment of a genetic system for M. adhaerens HP15 As part of a collaborative and concerted effort, M. adhaerens HP15 was taxonomically described as a novel member of the Marinobacter genus [Kaeppel et al. 2012], the genome sequence of M. adhaerens HP15 was determined and annotated [Gaerdes et al. 2010] and a powerful genetic system involving mutagenic approaches and reporter gene expression was established to allow for the precise manipulation of M. adhaerens HP15 [Sonnenschein et al. 2011].

Chapter 2

Attachment of M. adhaerens HP15 to T. weissflogii is not essential for the induction of transparent exopolymeric particle formation The role of M. adhaerens HP15 motility during its interaction with the diatom was investigated. By generating M. adhaerens HP15 flagellum- and MSHA type IV pilus- deficient mutants, we demonstrated that a fully-functional flagellum is a pre-requisite for the attachment of M. adhaerens HP15 to both an abiotic surface and to T. weissflogii cells. The MSHA type-IV pilus was also found to be important for attachment, albeit to a lesser extent. We additionally showed that although both cellular appendages are crucial for bacterial attachment to diatom surfaces, this attachment was not essential for inducing diatom-borne TEP production. It was suggested that additional yet-to-be determined mechanisms govern the induction of TEP formation following the initial cell-to-cell contacts mediated by bacterial flagella and pili [Seebah et al., in preparation].

Chapter 3

Combined effects of lowered pH and elevated temperature on diatom-bacteria interactions

In the final part of this work, we studied TEP production dynamics and aggregate formation in putative future oceanic scenarios. The results of our study cautiously suggested that the combination of ocean acidification and global warming impacted TEP production, marine aggregate formation and the sinking velocities of those aggregates. It was suggested that the transport of particulate organic carbon might be critically reduced in a future ocean [Seebah et al., in preparation].

Chapter 1

Taxonomic affiliation and establishment of a genetic system for M. adhaerens HP15

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

3.1.1 Marinobacter adhaerens sp. nov., prominent in aggregate formation with the diatom Thalassiosira weissflogii

The following manuscript was published in its present form in the International Journal of Systematic and Evolutionary Microbiology (2012) 62:124-128

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

Marinobacter adhaerens sp. nov., prominent in aggregate formation with the diatom Thalassiosira weissflogii

Eva C. Kaeppel1, Astrid Gärdes1 Shalin Seebah1, Hans-Peter Grossart2 and

Matthias S. Ullrich1*

1Jacobs University Bremen, School of Engineering and Science, Bremen, Germany 2 IGB-Neuglobsow, Dept. Limnology of Stratified Lakes, Stechlin, Germany

* Corresponding author: Jacobs University Bremen School of Engineering and Science Campus Ring 6 28759 Bremen Germany Tel: +49 421 200 3245 Fax: +49 421 200 3249 [email protected]

Running title: Description of the novel species Marinobacter adhaerens

Subject category: New taxa (Proteobacteria)

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence strain HP15T is AY241552.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

Summary

The Gram-negative, motile, and rod-shaped bacterial strain, HP15T, was isolated from particles sampled in surface waters of the German Wadden Sea. It was identified among 82 other marine isolates due to its high potential to induce production of transparent exopolymeric particles and aggregate formation while interacting with the diatom, Thalassiosira weissflogii. HP15T grew optimally at a range of 34-38 °C, a pH of 7-8.5, and was able to tolerate salt concentrations between 0.5-20 % (w/v) NaCl. HP15T was chemotaxonomically characterized by possessing ubiquinone-9 as the major respiratory lipoquinone as well as C16:0,

C18:1ω9c, and C16:1ω7Cc/ 15:0 iso 2-OH as predominant fatty acids. The G+C content of its DNA was 56.9 mol%. The closest relative by means of 16S rRNA sequence analysis was Marinobacter flavimaris with a similarity level of 99 %. The whole genome relatedness of HP15T to M. flavimaris, M. salsuginis, M. lipolyticus, and M. algicola was determined to be lower than 70 % by DNA-DNA hybridization. On the basis of phenotypic and chemotaxonomic properties as well as phylogenetic analyses, strain HP15T (=DSM 23420T = CIP 110141T) is proposed to represent the novel species, Marinobacter adhaerens sp. nov..

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

Introduction

The genus Marinobacter was established with the species Marinobacter hydrocarbonoclasticus in 1992 (Gauthier et al., 1992). A total of 26 further species have been described until today. These species are tolerant to various conditions as they were isolated from diverse locations - from the sediment (Gorshkova et al., 2003), the water column (Yoon et al., 2004), from coastal (Roh et al., 2008) and deep sea waters (Takai et al., 2005), from the Antarctic (Montes et al., 2008) and from the Red Sea (Antunes et al., 2007). Furthermore, representatives of this genus were isolated from oil-contaminated areas (Huu et al., 1999), hot springs (Shieh et al., 2003), and salines (Martin et al., 2003). Two species were identified based on their interactions with other organisms - M. algicola isolated from dinoflagellate cultures (Green et al., 2006) and M. bryozoorum derived from Bryozoa (Romanenko et al., 2005). The aggregation of phytoplankton cells is an important process in marine ecosystems leading to the sinking of particulate organic matter in form of marine snow. Heterotrophic bacteria were suggested to increase aggregation of microalgae and other particles (Decho, 1990). To study the interaction of diatoms with bacteria and its role in aggregate formation, a bilateral model system was established (Gärdes et al., 2010a). Among 82 bacterial isolates from aggregates (0.1-1 mm in diameter) sampled in surface waters of the German Bight (Grossart et al., 2004), strain HP15T was shown to induce highest transparent exopolymeric particle production and aggregate formation during its interaction with the diatom Thalassiosira weissflogii. Thus, strain HP15T proofed to be a suitable model organism to study bacteria microalgae interactions and its consequences for the organic matter sinking flux in the sea. The aim of the present study was to determine the taxonomic position of this species by analyzing its phenotypic properties and genotypic relatedness.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

For phenotypic examination, HP15T was grown aerobically on Marine Broth (MB) agar plates (5 g peptone, 1 g yeast extract, 0.1 g FePO4, 6 g agar in 750 ml of North Sea water and 250 ml of distilled water, pH adjusted to 7.4) at 28 °C for 48 h. The reference strains Marinobacter flavimaris DSM 16070T , Marinobacter salsuginis DSM18347T,Marinobacter lipolyticus DSM15157T, Marinobacter algicola DSM16394T, and Marinobacter hydrocarbonoclasticus synonym: aquaeolei) DSM11845 were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). The Gram staining reaction, cell morphology, and motility were examined by light microscopy and transmission electron microscopy (EM 900, Zeiss). Enzyme activities and carbon utilization were analyzed by using the API 20NE system (bioMérieux) and the BIOLOG GN2 system (Biolog, Hayward, CA, USA) in artificial seawater medium without carbon source using established procedures (Martinez & Butler, 2007) and as recommended by the manufacturer. The analysis of growth conditions were examined in MB medium at 37 °C and 250 rpm. For salinity tests, the indicated NaCl concentration was added to distilled water instead of using sea water. For pH tests, pH was adjusted by using NaOH or HCl. The temperature range analysis (4-60 °C) was performed in Marine Broth (Difco 279110) by DSMZ. The optimal growth temperature was defined by testing bacterial growth at the temperatures of 28, 30, 32, 34, 36, 38, 40, and 42 °C. The salinity range was studied between 0 and 35 % (w/v) NaCl and the pH range between 4 and 11. Cellular fatty acid composition and quinone analysis were carried out by DSMZ. Cells were grown on Marine Broth (Difco 2216) at 28 °C for 24 h. Fatty acid methyl esters were obtained from 40 mg cells scraped from Petri dishes by saponification, methylation, and extraction using minor modifications of the methods of Miller (1982) and Kuykendall et al. (1988), separated, and analysed using the Sherlock Microbial Identification System (MIS) (Version 4.5) (97 MIDI, Microbial ID, Newark, DE, USA) as described previously (Kämpfer & Kroppenstedt, 1996).

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

For quinone analysis, cells were grown in MB at 28 °C and 250 rpm to an OD of 0.3 and harvested. Extraction, separation, and analysis of lipoquinones was conducted as described by Tindall (1990a, b). DNA-DNA hybridization tests were conducted in duplicates between HP15T and the closest neighbouring strains derived from 16S rRNA analysis: M. flavimaris DSM 16070T, M. salsuginis DSM18347T, M. lipolyticus DSM15157T, and M. algicola DSM16394T. The hybridization was performed by DSMZ as described by De Ley et al. (1970) under consideration of the modifications described by Huss et al. (1983) in 2 x SSC with 5% formamid at 68 °C. For the phylogenetic analysis, the complete 16S rRNA gene of HP15T (AY241552, 1531 bp) was obtained from the genome sequence (GenBank accession no. CP001978) (Gärdes et al. 2010b). The analysis was performed using the ARB software package (Ludwig et al., 2004) and the reference alignment was provided by the Living Tree Project database (Yarza et al., 2008). The phylogenetic tree was based on the HP15T sequence, all type strains of the genus Marinobacter, and the type strains of Halospina denitrificans HGD 1-3T (DQ072719) and Salicola marasensis 7Sm5T (DQ019934) as outgroups. The G+C content of the HP15T genome was calculated using the complete genomic sequence (GenBank accession nos. CP001978, CP001979, and CP001980). The cells of strain HP15T were rod-shaped and motile by one polar flagellum (Supplementary Fig. S1). HP15T grew between 4 and 45 °C, at a pH from 5.5 to 10 and between 0.5-20% (w/v) NaCl. The API 20 NE test of HP15T was negative for nitrate reduction, indole production, arginine dihydrolase, urease, β-glucosidase, gelatinase, β-galactosidase, and the 123 utilization of D-glucose, L-arabinose, D124 mannose, D-mannitol, N-acetylglucosamine, maltose, D-gluconate, capric acid, adipic acid, and citric acid (Tab. 1). HP15T utilized malate and phenylacetate. The results of the BIOLOG GN2 plate were positive for the utilization of dextrin, Tween 40 and 80, pyruvic acid methyl ester, succinic acid mono-methyl-ester, cis-aconitic acid, β- hydroxybutyric acid,

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

γ-hydroxybutyric acid, α-keto glutaric acid, α-keto valeric acid, D,L-lactic acid, bromosuccinic acid, L-alaninamide, D-alanine, L-alanine, L-glutamic acid, L-leucine, and L-proline (Tab. 1). The API 20 NE and BIOLOG GN2 tests were conducted in parallel for HP15T, M. flavimaris strain DSM 16070T, M. salsuginis SD-14BT, M. algicola DG893T, and M. lipolyticus DSM15157T. HP15 differed in the ability to utilize specific substrates such as citric acid, D-gluconate, L-arginine, and malate in comparison to closely related Marinobacter type strains. Results for M. flavimaris DSM 16070T were identical to those reported by Yoon et al. (2004) and were clearly distinguishable for some substrates from those obtained for strain HP15T. Interestingly, the herein observed utilization pattern of M. lipolyticus DSM15157T for maltose as well as the lack of gelantinase and urease activities in M. salsuginis SD- 14BT and M. algicola DG893T, respectively, did not match those obtained by others (Martin et al., 2003; Antunes et al., 2007; Green et al., 2006). These discrepancies might be due to minor variations in cultivation conditions such as inocculum density, incubation temperature, or time of incubation. T The fatty acid profile of HP15 was composed of C16:0 (21.7 %), C18:1ω9 c (21.6 %),

C16:1ω7 c/iso C15:0 2-OH (14.6 %), C16:1ω9 c (9.0 %), C12:0 3-OH (7.9 %) and C12:0 (6.0 %). Thus, it is similar to that of other Marinobacter type strains by comparison of the most common fatty acids of the genus (Supplementary Tab. S1) although most of the previously published strains were grown under an array of diverse cultivation conditions (growth temperatures ranging from 15 to 37°C; incubation times ranging from 1 to 4 days prior to analyis). The predominant ubiquinone was ubiquinone-9, which is consistent with that of other Marinobacter species except M. lutaeoensis, which contained ubiquinone-8 (Shieh et al., 2003). Based on its 16S rRNA sequence, strain HP15T was affiliated to the Marinobacter genus of the Gammaproteobacteria. It is most closely related to the type strains of M. flavimaris (99 %), M. salsuginis (98 %), M. lipolyticus (98 %), and M. algicola (98 %) (Antunes et al., 2007; Green et al., 2006; Martin et al., 2003; Yoon et al., 2004). Beside M. lipolyticus, these type strains form a discrete cluster as evident in the phylogenetic tree (Fig. 1).

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

The G+C content of the genome of HP15T is 56.9 mol% (Tab. 1) and thus is similar to those of other Marinobacter species. As determined by DNA-DNA hybridization in duplicates, genomic DNA of HP15T showed similarities of 63.6 (68.7), 40.0 (38.0), 28.9 (26.0), and 28.2 (24.5) % to those of M. flavimaris, M. salsuginis, M. lipolyticus, and M. algicola, respectively. These similarities were below the generally accepted species differentiation limit of 70 % (Wayne et al., 1987). Their order of relatedness was the same as that for the 16S rRNA sequences. Due to DNA-DNA hybridization results, the type strain of M. flavimaris seemed to be closely related to the herein proposed species. However, both strains differed significantly in the following distinct characteristics: i) utilization of glycerol, D-fructose, DL-lactic acid, D- gluconate, L-alanine, phenylacetate, and L-glutamate; ii) the ability to reduce nitrate to nitrite (Tab. 1); and iii) colony pigmentation, for which HP15T exhibited brownish pigmentation on MB agar whereas colonies of M. flavimaris were cream-colored. Based on the herein determined specific phenotypic and phylogenetic characteristics and based on the genomic differences towards other Marinobacter type strains, strain HP15T should be placed in the genus Marinobacter and should be considered as a novel species. Due to its unique and characteristic attachment properties in the presence of marine particle surfaces, we propose for HP15T the name Marinobacter adhaerens sp. nov..

Description of Marinobacter adhaerens sp. nov. Marinobacter adhaerens [ad.hae'rens. L. part. adj. adhaerens: hanging on, sticking to]. The cells are motile by means of a single polar flagellum, Gram-negative, and non- spore-forming rods (0.6-0.8 x 1.7-2.4 m). Colonies on MB agar are brownish translucent and have a circular shape (1-2 mm in diameter) with smooth edges after 2 days of incubation at 28 °C. Colour intensity increased with time of incubation. HP15T grew between 4 and 45 °C with an optimum at 34-38 °C and at a pH ranging from 5.5 to 10 with an optimum of pH at 7 to 9. No growth was observed at a pH of 5 or lower and 10.5 or higher.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

The strain grew optimally at NaCl concentrations from 2 to 6 % (w/v). It resisted down to 0.5 % NaCl, but not 0 % and up to 20 % NaCl, but not 25 % NaCl. HP15T was negative for nitrate reduction, indole production, arginine dihydrolase, urease, β- glucosidase, gelatinase, and β-galactosidase activity. Malate, phenylacetate, dextrin, Tween 40 and 80, pyruvic acid methyl ester, succinic acid mono-methyl-ester, cis- aconitic acid, β-hydroxybutyric acid, γ-hydroxybutyric acid, α-keto glutaric acid, α- keto valeric acid, D,L-lactic acid, bromosuccinic acid, L-alaninamide, D-alanine, L- alanine, L-glutamic acid, L-leucine, and L-proline are utilized as sole carbon source. HP15T did not utilize α-cyclodextrin, N-acetyl-D-galactosamine, N-acetyl-D- glucosamine, adonitol, L-arabinose, D-arabitol, D-cellobiose, i-erythritol, D-fructose, L-fucose, D-galactose, gentiobiose, D-glucose, m-inositol, α-D-lactose, lactulose, maltose, D-mannitol, D-mannose, D-melibiose, β-methyl-D-glucoside, D-psicose, D- raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, adipic acid, capric acid, citric acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, α-hydroxybutyric acid, p- hydroxy phenylacetate, itaconic acid, malonic acid, propionic acid, quinic acid, D- saccharic acid, sebacic acid, glucuronamide, L-alanyl-glycine, L-aspartic acid, glycyl- L-aspartic acid, glycyl-L-glutamic acid, L-histidine, hydroxy-L-proline, L-ornithine, L-phenylalanine, L-pyroglutamic acid, D-serine, L-serine, L-threonine, D,L-carnitine, γ-amino butyric acid, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2- aminoethanol, 2,3-butanediol,glycerol, D,L-α-glycerol phosphate, α-D- glucose-1-phosphate, and D-glucose-6-phosphate. The major fatty acids are C16:0

(21.7 %), C18:1ω9 c (21.6 %), and C16:1ω7 c/iso C15:0 2-OH (14.6 %). The quinone system consists of quinone-9. The type strain is HP15T (=DSM 23420T =CIP 110141T). The G+C content is 56.9 mol%. The strain was isolated from marine aggregates (0.1-1 mm) of surface waters of the German Bight.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

Acknowledgements

The authors thank Pablo Yarza and Nikolaus Sonnenschein for help in computational analysis. This work was financially supported by Jacobs University Bremen, the Helmholtz Graduate School for Polar and Marine Research, and the Max Planck Society.

