proteomics analysis of the response of the marine ...bertani agar medium. bacterial cell numbers...

AQUATIC MICROBIAL ECOLOGYAquat Microb Ecol

Vol. 78: 65–79, 2016doi: 10.3354/ame01804

Published online December 21

INTRODUCTION

Despite their small size and inconspicuous appear-ance, diatoms contribute about one-fifth of global pri-mary production (Field et al. 1998). They do not onlyfix atmospheric carbon dioxide but also provide car-bon and other nutrients to their surroundings due to

© The authors 2016. Open Access under Creative Commons byAttribution Licence. Use, distribution and reproduction are un -restricted. Authors and original publication must be credited.

Publisher: Inter-Research · www.int-res.com

*Corresponding author: [email protected]

FEATURE ARTICLE

Proteomics analysis of the response of the marinebacterium 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

OPENPEN ACCESSCCESS

ABSTRACT: Bacteria are often associated withdiatoms through attachment or by being in closeproximity. Certain bacteria−diatom interactions areassumed to be mutualistic. Substrates determiningthese interactions have been identified, yet in-depthstudies on bacteria−diatom interactions re mainscarce. In the present study, we applied a pro-teomics approach to obtain a deeper perspective ofthe response of a bacterium to a diatom. A bi lateralmodel system was used, comprising the marine γ-proteobacterium Marinobacter adhaerens HP15 andthe diatom Thalassiosira weissflogii. In co-cultiva-tion experiments, the interacting partners werephysically separated by dialysis tubing allowing diffusion of compounds. The proteome of M. ad -haerens HP15 derived from cell lysates was sampledand compared with protein samples from diatom-free bacterial cultures. Proteome alterations werevisualized by 2-dimensional gel electrophoresis,and differentially expressed proteins were analyzedby mass spectrometry. We hypothesized that observ-able protein alterations will give information onsubstrates that determine a potential mutualisticinteraction. Our results suggested a differentiatednutrient supply for M. adhaerens HP15 during co-cultivation. The bacterium seemed to benefit fromthe 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-stantiate these results, a screening for suitablenutrient substrates of M. adhaerens was conductedrevealing that amino acids ap peared to be the pre-ferred carbon source of the bacterium, while varioustested sugars were not utilized.

Diatom cells (large) releasing Alcian blue-stained transpar-ent exopolymeric particles (blue) and surrounded by Mari-nobacter adhaerens cells (rod-shaped).

Photo: Veronika Will

KEY WORDS: Bacteria−diatom interaction · Mutual-ism · Marinobacter adhaerens HP15 · Thalassiosiraweissflogii · Carbon cycle · Transparent exopolymerparticles

Aquat Microb Ecol 78: 65–79, 201666

the release of exudates that may coagulate and formtransparent exopolymer particles (TEPs) (Passow2000, Passow et al. 2001). Depending on certain envi-ronmental parameters such as nutrient availability,up to 50% of carbon fixed during primary productionmay be re-allocated in the form of exudates (Beauvaiset al. 2006). Formed TEPs serve as valuable nutrientand carbon sources for other microbes, thus fuelingthe degradation and re-allocation of compounds inthe upper water column on a micro-scale level (Azam& Malfatti 2007). Indeed, algal bloom dynamics areclosely coupled to bacterial dynamics: depending onthe succession of a bloom, different bacterial taxaspecialized in degradation of specific substrates maypeak in abundance (Fandino et al. 2005, Rooney-Varga et al. 2005, Mayali et al. 2011, Teeling et al.2012). Therefore, primary production of diatoms andsubsequent interactions between both bacteria anddiatoms are important in carbon and nutrient cyclingon a global scale (Azam et al. 1994).

