Vol. 78: 65–79, 2016 AQUATIC MICROBIAL ECOLOGY Published online December 21 doi: 10.3354/ame01804 Aquat Microb Ecol OPEN ACCESS FEATURE ARTICLE Proteomics analysis of the response of the marine bacterium Marinobacter adhaerens HP15 to the diatom Thalassiosira weissflogii Antje Stahl, Matthias S. Ullrich* Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen, Germany ABSTRACT: Bacteria are often associated with diatoms through attachment or by being in close proximity. Certain bacteria−diatom interactions are assumed to be mutualistic. Substrates determining these interactions have been identified, yet in-depth studies on bacteria−diatom interactions re main scarce. In the present study, we applied a pro- teomics approach to obtain a deeper perspective of the response of a bacterium to a diatom. A bi lateral model system was used, comprising the marine γ- proteobacterium Marinobacter adhaerens HP15 and the diatom Thalassiosira weissflogii. In co-cultiva- tion experiments, the interacting partners were physically separated by dialysis tubing allowing diffusion of compounds. The proteome of M. ad - haerens HP15 derived from cell lysates was sampled Diatom cells (large) releasing Alcian blue-stained transpar- ent exopolymeric particles (blue) and surrounded by Mari- and compared with protein samples from diatom- nobacter adhaerens cells (rod-shaped). free bacterial cultures. Proteome alterations were Photo: Veronika Will visualized by 2-dimensional gel electrophoresis, and differentially expressed proteins were analyzed by mass spectrometry. We hypothesized that observ- KEY WORDS: Bacteria−diatom interaction · Mutual- able protein alterations will give information on ism · Marinobacter adhaerens HP15 · Thalassiosira substrates that determine a potential mutualistic weissflogii · Carbon cycle · Transparent exopolymer interaction. Our results suggested a differentiated particles nutrient supply for M. adhaerens HP15 during co- cultivation. The bacterium seemed to benefit from the release of amino acids by the diatom, as indi- cated by an up-regulation of several transporter ele- ments responsible for amino acid uptake. To sub- INTRODUCTION stantiate these results, a screening for suitable nutrient substrates of M. adhaerens was conducted Despite their small size and inconspicuous appear- revealing that amino acids appeared to be the pre- ance, diatoms contribute about one-fifth of global pri- ferred carbon source of the bacterium, while various mary production (Field et al. 1998). They do not only tested sugars were not utilized. fix atmospheric carbon dioxide but also provide car- bon and other nutrients to their surroundings due to © The authors 2016. Open Access under Creative Commons by *Corresponding author: [email protected] Attribution Licence. Use, distribution and reproduction are un - restricted. Authors and original publication must be credited. Publisher: Inter-Research · www.int-res.com 66 Aquat Microb Ecol 78: 65–79, 2016 the release of exudates that may coagulate and form A major benefit for bacteria is the availability of transparent exopolymer particles (TEPs) (Passow metabolites such as TEPs (Grossart 1999), which are 2000, Passow et al. 2001). Depending on certain envi- mainly composed of carbohydrate polymers and to a ronmental parameters such as nutrient availability, lesser content of nutrient sources such as proteins, up to 50% of carbon fixed during primary production peptides, amino acids, or lipids (Myklestad et al. may be re-allocated in the form of exudates (Beauvais 1989, Myklestad 1995, Biddanda & Benner 1997). et al. 2006). Formed TEPs serve as valuable nutrient Bacteria do not only metabolize but also may alter and carbon sources for other microbes, thus fueling TEP formation and release, resulting in increase or the degradation and re-allocation of compounds in decrease of TEP production (Bruckner et al. 2011, the upper water column on a micro-scale level (Azam Gärdes et al. 2012). Because TEPs may serve as a & Malfatti 2007). Indeed, algal bloom dynamics are stickiness-increasing ‘glue’ and therefore trigger closely coupled to bacterial dynamics: depending on particle formation and sinking, bacteria−diatom the succession of a bloom, different bacterial taxa interactions impact the biological carbon pump (All- specialized in degradation of specific substrates may dredge & Gotschalk 1989, Ducklow et al. 2001). peak in abundance (Fandino et al. 2005, Rooney- Diatoms are assumed to benefit from synergistic Varga et al. 2005, Mayali et al. 2011, Teeling et al. bacterial interactions due to the production of vita- 2012). Therefore, primary production of diatoms and mins by bacteria, with cobalamin (vitamin B12) being subsequent interactions between both bacteria and the