References

Antunes, A., Franca, L., Rainey, F. A., Huber, R., Nobre, M. F., Edwards, K. J. & DaCosta, M. S. (2007). Marinobacter salsuginis sp. nov., isolated from the brine seawater interface of the Shaban Deep, Red Sea. Int J Syst Evol Microbiol 57, 1035- 1040. De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. European Journal of Biochemistry 12, 133-142. Decho, A. W. (1990). Microbial exopolymer secretions in ocean environments: their role(s) in food webs and marine processes. In Oceanography and Marine Biology, vol. 28, pp. 73-153. Edited by H. Barnes. Oabn, Argyll, Scotland. Gärdes, A., Iversen, M., Grossart, H., Passow, U. & Ullrich, M. (2010a). Diatom- associated bacteria are required for aggregation of Thalassiosira weissflogii. ISME J, Advance online publication, 9 September 2010, Epub ahead of print. Gärdes, A., Kaeppel, E. C., Shehzad, A., Seebah, S., Teeling, H., Yarza, P., Glöckner, F. O., Grossart, H.-P. & Ullrich, M. S. (2010b). Complete genome sequence of Marinobacter adhaerens type strain (HP15), a diatom-interacting marine microorganism. Stand Genomic Sci 3, 97-107.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

Gauthier, M. J., Lafay, B., Christen, R., Fernandez, L., Acquaviva, M., Bonin, P. & Bertrand, J. C. (1992). Marinobacter hydrocarbonoclasticus gen. nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium. Int J Syst Evol Microbiol 42, 568-576. Gorshkova, N. M., Ivanova, E. P., Sergeev, A. F., Zhukova, N. V., Alexeeva, Y., Wright, J. P., Nicolau, D. V., Mikhailov, V. V. & Christen, R. (2003). Marinobacter excellens sp. nov., isolated from sediments of the Sea of Japan. Int J Syst Evol Microbiol 53, 2073-2078. Green, D. H., Bowman, J. P., Smith, E. A., Gutierrez, T. & Bolch, C. J. S. (2006). Marinobacter algicola sp. nov., isolated from laboratory cultures of paralytic shellfish toxin-producing dinoflagellates. Int J Syst Evol Microbiol 56, 523-527. Grossart, H. P., Schlingloff, A., Bernhard, M., Simon, M. & Brinkhoff, T. (2004). Antagonistic activity of bacteria isolated from organic aggregates of the German adden Sea. FEMS Microbiology Ecology 47, 387-396. Huss, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric etermination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184-192 Huu, N. B., Denner, E. 260 B. M., Ha Dang, T. C., Wanner, G. & Stan-Lotter, H. (1999). Marinobacter aquaeolei sp. nov., a halophilic bacterium isolated from a Vietnamese oil-producing well. Int J Syst Evol Microbiol 49, 367-375. Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989-1005. Kuykendall, L. D., Roy, M. A., O'Neill, J. J. & Devine, T. E. (1988). Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradyrhizobium japonicum. Int J Syst Evol Microbiol 38, 358. Ludwig, W., Strunk, O., Westram, R., Richter, L. & Meier, H. (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32, 1363. Martin, S., Marquez, M. C., Sanchez-Porro, C., Mellado, E., Arahal, D. R. & Ventosa, A. (2003). Marinobacter lipolyticus sp. nov., a novel moderate halophile with lipolytic activity. Int J Syst Evol Microbiol 53, 1383-1387.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

Martinez, J. S. & Butler, A. (2007). Marine amphiphilic siderophores: Marinobactin structure, uptake, and microbial partitioning. J Inorg Biochem 101, 1692-1698. Miller, L. T. (1982). Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids. J Clin Microbiol 16, 584. Montes, M. J., Bozal, N. & Mercade, E. (2008). Marinobacter guineae sp. nov., a novel moderately halophilic bacterium from an Antarctic environment. Int J Syst Evol Microbiol 58, 1346. Roh, S. W., Quan, Z. X., Nam, Y. D., Chang, H. W., Kim, K. H., Rhee, S. K., Oh, H. M., Jeon, C. O., Yoon, J. H. & other authors (2008). Marinobacter goseongensis sp. nov., from seawater. Int J Syst Evol Microbiol 58, 2866. Romanenko, L. A., Schumann, P., Rohde, M., Zhukova, N. V., Mikhailov, V. V. & Stackebrandt, E. (2005). Marinobacter bryozoorum sp. nov. and Marinobacter sediminum sp. nov., novel bacteria from the marine environment. Int J Syst Evol Microbiol 55, 143-148. Shieh, W. Y., Jean, W. D., Lin, Y. T. & Tseng, M. (2003). Marinobacter lutaoensis sp. nov. a thermotolerant marine bacterium isolated from a coastal hot spring in Lutao, Taiwan. Can J Microbiol 49, 244-252. Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688-2690. Takai, K., Moyer, C. L., Miyazaki, M., Nogi, Y., Hirayama, H., Nealson, K. H. & Horikoshi, K. (2005). Marinobacter alkaliphilus sp. nov., a novel alkaliphilic bacterium isolated from subseafloor alkaline serpentine mud from Ocean Drilling Program Site 1200 at South Chamorro Seamount, Mariana Forearc. Extremophiles 9, 17-27. Tindall, B. J. (1990a). Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66, 199-202. Tindall, B. J. (1990b). A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst Appl Microbiol 13, 128- 130.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Evol Microbiol 37, 463-464. Yarza, P., Richter, M., Peplies, J., Euzeby, J., Amann, R., Schleifer, K. H., Ludwig, W., Glöckner, F. O. & Rosselló-Móra, R. (2008). The All-Species Living Tree project: a 16S rRNA-based phylogenetic tree of all sequenced type strains. Syst Appl Microbiol 31, 241-250. Yoon, J. H., Yeo, S. H., Kim, I. G. & Oh, T. K. (2004). Marinobacter flavimaris sp. nov. and Marinobacter daepoensis sp. nov., slightly halophilic organisms isolated from sea water of the Yellow Sea in Korea. Int J Syst Evol Microbiol 54, 1799-1803.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

Figure legends

Fig. 1. Maximum likelihood phylogenetic tree based on 16S rRNA sequences of HP15T, all type strains of the genus Marinobacter and the type strains of Halospina denitrificans HGD 1-3T (DQ072719) and Salicola marasensis 7Sm5T (DQ019934) as out groups. The tree was inferred from 1531 alignment positions using the RAxML algorithm (Stamatakis, 2006). Support values from 1,000 bootstrap replicates were displayed above branches if larger than 50 %. Bar, 0.01 nucleotide substitutions per site.

Table 1. Phenotypic and genotypic differentiation between HP15T and the closest related Marinobacter type strains. Strains: 1, HP15T; 2, M. flavimaris DSM16070T; 3, M. salsuginis SD-14BT; 4, M. algicola DG893T; 5, M. lipolyticus DSM15157T; 6, M. aquaeolei DSM11845; 7, M. hydrocarbonoclasticus SP.17T. +, positive; -, negative; ND,

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII not determined. Data for growth temperature ranges and G+C contents of M. flavimaris (Yoon et al., 2004), M. salsuginis (Antunes et al., 2007), M. algicola DG893T (Green et al., 2006), and M. lipolyticus DSM15157T (Martin et al., 2003) were taken from the cited references. Additional data for growth temperature, G+C content, and the utilization of glycerol, D-fructose, DL-lactic acid, L-alanine, L- phenylalanine, and L-glutamate for M. aquaeolei DSM11845 (Huu et al., 1999) as well as all data for M. hydrocarbonoclasticus (Gauthier et al., 1992) were added as reported in the cited references. All other data were experimentally determined in the current study.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

Supplementary Fig. S1. Transmission electron micrograph of strain HP15T cultivated in MB for 24 h. Bar 250 nm.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

Supplementary Table S1. Cellular fatty acid composition (%) of Marinobacter type strains Strains: 1, HP15T; 2, M. flavimaris SW-145T (Yoon et al., 2004); 3, M. salsuginis SD- 14BT (Antunes et al., 2007); 4, M. algicola DG893T (Green et al., 2006); 5, M. guineae LMG 24048T (Montes et al., 2008); 6, M. lipolyticus SM19T (Martin et al., 2003); 7, M. sediminum R65T (Romanenko et al. 2005); 8, M. aquaeolei VT8T (Huu et al., 1999); 9, M. hydrocarbonoclasticus SP17T (Marquez & Ventosa, 2005). Values are percentages of total fatty acid content. ND, not detected.

*Rare fatty acids present are (%): iso-C13:0 (0.13); C16:0 N alcohol (3.19); iso-C17:0 10- methyl (1.09); C18:3 ω6c(6,9,12) (1.65); unknown peak 11.799 (0.2). Incubation temperature, 28 °C; length of incubation prior to analysis, 1 day.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

†Values of samples 2-9 were derived from the respective references cited above. In each of these studies, variable incubation temperatures (15-37 °C) and different lengths of incubation time (1-4 days) prior to analysis were used.

Supplementary References

Antunes, A., Franca, L., Rainey, F.A., Huber, R., Nobre, M.F., Edwards, K.J. & da Costa, M.S. (2007). Marinobacter salsuginis sp. nov., isolated from the brine- seawater interface of the Shaban Deep, Red Sea. Int J Syst Evol Microbiol 52, 1035- 1040. Green, D.H., Bowman, J.P., Smith, E.A., Gutierrez, T. & Bolch, C.J.S. (2006). Marinobacter algicola sp. nov., isolated from laboratory cultures of paralytic shellfish toxin-producing dinoflagellates. Int J Syst Evol Microbiol 56, 523-527. Huu, N.B., Denner, E.B.M., Ha, D.T., Wanner, G. & Stan-Lotter, H. (1999). Marinobacter aquaeolei sp. nov., a halophilic bacterium isolated from a Vietnamese oil-producing well. Int J Syst Evol Microbiol 49, 367-375 Marquez, M.C. & Ventosa, A. (2005). Marinobacter hydrocarbonoclasticus Gauthier et al. 1992 and Marinobacter aquaeolei Nguyen et al. 1999 are heterotypic synonyms. Int J Syst Evol Microbiol 55, 1349-1351. Martin, S., Marquez, M.C., Sanchez-Porro, C., Mellado, E., Arahal, D.R., & Ventosa, A. (2003). Marinobacter lipolyticus sp. nov., a novel moderate halophile with lipolytic activity. Int J Syst Evol Microbiol 53, 1383-1387. Montes, M.J., Bozal, N. & Mercade, E. (2008). Marinobacter guineae sp. nov., a novel moderately halophilic bacterium from an Antarctic environment. Int J Syst Evol Microbiol 58, 1346-1349. Romanenko, L.A., Schumann, P., Rohde, M. Zhukova, N.V., Mikhailov, V.V & Stackebrandt, E. (2005). Marinobacter bryozoorum sp. nov. and Marinobacter sediminum sp. nov., novel bacteria from the marine environment. Int J Syst Evol Microbiol 55, 143-148.

MARINOBACTER ADHAERENS SP. NOV., PROMINENT IN AGGREGATE FORMATION WITH THE DIATOM THALASSIOSIRA WEISSFLOGII

Yoon, J.H., Yeo, S.H., Kim, I.G. & Oh, T.K. (2004). Marinobacter flavimaris sp. nov. and Marinobacter daepoensis sp. nov., slightly halophilic organisms isolated from sea water of the Yellow Sea in Korea. Int J Syst Evol Microbiol 54, 1799-1803.

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

3.1.2 Complete genome sequence of Marinobacter adhaerens type strain (HP15), a diatom-interacting marine microorganism

The following manuscript was published in its present form in Standards in Genomic Sciences (2010) 3: 97-107.

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Complete genome sequence of Marinobacter adhaerens type strain (HP15), a diatom-interacting marine microorganism

Astrid Gärdes1, Eva Kaeppel1, Aamir Shehzad1, Shalin Seebah1, Hanno Teeling2,

Pablo Yarza3, Frank Oliver Glöckner2, Hans-Peter Grossart4 and Matthias S.

Ullrich1*

1Jacobs University Bremen, School of Engineering and Science, Bremen, Germany

2Max Planck Institute for Marine Microbiology, Microbial Genomics and Bioinformatics Group, Bremen, Germany

3Institut Mediterrani d’Estudis Avançats, Marine Microbiology Group, Esporles, Spain

4IGB-Neuglobsow, Dept. Limnology of Stratified Lakes, Stechlin, Germany

* Corresponding author: Jacobs University Bremen School of Engineering and Science Campus Ring 6 28759 Bremen Germany Tel: +49 421 200 3245 Fax: +49 421 200 3249 [email protected]

Keywords: marine heterotrophic bacteria, diatoms, attachment, marine aggregate formation

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Abstract

Marinobacter adhaerens HP15 is the type strain of a newly identified marine species, which is phylogenetically related to M. flavimaris, M. algicola, and M. aquaeolei. It is of special interest for research on marine aggregate formation because it showed specific attachment to diatom cells. In vitro it led to exopolymer formation and aggregation of these algal cells to form marine snow particles. M. adhaerens HP15 is a free-living, motile, rod-shaped, Gram-negative gammaproteobacterium, which was originally isolated from marine particles sampled in the German Wadden Sea. M. adhaerens HP15 grows heterotrophically on various media, is easy to access genetically, and serves as a model organism to investigate the cellular and molecular interactions with the diatom Thalassiosira weissflogii. Here we describe the complete and annotated genome sequence of M. adhaerens HP15 as well as some details on flagella-associated genes. M. adhaerens HP15 possesses three replicons; the chromosome comprises 4,422,725 bp and codes for 4,180 protein-coding genes, 51 tRNAs and three rRNA operons, while the two circular plasmids are ~187 kb and ~42 kb in size and contain 178 and 52 protein-coding genes, respectively.

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Introduction Strain HP15 (DSM 23420) is the type strain of the newly established species Marinobacter adhaerens sp. nov. and represents one of 27 species currently assigned to the genus Marinobacter [1]. Strain HP15 was first described by Grossart et al. in 2004 [2] as a marine particle-associated, Gram-negative, gammaproteobacterium isolated from the German Wadden Sea. The organism is of interest because of its capability to specifically attach in vitro to the surface of the diatom Thalassiosira weissflogii-inducing exopolymer and aggregate formation and thus generating marine snow particles [3]. Marine snow formation is an important process of the biological pump, by which atmospheric carbon dioxide is taken up, recycled, and partly exported to the sediments. This sink of organic carbon plays a major role for marine biogeochemical cycles [4]. Several studies reported on the formation and properties of marine aggregates [5-8]. Although it was shown that heterotrophic bacteria control the development and aggregation of marine phytoplankton [3], specific functions of individual bacterial species on diatom aggregation have not been explored thus far. A better understanding of the molecular basis of bacteria- diatom interactions that lead to marine snow formation is currently gained by establishing a bilateral model system, for which M. adhaerens sp. nov. HP15 serves as the bacterial partner of the easy-to-culture diatom, T. weissflogii [3]. Herein, we present a set of features for M. adhaerens sp. nov. HP15 (Table 1) together with its annotated complete genomic sequence, and a detailed analysis of its flagella-associated genes.

Classification and features M. adhaerens sp. nov. strain HP15 is a motile, Gram-negative, non-spore- forming rod (Figure 1). Based on its 16S rRNA sequence, strain HP15 was assigned to the Marinobacter genus of Gammaproteobacteria.

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Two other Marinobacter were based on their interactions with eukaryotes - M. algicola isolated from dinoflagellate cultures [20] and M. bryozoorum derived from Bryozoa [21]. The 16S rRNA gene of strain HP15 is most closely related to those of the type strains of M. flavimaris (99%), M. salsuginis (98%) and M. algicola (96%). These four type strains form a discrete cluster in the phylogenetic tree (Figure 2). In contrast, DNA-DNA hybridization experiements revealed that the genome of M. adhaerens sp. nov. HP15 showed about 64% binding to that of M. flavimaris [1], which is below the generally accepted species differentiation limit of 70% [25].

Figure 1: Transmission electron micrograph of M. adhaerens sp. nov. strain HP15

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Chemotaxonomy

Strain HP15 can grow in artificial seawater with a nitrogen-to-phosphorus ratio of 15:1 supplemented with glucose as the sole carbon source. In presence of diatom cells but without glucose, HP15 utilized diatom-produced carbohydrates as sole source of carbon. Furthermore, M. adhaerens sp. nov. HP15 differed from M. flavimaris and other Marinobacter species in a number of chemotaxonomic properties, such as utilization of glycerol, fructose, lactic acid, gluconate, alanine and glutamate [1]. Additionally, strain HP15 showed a unique fatty acid composition pattern.

Figure 2: Maximum likelihood phylogenetic tree based on 16S rRNA sequences of M. adhaerens type strain (HP15) plus all type strains of the genus Marinobacter and the type species of the neighbor order Pseudomonadales. Sequence selection and alignment improvements were carried out using the Living Tree Project database [22] and the ARB software package [23]. The tree was inferred from 1,531 alignment positions using RAxML [24] with GTRGAMMA model. Support values from 1,000 bootstrap replicates are displayed above branches if larger than 50%. The scale bar indicates substitutions per site.

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Table 1. Classification and general features of M. adhaerens sp. nov. HP15 according to MIGS recommendations [9].

Evidence codes – IDA: inferred from Direct Assay (first time in publication); TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property of the species, or anectodal evidence). These evidence codes are from the Gene Ontology project [19]. If evidence code is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Genome sequencing and annotation

Genome project history

M. adhaerens HP15 was selected for sequencing because of its phylogenetic position, its particular feature as a diatom-interacting marine organism [3], and its feasible genetic accessibility to act as a model organism. The respective genome project is deposited in the Genome OnLine Database [19] and the complete genome sequence in GenBank. The main project information is summarized in Table 2.

Table 2: Genome sequencing project information for M. adhaerens sp. nov. HP15

Growth conditions and DNA isolation M. adhaerens sp. nov. HP15 was grown in 100 ml Marine Broth medium [26] at 28°C. A total of 23 µg DNA was isolated from the cell paste using Qiagen

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

Genome sequencing and assembly The Marinobacter adhaerens sp. nov. HP15 genome was sequenced at AGOWA (AGOWA GmbH, Berlin, Germany) using the 454 FLX Ti platform of 454 Life Sciences (Branford, CT, USA). The sequencing library was prepared according to the 454 instructions from genomic M. adhaerens sp. nov. HP15 DNA with a final concentration of 153 ng/µl. Sequencing was carried out on a quarter of a 454 picotiterplate, yielding 258.645 reads with an average length of 405 bp, totaling to almost 105 Mb. These reads were assembled using the Newbler assembler version 2.0.00.22 (Roche), resulting in 253.285 fully and 4.763 partially assembled reads, leaving 932 singletons, 226 repeats and 371 outliers. The assembly comprised 112 contigs, with 40 exceeding 500 bp. The latter comprised more than 4.6 Mb, with an average contig size of almost 116 kb and a longest contig of more than 1.2 Mb. Gaps between contigs were closed in a conventional PCR-based gap closure approach, resulting in a fully closed circular chromosome of 4.421.911 bp, and two plasmids of 187.465 bp and 42.349 bp, respectively. Together all sequences provided 22.5x coverage of the genome. The error rate of the completed genome sequence is about 3 in 1,000 (99.7%).

Genome annotation Potential protein-coding genes were identified using GLIMMER v3.02 [27], transfer RNA genes were identified using tRNAScan-SE [28] and ribosomal RNA genes were identified via BLAST searches [29] against public nucleotide databases. The annotation of the genome sequence was performed with the GenDB v2.2.1 system [30].

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

For each predicted gene, similarity searches were performed against public sequence databases (nr, SwissProt, KEGG) and protein family databases (Pfam, InterPro, COG). Signal peptides were predicted with SignalP v3.0 [31, 32] and trans-membrance helices with TMHMM v2.0 [33]. Based on these observations, annotations were derived in an automated fashion using a fuzzy logic-based approach [34]. Finally, the predictions were manually checked with respect to missing genes in intergenic regions and putative sequencing errors, and the annotations were manually curated using the Artemis 11.3.2 program and refined for each putative gene [35].

Genome properties The genome of strain HP15 comprises three circular replicons: the 4,422,725 bp chromosome and two plasmids of ~187 kb and ~42 kb, respectively (Table 3A and Figure 3). The genome possesses a 56.9% GC content (Table 3B). Of the 4,482 predicted genes, 4,422 were protein coding genes, and 60 RNAs; 391 pseudogenes were also identified. The majority of the protein-coding genes (67.5%) were assigned with a putative function, while those remaining were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Table 3A. Genome composition for M. adhaerens HP15

§ Number of protein-coding genes: 4,180; ¶ Number of protein-coding genes: 178; * Number of protein-coding genes: 52

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Table 3B: Genome statistics for M. adhaerens HP15

a) The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome. b) Also includes 54 pseudogenes and 5 other genes

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Figure 3. Graphical circular maps of the genome and the two plasmids of HP15. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Table 4: Number of genes associated with the 21 general COG functional categories

a) The total is based on the total number of protein coding genes in the annotated genome

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Flagella-associated gene clusters of M. adhaerens HP15

M. adhaerens HP15 was experimentally shown to adhere to diatom cells. The gene clusters coding for secretion, assembly, and mechanistic function of the polar flagellum were analyzed in detail (Figure 4). Besides several other chemotactic mechanisms and various cell surface interactions, bacterial flagella and other cell appendages had previously been shown to be instrumental for chemotactic movement towards and adhesion to biotic surfaces [36, 37]. The amino acid sequences of proteins encoded by the three identified gene clusters showed significant similarities to orthologous and experimentally well-described gene products of P. aeruginosa PAO1 and various other bacterial species as determined by BLASTP algorithm comparison using the Blosum 62 substitution matrix [29]. Not surprisingly, hook and motor switch complex components were most conserved. However, gene products involved in flagellar filament formation encoded by Cluster II also showed 53 to 78% similarity to the Respective PAO1 proteins. Mutagenesis of flagella-associated genes of M. adhaerens HP15 will be carried out in the near future to study the role of flagella in bacteria-diatom interactions and to further our understanding of the cell-to-cell communication between those organisms.