Diatoms and other single-celled algae such asdinoflagellates are usually colonized by bacteria intheir phycosphere (Baker & Kemp 2014, Sison-Man-gus et al. 2014). The phycosphere is defined as thedirect surrounding of a micro-alga, in which bacterialgrowth is impacted due to the release of algal com-pounds (Bell & Mitchell 1972). Within this area, bac-teria may be attached to the cell surface or foundswarming in close proximity (Blackburn et al. 1998).Different diatom genera or even species harbor dis-tinct groups of bacteria (Grossart et al. 2005, Guannelet al. 2011, Eigemann et al. 2013, Sison-Mangus et al.2014), leading to the assumption that bacterial colo-nization is diatom species-specific. Individual algalexudates may selectively attract particular bacterialspecies (Seymour et al. 2009). An intimate co-evolu-tion of diatoms and colonizing bacteria has been sug-gested, based on the finding that several hundredgenes in the diatom Phaeodactylum tricornutum areof bacterial origin (Bowler et al. 2008). Although par-asitic or commensalistic relationships have beenobserved (Bratbak & Thingstad 1985, Paul & Pohnert2011), synergistic relationships are common, inwhich both partners profit from the presence of theother (Grossart 1999). A number of benefits areknown for bacteria and diatoms or other single-celled algae that are gained by both partners duringthe interaction (Amin et al. 2012). Past studies havefocused on e.g. TEP production dynamics, release ofhydrolytic enzymes, or growth behavior (Grossart1999, Gärdes et al. 2012). However, in-depth studiesthat use advanced metabolomics, molecular genetics,or proteomics approaches remain scarce.

A major benefit for bacteria is the availability ofmetabolites such as TEPs (Grossart 1999), which aremainly composed of carbohydrate polymers and to alesser content of nutrient sources such as proteins,peptides, amino acids, or lipids (Myklestad et al.1989, Myklestad 1995, Biddanda & Benner 1997).Bacteria do not only metabolize but also may alterTEP formation and release, resulting in increase ordecrease of TEP production (Bruckner et al. 2011,Gärdes et al. 2012). Because TEPs may serve as astickiness-increasing ‘glue’ and therefore triggerparticle formation and sinking, bacteria−diatominteractions impact the biological carbon pump (All-dredge & Gotschalk 1989, Ducklow et al. 2001).

Diatoms are assumed to benefit from synergisticbacterial interactions due to the production of vita-mins by bacteria, with cobalamin (vitamin B12) beingthe most often discussed candidate in this context(Droop 1970, Cole 1982, Croft et al. 2005). A widerange of algae are auxotrophic for cobalamin (Croft etal. 2005), whereas auxotrophy towards other vitaminssuch as thiamine (vitamin B1) or biotin (vitamin B7) isless widespread but is also a potential trigger for synergistic relationships (Croft et al. 2006, Wagner-Döbler et al. 2010). Another valuable ‘traded item’between bacteria and diatoms is iron. Iron is alimiting growth factor for phytoplankton in certainparts of the ocean (Behrenfeld et al. 1996) and couldbe provided by algae-associated bacteria via theirability to form siderophores (Amin et al. 2009). Nitro-gen is a further ‘traded item’ provided by bacteria, asshown for cyanobacteria (Foster et al. 2011) or for aSulfitobacter strain (Amin et al. 2015). The latter bacterium did not only provide ammonia, but alsoproduced the plant hormone indole-3-acetic acid(IAA), thus generating a growth-promoting effect inits co-cultivated diatom partner Pseudo-nitzschiamultiseries (Amin et al. 2015). Bruckner et al. (2011)applied a proteomics approach to an in vitro co-culti-vation of the diatom P. tricornutum and Escherichiacoli K12. These authors studied diatom growth be-havior, TEP formation, and amino acid release andfurther focused on the extracellular proteome. Theyfound bacterial extracellular proteins induced duringco-cultivation that were associated with protein-binding, transport, biofilm formation, and carbo -hydrate turnover. In a co-cultivation experiment ofThalassiosira pseudonana and a member of theRoseobacter clade, Dinoroseobacter shibae, Paul etal. (2013) used metabolomic techniques for the analysis of intracellular diatom metabolites. They ob-served an increase in amino acid concentration in T.pseudonana during co- cultivation. Additionally, long-

Stahl & Ullrich: Bacterial response to diatoms analyzed by proteomics

chain fatty acids and undefined carbohydrates wereincreased in the diatom in the presence of bacteria.