most often discussed candidate in this context diatoms are important in carbon and nutrient cycling (Droop 1970, Cole 1982, Croft et al. 2005). A wide on a global scale (Azam et al. 1994). range of algae are auxotrophic for cobalamin (Croft et Diatoms and other single-celled algae such as al. 2005), whereas auxotrophy towards other vitamins dinoflagellates are usually colonized by bacteria in such as thiamine (vitamin B1) or biotin (vitamin B7) is their phycosphere (Baker & Kemp 2014, Sison-Man- less widespread but is also a potential trigger for gus et al. 2014). The phycosphere is defined as the synergistic relationships (Croft et al. 2006, Wagner- direct surrounding of a micro-alga, in which bacterial Döbler et al. 2010). Another valuable ‘traded item’ growth is impacted due to the release of algal com- between bacteria and diatoms is iron. Iron is a pounds (Bell & Mitchell 1972). Within this area, bac- limiting growth factor for phytoplankton in certain teria may be attached to the cell surface or found parts of the ocean (Behrenfeld et al. 1996) and could swarming in close proximity (Blackburn et al. 1998). be provided by algae-associated bacteria via their Different diatom genera or even species harbor dis- ability to form siderophores (Amin et al. 2009). Nitro- tinct groups of bacteria (Grossart et al. 2005, Guannel gen is a further ‘traded item’ provided by bacteria, as et al. 2011, Eigemann et al. 2013, Sison-Mangus et al. shown for cyanobacteria (Foster et al. 2011) or for a 2014), leading to the assumption that bacterial colo- Sulfitobacter strain (Amin et al. 2015). The latter nization is diatom species-specific. Individual algal bacterium did not only provide ammonia, but also exudates may selectively attract particular bacterial produced the plant hormone indole-3-acetic acid species (Seymour et al. 2009). An intimate co-evolu- (IAA), thus generating a growth-promoting effect in tion of diatoms and colonizing bacteria has been sug- its co-cultivated diatom partner Pseudo-nitzschia gested, based on the finding that several hundred multiseries (Amin et al. 2015). Bruckner et al. (2011) genes in the diatom Phaeodactylum tricornutum are applied a proteomics approach to an in vitro co-culti- of bacterial origin (Bowler et al. 2008). Although par- vation of the diatom P. tricornutum and Escherichia asitic or commensalistic relationships have been coli K12. These authors studied diatom growth be- observed (Bratbak & Thingstad 1985, Paul & Pohnert havior, TEP formation, and amino acid release and 2011), synergistic relationships are common, in further focused on the extracellular proteome. They which both partners profit from the presence of the found bacterial extracellular proteins induced during other (Grossart 1999). A number of benefits are co-cultivation that were associated with protein- known for bacteria and diatoms or other single- binding, transport, biofilm formation, and carbo - celled algae that are gained by both partners during hydrate turnover. In a co-cultivation experiment of the interaction (Amin et al. 2012). Past studies have Thalassiosira pseudonana and a member of the focused on e.g. TEP production dynamics, release of Roseobacter clade, Dinoroseobacter shibae, Paul et hydrolytic enzymes, or growth behavior (Grossart al. (2013) used metabolomic techniques for the 1999, Gärdes et al. 2012). However, in-depth studies analysis of intracellular diatom metabolites. They ob- that use advanced metabolomics, molecular genetics, served an increase in amino acid concentration in T. or proteomics approaches remain scarce. pseudonana during co- cultivation. Additionally, long- Stahl & Ullrich: Bacterial response to diatoms analyzed by proteomics 67 chain fatty acids and undefined carbohydrates were and plating of culture aliquots on MB and Luria- increased in the diatom in the presence of bacteria. Bertani agar medium. Bacterial cell numbers during The aim of the present study was to contribute to the experiment were determined by serial dilution an in-depth understanding of bacteria−diatom inter- plating in triplicate, and diatom numbers were deter- actions by focusing on the response of a bacterium to mined with a Sedgwick-Rafter counting chamber, a diatom. A proteomics approach was applied to a using a Zeiss Axiostar plus microscope (Carl Zeiss) at model interaction system comprising the diatom T. 100× magnification. weissflogii and the marine bacterium Marinobacter adhaerens HP15. This model system had been established in the past in order to investigate bacter- Co-cultivation of M. adhaerens HP15 ial colonization of diatoms and formation of marine with T. weissflogii aggregates (Sonnenschein et al. 2011). It has since successfully served in a set of studies dissecting