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Figure 4: Schematic presentation of the three flagella-associated gene clusters of M. adhaerens HP15 coding for the basal body, the filament, and the hook and motor switch complex. Identities to the respective orthologs in the genome of P. aeruginosa PAO1 are indicated by gray-scale code. Numbers of CDS are shown below gene names.

COMPLETE GENOME SEQUENCE OF MARINOBACTER ADHAERENS TYPE STRAIN (HP15), A DIATOM-INTERACTING MARINE MICROORGANISM

Acknowledgements

We thank Yannic Ramaye for help with TEM operation and Christian Quast for computer support. The work was financially supported by the Max-Planck Society, the Helmholtz Foundation and Jacobs University Bremen

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DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

3.1.3 Development of a genetic system for Marinobacter adhaerens HP15 involved in marine aggregate formation by interacting with diatom cells

The following manuscript was published in its present form in the Journal of Microbiological methods (2011) 87(2): 97-107.

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

Development of a genetic system for Marinobacter adhaerens HP15 involved in marine aggregate formation by interacting with diatom cells

Eva C. Sonnenschein1#, Astrid Gaerdes1#, Shalin Seebah1, Ingrid Torres-Monroy1,

Hans-Peter Grossart2 and Matthias S. Ullrich1*

# E.C.S and A.G contributed equally

1Jacobs University Bremen, School of Engineering and Science, Bremen, Germany

2 IGB-Neuglobsow, Dept. Limnology of Stratified Lakes, Stechlin, Germany

* Corresponding author: Jacobs University Bremen School of Engineering and Science Campus Ring 6 28759 Bremen Germany Tel: +49 421 200 3245 Fax: +49 421 200 3249 [email protected]

Keywords: Marinobacter, marine aggregates, genetic toolbox, mutagenesis, bacterial motility

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

ABSTRACT

Diatom aggregation is substantial for organic carbon flux from the photic zone to deeper waters. Many heterotrophic bacteria ubiquitously found in diverse marine environments interact with marine algae and thus impact organic matter and energy cycling in the ocean. In particular, Marinobacter adhaerens HP15 induces aggregate formation while interacting with the diatom, Thalassiosira weissflogii. To study this effect at the molecular level, a genetic tool system was developed for strain HP15. The antibiotics susceptibility spectrum of this organism was determined and electroporation and conjugation protocols were established. Among various plasmids of different incompatibility groups, only two were shown to replicate in M. adhaerens. 1.4 x 10-3 transconjugants per recipient were obtained for a broad-host-range vector. Electroporation efficiency corresponded to 1.1 x 105 CFU per µg of DNA. Transposon and gene-specific mutageneses were conducted for flagellum biosynthetic genes. Mutant phenotypes were confirmed by swimming assay and microscopy. Successful expression of two reporter genes in strain HP15 revealed useful tools for gene expression analyses, which will allow studying diverse bacteria-algae interactions at the molecular level and hence to gain a mechanistic understanding of microscale processes underlying ocean basin-scale processes. This study is the first report for the genetic manipulation of a Marinobacter species which specifically interacts with marine diatoms and serves as model to additionally analyze various previously reported Marinobacter-algae interactions in depth.

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

INTRODUCTION

Marine heterotrophic bacteria interacting with micro-algae play an important role in the formation of marine snow particles and are thus important for the carbon cycling in marine pelagic systems (Grossart et al., 2006a; Sapp et al., 2008; Geng and Belas, 2010). Besides their role in degradation of organic carbon and re-mineralization of nutrients (Cole, 1982), these bacteria promote aggregation of phytoplankton cells (Decho, 1990) and are thus important for the biological carbon pump (Longhurst & Harrison, 1989). Understanding their impact during the interaction with micro-algae is essential to gain knowledge about the ecological relevance of these bacteria on the growth of algae in natural habitats. Bacteria interacting with algal cells might feed on them or their products, or support their growth by re-mineralization of nutrients (Grossart and Simon, 2007). Since various scenarios can be envisioned, it remained to be determined whether bacteria enhancing aggregate formation inhibit or promote the metabolism and growth of algae and how they accomplish that. Most previous studies focused on bacterial communities associated with phytoplankton at the ecological level (Grossart et al., 2006b; Sapp et al., 2008), which did not allow to distinguish between the algal and bacterial contribution to specific ecosystem processes. Consequently, very little is known about the genetic characteristics and functional strategies that algae-associated bacteria have adopted to cope with environmental parameters and phytoplankton cells. The genus Marinobacter is one of the most ubiquitous in the oceans and assumed to significantly impact various biogeochemical cycles (Singer et al., 2011; Gauthier et al., 1992; Rotani et al., 2003; Gorshkova et al., 2003). Due to their high functional diversity, different Marinobacter species have gained intense attention by the research community. Members of the Marinobacter genus were frequently isolated from algal samples, corroborating the hypothesis that several species of Marinobacter are frequently associated with phytoplankton (Green et al., 2006, Amin et al., 2009, Alavi et al., 2001, Hold et al., 2001; Gärdes et al., 2011).

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

Genome data of algae-associated Marinobacter species suggested tight relationships to their algal partners since a number of genes coding for proteins and secretion systems typical for bacterial pathogens or symbionts have been identified in M. algicola DG893 (Amin et al., 2009) and M. adhaernes HP15 (Gärdes et al., 2010) as well as in genomes of other algae-associated bacteria (Worden et al., 2006). For an in- depth molecular analysis of diatom-bacteria interactions and for determining its actual nature and mechanism(s), a bilateral model system consisting of the unicellular diatom, Thalassiosira weissflogii, and the bacterial strain, Marinobacter adhaerens HP15, was established recently (Gärdes et al., 2011; Kaeppel et al., 2011). M. adhaerens HP15 had been isolated from marine particles taken from surface water samples of the German Wadden Sea (Grossart et al., 2004). Close and specific interaction of HP15 and T. weissflogii was demonstrated in vitro by attachment and aggregate formation assays as well as determination of transparent exopolymer particle (TEP) production concluding that strain HP15 plays an important role in T. weissflogii aggregation dynamics (Gärdes et al., 2011). Interestingly, this type of interaction required photosynthetic activity of diatom cells and led to improved growth of both interaction partners. This prompted the cautious assumption that the interaction might be symbiotic and not purely saprophytic. Hence, the actual nature of this symbiosis still remains to be elucidated. The genome sequence of M. adhaerens HP15 was determined exhibiting interesting features known from other gram-negative bacteria interacting with eukaryotic hosts (Gärdes et al., 2010). M. adhaerens HP15 was taxonomically established as a novel member of the Marinobacter genus (Kaeppel et al., 2011). Other members of the genus Marinobacter were found in various marine habitats (Gauthier et al., 1992; Rotani et al., 2003; Gorshkova et al., 2003) as well as in interactions with eukaryotic organisms such as Bryozoa or dinoflagellates (Green et al., 2006; Romanenko et al., 2005). Genetic studies with M. adhaerens HP15 have the potential to dissect cell-to-cell interactions of this organism as well as other Marinobacter species with phytoplankton cells at the molecular level. This might lead to the identification of novel processes of sensing, cellular

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

communication, and nutrient exchange and might thus help us to better understand globally important processes and biogeochemical cycles such as marine aggregate formation. As previously shown for other environmentally important bacterial species (Bakersmans et al., 2009; Piekarski et al., 2009; Wöhlbrand and Rabus, 2008), establishment of the genetic accessibility of individual strains represents the pivotal base for detailed and accelerated research on these organisms. Herein, for the first time the genetic accessibility of a Marinobacter species was comprehensively analyzed. The suitability of M. adhaerens HP15 for molecular studies was demonstrated by transfer of plasmids via electroporation and conjugation and by two types of mutagenesis. As proof-of-principle, motility-deficient mutants were generated by transposon insertion as well as by gene-specific mutagenesis using homologous recombination. Expression of reporter genes such as enhanced green fluorescent protein and β-galactosidase was successfully demonstrated for strain HP15.

MATERIALS AND METHODS

Bacterial strains, plasmids and media The bacterial strains and plasmids used are listed in Table 1. Oligonucleotide primers used are listed in Table 2. M. adhaerens HP15 was isolated from marine particles collected from surface waters of the German Bight (Grossart et al., 2004). Marinobacter cells were cultivated in marine broth (MB) medium (5 g peptone, 1 g yeast extract, 0.1 g FePO4, 6 g agar in 750 ml of North Sea water and 250 ml of distilled water, pH 7.4). For electroporation, cells were cultivated on MB agar medium overnight at 37 °C. Escherichia coli strains were maintained in Luria-Bertani (LB) agar medium. For conjugation, Marinobacter cells were grown in 100 ml MB liquid culture at 250 rpm overnight at 28°C. The donor strain E. coli ST18 was grown in LB medium containing 50 g ml-1 5-aminolevulinic acid (ALA). The following antibiotics were added to media when needed (in g ml-1): chloramphenicol, 25;

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

kanamycin, 500; and ampicillin, 50. To analyze the antibiotics susceptibility as selection marker for transformation, strain HP15 was grown in MB medium at 28°C

to an OD600 of 1, and 20 µl of cell suspensions were spotted on MB agar medium containing various concentrations of ampicillin, chloramphenicol, gentamycin, kanamycin, spectinomycin, or tetracycline. The MICs for these antibiotics in MB were determined by the micro-dilution assay as described previously (Burse et al., 2004).

DNA procedures Plasmids were isolated using the NucleoSpin® Plasmid kit (Macherey-Nagel, Düren, Germany). Restriction enzymes and DNA-modifying enzymes were used as recommended by the manufacturer (Fermentas, St. Leon-Rot, Germany). DNA fragments were resolved in 1% agarose gel and extracted with NucleoSpin® Extract kit (Macherey-Nagel). Preparation of genomic DNA was conducted with NucleoSpin® Tissue kit (Macherey-Nagel).

Plasmid conjugation Recombinant plasmids were introduced to the recipient M. adhaerens HP15 by biparental conjugation with E. coli ST18 as a donor. Additionally, triparental mating with the plasmid-mobilizing helper strain E. coli HB101 (pRK2013) was performed.

Bacterial strains were grown as described above overnight and the OD600 was adjusted to 0.1 (~ 3 x 107 cells ml-1). 107-108 cells of donor and recipient were mixed in a ratio of 1:2. For triparental mating, recipient, donor, and helper strain were mixed in a ratio of 3:1:1. For both types of mating, cells were re-suspended in 500 l of LB medium supplemented with ALA, spotted on LB agar plates supplemented with ALA, and incubated for 24, 48, or 72 h at 28°C. After incubation, the cell mass was scraped off the agar plates and re-suspended in MB medium for subsequent dilution plating. Transconjugants were selected on MB agar supplemented with chloramphenicol after incubation for 2-5 days at 28°C. All experiments were conducted in triplicates.

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

Electroporation Electro-competent Marinobacter cells were prepared directly before electroporation and kept on ice during all steps of the washing procedure. The cell mass of two fully covered MB agar plates was re-suspended in 1 ml of pre-cooled 300 mM sucrose and washed two times with 1 ml of cold 300 mM sucrose using centrifugations at 13,000 rpm and 4°C for 3 min. The final pellet was re-suspended in 200 µl of 300 mM sucrose to obtain a dense suspension (OD600 of ~30). 50 µl of cell suspension was mixed with 0.3 to 1.5 µg of plasmid DNA for electroporation (cuvette width 0.2 cm, resistance 200 , capacitance 25 µF, pulse 2.5 kV for ~5 ms). Immediately after the pulse, 950 ml of SOC medium was added to the cuvette. The cell suspension was transferred to a sterile 1.5-ml tube and incubated by shaking for 15-20 hrs at 37°C. 50 to 400 µl of suspensions were subsequently plated on MB agar medium supplemented with the appropriate antibiotics and incubated at 37°C. Electro-transformation of strain HP15 was tested in triplicates with the following plasmids: pBBR1MCS, pSUP106, pWeb-Cm, pGEM.Km, pEx18Tc, pK18mob, pLAFR3, pKnock-Cm, pPH1JI, pRK415, and pSU18 (Table 3).

Transposon mutagenesis Plasmids pBK-miniTn7-gfp1, pEP4351, and pRL27 (Table 1) containing different transposons were tested for transposon mutagenesis efficiency in HP15 using electroporation. Resulting mutant colonies were grown in MB medium supplemented with kanamycin in 96-well microtiter plates overnight, re-suspended in 15% glycerol, and stored at -80°C. For screening of flagellum-deficient mutants, mutant cells were grown in MB medium containing kanamycin and picked on 10-fold diluted MB soft agar plates (0.3% agar). Swimming-deficient mutants were identified by lack of the typical motility pattern of the HP15 wild type. The genomic DNA of promising mutants was extracted, treated with the restriction enzyme NcoI, re-ligated with T7 DNA ligase, and introduced to E. coli DH5α λ-pir by electroporation. Nucleotide sequencing of transposon-flanking regions was conducted with the primers TnF and

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

TnR. The obtained sequence data were aligned with the GenBank sequence database entries using BlastX (Altschul et al., 1990).

Gene-specific mutagenesis by homologous recombination As a candidate gene for gene-specific mutagenesis, the flagellin-encoding gene, fliC, was selected using the M. adhaerens HP15 genome sequence (GenBank accession no. CP001978) (Gärdes et al., 2010), GenDB 2.2, and BlastN analysis (Altschul et al., 1990). A 1,002-bp upstream and a 1,236-bp downstream flanking regions of fliC were amplified using the primer pairs FliCupF/FliCupR and FliCdownF/FliCdownR, respectively. Both fragments were sub-cloned to vector pGEM®-T Easy (Promega, Mannheim, Germany) resulting in plasmids, pAS3 and pAS4. A chloramphenicol resistance cassette was excised from pFCM1 with a KpnI restriction digest and inserted into KpnI-treated pAS3 yielding plasmid pAS5. Plasmid pAS5 was treated with the restriction enzymes BamHI and SpeI, the fragment was purified, and ligated into the BamHI-SpeI-treated plasmid pAS4, resulting in plasmid pAS6, which contained the 6,338-bp knock-out fragment consisting of the chloramphenicol resistance gene flanked by fliC upstream and downstream fragments. The knock-out fragment was excised with enzyme EcoRI and ligated to the EcoRI-treated suicide vectors pEX18Ap and pK19mobsacB, respectively, generating pAS7 and pAS8 as mutagenic constructs. After biparental conjugation and subsequent homologous recombination, correct insertion of knock-out fragments in the M. adhaerens HP15 chromosome by double crossover was confirmed by antibiotics selection and PCR with primer pairs FliCF/FliCR and CmF/CmR, respectively.

Determination of mutant phenotype by swimming assay and transmission electron microscopy Flagellum-deficient mutants and the wild-type strain HP15 were grown overnight in MB medium containing – when needed – kanamycin or chloramphenicol, respectively, inoculated to 10-fold diluted MB soft agar plates (0.3% agar) with a sterile toothpick, and incubated for 48 h. For transmission electron microscopy, cells

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

were grown in MB medium as described above. A 300-µm-mesh carbon-coated copper grid (Plano, Wetzlar, Germany) was incubated for 30 s in 20 µl of cell suspension, excess liquid was removed, adhering cells were stained with 1% uranyl acetate, washed with distilled water, and dried. The stained cells were visualized using an EM900 transmission electron microscope (Zeiss, Jena, Germany).

Expression of enhanced green fluorescent protein and ß-galactosidase in M. adhaerens HP15 Plasmid pBBR.EGFP carries the egfp gene encoding enhanced green fluorescent protein in pBBR1MCS downstream of the promoter of lacZ′. pBBR.EGFP was introduced to strain HP15 by electroporation. Expression of egfp in single cells was visualized using a LSM510 META confocal laser scanning microscope (Zeiss). The wild type of HP15 carrying the pBBR1MCS vector served as a negative control. The E. coli lacZ gene was amplified from plasmid pMC1871 with primers LacZF and LacZR, each containing a recognition site for KpnI. The resulting 3,057-bp fragment was treated with KpnI and was ligated to KpnI-treated pBBR1MCS in both orientations resulting in plasmids, pITM1 or pITM2. In pITM1, lacZ is in opposite direction to the lacZ’ promoter, whereas in pITM2 it is under the control of the lacZ’ promoter. Both plasmids were introduced to HP15 via electroporation. Transformants were selected on MB agar plates containing chloramphenicol and X-Gal.

RESULTS

Antibiotics susceptibility of M. adhaerens HP15 Growth of M. adhaerens HP15 was inhibited by a number of commonly used antibiotics (Table 3). Minimal inhibitory concentrations (MIC) were generally higher on agar than those observed in liquid medium. The highest susceptibility of strain HP15 on agar medium with MICs of 25 µg ml-1 was observed for ampicillin and chloramphenicol, the latter one being further used as selection marker for

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

transformation. Furthermore, dense M. adhaerens HP15 cell suspensions with an optical density at 600 nm (OD600) of ~30 were plated on MB agar plates supplemented with either 25 g ml-1 of chloramphenicol and ampicillin, respectively, or with 100 g ml-1 of kanamycin, respectively. Not a single spontaneously resistant colony could be obtained (Data not shown) indicating that chloramphenicol, ampicillin, and kanamycin are suitable resistance markers for strain HP15.

Transformation efficiency and expression of reporter genes From various vectors tested, only plasmids pBBR1MCS and pSUP106 were found to replicate in M. adhaerens HP15. Other plasmids, such as pWEB-Cm, pGEM-Km, pLAFR3, pPH1JI, pRK415, and pSU18 (Table 1) could not be transformed or did not replicate in strain HP15. Highest conjugation efficiencies were obtained via biparental mating at a donor-to-recipient ratio of 1:2 and after 24 hrs of mating time (Table 4). For plasmid pBBR1MCS, 1.4 x 10-3 transconjugants per number of recipients and for plasmid pSUP106 2.7 x 10-4 transconjugants per number of recipients were obtained. Using electroporation, transformation efficiencies of 5.1 x 10-5 transformants per number of recipients for pBBR1MCS and 9.2 x 10-7 transformants per number of recipients for pSUP106 were observed. These values corresponded to 1.1 x 105 CFU µg-1 DNA for pBBR1MCS and 1.6 x 103 CFU µg-1 DNA for pSUP106 (Table 4). When plasmid pBBR.EGFP carrying the egfp gene encoding enhanced green fluorescent protein was introduced to strain HP15, transformants exhibited fluorescence when excited at a wavelength of 488 nm, thus demonstrating that egfp was expressed (Fig. 1A). In contrast, no fluorescence was observed for strain HP15 carrying vector pBBR1MCS (Fig. 1B) suggesting that egfp is a suitable reporter gene for this bacterium. Colonies of HP15 wild type were white-brownish on MB agar. HP15 transformants harboring plasmid pITM1, which contains the ß-galactosidase gene lacZ in opposite direction to the Plac promoter, were white-brownish on MB agar containing X-Gal similar to the wild type (Fig. 2B). However, transformants containing pITM2, which harbors lacZ under control of the Plac promoter, grew in form of blue-colored colonies

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

on MB agar containing X-Gal thus expressing the reporter gene lacZ (Fig. 2A). Next, plasmid pITM2 was isolated from blue transformants of strain HP15. Multiple restriction enzyme treatments of this plasmid extract proved a correction orientation of lacZ in the recovered plasmid.