The aim of the present study was to contribute toan in-depth understanding of bacteria−diatom inter-actions by focusing on the response of a bacterium toa diatom. A proteomics approach was applied to amodel interaction system comprising the diatom T.weissflogii and the marine bacterium Marinobacteradhaerens HP15. This model system had been established in the past in order to investigate bacter-ial colonization of diatoms and formation of marineaggregates (Sonnenschein et al. 2011). It has sincesuccessfully served in a set of studies dissecting thisinteraction (Gärdes et al. 2011, 2012, Sonnenscheinet al. 2012). For the current proteomics approach, aninteraction was chosen whereby both partners werephysically separated but could exchange signal molecules and other chemical compounds. Bacterialproteins were thus identified, that were altered inexpression due to the exchange of such compounds.The focus was set on cytoplasmic proteins, takingadvantage of their hydrophilic character that makesthem suitable for the applied methodical approaches.We further assumed that most proteomic changes inresponse to the interaction would be recognizablewithin the cytoplasmic proteome. The potential roleof altered expression of proteins during the inter -action was experimentally supported by a screeningfor carbon sources that the bacterium is able tometabolize.

MATERIALS AND METHODS

Culture conditions for Marinobacter adhaerensHP15 and Thalassiosira weissflogii

M. adhaerens HP15 (Grossart et al. 2004, Kaeppelet al. 2012) was cultured in 500 ml Erlenmeyer flasksin liquid marine broth (MB) medium (Sonnen -schein et al. 2011) at 18°C under constant shaking at250 rpm. Axenic T. weissflogii diatom cultures(CCMP 1336; received from the Provasoli-GuillardNational Center for Culture of Marine Phytoplank-ton) were grown in 2000 ml Erlenmeyer flasks in f/2medium prepared from filtered and autoclavedNorth Sea water (NSW; 15°C, 12:12 h light:darkperiod at 150 µm photons m−2 s−1). In detail, f/2medium was supplemented with 8.82 × 10−4 MNaNO3, 3.62 × 10−5 M NaH2PO4 · 2H2O, 1.06 × 10−4 MNa2SiO3 · 9 H2O, 1 ml l−1 trace element and vitaminsolution according to Guillard (1975). The cultures’axenity was constantly checked by light microscopy

and plating of culture aliquots on MB and Luria-Bertani agar medium. Bacterial cell numbers duringthe experiment were determined by serial dilutionplating in triplicate, and diatom numbers were deter-mined with a Sedgwick-Rafter counting chamber,using a Zeiss Axiostar plus microscope (Carl Zeiss) at100× magnification.

Co-cultivation of M. adhaerens HP15 with T. weissflogii

M. adhaerens HP15 cells were harvested from liquid culture in exponential growth phase (OD6000.5−1.0) by centrifugation (3000 × g, 15 min at 4°C).Cells were washed twice in 75% (v/v) NSW, sus-pended, and starved for 3 h in 75% NSW at 18°Cunder constant shaking (250 rpm). Cells were thenpelleted and suspended in 75% NSW supplementedwith 3.4 × 10−2 M glutamate as the sole carbon andnitrogen source and 2.12 × 10−3 M phosphate (NaH2PO4· 2H2O). The phosphate content was chosenaccording to the nitrogen molarity provided by gluta-mate and the resulting favorable Redfield ratio of16:1 N:P (Hecky et al. 1993). The initial start OD600for the bacterial culture was adjusted to 0.05 (~7 × 106

cells ml−1).Three individually grown T. weissflogii pre-cul-

tures in equal growth state (TW I−III) were splitinto 2 parts (Fig. 1). Half of each culture was usedfor sampling of the culture supernatant. For this,cultures were centrifuged under mild conditions(1250 × g, 10 min at 4°C) to avoid cell lysis, andthe supernatant was filtered through 0.2 µm poresize filters (hydrophilic). The second half was usedfor direct co-cultivation of T. weissflogii with thebacterium.

Two treatments were chosen, in which M.adhaerens HP15 was cultivated either with thesupernatant of the diatom culture, serving as a refer-ence for the 2-dimensional protein gel analysis (REFI−III) or was co-cultivated with the diatom cultureitself, serving as the actual treatment (TRE I−III,Fig. 1). The reference was prepared with diatomsupernatant in order to (1) normalize effects of differ-ential nutrient status that create artifacts in the pro-teome (as observed in pilot experiments conductedwithout diatom culture supernatant) and (2) to iden-tify those proteomic shifts in the treatment that exclu-sively appear due to the active response of thediatom to the bacterium. For these cultivations, 100ml of diatom culture supernatant or diatom culture,respectively, were loaded into flexible dialysis tubing