Transposon and gene-specific mutagenesis of M. adhaerens HP15 Transposon-carrying plasmids pBK-miniTn7-gfp1, pEP4351, and pRL27 were assayed for their potential to be used for transposon insertion mutagenesis of M. adhaerens HP15 via electroporation. Transformation with pBK-miniTn7-gfp1 and pEP4351 did not yield in transposon mutants. In contrast, transformation of strain HP15 with plasmid pRL27 carrying transposon Tn5 resulted in an efficiency of 6.8 x 102 CFU µg-1 DNA (1.8 x 10-7 mutants per number of recipients). A group of 18 randomly chosen mutants was subjected to cloning of the transposon insertion regions. Subsequent nucleotide sequencing of the transposon-flanking regions revealed 18 distinct and unique insertion sites (Data not shown) thus confirming the randomness of transposon insertions. Testing a total of 768 transposon mutants by soft agar swimming assay revealed two swimming-deficient mutants. For these HP15 mutants, nucleotide sequencing of the transposon-flanking DNA regions revealed that their phenotype correlated to individual transposon insertions in the motility- associated genes fliG and fliR (Data not shown). A mutant with the transposon insertion in fliG termed HP15-fliG::tn5 was used for further phenotypic analysis. Gene-specific mutagenesis was conducted by introducing the suicide plasmids pAS7 and pAS8, respectively, harboring the fliC mutagenic construct by biparental conjugation. Transconjugants were selected on MB agar plates supplemented with chloramphenicol, and double crossover of the chloramphenicol resistance cassette in the fliC gene of strain HP15 was demonstrated by PCR with primers FliCF and FliCR yielding the expected 1,734-bp fragment. In contrast, PCR with the HP15 wild type using the same primer set yielded an intact fliC amplification of 2,487 bp. Absence of plasmids pAS7 and pAS8, respectively was confirmed by lack of recombinant

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

plasmids in extractions from the transconjugants (Data not shown). One of the mutants was designated HP15-fliC. The results confirmed a successful gene-specific mutagenesis using homologous recombination in M. adhaerens HP15. Conjugation of the respective vectors, pEX18Ap and pKmobsacB, without insert DNA homologous to genes of strain HP15 did not yield antibiotics-resistant HP15 transformants.

Phenotypic characterization of M. adhaerens HP15 mutants In contrast to the HP15 wild type, motility-deficient mutants HP15-∆fliC and HP15- fliG::tn5 were not motile on soft agar demonstrating that genes fliC and fliG were essential for flagellar movement of HP15 (Fig. 3). Furthermore, transmission electron microscopy revealed that HP15 wild type possessed one polar flagellum (Fig. 4A) while mutant HP15-fliC did not produce a visible flagellum but retained the flagellar hook (Fig. 4B) demonstrating the accurate gene-specific mutation. In contrast, transposon insertion in the hook-associated fliG gene led to a total loss of the flagellum as seen for mutant HP15-fliG::tn5 (Fig. 4C).

DISCUSSION

In contrast to well-established bacterial model organisms in medical, veterinary or plant pathology as well as in microbial biotechnology, environmentally important microbes - particularly of marine origin - are often not readily accessible for molecular laboratory work. However, in order to understand the molecular basis of microbial processes in the oceans, genetically accessible model systems are needed. The current study was part of a concerted action, in which the pivotal role of M. adhaerens in marine aggregate formation was demonstrated (Gärdes et al., 2011), its genome analyzed (Gärdes et al., 2010), and its taxonomic affiliation as a new species determined (Kaeppel et al., 2011). For the first time, we show that a single marine bacterial species being directly and specifically involved in marine aggregate formation (Gärdes et al., 2011) is genetically accessible in terms of transformation, transposon and gene-specific mutagenesis, as well as reporter gene expression.

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

This study is distinctive from that of Kato et al. (1998), who established a genetic transformation system for algae-lysing Alteromonas strains. These bacteria were shown to lyse different species of diatoms including Thalassiosira sp. In the future, a comparative functional analysis of algae-aggregating and algae-lysing bacteria based on mutagenic approaches and gene expression analyses might reveal important new insights into the mechanisms of their interactions with diatoms. The genus of Marinobacter is assumed to contribute significantly to different marine biogeochemical cycles (Singer et al., 2011). Various ubiquitously distributed and environmentally prominent representatives of the Marinobacter genus have been under research for almost 20 years in terms of their oil-degrading capacity (Gauthier et al., 1992; Yakimov et al., 2007), wax ester production (Rontani et al., 2003), siderophores (Barbeau et al., 2002; Martinez and Butler, 2007), particle colonization (Grossart et al., 2003), and interactions with phytoplankton (Jasti et al., 2005; Sher et al., 2011; Gärdes et al., 2011). The currently available genome sequences of four Marinobacter species are highly similar to each other (Gärdes et al., 2010; Singer et al., 2011). Consequently, the herein developed genetic tool box for M. adhaerens will assist researchers studying specific functional traits in other Marinobacter species. Essential methods to allow molecular analyses of a given bacterium are plasmid transformation techniques, different types of mutagenesis, and reporter gene expression. Herein, plasmid introduction to M. adhaerens HP15 by electroporation and conjugation, random and gene-specific mutagenesis, as well as expression of reporter genes were reported as a first proof-of-principle. With the established techniques, it is now possible to identify the particular role of genes and to quantify gene products important for the interaction of this bacterium with diatom cells. In turn, this might lead to the identification of molecular signals and environmental patterns underlying this interaction. The current study was conducted with a marine diatom-associated γ-proteobacterium and thus is complementary to but also clearly distinctive from very impressive approaches with representatives of the Roseobacter clade of α-proteobacteria, which are living in symbiosis with heterotrophic dinoflagellates, such as Pfiesteria piscicida

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

(Miller et al., 2006; Geng et al., 2008; Geng and Belas, 2010). On the one hand – to be highly effective – genetic tools and protocols need to be specific and need to be optimized for bacteria phylogenetically belonging to different proteobacterial sections, i.e. α- and γ-proteobacteria (Davidson, 2002). On the other hand, our future molecular analyses of M. adhaerens might reveal fundamentally novel mechanisms of the biotrophic interaction of this bacterium with a photosynthetic marine eukaryote, T. weissflogii. The determined antibiotics susceptibility spectrum of M. adhaerens HP15 allowed selection of transformants or mutants by antibiotics resistance markers, i.e. chloramphenicol, ampicillin, and kanamycin. The relative low susceptibility of strain HP15 to other antibiotics might be due to the high salt concentration in the used medium as concluded previously for other marine organisms (Piekarski et al., 2009). Resistance to different antibiotics was earlier claimed to be a suitable taxonomic marker for marine bacteria (Gorshkova and Ivanova, 2001). Herein obtained data are comparable to those for M. aquaeolei (Huu et al., 1999) but not to those of M. vinifirmus and M. alkaliphilus (Liebgott et al., 2006; Takai et al., 2005) and thus did not result in a clear genus-specific pattern. Recombinant plasmids of different incompatibility groups were tested for replication in M. adhaerens HP15. Interestingly, transformation with plasmids of the incompatibility group IncQ was successful whereas plasmids of incompatibility groups IncP, IncX, colE1, or pMB1 did not replicate, could not be introduced to strain HP15, or did not allow for the expression of the respective resistance gene. It remains to be analyzed whether the two native plasmids of strain HP15 with molecular sizes of 42 and 187 kb (Gärdes et al, 2010), respectively, possibly interfere with replication of the latter plasmid groups. The herein obtained electroporation efficiency was comparable to that of the marine γ-proteobacterium Pseudoalteromonas (Kurusu et al., 2001) but was lower than that described for Alteromonas (Kato et al., 1998). Plasmid conjugation efficiency for strain HP15 was found to be similar to those of other marine γ-proteobacteria (Dahlberg et al., 1998) or α-proteobacteria of the Roseobacter clade

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

(Piekarski et al., 2009). The reporter genes egfp and lacZ were introduced in trans to strain HP15 and showed a clear phenotypic expression making both genes suitable for in vivo labeling and for reporter gene analyses in future studies. The transposon delivery plasmid pRL27 (Larsen et al., 2002) was used to generate a library of mutants of M. adhaerens HP15. Efficiency of mutagenesis was lower than that for the close relative, Pseudomonas stutzeri (Larsen et al., 2002). However, it was sufficient to readily generate a library characterized by a high degree of randomness. For homologous recombination, derivatives of the mobilizable vectors pEX18Ap and pK18mobsacB were used due to their inability to replicate in non-enterobacterial species (Hoang et al., 1998; Schäfer et al., 1994). As expected, conjugation of these vectors without insert DNA homologous to genes of strain HP15 did not yield HP15 transformants indicating that they could be used as suicide vectors. To demonstrate the ability to knock-out any specific gene, motility of obtained transposon mutants was screened. The flagellum-deficient transposon mutants HP15- fliG::tn5 and HP15-fliR::tn5, as well as the gene-specific mutant HP15-fliC were non-motile in soft agar in contrast to the HP15 wild type. As expected, in mutant HP15-fliG::tn5 the flagellum was not formed at all since this gene is required for the flagellar hook formation as described earlier for Salmonella enterica (Thomas et al., 2001). In contrast, mutant HP15-fliC exhibited the flagellar hook but was missing the flagellar filament confirming previous data obtained for Heliobacter pylori and other bacteria (Macnab, 2003; Seong et al., 1999). These results demonstrated that the flagellar filament of M. adhaerens HP15 is encoded by a flagellin gene. The flagellum-deficient mutants will next be tested during their interaction with diatoms to study the role of bacterial motility in chemotaxis and attachment.

CONCLUSIONS

An easy-to-work-with and powerful genetic toolbox for M. adhaerens HP15 was established, which renders this bacterium a suitable model organism for molecular analysis of diatom-bacteria interactions. This genetic toolbox can be used for other

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

members of the Marinobacter clade involved in phytoplankton interactions and oceanic biogeochemical cycles. Herein tested and established methods and procedures will be applied to knock-out and functionally analyze genes involved in i.e. motility, surface attachment, chemotaxis, biofilm formation, as well as nutrient sensing and acquisition. Use of reporter genes will serve in differential gene expression studies and in a currently being established in vivo expression technology screen (Slauch et al., 1994) allowing the identification of novel genes important for the biotrophic interaction of M. adhaerens with its diatom host. As shown by previous studies, which established genetic systems for other environmentally important bacteria (Bakersmans et al., 2009; Piekarski et al., 2009; Wöhlbrand and Rabus, 2008), the current study has built the technical base for intense future research on a globally important process: bacteria-induced formation of diatom aggregates and thus their sinking behavior in the ocean. Improving our understanding of specific cell-to-cell interactions at the molecular level provides the basis for a mechanistic understanding of the “biological carbon pump” and is crucial to identify specific environmental parameters and cellular factors contributing to or triggering the ecological consequences of a globally changing world.

Acknowledgements We thank Helge Weingart, Sabrina Thoma, William Metcalf, and Ingo Leibiger for providing bacterial strains and plasmids. This work was financially supported by Jacobs University Bremen, the Max Planck Society and the Helmholtz Graduate School for Polar and Marine Research.

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

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Yakimov, M.M., Timmis, K.N., Golyshin, P.N., 2007. Obligate oil-degrading marine bacteria. Curr. Opin. Biotechnol. 18, 257-266

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

Table 1. Bacterial strains and plasmids used in this study

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

Table 2. Oligonucleotide primers used in this study. The underline marks the restriction enzyme recognition sites.

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

Table 3. Minimal inhibitory concentration for strain HP15 on 1.2 % MB agar and in MB medium

Table 4. Conjugation efficiencies for plasmids pBBR1MCS and pSUP106 in Marinobacter adhaerens HP15

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

Figure 1. Fluorescence microscopy photographs of Marinobacter adhaerens HP15 harboring the reporter gene-carrying plasmid pBBR.EGFP (A) or the vector pBBR1MCS as control (B) excited at 488 nm

Figure 2. Colony phenotypes of Marinobacter adhaerens HP15 carrying pITM2 (A) and pITM1 (B) on MB agar supplemented with X-Gal.

DEVELOPMENT OF A GENETIC SYSTEM FOR MARINOBACTER ADHAERENS HP15 INVOLVED IN MARINE AGGREGATE FORMATION BY INTERACTING WITH DIATOM CELLS

Figure 3. Phenotypic characterization of flagellum-deficient Marinobacter adhaerens HP15 mutants by 0.3 % soft agar assay after 2 days of incubation: (A) HP15 wild type; (B) HP15-∆fliC; and (C) HP15-fliG::Tn5.

Figure 4. Phenotypic characterization of flagellum-deficient Marinobacter adhaerens HP15 mutants by transmission electron microscopy: (A) HP15 wild type showing a full flagellum; (B) HP15-∆fliC carrying the flagellar hook only; and (C) HP15- fliG::Tn5 lacking both, flagellar hook and flagellum.

Chapter 2

Attachment of Marinobacter adhaerens HP15 to Thalassiosira weissflogii is not essential for the induction of transparent exopolymeric particle formation

ATTACHMENT OF MARINOBACTER ADHAERENS HP15 TO THALASSIOSIRA WEISSFLOGII IS NOT ESSENTIAL FOR THE INDUCTION OF TRANSPARENT EXOPOLYMERIC PARTICLE FORMATION

3.1.4 Attachment of Marinobacter adhaerens HP15 to Thalassiosira weissflogii is not essential for the induction of transparent exopolymeric particle formation

The following manuscript is in preparation

ATTACHMENT OF MARINOBACTER ADHAERENS HP15 TO THALASSIOSIRA WEISSFLOGII IS NOT ESSENTIAL FOR THE INDUCTION OF TRANSPARENT EXOPOLYMERIC PARTICLE FORMATION

Attachment of Marinobacter adhaerens HP15 to Thalassiosira weissflogii is not essential for the induction of transparent exopolymeric particle formation

Shalin Seebah1, Uta Passow2 and Matthias S. Ullrich1*

1Molecular Life Science Research Center, Jacobs University Bremen, Bremen, Germany

2 Marine Science Institute, University of California Santa Barbara, CA, USA

Running title: Bacterial attachment not essential for diatom TEP production

* Corresponding author: Jacobs University Bremen School of Engineering and Science Campus Ring 6 28759 Bremen Germany Tel: +49 421 200 3245 Fax: +49 421 200 3249 [email protected]

Keywords: Flagella, MSHA Type-IV pili, TEP, diatom-bacteria interactions, ocean carbon cycle

ATTACHMENT OF MARINOBACTER ADHAERENS HP15 TO THALASSIOSIRA WEISSFLOGII IS NOT ESSENTIAL FOR THE INDUCTION OF TRANSPARENT EXOPOLYMERIC PARTICLES FORMATION

ABSTRACT

Transparent exopolymeric particles (TEP) play a critical role in the formation of marine aggregates. Although predominantly formed by the abiotic assembly of TEP precursors, the interactions between phytoplankton and certain specific bacterial species have been shown to directly impact the production of TEP and the aggregation of phytoplankton. We hypothesized that motility-mediated attachment of bacteria to phytoplankton cell surfaces impacts the production of TEP. In this study, the role of the marine gammaproteobacterium, Marinobacter adhaerens HP15, and its flagellum- or MSHA type IV pilus-deficient mutants were investigated with respect to attachment to abiotic surface as well as to the surface of the diatom Thalassiosira weissflogii. Our results demonstrated that a fully-functional flagellum is a pre-requisite for the attachment of M. adhaerens HP15 to both, abiotic and biotic surfaces. The MSHA type-IV pilus was also found to be important for attachment, yet to a lesser extent. Although both cellular appendages are crucial for bacterial attachment to diatom surfaces, herein obtained results also showed that this attachment is not essential for inducing diatom-borne TEP production. This suggested additional yet-to-be determined mechanisms governing the induction of TEP formation following the initial cell-to-cell contacts mediated by bacterial flagella and pili.

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INTRODUCTION Due to their gelatinous and sticky nature, transparent exopolymeric particles (TEP) play a critical role in the formation of marine aggregates (Alldredge et al. 1993, Passow and Alldredge 1994). These ubiquitously distributed and abundant particles have been found in all marine aggregates investigated to date (Alldredge et al. 1993, Passow and Alldredge 1994, Passow 2002). TEP are formed by a variety of pathways, but in marine pelagic systems, they are predominantly formed by the abiotic assembly of dissolved organic carbon (DOC) polymers which subsequently coagulate into nano-, micro- and ultimately macrogels which can be up to several centimeters in size (Chin et al. 1998, Verdugo et al. 2004, Verdugo 2012). DOC polymers which ultimately coalesce to form TEP are released by a variety of organisms including phytoplankton, bacteria, macroalgae, and e.g. the jellyfish Aurelia aurita. (Stoderegger & Herndl 1999, Passow 2000, Ramaiah et al. 2001, Dicker 2011). Phytoplankton exudates significantly contribute to the TEP pool, and exudation depends on prevailing environmental conditions (Alldredge 1995, Gaerdes et al. submitted) and on phytoplankton-associated bacterial interactions (Grossart 1999, Gaerdes et al 2011). Phytoplankton-bacterial associations have been investigated in different aquatic systems (Bratbak and Thingstad 1985, Reche et al. 1997, Danger et al. 2007, Gaerdes et al. 2011). The actual degree of bacterial colonization of algal cells may vary greatly and seems to depend on the particular phytoplankton life stage (Vaque et al 1989, Smith et al. 1995, Kaczmarska et al. 2005, Graff et al. 2011). It appears that phytoplankton-associated bacteria are usually species-specific, benefit from the interaction, and influence the secretion of extracellular polymeric substances (EPS) (Grossart 1999, Bruckner et al. 2008, Bruckner et al. 2011, Gaerdes et al 2011). Phytoplankton-associated bacteria have also been hypothesized to directly impact bloom dynamics, community succession, primary productivity, the microbial loop and ultimately the marine global carbon cycle (Azam and Malfatti 2007, Graff et al. 2011). However, the underlying mechanisms mediating phytoplankton-bacteria interactions are not well understood.

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In a recent study, Gaerdes et al. (2011) systematically tested 85 marine bacterial isolates for their ability to attach to the diatom Thalassiosira weissflogii and for their impact on TEP formation. From this pool, only four bacterial strains, belonging to the gammaproteobacteria, flavobacteria, or firmicutes, were shown to directly enhance TEP production and aggregation of diatom cells (Gaerdes et al. 2011). This led us to speculate that specific interaction-relevant bacterial genes might be responsible for inducing diatom-borne TEP production. In order to mechanistically investigate the interaction at the molecular level, a bilateral model system consisting of the diatom, T. weissflogii, and the genetically accessible marine gammaproteobacterium, Marinobacter adhaerens HP15, has been established (Gaerdes et al. 2010, Gaerdes et al. 2011, Sonnenschein et al. 2011, Kaeppel et al. 2012). In the context of identifying diatom-bacteria interaction-relevant genes, it was hypothesized that bacterial attachment to the diatom surfaces might be essential for the interaction. Furthermore it was hypothesized that the attachment is (a) mediated by bacterial motility determinants and (b) crucial for the induction of diatom-borne TEP production. Bacterial attachment to biotic surfaces such as phytoplankton cells and to abiotic surfaces such as marine snow particles might enable associated bacteria to obtain nutrients and substrate, thereby providing a competitive advantage over free- living bacteria. The adherence of bacteria to surfaces has been investigated in various studies (Kogure et al. 1998, Morisaki et al. 1999, Dalisay et al. 2006, Wong et al. 2012), and the genetic basis of attachment and colonization of surfaces has often been attributed to genes coding for the bacterial flagellum or different type IV pili (O' Toole & Kolter 1998, Dalisay et al. 2006, Martinez et al. 2010). In the marine environment, the symbiotic interaction of Vibrio fischeri with the squid Euprymna scolopes has been shown to depend on the flagellum-mediated attachment of the bacterium to its host cells, and bacteria deficient in flagellum are unable to cause bioluminescence within the squid’s light organ, an important survival strategy for this bacterial species (Nyvholm & McFall-Ngai 2004). The attachment of the marine bacterium Pseudoalteromonas tunicata to both abiotic substrata and cellulose- containing surfaces of the green alga Ulva australis has been shown to be mediated by the type IV mannose-sensitive haemagglutinin (MSHA) pilus (Dalisay et al. 2006),

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as has the colonization of plankton surface by V. cholerae (Hsiao et al. 2006). Although there is ample evidence of bacterial interactions with biotic surfaces in the environment, the impact of bacterial attachment on the mediation of TEP production has been less scrutinized. In this study, the attachment of flagellum- or MSHA type IV pilus-deficient M. adhaerens HP15 mutants to abiotic and diatom surfaces have been investigated. Furthermore, it was tested whether bacterial attachment to the diatom impacted TEP production by quantifying the TEP amount produced in co-cultures of the diatom with the wild type and its motility-impaired mutants.

MATERIALS AND METHODS Microbial strains, plasmids and culture conditions Axenic cultures of the diatom Thalassiosira weissflogii (CCMP 1336) were obtained from the Provasoli-Guillard National Center for Marine Algae and Microbiota (Maine, USA). The cultures were grown at 16°C in f/2 medium (Guillard & Ryther 1962, Guillard 1975), using a 12-hr light period at 115 mmol photons m-2 s- 1. F/2 medium was prepared with pre-filtered (0.2 µm pore size) and autoclaved North-Sea water. Diatom cell abundance was monitored daily by counting cells in a Sedgwick-Rafter Cell S50 (SPI Supplies, West Chester, PA, USA) using an inverted Axiovert 200 microscope (Zeiss, Jena, Germany). The axenicity of the diatom culture was regularly checked by plating on marine broth (MB) (Zobell 1941) plates and by epifluorescence microscopy after staining with the dye 4′, 6-diamidino-2-phenylindol (DAPI). All bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were routinely grown in Luria-Bertani (LB) medium at 37°C with shaking at 250 rpm and supplemented with 25 µg ml-1 chloramphenicol when needed. M. adhaerens HP15, previously isolated from marine particles collected from the surface waters of the German Bight (Grossart et al. 2004), was routinely grown in MB at 28°C with shaking at 250 rpm. HP15 flagellum-impaired mutants fliG::Tn5 and ∆fliC had previously been created by transposon and gene-specific directed mutagenesis, respectively (Sonnenschein et al. 2011). Mutant fliG::Tn5 is totally deficient in

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synthesis of the flagellum. Mutant ∆fliC was created by the targeted knock-out of the structural biosynthetic gene fliC coding for flagellin and is deficient in the flagellar filament but still possesses an intact hook structure (Sonnenschein et al. 2011). The M. adhaerens HP15 MSHA type IV pilus-deficient mutant, ∆mshB, was created in the current study. All HP15 mutants were grown as described above in cultures supplemented with 25 µg ml-1 chloramphenicol for mutants ∆fliC and ∆mshB and with 500 µg ml-1 kanamycin for mutant fliG::Tn5.

Table 1: Bacterial strains and plasmids used in this study Strains and plasmids Characteristics Source or reference Bacterial strains M. adhaerens HP15 Wild type strain Grossart et al. 2004 - + Escherichia coli DH5α F'/endA1 hsdR17(rk mk ) relA1 Raleigh et al. 1989 supE44 thi-1 recA1 gyrA (NaIr) ∆(lacIZYA- argF) U169 deoR (Φ80dlac∆(lacZ)M15 ∆ fliC fliC gene deletion mutant of HP15, Sonnenschein et al. 2011 CmR fliG::Tn5 Transposon insertion mutant of fliG of Sonnenschein et al. 2011 HP15, KmR ∆mshB mshB gene deletion mutant of HP15, This study CmR Plasmids pGEM®-T Easy colE1, lacZ, AmpR Promega GmbH, Mannheim, Germany pMshB-up pGEM®-T Easy containing 1003 bp This study upstream of the mshB gene, AmpR pMshB-down pGEM®-T Easy containing 1001 bp This study downstream of the mshB gene, AmpR pFCM1 AmpR, CmR Choi and Schweizer 2005 pMshB-up-Cm pMshB-up containing the This study chloramphenicol cassette from pFCM1, AmpR, CmR mshB mutagenic construct pMshB-up-Cm containing 1001 bp This study downstream of the mshB gene, AmpR, CmR

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In silico search for genes encoding MSHA Type IV pili The complete M. adhaerens HP15 genome deposited at GenBank/EMBL/DDBJ under accession number CP001978 (Gaerdes et al. 2010), was searched for genes encoding for proteins annotated as MSHA family proteins using the BLAST search algorithm (Altschul et al. 1990) available at the NCBI site (http://www.ncbi.nlm.nih.gov/BLAST/). To determine sequence similarities of the thereby predicted M. adhaerens HP15 MSHA type IV cluster, the deduced amino acid sequences were compared to those of the well-characterized MSHA clusters of V. cholerae O1 biovar El Tor strain N16961 and that of P. tunicata D2 (Heidelberg et al. 2000, Moran et al. 2006). For this, the MSHA type IV pilus clusters from choromosome I of V. cholerae O1 biovar El Tor strain N16961 (Accession NC_002505, Heidelberg et al. 2000) and that of P. tunicata D2 (Accession NZ_AAOH01000003, Moran et al. 2006) were used. Furthermore, the putative major MSHA pilin structural subunit was identified by searching for the presence of an N- terminal signal peptide sequence and the consensus FTLIELVV pilin motif characteristic for pilin encoded in the MSHA cluster of V. cholerae O1 biovar El Tor strain N16961 (Heidelberg et al. 2000).

Gene-specific mutagenesis by homologous recombination As candidate gene for the gene-specific mutagenesis of the MSHA type IV pilus, the MSHA pilin-encoding gene mshB was selected from the predicted M. adhaerens HP15 MSHA cluster (Fig. 1). The gene-specific mutagenesis by homologous recombination was performed according to Hoang et al. (1998). The sequences of the primers used in this study are listed in Table 2. A mutagenic construct containing a chloramphenicol cassette bordered by upstream and downstream flanking regions of the mshB gene was created as follows. A 1,003-bp upstream and a 1,001-bp downstream region of the mshB gene were PCR amplified using the primers MshBupF/MshBupR and MshBdownF/MshBdownR, respectively. Both fragments were sub-cloned into the pGEM®-T Easy vector (Promega, Manheim, Germany) resulting in plasmids pMshB-up and pMshB-down, respectively.

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A 1,300-bp DNA fragment containing the chloramphenicol resistance cassette was PCR amplified from plasmid pFCM1 using primers CmF/CmR. The fragment was treated with NheI and sub-cloned into NheI-treated pMshB-up resulting in plasmid pMshB-up-Cm. The latter plasmid was then treated with enzymes AvrII and Eco81I, and the resulting fragment was ligated into AvrII- and Eco81I- treated plasmid pMshB-down resulting in a plasmid containing the final 6,319-bp mshB mutagenic construct. The mutagenic construct was electroporated into competent wild type M. adhaerens HP15 cells according to Sonnenschein et al. (2011), and the resulting mutants were selected on 25 µg ml-1 chloramphenicol containing MB agar plates. A successful double cross-over event for the resulting ∆mshB mutant was confirmed by PCR using a combination of primers listed in Table 2 resulting in the expected fragments for the wild type and the mutated mshB gene, respectively.

Table 2: Oligonucleotide primers used in this study. Underlined are restriction recognition sites

Primer name Sequence 5′′′ – 3′′′ Primers for the creation of the mshB mutagenic construct MshBupF ACCACACCCGCCAGGGAA MshBupR CCTNAGGCCTAGGGCTAGCCCTGTTTGCCAGCCGCTC MshBdownF GACCTAGGCCGTTCTTCCTGCTCCCG MshBdownR GACCTNAGGAACAGGGGCGGCTGACCT CmupF AGCTGGCTAGCGGATGTGCTGCAAGGCGA CmupR AGCTGGCTAGCGCCAAGCTTGCATGCCTG Primers for the confirmation of the ∆mshB mutant MshBF TATTGGTGACCACAGAGC MshBR TGATGCAGTACGACAGGA CmF AGCTCGAATTGGGGATCT CmR AAGATCCCCTGATTCCCT MshBupCmF GGCCACACTGATAATCAC MshBupCmR CGGTGGTATATCCAGTGA MshBdownCmF CGCAAGGCGACAAGGTGC MshBdownCmR CGTGCTGGGCGTTCTGTG

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Figure 1: MSHA cluster in M. adhaerens HP15 Schematic representation of M. adhaerens HP15 MSHA type IV pilus cluster as compared to (A) P. tunicata D2 and (B) V. cholerae O1 biovar El Tor str. N16961. The entrire locus of M. adhaerens HP15 is 16.6 kb in length and consists of 16 continuous ORFs (mshI1I2JKLMNEGFBCDOPQ). The scale bar represents approximately 2 kb. The gray-scale codes depict the percentages of protein similarity with black shading representing > 70% similarity, dark grey > 60%, pale grey > 40% and white representing no similarity.

Phenotypic mutant characterization by swimming assay and by in vitro biofilm assays The swimming behavior of M. adhaerens HP15 and its mutants ∆fliC, fliG::Tn5 and ∆mshB was investigated by soft-agar assay as described by Sonnenschein et al. (2012). In vitro biofilm assays were performed according to O’Toole (2011) with minor modifications as follows. Bacterial cultures were grown overnight at 28 °C in MB medium with shaking at 250 rpm. Subsequently, cultures were tenfold diluted with MB and their optical densities (OD600) adjusted to ensure similar starting values. 600-µl aliquots of the diluted cultures were then incubated in 1.5 ml polypropylene microtubes for 24 hrs at 37°C without shaking.

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The tubes were subsequently thoroughly washed with distilled water, air-dried, and the wall-attached cells were stained with 700 µl of 0.1% crystal violet for 20 mins. After staining, tubes were thoroughly washed with distilled water and photographed. Finally, attached and stained cells were washed off the walls with 95% ethanol for spectrophotometric quantification at 600 nm.

Attachment assay with axenic T. weissflogii To quantify the percentage of bacterial cells attached to the diatom T. weissflogii, attachment assays were performed according to Gaerdes et al. (2011) with some modifications. The axenic diatom cultures were grown in f/2 medium and harvested at the stationary growth phase. Approximately 5,000 diatom cells ml-1 were incubated with approximately 1x106 bacterial cells ml-1 for 24 and 48 hrs at room-temperature in darkness. After incubation, the culture was gently mixed and passed through a 10-µm pore size sieve (Sefar, Heiden, Switzerland) to separate diatom-attached bacteria from the non-attached fraction. The enumeration of bacteria was performed by dilution plating and counting of the colony-forming units (CFU ml-1) for both the attached and non-attached bacteria. The experiment was conducted in three replicates in three independent experiments.

Quantification of TEP production TEP production was measured colorimetrically in triplicates by filtration of samples onto 0.4-µm pore size polycarbonate filters (Sartorius, Goettingen, Germany) and subsequent staining with Alcian blue following the procedure described by Passow and Alldredge (1995). The staining solution was calibrated using Gum Xanthan, and TEP was expressed as Gum Xanthan equivalents per liter (GXeq L-1). The Alcian blue-stained filters were soaked in 80% sulphuric acid for 2 hrs and mixed every 30 minutes. The Alcian blue from the filters were spectrophotometrically measured at 787 nm.

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RESULTS The putative MSHA type IV pili biogenesis locus of M. adhaerens HP15 Analysis of the M. adhaerens HP15 genome revealed a putative MSHA type IV pilus gene cluster which was found to be conserved with respect to the homology of the predicted amino acid sequences and with respect to the similarity in the organization, orientation and arrangement of its genes to those of the MSHA type IV pilus clusters found in the genomes of V. cholerae O1 biovar El Tor strain N16961 and P. tunicata D2 (Fig. 1). The genetic locus required for the assembly and secretion of M. adhaerens HP15 MSHA type IV pilus was localized to a 16.6-kb region of the M. adhaerens HP15 chromosome and contains 16 MSHA-related genes termed mshI1I2JKLMNEGFBCDOPQ. With exception of the mshA gene, M. adhaerens HP15 contains all MSHA-related genes present in the MSHA clusters of P. tunicata D2 and V. cholerae O1 biovar El Tor strain N16961 (Fig. 1). A comparative analysis of the M. adhaerens HP15 MSHA cluster with the genome of M. aquaeolei VT8 (Accession No. CP000514, Copeland et al. 2006) revealed that both Marinobacter species possess highly homologous msh genes (data not shown).

Mutagenesis of mshB by homologous recombination Electroporation of the mshB mutagenic construct into M. adhaerens HP15 cells yielded three transformants on MB agar plates supplemented with chloramphenicol. Transformants were checked with a combination of primer pairs (Table 2) to distinguish transformants that had either undergone a single or a double cross-over event. Mutant ∆mshB was created from a successful double cross-over event where both the upstream and downstream flanking regions of the mshB gene have undergone homologous recombination with the wild type chromosome and the mshB gene was replaced by the chloramphenicol resistance cassette. The primer pair MshBF/MshBR contained intragenic primers of mshB and as expected, no PCR product was obtained with ∆mshB whereas mshB gene with an amplicon size of 510 bp was amplified in the wild type.

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In contrast, CmF/CmR amplifies the chloramphenicol cassette and as expected, ∆mshB yields a 1,052-bp product whereas the wild type of M. adhaerens HP15 did not (data not shown). Two additional sets of primers, MshBupCmF/MshBupCmR and MshBdownCmF/MshBdownCmR, were used to yield expected PCR product sizes thus confirming the genotype of mutant ∆mshB (data not shown). Mutant ∆mshB, the wild type of HP15, and the two previously generated mutants ∆fliC and fliG::Tn5 were subsequently incubated as diatom-free batch cultures in MB broth as well as in f/2 medium revealing no significant growth differences among each other in either of the media (data not shown).

Phenotypic mutant characterization by swimming assay and by in vitro biofilm assays Previously it had been demonstrated that HP15 mutants ∆fliC and fliG::Tn5 were deficient in their swimming ability by soft-agar assays (Sonnenschein et al. 2011). The phenotypes of both mutants could be confirmed. As expected, the swimming ability of mutant ∆mshB, which carries a functional flagellum, was not affected and resembled that of the wild type (data not shown). In vitro biofilm assays conducted on polypropylene microtubes acting as abiotic surfaces revealed that all three motility mutants were impaired in attachment and biofilm formation (Fig. 2A). Interestingly, HP15 mutants ∆fliC and fliG::Tn5 were found to be more severely impacted as compared to mutant ∆mshB when attachment was quantified (Fig. 2B). Samples of the crystal-violet-stained M. adhaerens HP15 wild type displayed an average absorbance of 1.9 ± 0.08, which was approximately three-fold higher than those of the crystal-violet-stained samples of mutants ∆fliC and fliG::Tn5 and approximately two-fold higher than samples of the crystal-violet- stained ∆mshB mutant (Fig. 2B) thus confirming the visual estimations and suggesting a stronger role of the flagellum for attachment as compared to the MSHA type IV pilus.

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(A)

(B)

Figure 2. Biofilm formation of M. adhaerens HP15 and its motility deficient mutants Biofilm formation phenotype of M. adhaerens HP15 and its motility deficient mutants ∆fliC, fliG::Tn5 and ∆mshB. Bacterial strains were incubated on polypropylene surfaces for 24hrs at 37°C. (A) Visualization of attached bacterial cells stained with 0.1 % crystal violet (B) Quantification of crystal violet dissolved in 96% ethanol and absorbance measured spectrophotometrically at a wavelength of 600 nm. Data are from the average of 12 samples from 4 independent experiments. Bars represent standard errors. All the bacterial strains are significantly different from the MB only control with P < 0.01. Compared to the wild type HP15, all the mutants are significantly different with P < 0.01. ∆fliC and fliG::Tn5 values are not significantly different with P > 0.01. ∆mshB is significantly different from both ∆fliC and fliG::Tn5 mutants with P < 0.01.

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Flagellum and MSHA Type IV pilus promote bacterial attachment to T. weissflogii In order to analyze a potential impact of the cellular appendages on the cell-to-cell interaction of M. adhaerens HP15 and the diatom, T. weissflogii, attachment assays were conducted. Bacterial abundances of HP15 wild type and its mutants expressed as CFU ml-1 did not statistically differ at the start of the experiment (Table 3). After 24 hrs of co-incubation with the diatom cells, however, the total cell numbers (sum of attached and free-living cells) for the ∆fliC and fliG::Tn5 mutants had significantly increased, whereas those of the wild type and ∆mshB had not (Table 3). Interestingly, the percentage of bacterial cells attached to the diatoms was significantly reduced for the ∆fliC and fliG::Tn5 mutants as compared to those of the wild type and the mshB mutant (Fig. 3). The percentage of attached ∆mshB mutant cells was also significantly reduced with respect to the HP15 wild type, however to a lesser extent. After 24 hrs, the fraction of bacterial cells attached to the diatom was 15.27 ± 2.21 % for the wild type, only 1.85 ± 0.47 % and 1.65 ± 0.36 % for the ∆fliC and fliG::Tn5 mutants, respectively, and 5.31 ± 0.9 % for mutant ∆mshB (Fig. 3A) thus confirming data of the in vitro biofilm formation assay. Results of the experiment after 48 hrs of incubation did not differ from those obtained after one day of incubation (Fig. 3B). The finding that phenotypes did not differ between the two flagellum mutants indicated that the flagellar hook structure is not sufficient to allow bacterial attachment and that a fully functional flagellum is a pre-requisite for attachment of M. adhaerens HP15 to abiotic and biotic surfaces.