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(25 mm ZelluTrans hose, molecular weight cut-off12 000−14 000; Carl Roth). Filled tubes were placedinto a 200 ml suspension of M. adhaerens HP15 cells,prepared in a 1000 ml Erlenmeyer flask. M. ad -haerens HP15 cells had previously been starved, pel-leted, and suspended in corresponding medium to anOD600 of 0.05. All treatments were further supple-mented with trace elements, vitamins, and nutrients,as done for f/2 medium. Treatments were carried outin triplicate. Culture conditions used were those ap -plied for diatom cultivation (15°C; 12:12 h light: darkperiod at 150 µm photons m−2 s−1). Cultures weregently stirred (50 mm magnetic stirring bar, 60 rpm)to guarantee diffusion of chemical signal moleculesthrough the dialysis tubing.

Protein sampling, 2-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis

(2D-SDS-PAGE), and data analysis

Bacterial cells were harvested after 66.5 h of co-cultivation, marking the late exponential phase, from200 ml of culture by centrifugation at 3000 × g. Thecell pellet was suspended in chilled sonication buffer(100 mM Tris-HCl, pH 7.5; 50 mM NaCl; 0.5 mMdithiothreitol [DTT]; Protease Inhibitor Cocktailaccording to the manufacturer’s instructions [ThermoScientific]). Cell suspensions were sonicated on ice(10 × 1 s, 4 s break, 3 repeats; amplitude 70%; ActiveMotif® sonicator), and cell debris was centrifuged offafterwards (16 100 × g, 30 min at 4°C). Supernatants

Fig. 1. Experimental set-up of the co-cultivation experiment. Three individual pre-cultures of Thalassiosira weissflogii werecultivated (TW I−III). Each culture was split in 2 parts: one was used for sampling of the culture supernatant by centrifugalremoval of the diatom cells; the other was directly used for co-cultivation. Both culture supernatant (‘reference’) and directdiatom culture (‘treatment’) were loaded into flexible dialysis tubing and combined with a Marinobacter adhaerens HP15 culture. M. adhaerens HP15 was previously prepared in f/2 medium supplemented with 3.4 × 10−2 M glutamate (OD600 = 0.05).Both references and treatments were conducted in triplicate (REF I−III; TRE I−III). Cultures were gently stirred during

incubation to support diffusion of compounds through the dialysis tubing


were immediately used or kept at −80°C. Proteincontent of the supernatant was determined via abicinchoninic acid test according to the manufac-turer’s instructions (Thermo Scientific).

To conduct isoelectric focusing, 80 µg of proteinwere applied to immobilized pH gradient (IPG) strips(7 cm, pH 4−7; Biorad). Corresponding amounts ofprotein were precipitated and cleaned via acetoneprecipitation. In detail, 1 volume of protein suspen-sion was mixed with 4 volumes of ice-cold acetone(−20°C). Samples were kept at −20°C for at least 3 h.A protein pellet was harvested afterwards by centri -fugation (16 100 × g, 30 min at 4°C). The supernatantwas decanted and the protein pellet briefly air-dried.The pellet was suspended in rehydration buffer (2 Mthiourea, 6 M urea, 16.2 × 10−3 M CHAPS, 25.9 ×10−3 M DTT) and supplemented with ampholytesaccording to the manufacturer’s specifications (Bio-rad). IPG-strips were soaked with protein suspensionfor ~14 h. Isoelectric focusing was carried out on aBiorad Protean®i12™ IEF Cell (50 V, 70 min; 150 V,20 min; 300 V, 15 min; gradient to 600 V, 10 min;600 V, 15 min; gradient to 1500 V, 10 min; 1500 V,30 min; gradient to 3000 V, 20 min; 3000 V, 210 min;pause on 50 V). Strips were afterwards either kept at−80°C until further use or used immediately for second-dimension separation. IPG-strips were equil-ibrated beforehand for 15 min in 6.48 × 10−2 M DTTand then for 15 min in 0.216 M iodoacetamide solu-tion, dissolved in equilibration buffer (6 M urea, 30%[w/v] glycerol, 69.2 × 10−3 M SDS in 0.05 M Tris-HClbuffer, pH 8.8). Molecular weight separation wasconducted on a Biorad Mini-Protean®Tetra System(50 V, 10 min, 110 V thereafter) via a 12.5% acryl-amide concentrated polyacrylamide gel. The result-ing gel was Coomassie® stained for 20 min (45%[v/v] methanol, 10% acetic acid, 2.93 × 10−3 MCoomassie® Brilliant Blue G-250) and further treatedin corresponding destaining solution (10% [v/v] aceticacid, 5% [v/v] 2-propanol). Destained gels werescanned (600 dpi resolution) and analyzed for differ-ential protein patterns using Delta 2D software(Decodon). Gels resulting from bacterial co-cultiva-tion with diatom supernatant were set as referencegels (REF I−III), and those gels resulting from co- cultivation with diatoms (treatment; TRE I−III) werecompared to the reference gels (Fig. 1). Only signifi-cant results (p < 0.05) based on 3 replicates with atleast 2-fold changes were considered (2-fold up- regulation, 0.5-fold down-regulation). Statistical ana -lysis was conducted using the Delta 2D softwarepackage. All results were manually reviewed withrespect to correct spot definition and warping.