Table 3: Bacterial cell dynamics for T. weissflogii attachment assay Bacterial cell Avg ± SE abundance ( x 106 ) CFU ml-1 Wild type ∆ fliC fliG::Tn5 ∆mshB t = 0, total 0.81 ± 0.23 1.35 ± 0.40 1.23 ± 0.32 0.87 ± 0.22 t = 24 hrs, total 0.54 ± 0.09 3.59 ± 0.48 * 3.16 ± 0.52 * 0.97 ± 0.08 t = 48 hrs, total 1.02 ± 0.21 4.00 ± 0.24 * 3.19 ± 0.48 * 1.11 ± 0.16 * P < 0.01

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(A)

(B)

Figure 3. Percentage of bacterial cells attached to T. weissflogii % of M. adhaerens HP15 wild type cells and the mutants ∆fliC, fliG::Tn5 and ∆mshB attached to stationary phase T. weissflogii diatoms after (A) 24hrs and (B) 48 hrs of incubation in f/2 medium. Data are from triplicates from 3 independent experiments.

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Quantification of TEP production To test whether cellular appendages-mediated bacterial attachment impacted algal TEP formation, TEP concentrations of the diatom cultures incubated with either, the wild type of M. adhaerens HP15, or its ∆fliC, fliG::Tn5 and ∆mshB mutants were analyzed. The TEP concentrations of all treatments at the start of the experiment were not statistically different from each other (Fig. 4). After 24 or 48 hrs of co-incubation TEP concentrations increased approximately two- to three-fold in all treatments without any statistically significant difference among treatments (P > 0.01). This surprising result suggested that the observed differences in cellular attachment of the different bacterial mutants did not impact TEP production at all.

Figure 4. TEP quantification of the different treatments

TEP concentration in diatom cultures incubated with M. adhaerens HP15 and the motility-impaired mutants. The average TEP concentration at the start of the experiment did not significantly differ between the treatments (P > 0.01). Approximately 2-3 fold increase in TEP concentration was observed in all the treatments after both 24 hrs and 48 hrs of incubation (P < 0.01). TEP production between the samples however did not statistically differ after either of the incubation times (P > 0.01).

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DISCUSSION In this study, a MSHA type IV pilus gene locus comprised of the genes mshI1I2JKLMNEGFBCDOPQ was identified in the genome of M. adhaerens HP15, which showed a similar genetic organization and high protein sequence similarities to homologous clusters of V. cholerae O1 biovar El Tor str. N16961 and P. tunicata D2 (Heidelberg et al. 2000, Moran et al. 2006). The genome of M. aquaeolei also seems to contain similarily conserved MSHA type IV pili clusters. The MSHA type IV pilus cluster was first described in marine Vibrio species (Marsh & Taylor 1999, Shime- Hattori et al. 2006) and thereafter identified in Shewanella oneidensis MR-1 (Thormann et al. 2004) and in P. tunicata D2 (Dalisay et al. 2006). The lack of significant sequence similarities of the herein detected cluster with that of S. oneidensis MR-1 might reflect a further taxonomic divergence. Vibrio mutants defective in the formation of the MSHA pili have been shown to be defective in adherence to zooplankton and crustaceans (Chiavelli et al. 2001). Transposon mutagenesis of P. tunicata D2 confirmed that mutants defective in the biosynthesis of the MSHA type IV pilus exhibited severe impairment of the bacterial attachment to the algae Ulva australis (Dalisay et al. 2006). These previous observations suggested that the MSHA type IV pilus might play a significant role for attachment of the investigated microbes to biotic surfaces. Likewise, MSHA type IV pilus-mediated attachment to abiotic surfaces was demonstrated for S. oneidensis MR- 1 (Thormann et al. 2004). Our data are in line with either of these findings since the M. adhaerens HP15 ∆mshB mutant showed significant reduction in attachment to both the abiotic and the diatom surface. It was observed that the impact of the flagellum in attachment is more pronounced than that of the MSHA type IV pilus. By testing the attachment of two individual M. adhaerens HP15 flagellum mutants, ∆fliC and fliG::Tn5, it was shown that the bacterial hook structure is not sufficient for the bacteria to attach to surfaces and that a fully-functional flagellum is imperative for this attachment. These results are in line with observations previously made by O'Toole and Kolter (1998) who had demonstrated the importance of flagellar motility by comparing attachment of motile and non-motile P. aeruginosa strains to plastic surfaces under static biofilm culture conditions.

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The current study showed that the interaction of M. adhaerens HP15 to both abiotic and T. weissflogii surfaces initially depended on the attachment of the bacteria through its flagellum. It is tempting to speculate that sustained attachment might thereafter be mediated by i.e. the MSHA type IV pilus. To substantiate this idea, future studies will focus on estimating the differential expression of msh genes throughout the course of the interaction. Not easy to interpret is the puzzling finding that – despite significant lower number of attached cells – the total cell numbers of the flagellum-deficient mutants at the end of the diatom attachment assays were higher than those of the wild type. Since all mutants showed a growth pattern indistinguishable from that of the wild type in diatom-free batch cultures, at least two possible scenarios may be envisioned: a) non- attaching cells are replicating faster than attached cells; or b) there are technical difficulties separating attached cells from the diatom surfaces or from each other thus yielding lower CFU ml-1 numbers. Should the later scenario hold true, the actual number of attached wild type cells were higher than determined. Therefore, future studies will emphasize on direct three-dimensional microscopic investigations using confocal laser scanning microscopy and fluorescently labeled bacterial cells. Algal carbon exudation in form of TEP has been shown to increase in presence of specific bacteria (Grossart et al. 1999, Gaerdes et al. 2011). Herein, it was hypothesized that the induction of diatom-borne TEP depended on the attachment of M. adhaerens HP15 to T. weissflogii. Results of our study, however, did not support this hypothesis since neither, disruption of the flagellum and the MSHA type IV pilus, nor decreased attachment of the respective mutants led to significant differences in TEP formation as compared to that induced by the wild type. Although it cannot be ruled out that attachment of M. adhaerens HP15 to the diatom may induce a different type(s) of carbon exudates not detectable with the currently applied method, it may be additionally speculated that other yet-to-be-determined genetic traits are responsible for the induction of TEP formation in the investigated interaction.

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In conclusion, results of this study hint at a potentially high complexity of the marine diatom-bacteria interactions and prompt further investigations on the nature of carbon exudates produced as well on additional bacterial genes required for its induction. Consequently, our future attempts will focus on identifying M. adhaerens HP15 gene products specifically expressed during the interaction using in vivo expression technology (Darwin 2005) and on the development of additional carbon exudate detection methods using i.e. differential lectin staining approaches (Wigglesworth- Cooksey and Cooksey 2005).

ACKNOWLEDGEMENTS The authors thank Caitlin Fairfield for valuable technical help and suggestions. This work was funded by the Helmholtz Graduate School for Polar and Marine Research and the Marine Science Institute, University of California Santa Barbara.

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Chapter 3

Combined effects of lowered pH and elevated temperature on diatom-bacteria interactions

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3.1.5 Combined effects of lowered pH and elevated temperature on diatom-bacteria interactions

The following manuscript is in preparation

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Combined effects of lowered pH and elevated temperature on diatom-bacteria interactions

Shalin Seebah1, Caitlin Fairfield 2 , Matthias S. Ullrich1 and Uta Passow2*

1Molecular Life Science Research Center, Jacobs University Bremen, Bremen, Germany

2 Marine Science Institute, University of California Santa Barbara, CA, USA

* Corresponding author: Marine Science Institure University of California Santa Barbara CA 93106 USA Tel: +49 421 200 3245 [email protected]

Keywords: ocean acidification, temperature, TEP, marine aggregates, climate change, diatom, Thalassiosira weissflogii, Marinobacter adhaerens HP15

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ABSTRACT

As atmospheric carbon dioxide concentrations continue to rise, the question of how marine pelagic ecosystems and biogeochemical cycling of elements will react to increasing ocean acidification and elevated temperatures has become a subject of intense investigations. In this study, the combined effects of a lowered pH and elevated temperature on transparent exopolymeric particle and marine aggregate formation were studied in roller tank experiments. Aggregates were formed either from axenic cultures of the diatom Thalassiosira weissflogii or with the diatom cultures co-inoculated with the marine bacterium M. adhaerens HP15. The carbonate system was manipulated to reflect present-day conditions with a pH range of 8.0 – 8.2 and two different future ocean scenarios with a pH range of 7.6 – 7.8 and 7.4 – 7.6 respectively. The experiments were conducted at 15 °°°C or 20 °°°C. Our results show that the growth of the diatom T. weissflogii is not significantly impacted by the tested levels of ocean acidification, but that its growth is favored under elevated temperatures. Furthermore, it was shown that synergistic effects of ocean acidification and temperature may have a pronounced impact on TEP production in axenic cultures of the diatoms, but not in presence of bacteria. We further show that the combined effects of decreasing pH and elevated temperatures substantially reduce marine aggregate formation and the sinking velocities of aggregates. We conclude that the vertical export of particulate organic matter through marine aggregates may be severely impacted in a future ocean, depending on the magnitude and on the vertical depth penetration of warming in the ocean.

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INTRODUCTION

The majority of particulate organic carbon (POC) sinks out of the marine euphotic zone in the form of marine snow (Fowler and Knauer 1986). Marine snow can either be produced de novo by marine plankton or by the physical coagulation of smaller particles (Alldredge and Silver 1988). The coagulation of particles to form larger marine aggregates is enhanced by the presence and abundance of transparent exopolymeric particles (TEP) and specific bacteria (Alldredge et al. 1993, Passow and Alldredge 1994, Gaerdes et al. 2011). These ubiquitously distributed and abundant particles have been found in all marine aggregates investigated to date (Alldredge et al. 1993, Passow and Alldredge 1994, Passow 2002). TEP are formed by a variety of pathways, but in marine pelagic systems, they are predominantly formed by the abiotic assembly of dissolved organic carbon (DOC) polymers which subsequently coagulate into nano-, micro- and ultimately macrogels which can be up to several centimeters in size (Chin et al. 1998, Verdugo et al. 2004, Verdugo 2012).

Since the age of the industrial revolution, CO2 emissions from the burning of fossil fuels and changes in land use have increased atmospheric CO2 concentrations from pre-industrial values of 280 ppm to currently 390 ppm (http://www.esrl.noaa.gov/gmd/ccgg/trends, data by Tans and Keeling, NOAA/ESRL). Values are expected to rise to 750 ppm (IPCC scenario IS92a, IPCC 2007) or even beyond 1,000 ppm by the end of this century (Raupach et al. 2007). As atmospheric CO2 concentrations continue to rise, the question of how the marine pelagic ecosystems and biogeochemical cycling of elements will react to increasing ocean acidification has become a subject of intense investigations. Ocean acidification results from an increase of dissolved inorganic carbon (DIC) and a concomitant decrease in pH (Zeebe and Wolf-Gladrow 2001). The increasing atmospheric CO2 concentrations do not solely result in ocean acidification but are coupled to intensified radiative forcing which results in higher sea-surface temperatures (Houghton et al. 1995).

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Studies addressing the impact of elevated atmospheric CO2 on TEP production have revealed very different results with partially contradicting conclusions. In their mesocosm studies, for example, Engel et al. (2004) showed that the net production of TEP in the presence of the phytoplankton Emiliana huxleyi increased with an increased pCO2 level of 710 µatm. In contrast, Egge et al. (2009) showed no significant change in net TEP production with increasing pCO2. Furthermore, Wetz et al. (2009) reported opposing trends in a long-term monitoring of TEP distributions in an estuary. They observed that TEP concentrations showed clear seasonal and spatial patterns, and that TEP distributions were positively correlated with pH but negatively correlated with temperature. Further studies in this discipline have largely focused on microbial community dynamics in mesocosms (Riebesell 2008) and on specific investigations looking at the impact of calcification on calcifying organisms such as coccolithophores (Biermann and Engel 2010). It had been argued that the expected changes in pH will have little impact on non-calcifying marine microbes (Berge et al. 2010, Joint et al. 2010) although this assumption was been disputed (Liu et al. 2010). Despite the striking differences in the above studies, it became clear that TEP concentrations in the environment are not in steady state but vary with changes in environmental conditions depending on the group of organisms being studied. Although there is a lot of studies in this field, investigations on the combined impact of ocean acidification and temperature increases remained scarce. To our knowledge, no investigations have yet been conducted on the impact of future ocean scenarios on microbial interactions, especially on diatom-bacteria interactions. Certain specific bacterial strains have been shown to directly induce diatom-borne TEP production and aggregate formation of the diatom Thalassiosira weissflogii (Gaerdes et al. 2011). This observation has led to the establishment of a genetically accessible bilateral model system consisting of that diatom and the marine gammaproteobacterium Marinobacter adhaerens HP15 (Gaerdes et al., 2010, Gaerdes et al. 2011, Sonnenschein et al. 2011, Kaeppel et al. 2012).

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In the current study, the combined effects of changing the carbonate chemistry and temperature on this established bilateral model system was investigated. Thus, we present results on the impact of future ocean scenarios on TEP production and aggregate formation in an in vitro established diatom-bacteria model system.

MATERIALS AND METHODS Experimental design Six experimental set-ups were conducted to investigate the combined effects of changing marine carbonate chemistry and temperature on TEP production and marine aggregate formation. The experiments focused on the established bilateral model system consisting of the diatom T. weissflogii and the marine bacterium M. adhaerens HP15. Axenic diatom cultures were included as controls to investigate the effects of the presence of bacteria on diatom aggregation and TEP formation. Three different carbonate chemistry regimes were selected to reflect: (i) the present-day conditions, with the partial pressure of CO2 (pCO2) ranging between 300-350 µatm (termed ambient) and (ii) two future ocean scenarios with pCO2 ranging from 750-850 µatm (designated future 1) and 1000-1250 µatm (referred to as future 2). For each carbonate chemistry regime, two temperatures were chosen, 15 °C and 20 °C, respectively. (Table 1). Prior to conducting the experiments, the diatom and bacterial cultures were acclimatized to the different temperature and carbonate chemistry regimes for more than 8 generations (for details see below). After acclimatization, experiments were conducted in duplicates in roller tanks at 15 °C and 20 °C, respectively, in darkness. Roller tanks were 1.15-L plexiglass cylinders with a diameter of 14 cm and a depth of 7.47 cm rotated on a roller table with three rotations per minute (rpm), which assured that growing aggregates remained suspended at all times. The carbonate chemistry of the experimental media was perturbed by manipulating pH and dissolved inorganic carbon (DIC) concentrations. Total alkalinity (TA), pH and DIC were monitored during both the acclimatization and experimental phases. TEP concentrations were quantified at the onset of the experiments. The experiments were terminated after 96 hrs of incubation and the number and size classes of the

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formed aggregates determined in each tank. The aggregates were then removed, their sinking velocities determined and the aggregates mixed in a known volume of sterile seawater. The aggregate fraction will henceforth be called aggregated slurry. The volume of the aggregated slurry was the difference between the known volume of sterile seawater and the new volume once all the aggregates were added and shaken thoroughly to produce a homogeneous suspension for further sampling. After removal of aggregates, the remaining seawater will henceforth be termed as surrounding seawater (SSW). TEP concentrations from both the aggreggated slurry and the SSW were quantified with four replicates each. The carbonate system was determined in SSW.

Experiment # Carbonate chemistry Temperature Treatments

1 ambient 15 °C Tw only, Tw + HP15

2 future 1 15 °C Tw only, Tw + HP15

3 future 2 15 °C Tw only, Tw + HP15

4 ambient 20 °C Tw only, Tw + HP15

5 future 1 20 °C Tw only, Tw + HP15

6 future 2 20 °C Tw only, Tw + HP15

Table 1. Experimental design showing the different combinations of carbonate chemistry, temperature and microbial combinations in the respective experiments. Tw: T. weissflogii and HP15: M. adhaerens HP15

Experimental media In the course of preliminary tests, it was observed that filtration of natural seawater through 0.2 µm pore-sized filters (Millipore, MA, USA) did not satisfactorily remove any bacterial contaminants such as nanobacterial cells passing through the filters and forming colonies on test agar plates (data not shown). Since the carbonate chemistry is severely impacted by de-gassing, sterilization of natural seawater by autoclaving was not an option (Riebesell et al. 2010, pers. observations). Consequently, artificial

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seawater (ASW) was prepared according to Kester et al. (1967) with modifications to control the carbonate system. Preparation of the base media was done as follows: Per kg of ASW, two separate solutions of salts were prepared. The first solution

comprised of the anhydrous salts NaCl, Na2SO4, KCl, KBr and H3BO3 while the second solution contained the hydrous salts MgCl2.6H20, CaCl2.2H20 and SrCl2. Salt concentrations were according to Kester et al. (1967). Both solutions were prepared with MilliQ water and autoclaved separately. When cooled, both solutions were mixed and 0.172 g of NaHCO3 was added per kg of ASW to yield DIC concentrations for ambient seawater at 2,050 µmol kg-1. The base experimental medium was thereafter supplemented with vitamins and trace metal solutions as in F/2 medium (Guillard & Ryther 1962, Guillard 1975), and macronutrients had final concentrations of 59 µM nitrate, 3.6 µM phosphate, and 53.5 µM silicic acid.

Carbonate chemistry perturbations

Increasing atmospheric CO2 concentrations alter pCO2, pH and DIC but not the TA of the surface ocean. These changes can be experimentally mimicked either by bubbling seawater with pCO2-adjusted air or by chemically altering the seawater using the closed system approach (Rost et al. 2008). Since TEP production has been shown to be impacted by bubbling (Mopper et al. 1995, Schuster and Herndl 1995, Zhou et al. 1998), the carbonate system was chemically perturbed. To mimic future ocean -1 -1 conditions, appropriate amounts of 0.1 M HCl (mL kg ), 0.1 M NaHCO3 (mL kg ) -1 and 0.001 M Na2CO3 (mL kg ) were added. The respective volumes needed for additions were calculated using CO2Sys (Lewis and Wallace 1998) with detailed steps as described in Passow (2011). Measurements of pH and TA confirmed that our perturbations changed the system as expected and that changes reflected those anticipated in the future ocean.

Microbial cultures and their acclimatization Axenic cultures of Thalassiosira weissflogii (CCMP 1336) were obtained from the Provasoli-Guillard National Center for Marine Algae and Microbiota (Maine, USA).

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The cultures were acclimatized by incubation for more than 8 generations at 15 °C or 20 °C, respectively, in the manipulated media to reflect the ambient or two different future treatments. Diatoms were kept in the exponential phase of growth, and regular dilutions of the cultures were performed to keep the cell density below 60, 000 cells mL-1. Cell concentrations, pH and TA of the cultures were regularly monitored to ensure that the carbonate chemistry did not change significantly during pre-cultures. The cultures were maintained at 50 µE s-1 with a 12-hr light period in 2-L Fernbach flasks with narrow openings to minimise carbonate system changes due to air exchange. Diatom cell abundance was monitored daily by counting cells in a Sedgwick-Rafter Cell S50 (SPI Supplies, West Chester, PA, USA) using an inverted Axiovert 200 microscope (Zeiss, Jena, Germany). The axenicity of the diatom culture was intermittently checked by epifluorescence microscopy after staining with the dye 4′, 6-diamidino-2-phenylindol (Porter and Feig 1980). M. adhaerens HP15 previously isolated from marine particles collected from the surface waters of the German Bight (Grossart et al. 2004) was acclimatized by growing cells overnight in marine broth prepared with ASW, which had been adjusted to reflect the different pCO2 treatments, either at 15 ºC or 20 ºC in sterile culture flasks with aeration of approximately 250 rpm. After the acclimatization phase, roller tank experiments were set-up with diatom cells in a final concentration of approximately 3 x 103 cells ml-1 and bacterial cells, where present, at a final concentration of approximately 3 x 105 cells ml-1. The microbial cultures were added to the media prepared as described above and the roller tanks filled, bubble-free under sterile conditions. Prior to inoculation with the diatom culture, the bacterial cells were washed twice in sterile seawater to minimize carry- over of nutrients or bacterial growth-derived matter into the diatom cultures.

Carbonate chemistry analysis The carbonate system of the experiments was monitored by measuring pH, TA and DIC. Samples for pH were collected bubble-free in 20-mL scintillation vials and the pH (total scale) was measured with a spectrophotometer using the indicator dye m- cresol purple (Sigma-Aldrich) within 1-2 hours of sampling.

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The measurement temperature was held at 25 ºC and the absorbance measured at 730 nm, 578 nm and 434 nm before and after dye addition (Clayton and Byrne 1993,

Fangue et al. 2010). pH was calculated following the standard of operating procedure (SOP) 7 ″Determination of the pH of seawater using the indicator dye m-cresol purple″ (Dickson and Goyet 1994). Samples for TA and DIC measurements were taken following SOP1 ″Water sampling for the parameters of the oceanic carbon dioxide system″ (Dickson and Goyet 1994). Samples were poisoned with 0.02% saturated HgCl2 by volume and sent for analysis to the Dickson Laboratory at the Scripps Institution of Oceanography, UCSD.

The program CO2Sys (Lewis and Wallace 1998) was used to calculate the carbon system from TA and pH. The dissociation constants K1 and K2 from Roy et al. (1993) were used since it has been described as the most appropriate for ASW (Zeebe and Wolf-Gladrow 2001) and KHSO4 according to Dickson. Any two of the main carbonate parameters (pH, TA, DIC, pCO2) describe the carbonate system sufficiently and the other parameters can be calculated from the measured ones. In 50 different samples, we measured three carbonate parameters (pH, TA and DIC) to over- determine the carbonate system.

Sinking velocity The sinking velocity of aggregates was measured by gently transferring individual aggregates from the roller tanks using a wide bore pipette to a high cylinder containing sterile experimental media. Prior to measuring sinking velocity of aggregates, the analysis medium was incubated overnight at either 15 °C or 20 °C to ensure that aggregates experience no change in environmental conditions during the subsequent sinking velocity determinations. The time taken for each aggregate to sink a defined distance was recorded. The dimensions of the aggregate axes (x, y, and z direction) were measured under a dissecting microscope, using a grid paper and ruler, and the aggregated volume was calculated by assuming an ellipsoid shape. The equivalent spherical diameter (ESD) was calculated. The sinking velocity was determined for approximately 10 aggregates per tank, and the sizes of all visible aggregates measured. The aggregates were classified into two size classes: large

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aggregates of size ≥ 5 mm ESD and smaller aggregates of size < 5mm ESD.

Quantification of TEP TEP concentrations were measured colorimetrically by filtration of samples onto 0.4- µm pore size polycarbonate filters (Millipore, MA, USA) and subsequent staining with Alcian blue following the procedure described by Passow and Alldredge (1995). The staining solution was calibrated using Gum Xanthan, and TEP was expressed as Gum Xanthan equivalents per liter (GXeq L-1). The Alcian blue-stained filters were soaked in 80% sulphuric acid for 2 hrs and mixed every 30 minutes. The Alcian blue from the filters was spectrophotometrically measured at 787 nm. TEP was measured in four replicates per tank at the start of the experiment and after 96 hrs from both, the aggregate slurry and the SSW fractions of all treatments.

RESULTS Over-determination of the carbonate chemistry The carbonate system was over-determined by simultaneously measuring the pH, DIC

and TA in 50 independent samples. The pCO2 concentrations were then calculated using all three possible combinations: TA and DIC, pH and DIC as well as pH and

TA. Fig. 1 shows the pCO2 based on pH and TA fitted with that calculated from DIC and TA. DIC and pH- based calculations gave slightly different pCO2 values and the correlation coefficient with those calculated from TA and either pH or DIC, respectively, was slightly lower.

Acclimatization phase T. weissflogii cultures were acclimatized in media manipulated to reflect the ambient and two future scenarios, at two different temperatures. Diatom growth rates and pH measurements during the acclimatization phases are shown in Table 2. The maintenance of the low cell density ensured that the pH was maintained within the limits targeted for all the treatments. Throughout the acclimatization phase, the average TA was 2351 ± 7 µmol kg-1 and did not significantly vary between any of the

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treatments (p > 0.01), thereby demonstrating that the carbonate system during the acclimatization phase remained within the targeted parameters. COMBINED EFFECTS OF LOWERED pH AND ELEVATED TEMPERATURE ON DIATOM- BACTERIA INTERACTIONS

Fig. 1. Results of the over-determination of the carbonate system. pCO2 concentrations calculated from pH and TA were identical to values calculated from TA and DIC (y = 1.000x – 4.3123, r2 = 1.0000), whereas those calculated from pH and DIC differed slightly from those derived from pH and TA (y = 0.9838x – 42.1785, r2 = 0.9890).

Treatment µ (d -1) pH No. of days acclimatized

15 °C ambient 0.51 7.93 – 8.21 11 future 1 0.52 7.57 – 7.76 11 future 2 0.49 7.45 – 7.66 11

20 °C ambient 0.86 8.04 - 8.23 8 future 1 0.86 7.61 - 7.84 8 future 2 0.82 7.46 – 7.67 8

Table 2: Diatom growth rates and adjusted pH range during the acclimatization phase

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Experimental phase Carbonate system During the experimental phase, the carbonate system was determined at the onset and

after 96 hrs of incubation in the roller tanks. pCO2 and pH data are shown in Figs. 2- 3. The results show a clear distinction between the starting conditions of the ambient and two future scenarios. In all the treatments and irrespective of bacterial presence, the pCO2 increased after 96 hrs. Although similar trends were observed for both temperatures, the change in pCO2 (Fig. 2A-B) was largest in the two future treatments at 20 ºC, reflecting higher respiration at the higher temperature and a weaker buffering system under future carbonate chemistry conditions.

Aggregate characteristics and total aggregated volume Between 33 and 52 aggregates per tank were generated in both treatments of experiments 1-3 at 15 °C and in the axenic cultures of the diatoms treatment of experiment 4, at 20 °C (Fig. 4A-B). In contrast at 20 °C, both treatments of the future experiments 5 and 6 as well as the ambient experiment 4 with bacteria-containing treatment generated less than 20 aggregates per tank (Fig. 4B). The highest number of aggregates was formed at 15 °C under ambient conditions and in the presence of bacteria. Although more aggregates were produced under this condition, the presence of bacteria seemed to lead to a higher proportion of smaller aggregates (> 5mm). Under future scenarios at 15 °C however, an opposite trend was found. The comparison of axenic versus xenic cultures showed an increase in the formation of large aggregates in presence of bacteria. Despite slight differences in the number and size distributions of aggregates formed in the different treatments at 15 °C, the overall trend was that the total number of aggregates or the sizes of the aggregates did not drastically differ between the treatments. However, when the same treatments were compared with those incubated at the elevated temperature of 20 °C, striking differences were observed. With exception of the axenic diatom cultures incubated under ambient conditions, a drastic reduction in both the number and size of

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aggregates was observed at 20 °C (Fig. 4B).

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(A)

(B)

Fig. 2. Determination of pCO2 concentrations at the initial measurement and after 96 hrs of incubation. (A) pCO2 at 15 °C (B) pCO2 at 20 °C. The values for t = 96 hrs are averages from duplicate tanks and standard deviations are depicted by error bars.

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(A)

(B)

Fig. 3. Determination of pH values at the initial measurement and after 96 hrs of incubation. (A) pH at 15 °C (B) pH at 20 °C. The values for t = 96 hrs are averages from duplicate tanks and standard deviations are depicted by error bars.

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(A)

(B)

Fig. 4. Numbers and size distribution of aggregates formed after 96 hrs in roller tanks at (A) 15 °C and (B) 20 °C. Aggregate sizes were expressed as equivalent spherical diameters (ESD) in mm.

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When expressed in terms of total aggregated volumes, we observed no appreciable change among any of the treatments conducted at 15 °C, with the exception of axenic cultures incubated under the future 2 scenario (Fig. 5). This observation suggested that there is no significant impact of a changed carbonate chemistry on total aggregated volumes. The impact of the presence of bacteria on the total aggregated volumes also appeared small, with no appreciable change observed in any of the six experiments containing bacteria. In striking contrast however, was the finding that in both future treatments at 20°C, a drastic reduction in the total aggregated volume was observed (Fig. 5), suggesting a interactive effect of elevated temperature with a changed carbonate chemistry.

Fig. 5. Total aggregated volume in the different treatments after 96 hrs in roller tanks.

Sinking velocity Sinking velocities were determined for aggregates at the end of the roller tank

experiments. Sinking velocities did not seem to be impacted by pCO2 but aggregate size and temperature had an effect: At 15°C, sinking velocity generally increased with increasing sizes, reaching approximately 50 m d-1 for aggregates with an ESD of approximately 5 mm, and 100 m d-1 for aggregates with an ESD of approximately 13 mm (y = 22.607x + 70.33 , n = 68, r2 = 0.7997) (Fig. 6A).

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Since fewer aggregates were formed in the roller tanks at 20 °C, fewer sinking velocity determinations were conducted. Although fewer larger-sized aggregates were formed at 20 °C, the tendency of increased velocity with increasing sizes hold true (y = 2.404x + 16.08, n = 45, r2 = 0.5902). However, the overall sinking velocity at 20°C was lower for the same ESD compared to those formed at 15°C. For example, an aggregate with an ESD of approximately 7 mm at 15°C had a sinking velocity of approximately 60 m d-1. The same sized aggregate from 20 °C treatments sank with approximately 30 m d-1. Furthermore, the slope of the size versus sinking velocity relationship was much smaller, by almost a factor of 10, for aggregates formed at 20 °C compared to those at 15°C. As an example, two aggregates with ESD of approximately 7 mm and 14 mm, respectively, had very similar sinking velocities of approximately 30 m d-1 (Fig. 6B).

(A)

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(B)

Fig. 6. Determination of sinking velocities of aggregates from (A) 15 °C (y = 22.607x + 70.33 , n = 68, r2 = 0.7997) and (B) 20 °C (y = 2.404x + 16.08, n = 45, r2 = 0.5902)

TEP concentration dynamics With p-values > 0.01, the initial TEP concentrations did not significantly differ between the treatments of the six experiments (Fig. 7., Table 3.). In all the experiments incubated at 15 °C, TEP concentrations increased approximately 3-fold after 96 hrs of incubation in the roller tanks and approximately 3-4 fold at 20 °C (Fig. 7.). The total TEP concentration expressed as µg Xeq. tank -1 was the sum of TEP in the aggregate fraction and in the surrounding seawater. The increases in total TEP concentration after 96 hrs were not statistically different between the axenic and xenic cultures of all the 6 experiments (Table 3). A statistically significant increase (P < 0.05) in total TEP production was however observed when the axenic treatments were

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Compared in terms of the two temperatures (Table 3.). This comparison of the two temperature settings revealed that the total TEP concentrations significantly increased (P < 0.05), in both future scenarios at 20 °C when diatoms were incubated axenically

(Table 3.). Thus, neither pCO2, nor the presence of bacteria, nor temperature had a clear impact on net TEP production during the 96 hrs experiments, but the combination of increased temperature and ocean acidification conditions appeared to result in higher TEP concentrations of the axenic cultures. When separating the total TEP concentrations for the aggregate slurry and the SSW, respectively, it was observed that 20-45 % of all TEP were found in the aggregate slurry (Table 4.). At both temperatures the highest fraction of TEP assignable to the aggregates was detected in axenic diatom cultures incubated under ambient conditions and amounted to 28 % at 15 °C and 45 % at 20 °C, respectively. There also seemed to be a tendency of decreasing proportions of TEP occuring in the aggregate slurry under both future scenarios and at both temperatures, and irrespective of the presence of bacteria (Table 4). We further analyzed whether total TEP concentrations correlated with to the total aggregated volumes. However, results given in Fig. 8, show no such obvious correlation between the abundance of TEP produced and the total aggregated volume.

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(A)

(B)

Fig. 7. TEP concentration dynamics at the start of the experiment and after 96 hours in roller tanks.

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Table 3. Results of statistical tests (ANOVA) comparing total TEP concentrations between experiments and between treatments

Experiment # Treatment Difference Initial t = 0 hr 1 Tw ambient vs Tw + HP15 ambient Not. sig. diff. 2 Tw future 1 vs Tw + HP15 future 1 Not. sig. diff. 3 Tw future 2 vs Tw + HP15 future 2 Not. sig. diff. 4 Tw ambient vs Tw + HP15 ambient Not. sig. diff. 5 Tw future 1 vs Tw + HP15 future 1 Not. sig. diff. 6 Tw future 2 vs Tw + HP15 future 2 Not. sig. diff. Total TEP t = 96 hrs at 15 °°°C 1 Tw ambient vs Tw + HP15 ambient Not. sig. diff. 2 Tw future 1 vs Tw + HP15 future 1 Not. sig. diff. 3 Tw future 2 vs Tw + HP15 future 2 Not. sig. diff. 1 vs 2 Tw ambient vs Tw future 1 Not. sig. diff. 1 vs 3 Tw ambient vs Tw future 2 Not. sig. diff. 1 vs 2 Tw + HP15 ambient vs Tw + HP15 future 1 Not. sig. diff. 1 vs 3 Tw + HP15 ambient vs Tw + HP15 future 2 Not. sig. diff. Total TEP t = 96 hrs at 20 °°°C 4 Tw ambient vs Tw + HP15 ambient Not. sig. diff. 5 Tw future 1 vs Tw + HP15 future 1 Not. sig. diff. 6 Tw future 2 vs Tw + HP15 future 2 Not. sig. diff. 4 vs 5 Tw ambient vs Tw future 1 P < 0.05 4 vs 6 Tw ambient vs Tw future 2 Not. sig. diff. 4 vs 5 Tw + HP15 ambient vs Tw + HP15 future 1 Not. sig. diff. 4 vs 6 Tw + HP15 ambient vs Tw + HP15 future 2 Not. sig. diff. Total TEP t = 96 hrs 15 °°°C versus 20 °°°C 1 vs 4 Tw ambient Not. sig. diff. 1 vs 4 Tw + HP15 ambient Not. sig. diff. 2 vs 5 Tw future 1 P < 0.05 2 vs 5 Tw+ HP15 future 1 Not. sig. diff. 3 vs 6 Tw future 2 P < 0.05 3 vs 6 Tw + HP15 future 2 Not. sig. diff.

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Table 4. Total TEP concentrations in the different treatments and the % partitioned into the aggregate slurry.

Sample Total TEP concentrations µµµg Xeq. Tank-1 (Avg ±±± SE)

15 °°°C 20 °°°C

Total TEP % in Agg slurry Total TEP % in Agg slurry

Tw amb 1286 ± 141 28 1361 ± 66 45

Tw fut 1 1439 ± 2 24 1697 ± 6 30

Tw fut 2 1160 ± 55 22 1584 ± 47 28

Tw+HP15 amb 1383 ± 195 24 1360 ± 125 34

Tw+HP15 fut 1 1321 ± 109 20 1639 ± 271 26

Tw+HP15 fut 2 1272 ± 189 21 1627 ± 123 21

Fig. 8. Total TEP concentrations in the tank as a function of total aggregated volume

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DISCUSSION Perturbation of the carbonate system Ocean acidification experiments rely on the accurate perturbation of the carbonate system. Although, any two of the main carbonate parameters (pH, TA, DIC, and

pCO2) can describe the carbonate system sufficiently, each of the measured parameters is associated with uncertainties in their estimations (Hoppe et al. 2012).

Due to these uncertainties, discrepancies in calculated pCO2 values can vary between 10 and 30 % (Riebesell et al. 2010, Hoppe et al. 2012). Over-determinination of the carbonate system of the herein tested samples showed that overall our carbonate chemistry was solid and consistent.

Acclimatization As phytoplankton photosynthesize and cell culture densities increase correspondingly,

CO2 is removed from the medium leading to an increase in pH. By frequent dilutions of the diatom cultures before the cell density reached 60 000 cells ml-1, it was possible to maintain the pH of the carbonate system within a narrow range during the acclimatization phase. Since the TA was nearly constant during this phase, it coule be confirmed that the carbonate system was satisfactorily controlled throughout the acclimatization phase. Diatom cultures were acclimatized for a period of over 8 generations lasting between 8 and 11 days. Under the herein tested carbonate chemistry regimes, diatom growth rates were not significanlty impacted. These results are in line with studies conducted on other Thalassiosira species (Chen and Durbin 1994) and various other phytoplankton species (Berge et al. 2010). Both previous studies had shown that algal growth rates were statistically similar at a pH ranging from 7.0 to 8.5. The elevated temperature of 20 °C significantly favored diatom growth. This positive correlation between microbial growth rate and temperature is a commonly observed phenomenon and reflects enhanced metabolic activities with increasing temperature.

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Marine aggregate formation in a future ocean Noticeably more aggregates were formed in the 15 °C roller tank experiments. Despite weak differences in the number and size distribution among aggregates formed in the different treatments at this temperature, the general trend was that the total number of aggregates or the aggregate sizes did not drastically differ. These results suggested that in future ocean scenarios, ocean acidification may not significanlty impact the formation of marine aggregates if water temperatures stayed low. However, as soon as the temperature was increased a drastic reduction in both the number and sizes of aggregates was observed in all treatments except those containing axenic diatom cultures at ambient conditions. Generally, these results suggested that the presence of bacteria might not significantly impact aggregate formation in a future ocean. Additionally, it could be speculated that synergistic effects of elevated temperature and ocean acidification may drastically reduced the formation of marine aggregates per se as well as the total aggregated volumes. Furthermore, results of the sinking velocity determination showed that there is generally a higher sinking velocity for larger aggregates. However, this might only hold true if temperatures did not increase since the absolute sinking velocity of aggregates formed under future ocean scenarios at 20 °C was nearly half of that of similarly sized aggregates formed at 15 °C. These results suggested that the vertical transport of particulate organic matter via marine aggregates may become severely impacted in a future ocean and will depend on the magnitude and on the vertical depth penetration of warming in the ocean.

Synergistic effects of changes of temperature and carbonate chemistry enhance TEP production of axenic diatom cultures TEP production by diatoms in all experiments conducted at 15 °C was not significantly impacted suggesting that ocean acidification only may not become relevant for this process regardless whether bacteria are present or not. However and interestingly, both future scenarios conducted at elevated temperature exhibited a significant increase in TEP production when diatoms were incubated axenically.

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This result might indicate that potential consumption of TEP by bacteria at elevated temperatures resulted in less TEP in the acidified ocean. However, the actual high diversity of microbial interactions in the sea makes such a scenario rather unlikely. For diatom-bacteria interactions, it is very cautiously suggested that they might not impact total TEP production in a future ocean.