Matrix-assisted laser desorption/ionization-time offlight mass spectrometry (MALDI-TOF MS) analysis

Protein spots of interest were excised from SDSgels, chopped into small pieces, and washed twicefor 15 min (2 × 15 min) in 100 µl of 0.05 M ammoniumbicarbonate buffer, containing 50% acetonitrile (v/v).Gel pieces were finally dehydrated by addition of500 µl acetonitrile. After decanting and short air- drying, samples were further treated with trypsin digestion buffer (Promega) according to Shevchenkoet al. (2006). Fast tryptic digest was carried out by incubation of samples at 55°C for 30 min (Shev -chenko et al. 2006). Sample supernatant was directlyused for MALDI-TOF MS analysis. The supernatantwas mixed in a 1:1 ratio with a saturated α-cyano-4- hydroxycinnamic acid solution beforehand (pre-pared in 30% acetonitrile, 0.5% TFA; non-dissolvedmatrix solids were removed by centrifugation(15 min, 16 100 × g). An AutoflexII TOF/TOF massspectrometer (Bruker Daltonics) was used accordingto standard parameters (acquisition range 840−4000 Da; signal-to-noise ratio = 6, in specific cases 3;error range 50 ppm; allowed mis-cleavages = 1;potential modifications ‘Oxidation (M)’). Peptidemasses derived from trypsin auto digestion wereused for calibration (842.50940; 1045.56370; 1713.80840; 1774.89750; 2083.00960; 2211.10400; 2283.18020 Da). Obtained mass lists were identified usingthe MASCOT search engine (Perkins et al. 1999).The presence of 2 proteins within 1 protein gel spotwas tested and ruled out as follows. When peptidemasses were identified in the mass spectrum thatcould not be assigned to the obtained protein identi-fication, these unassigned peptides were re-searchedvia the MASCOT search engine, thus potentiallyidentifying any second protein in the spot. All resultswere reviewed with re spect to the identified organ-ism and expected molecular weight as retrieved fromSDS-PAGE.

Screening for metabolizable carbon sources inliquid and on solid medium

The use of potential carbon sources of M. ad -haerens HP15 was tested both in liquid and on solidmedium. We prepared 5 g l−1 of a compound in 75%NSW, containing additions of 3.62 × 10−2 M NaH2PO4· 2H2O, 1 × 10−3 M NH4Cl, and trace elements as usedfor f/2 medium. The pH was adjusted to 7.6 with 1 MNaOH solution. Beforehand, bacterial cells weregrown, harvested, and washed as described above.

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Liquid incubations were started at an OD of 0.05. Anegative control containing no carbon source wasprepared as a reference. Medium as described abovecontaining glutamate was used as a positive control.In the case of solid medium, 12 g l−1 agar was added.Streaks on agar plates were conducted with pre-washed cells as described above (OD ~0.05).