Lack of correlation between TEP production and marine aggregate formation Although TEP production as well as marine aggregate formation dynamics were found to be affected at variable intensity by the tested future ocean scenarios, no direct correlation between TEP production and aggregate formation was observed. This finding is in line with studies conducted by Bhaskar et al. (2005), in which no correlation between macroaggregate size and TEP abundance had been detected for the interaction of the diatom Skeletonema costatum and Marinobacter sp.. In contrast, our own previous data obtained from studies on the interaction of T. weissflogii and M. adhaerens HP15 (Gaerdes et al. 2011) indicated a positive linear correlation between TEP concentrations and aggregate formation. Although this hard-to- interprete discrepancy might reflect the high level of complexity of diatom-bacteria interactions and unknown processes that govern the transformation of dissolved organic matter into particulate organic matter, it should cautiously be noted that a number of parameters such as light exposure and preparation of the seawater medium were different in both of our studies.

Conclusions Our results showed that growth of the diatom T. weissflogii is not significantly impacted by the projected level of ocean acidification, but that its growth is likely to be favored under elevated seawater temperatures. Furthermore, it was demonstrated that synergistic effects of ocean acidification and a temperature increase might lead to a more pronounced impact on TEP production, irrespective of the presence of bacteria. In line with our findings, Engel (2004) showed that the net production of

TEP by the phytoplankton Emiliana huxleyi increased with an increased pCO2 level of 710 µatm. This elevation was close to the herein predicted future 1 scenario

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(750-850 µatm). In contrast, other ocean acidification studies showed no significant impact in net TEP production with increasing pCO2 (Egge et al. 2009). These conflicting results support the assumption that yet-unknown additional processes might significanlty contribute to the transformation of DOM into POM. Results of the current study indicate that potential detrimental effects of ocean acidification and elevated seawater temperature might affect the formation of aggregates in the first place. It is therefore concluded that the vertical export of POM via marine aggregates may become severely impacted in a future ocean and might highly depend on the magnitude and the vertical depth penetration of warming in the ocean.

ACKNOWLEDGEMENTS The authors would like to thank Eva Sonnenschein and the Bio workshop personnel of UCSB for technical assistance. This work was financially supported by the Helmholtz Graduate School for Polar and Marine Research, the Marine Science Institute, University of California Santa Barbara and Jacobs University Bremen

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Discussion

Using a polyphasic approach consisting of a combination of genomic and phenotypic analyses, in the first part of this study, M. adhaerens HP15 was described as a novel member of the Marinobacter genus [Kaeppel et al. 2012]. Genomic information was gathered from the analysis of bacterial 16S rRNA sequences, whole genome DNA- DNA relatedness and G+C contents of strain HP15 and its phylogenetic relatives. 16S rRNA gene sequences are routinely used molecular markers for phylogenetic analyses [Stackebrandt et al. 1985, Ludwig and Schleifer 1994, Rossello-Mora and Amann 2001]. The 16S rRNA gene sequence of HP15T (GenBank Accession no. AY241552) was analysed using the ARB software package [Ludwig et al. 2004] and the reference alignment was provided by the Living Tree Project database [Yarza et al. 2008]. Results of the analysis distinctly showed that M. adhaerens HP15 clusters with other Marinobacter species and that M. adhaerens HP15 is most closely related to the type strains of M. flavimaris (99 %), M. salsuginis (98 %), M. lipolyticus (98 %) and M. algicola (98 %) [Antunes et al. 2007, Green et al. 2006, Martin et al. 2003, Yoon et al. 2004]. The recommended boundary for demarcating species based on 16S rRNA sequence is 97 % [Stackebrandt and Goebel 1994]. However, it has come to light that in certain cases – also observed for the above tested Marinobacter strains – 16S rRNA lacks resolving power at the species level [Rossello-Mora and Amann 2001]. Therefore, aside from 16S rRNA analyses, whole genome DNA-DNA relatedness and G+C content analyses were performed. Once denaturated, complementary DNA strands can re-associate to form native duplex structures under stringent experimental conditions. This characteristic property of DNA forms the basis of the whole genome DNA-DNA relatedness technique [Rosselo-Mora and Amann 2001]. In a mixture of two different DNAs for example, the amount of re-association depends on the degree of identity between the DNAs. Based on numerous studies with well-defined prokaryotic species, it has been suggested that values of 70 % or above are reasonable borders for species differentiation [Wayne et al. 1987]. The genomic DNA of HP15T showed similarities of 63.6 (68.7), 40.0 (38.0), 28.9 (26.0), and 28.2 (24.5) % to those of M. flavimaris, M. salsuginis, M. lipolyticus and M. algicola, respectively [Kaeppel et al. 2012].

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These similarities were below the accepted boundary for species differentiation and thus proved that M. adhaerens represents a novel species. Among prokaryotes, G+C contents vary between 20 and 80 mol% [Tamaoka 1994]. The greater the difference between two organisms, the less closely related they are. Empirical data showed that organisms that differ by more than 10 mol% do not belong to the same genus and that 5 mol% is the common range found within species [Rosselo-Mora and Amann 2001]. This observation also held true for the Marinobacter genus and with 56.9 mol%, the G+C content of M. adhaerens HP15 falls well within the accepted range of variation in G+C contents of six other tested Marinobacter species with G+C contents ranging from 52.7 and 58.0 mol% [Kaeppel et al. 2012] thus confirming that strain HP15 represents a novel Marinobacter species. For taxonomic purposes, a combined genomic and phenotypic description is essential for the delineation of new species in prokaryotes [Wayne et al. 1987]. Although M. flavimaris was most closely related to M. adhaerens HP15 in our genomic analyses, the phenotypic characterization clearly demarcated the two species [Kaeppel et al. 2012]. The two strains differed in their pigmentation, with M. adhaerens HP15 exhibiting a brownish pigmentation while M. flavimaris colonies were cream-colored. Furthermore, the two strains differed significantly in their utilization of glycerol, D- fructose, DL-lactic acid, D-gluconate, L-alanine, phenylacetate, and L-glutamate as well as in their ability to reduce nitrate to nitrite [Kaeppel et al. 2012]. On the basis of our detailed polyphasic approach, the bacterial partner of the selected bilateral model system, strain HP15T (=DSM 23420T = CIP 110141T), was confirmed to represent a novel species and was named as Marinobacter adhaerens [ad.hae'rens. L. part. adj. adhaerens: hanging on, sticking to] [Kaeppel et al. 2012]. The genus Marinobacter was established with the species M. hydrocarbonoclasticus in 1992 [Gauthier et al. 1992]. A total of 31 further species have been described until today. In addition to our research interest on its potential to induce TEP production and aggregate formation, Marinobacter have attracted increasing interest in the field of petroleum microbiology and hydrocarbon degradation. Together with the genera Alcanivorax, Thallassolituus, Cycloclasticus and Oleispira, certain representatives of the genus Marinobacter form the obligate hydrocarbonoclastic group of bacteria recognized to play a significant role in the biological removal of petroleum hydrocarbons from polluted marine waters [Gauthier et al. in 1992, Yakimov et al. 2007]. Marinobacter are tolerant to various conditions and have been isolated from a

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variety of marine environments ranging from oil-contaminated environments to sediments, surface and deep sea waters as well as in polar regions [Huu et al. 1999, Gorshkova et al. 2003, Yoon et al. 2004, Grossart et al. 2004, Takai et al. 2005, Montes et al. 2008, Roh et al. 2008]. Furthermore, representatives of this genus have been identified based on their interactions with other organisms: M. algicola was originally obtained from dinoflagellate cultures, M. bryozoorum was found associated with bryozoa and M. xestospongiae was isolated from a marine sponge [Romanenko et al. 2005, Green et al. 2006, Lee et al. 2011]. The abundance of representatives of the Marinobacter genus varies between 2-9 % in the Adriatic Sea [Grossart HP, personal communication] and was found to represent up to 16% of proteobacterial signals in fluorescent in situ hybridization experiments conducted with samples from Helgoland Roads (Fuchs B, personal communication). Considering the great diversity of marine microbes in the ocean, these amounts are significant, and further strengthen the potential ecological importance of this genus in certain areas of the ocean. As previously shown for other environmentally important bacterial species [Bakersmans et al. 2009, Piekarski et al. 2009, Wöhlbrand and Rabus 2008], establishment of the genetic accessibility of individual strains represents the pivotal base for detailed and accelerated research on these organisms. Therefore, we focused on the establishment of a genetic system to allow for the precise molecular manipulation of M. adhaerens HP15. Herein, (i) the genome of M. adhaerens HP15 was sequenced and annotated [Gaerdes et al. 2010] and (ii) protocols for the manipulation of M. adhaerens HP15 at the molecular level were identified and optimized [Sonnenschein et al. 2011]. The M. adhaerens HP15 genome was sequenced using the 454 FLX Ti platform of 454 Life Sciences (Branford, CT, USA) and the annotated genome sequence was deposited in GenBank under the accession number CP001978 for the chromosome and CP001979 and CP001980 for the two indigenous circular plasmids pHP-42 and pHP-187, respectively [Gaerdes et al. 2010]. M. adhaerens HP15 possesses three replicons: (i) a ~4.4 Mb chromosome encoding for 4,180 protein-coding genes, 51 tRNAs and three rRNA operons; (ii) pHP-42, a 42-kb plasmid encoding for 52 protein-coding genes; and (iii) pHP-187, a 187-kb plasmid encoding for 178 protein- coding genes [Gaerdes et al. 2010]. From the genome and plasmid sequences, the design of precise PCR primers needed for experiments such as gene-specific

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mutagenesis and microarray analysis can be accomplished. Beyond the identification of genes and design of PCR primers, the complete genome sequence represents a powerful basis to allow for functional as well as comparative genomic studies [Fraser et al. 2002]. So far, the genome sequences of M. algicola DG893 [Green et al. 2007], M. aquaeolei VT8 [Copeland et al. 2006] and M. adhaerens HP15 [Gaerdes et al. 2010] are publicly available. A potential comparative genomic study could be based on the genomic comparison of M. algicola DG893 – isolated from dinoflagellate cultures – to that of M. adhaerens HP15. Since both Marinobacter strains have been shown to interact with phytoplankton, this study could potentially provide biological insights and reveal functional genes that are important in the mediation of phytoplankton-bacteria interactions. Furthermore, ecological experiments testing the specificity of the Marinobacter genus in inducing TEP production and aggregate formation could be conducted with other diatom species. In this respect, it will be interesting to conduct experiments with diatom species whose genome sequences are publicly available. Two candidates that could potentially serve this purpose are the ubiquitously found organisms, T. pseudonana and Phaeodactylum tricornutum. Dual transcriptomics studies with those diatoms and M. adhaerens could further provide interesting biological insights on diatom-bacteria interactions. Genetic methods that allow for the manipulation of M. adhaerens HP15 at the molecular level were identified and optimized [Sonnenschein et al. 2011]. Protocols for the efficient transformation of two replicable plasmids, pBBR1MCS [Kovach et al. 1994] and pSUP106 [Priefer et al. 1985], were established both for conjugal transfer and electroporation [Sonnenschein et al. 2011]. The antibiotic susceptibility spectrum was determined. Additionally, two reporter genes encoding for green fluorescent protein and ß-galactosidase, respectively, were introduced and successfully expressed in M. adhaerens HP15 thus yielding in powerful tools for gene expression analyses. In combination with the genome sequence, these tools offered the possibility to investigate the roles of specific genes during the diatom-bacteria interaction. In the second part of this work, the established genetically accessible model system was employed to test the hypothesis that M. adhaerens HP15 motility appendages were crucial for its attachment to the diatom T. weissflogii. M. adhaerens HP15 flagellum- and MSHA type IV pilus-deficient mutants were generated by transposon insertion as well as by gene-specific mutagenesis using homologous recombination

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[Sonnenschein et al. 2011, Seebah et al. manuscript in preparation]. By conducting in vitro biofilm assays and attachment assays with diatom cells, our results clearly demonstrated that a fully-functional flagellum was a pre-requisite for the attachment of M. adhaerens HP15 to both abiotic and to the diatom surfaces. The MSHA type-IV pilus was also found to be important for attachment, albeit to a lesser extent. These results are in line with observations previously made by O' Toole and Kolter [1998] who had demonstrated the importance of flagellar motility by comparing attachment of motile and non-motile P. aeruginosa strains to plastic surfaces under static biofilm culture conditions. Our data are also in line with studies where mutants defective in the biosynthesis of the MSHA type IV pilus exhibited severe impairment of P. tunicata attachment to the algae Ulva australis [Dalisay et al. 2006]. Likewise, MSHA type IV pilus-mediated attachment to abiotic surfaces was demonstrated for S. oneidensis MR-1 [Thormann et al. 2004]. TEP are both ubiquitous and abundant in the ocean and have been found in all aggregates investigated to date [Alldredge et al. 1993; Passow and Alldredge 1994; Passow 2002]. However, the underlying molecular mechanisms that govern TEP production remained unknown. We predicted that the attachment of bacteria to diatom surfaces could potentially influence diatom-borne TEP production. In order to test this, M. adhaerens HP15 and its flagellum- or MSHA type IV pilus-defective mutants were co-incubated with axenic cultures of T. weissflogii. Thereafter, the amount of TEP produced by the diatom cells was quantified. With TEP concentrations occurring in similar amounts in all co-cultures, our findings showed that although bacterial motility appendages are crucial for bacterial attachment to diatom surfaces, this attachment is not essential for inducing diatom-borne TEP production. Additional yet- to-be determined mechanisms appear to govern the induction of TEP formation following the initial cell-to-cell contacts mediated by bacterial flagella and pili [Seebah et al. manuscript in preparation]. From the molecular perspective, the bacterial determinants responsible for inducing diatom-borne TEP production still remain unknown. Consequently, future attempts will focus on identifying M. adhaerens HP15 gene products specifically expressed during the interaction using in vivo expression technology (IVET) [Mahan et al. 1993]. This technique offers the possibility of identifying gene products specifically expressed during the interaction. The identified genes expressed in vivo could then be mutagenized and their impact on inducing diatom-borne TEP production assessed.

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TEP production and diatom aggregation play pivotal roles in the oceanic carbon cycle. In order to evaluate potential feedback mechanisms of marine biogeochemical cycles in response to climate change, it is essential to investigate the effects of projected changes in ocean temperature and acidification on TEP and marine aggregate formation dynamics. The final part of this thesis therefore focused on ecological experiments where TEP and aggregate formation dynamics were tested in conditions mimicking future ocean scenarios. Prior to conducting the respective experiments, we observed that the routinely used technique of filtering natural seawater through 0.2 µm sieves did not satisfactorily remove all bacterial contaminants such as nanobacterial cells passing through the filters and forming colonies on agar plates (data not shown). Since sterilization of natural seawater by autoclaving severely impacts the carbonate system [Riebesell et al. 2010, pers. observation], we established a new protocol for the preparation of experimental media, which is appropriate for the manipulation of the carbonate system [Seebah et al. manuscript in preparation]. By investigating the impact of TEP production and aggregate formation under future ocean carbonate chemistry and temperature regimes, the results of our study cautiously suggested that the combined effect of ocean acidification and increased temperature leads to a pronounced reduction of marine aggregate formation. Furthermore, we show that aggregates formed under these conditions had slower sinking velocities than aggregates formed under present-day conditions. We therefore suggested that the vertical export of particulate organic matter through marine aggregates may be severely impacted in a future ocean, depending on the magnitude, and on the vertical depth penetration of warming in the ocean. The results of our study further showed that TEP production was not significantly impacted under the tested ocean acidification conditions. However, the synergistic effect of an elevated temperature and ocean acidification favored TEP production in axenic cultures of the diatom T. weissflogii. The results of our study, suggest that bacteria do not significantly impact diatom-borne TEP production. In cultures containing both diatoms and bacteria, no significant differences were observed in TEP production. These results are contradictory to the previous finding where M. adhaerens HP15 was shown to directly enhance TEP production in axenic cultures of T. weissflogii [Gaerdes et al. 2011]. This observation highlights the complexity of

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TEP production by phytoplankton and suggests that other unknown factors govern the transformation of dissolved organic matter into particulate organic matter. One possible factor that could be tested in future studies is the effect of light on TEP production. The rolling tank experiments conducted in this study were carried out in un-interrupted darkness. In contrast, Gaerdes et al. (2011) had used rolling tanks subjected to two hours of light daily. It is possible that this short-term light exposure contributed to a higher exudation of the diatom-borne photosynthesis products and hence more TEP was produced. The integration of results obtained in this work revealed that a set of genetic tools and a workflow for the precise manipulation of M. adhaerens HP15 was successfully established. The developed techniques are easily transposable and offer the possibility of manipulating other Marinobacter species at the molecular level and can be used to investigate hypothesis-driven questions. We investigated the role of M. adhaerens HP15 motility during its interaction with the diatom T. weissflogii and concluded that although the bacterial cell appendages were crucial to mediate the attachment to the diatom, this attachment was not crucial for inducing TEP production. The interesting finding that marine aggregation was severely impacted under conditions mimicking future ocean scenarios, confirmed numerous predictions of the detrimental effects of an increased level of anthropogenic CO2 emissions. In conclusion, this work shed light on diatom-bacteria interactions, TEP production and aggregate formation both, from a molecular and ecological perspective. Not covered by experiments reported in this thesis, but nevertheless an interesting observation was made from scanning electron micrographs [Figure 17] of T. weissflogii and M. adhaerens HP15. In this micrograph, it would appear that M. adhaerens HP15 is entangled in the surrounding network of fibrils around the diatom T. weissflogii. This observation suggests that M. adhaerens HP15 might i.e. attach to the spines of the diatom. The spines of T. weissflogii are made up of β-chitin fibrils (Durkin et al. 2009). Whether M. adhaerens HP15 interacts with the diatom to use TEP as a carbon source or possesses chitinases to breakdown β-chitin fibrils for use as a carbon source, or both, remains at this stage an intriguing question. A preliminary scan of the M. adhaerens HP15 sequenced genome did not reveal the presence of chitinases encoding genes (data not shown). However, due to the inherently diverse nature of chitinase genes (LeCleir and Hollibaugh 2006), we cannot exclude the

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possibility that the bacterium might in effect possess chitinases encoding genes. A thorough bioinformatics analysis will be in the future needed. M. flavimaris str. SW- 145 - the closest relative of M. adhaerens HP15 – has been reported to possess chitinolytic activity (LeCleir and Hollibaugh 2006), which further suggests the possibility that M. adhaerens HP15 could also possess chitinolytic activity. In order to substantiate or exclude this possibility, it would be an interesting future project to test the chitinolytic activity of the bacterium. By growing M. adhaerens HP15 on media with β-chitin as the sole carbon source, and thereafter analysing whether chitinases appear in the protein profiles by matrix-assisted laser desorption/ionization time-of- flight mass spectrometry, this project could potentially offer new insights of the interaction. In addition, if found, the mutagenesis of putative chitinases could be achieved with the established genetic tools. Testing these mutants by attachment assays and by microscopy could potentially confirm whether the bacterium preferentially attaches to and takes advantage of the β-chitin fibrils of the diatom.

Figure 17

Scanning electron micrograph depicting M. adhaerens HP15 entangled in the spines of the diatom T. weissflogii [Micrograph courtesy of Astrid Gaerdes and Yannic Ramaye, Ullrich Laboratory, Jacobs University Bremen].

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Declaration

I hereby declare that this thesis is my own work and effort and that it has not been submitted to another university for the conferral of a degree. Where other sources of information have been used, they have been acknowledged.

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