We tested 21 carbohydrates, 17 amino acids, 2 protein-associated compounds, and 12 further com-pounds as potential carbon sources. Specifically,10 monosaccharides (L(+)-arabinose, galactose, N-acetyl-D-glucosamine, fructose, D(+)-fucose, D-mal-tose, D(+)-mannose, N-acetyl-D-mannosamin, L(+)-rhamnose, D(+)-xylose), 3 disaccharides (lactose,D-maltose, and D(+)-sucrose), 1 trisaccharide (raffi-nose), and 7 polysaccharides (alginic acid, car-boxymethyl-cellulose, carrageenan, cellulose, chitin,dextrin, and xylan) were tested. The tested aminoacids and protein-associated compounds were ala-nine, L-arginine, L-asparagine, cystein, glutamate,glutamine, glycine, isoleucine, histidine, leucine,lysine, methionine, proline, phenylalanine, serine, L-threonine, and valine, as well as bovine serum albu-min V and a casamino acid mix. Further compoundstested were acetate, butyrate, citrate, lecithin,malate, nicotininc acid, octanoic acid, putrescine,propionate, pyruvate, spermidine, and succinate. Foramino acids, experiments were additionally con-ducted under supplementation of another carbonsource known to support growth (5 g l−1 glutamate).This additional test aimed to determine any potentialtoxic effects on the bacterium caused by the providedamino acid.

Growth behavior was determined after 72 h ofincubation. In liquid culture, visible growth wasdetermined as an increase in OD in comparison tothe negative control. To verify actual growth of M.adhaerens HP15 in liquid culture (and to rule outcontamination), test streaks on MB medium of allpositive samples were further carried out, and theexpected morphology was observed after a repeatedincubation and colony formation. On solid medium,positive growth was determined by the formation ofcolonies, showing the expected morphology of M.adhaerens HP15.

Databases used for protein and sequence interpretation

Information on available metabolic pathways anduptake systems of M. adhaerens HP15, as well asgene cluster analysis, was retrieved from the Kyoto

Encyclopedia of Genes and Genomes (KEGG) web-site (Kanehisa & Goto 2000). Proteins and gene loci ofthe bacterium were obtained from the National Cen-ter of Biotechnology Information (NCBI) website,where the full genome sequence of the bacterium isavailable (CP001978.1 [chromosome]; CP001979.1[plasmid pHP-42]; CP001980.1 [plasmid pHP-187]).Gene and protein similarity searches were con-ducted, using the Basic Local Alignment Search Tool(BLAST) program offered by NCBI. Further informa-tion on proteins of M. adhaerens HP15 involved incarbohydrate utilization was derived from the carbo-hydrate-active enzymes (CAZy) database (Cantarelet al. 2009, Lombard et al. 2014).

RESULTS

Cell numbers after termination of co-cultivation experiment

Marinobacter adhaerens HP15 cells were incu-bated with the supernatant of a diatom culture (‘reference’) or the diatom culture itself (‘treatment’)(Fig. 1) for 66.5 h. At time point 0, the cell numberswere 6.0 × 108 cells ml−1 (±SD of 6.0 × 107 cells ml−1)for the references and 5.4 × 108 cells ml−1 (± 1.1 ×108 cells ml−1) for the treatments. The obtained cellnumbers upon sampling of both protocols did not dif-fer significantly (2-sample t-test; p < 0.05), with 2.6 ×109 cells ml−1 (± 7.1 × 108 cells ml−1) for the referencesand 3.6 × 109 cells ml−1 (± 3.3 × 108 cells ml−1) for thetreatments. The growth status marked the late expo-nential to early stationary phase. The high bacterialnumber was chosen to obtain a sufficient amount ofprotein, and should be kept in mind when drawingconclusions on environmental aspects. The culturevolume of ~200 ml was used for extraction of bac -terial proteins. Diatom cultures grew from 7.62 ×105 cells ml−1 (±4 × 104 cells ml−1) at the beginning ofthe incubation to 1.92 × 106 cells ml−1 (±7.4 × 105 cellsml−1) after 66.5 h.

Changes in the bacterial protein pattern during co-cultivation

Glutamate was added to the experimental set-up toprovide sufficient carbon and nitrogen for the denovo synthesis of proteins by M. adhaerens HP15in response to the diatom. The exposure of M.adhaerens HP15 towards Thalassiosira weissflogiiled to a set of significant changes in certain protein

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abundances. In summary, 10 proteins were at least 2-fold up- or down-regulated during co-cultivationof bacteria with diatom cells (treatment, Fig. 1) in comparison to the reference (Table 1; see alsoTable S1 and Fig. S1 in the Supplement at www.int-res.com/articles/suppl/a078p065_supp.pdf). Thisrather low yield of altered proteins was assumed tobe the effect of (1) the 2D-gel parameters used (smallSDS gels of 7 cm width, Coomassie® staining as amoderately sensitive staining method), (2) the gen-eral challenge of identifying significant proteomicalterations on 2D-gels obtained from biological sys-tems, and (3) the focus on the cytoplasmic proteinfraction (as opposed to e.g. the membrane fraction orthe secretome in addition).

Gene products of M. adhaerens HP15 encoding2 alcohol dehydrogenases (ADP98924.1 and ADP -98928.1) and 1 aldehyde dehydrogenase (ADP98908.1)were down-regulated when diatom cultures werepresent. Notable for these 3 genes is their close prox-imity in the genome sequence. The aldehyde dehy-drogenase gene is separated from the 2 loci compris-ing the alcohol dehydrogenase genes by 15 genes,while the latter 2 genes are separated by only 3 addi-tional genes (Table 1 and Fig. S2, the latter in theSupplement). Due to close proximity of these genes,a cluster analysis for this genomic region was con-ducted to eventually deduce functional genomicembedment of the respective gene products. Thecluster analysis function of the KEGG website wasused for this purpose. Results showed that the corre-

sponding genomic region is separated into 6 differ-ent clusters. Importantly, the 3 genes encoding thedown-regulated proteins are not encoded in thesame cluster (Fig. S2). However, a number of genesin this locus encode proteins that are associated withmetabolic functions on, and uptake systems for,nitrogen-rich compounds. Examples of this are ureacarboxylase-associated proteins (ADP98910.1, ADP -98911.1) and a urea amidolyase-like protein (ADP -98913.1), a glutamate-ammonia ligase (ADP98920.1),a spermidine/ putrescine ABC transporter ATPase(ADP98923.1), and the homoserine O-acetyl -transferase MetX involved in cysteine and methion-ine metabolism (ADP98921.1) (Table S2 in the Supplement).

Three additional genes that were down-regulatedin M. adhaerens HP15 when encountering diatomcells code for an isocitrate lyase potentially involvedin the glyoxylate cycle (ADP98998.1), a 3-hydroxy-isobutyrate dehydrogenase putatively involved invaline catabolism (ADP98998.1), and a transportercomponent which may be involved in short-chainamide uptake, possibly also branched amino aciduptake (ADP98645.1) (Table 1). By additional BLASTanalysis, the latter transporter component was identi-fied as the UrtA component of a urea transporter.

Four M. adhaerens HP15 proteins were identifiedas being up-regulated during co-cultivation withthe diatom (Table 1). Two of these proteins areencoded by genes located in close proximity to eachother on the genome, suggesting their functional

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Identified protein (annotation according to NCBI) Additional information Accession no. EC no.

Down-regulated Quinoprotein alcohol dehydrogenase Alcohol aldehyde ADP98924.1 1.2.1.8Low-quality protein: quinoprotein alcohol dehydrogenase Alcohol aldehyde ADP98928.1 1.2.1.8NAD-dependent aldehyde dehydrogenase Aldehyde alcanic acid ADP98908.1 1.2.1.–Isocitrate lyase Isocitrate -> glyoxylate + succinate ADP98998.1 4.1.3.1

(glyoxylate cycle)3-hydroxyisobutyrate dehydrogenase Valine catabolism ADP96674.1 1.1.1.31Urea short-chain amide or branched-chain UrtA component of ADP98645.1 –amino acid uptake ABC transporter, urea transporterperiplasmic solute-binding protein

Up-regulated Phosphonate ABC transporter, PhnD unit of phosphonate ADP97492.1 –periplasmic phosphonate-binding protein transporter

Extracellular solute-binding protein, family 3 AotJ unit of an arginine/ornithine ADP98795.1 –amino acid transporter

TRAP dicarboxylate family transporter, DctP subunit – ADP98792.1 –Amino acid binding protein LivK unit of branched amino acid ADP99862.1 –

uptake transporter, periplasmic

Table 1. Proteins of Marinobacter adhaerens HP15 cell lysate, found to be at least 2-fold up- or down-regulated during co-cultivation with the diatom Thalassiosira weissflogii

http://www.int-res.com/articles/suppl/a078p065_supp.pdfhttp://www.int-res.com/articles/suppl/a078p065_supp.pdf


coupling. These genes encode for a DctP subunit ofa tripartite ATP-independent periplasmic (TRAP)dicarboxylate family transporter (ADP98792.1) anda protein annotated as extracellular solute-binding(ADP98795.1). Analysis of the latter protein usingthe KEGG database assigned it as the AotJ compo-nent of an arginine/ornithine amino acid trans-porter. Its potential function is substantiated by thepresence of surrounding genes, encoding the sub-unit of a histidine-lysine-arginine-ornithine trans-porter (HisP; ADP98794.1) and 2 inner membranepermeases of arginine- and histidine-associatedABC transporters (ArtQ, ADP98796.1; and ArtM,ADP98797.1).

The third bacterial protein up-regulated during co-cultivation with diatoms was annotated as a peri -plasmic substrate-binding protein of an ABC trans-porter and might be a homolog of the PhnD unit of anABC phosphonate transporter (ADP97492.1). Thisassumption is supported by the presence of genesencoding for the 2 other transporter componentsPhnC (ADP97493.1) and PhnE (ADP97494.1) withinthe same gene cluster.

The fourth of the up-regulated M. adhaerens HP15proteins was annotated as an amino acid-bindingprotein (ADP98795.1), likely being a homolog of theperiplasmic LivK unit of a branched amino acid up -take transporter. Again, a functional involvement ofthis protein in branched amino acid transport is sup-ported by the close proximity of genes, encoding theinner membrane translocators LivH and LivG as wellas the ATP-binding components LivM and LivF(Adams et al. 1990).

In summary, the proteomics analysis suggestedthat genes encoding for proteins involved in aminoacid uptake or utilization were affected in theirexpression when M. adhaerens HP15 was co-culti-vated with diatom cells.

Analysis of carbon source utilization of M. adhaerens HP15

In order to experimentally support some of the pro-teomics findings, a set of organic compounds such asdiverse carbohydrates, amino acids, and other mole-cules like short-chained fatty acids were tested aspotential sole carbon sources of M. adhaerens HP15.

Among all tested carbohydrates, fructose was theonly one that facilitated growth of the bacterium inboth liquid and solid medium (Table 2). The analysisof data retrieved from KEGG showed that themajority of classical mono- or oligosaccharide trans-

porters could not be identified in M. adhaerensHP15 (Tables S3 & S4 in the Supplement). Interest-ingly, genes encoding a fructose uptake systemwere also not identified (Table S4), probably assum-ing the presence of a different system for fructoseuptake. To test if potential genes were missed bythe applied KEGG program, fructose uptake systemgenes of other proteobacteria were directly blastedagainst the genome of M. adhaerens HP15 but didnot reveal any similarities. Focusing on phospho-transferase systems, genes encoding for phospho-transferases are only present in the case of a fruc-tose phosphatase system but not in other saccharidephosphatase systems (Table S5 in the Supplement).This information might support the experimentalfinding that exclusively fructose and no other carbo-hydrate was metabolized by the bacterium.

Subsequently, using the CAZy database, wescreened for genes associated with carbohydrate uti-lization. In total, 64 proteins encoded within the M.adhaerens HP15 genome were identified to be asso-ciated with carbohydrate utilization and sub-classi-fied as glycoside hydrolases, glycosyl transferases,polysaccharide lyases, carbohydrate esterases, andthe carbohydrate-binding module family (Table 3).For comparison, we additionally retrieved the infor-mation for carbohydrate-utilizing proteins of the mar-ine bacterium Flavobacterium johnsoniae UW101(McBride et al. 2009), a representative of the Bac-teroidetes group known to degrade algal polysaccha-rides. For this bacterium, a total of 279 carbohydrateutilization-associated genes were found (Table 3).This suggests that the high number of carbohydrate-active enzymes in F. johnsoniae UW101 fairly wellrepresents the metabolic profile and capabilities ofthis bacterium as an algae polysaccharide-associatedorganism, whereas M. adhaerens HP15 appears lessadapted to polysaccharide catabolism.

Whilst the majority of carbohydrates were appar-ently not metabolized by M. adhaerens HP15, cer-tain amino acids and protein-associated compoundsturned out to be growth-supporting for the bac-terium. These amino acids were alanine, L-arginine,L-asparagine, glutamic acid, glutamine, isoleucine,leucine, lysine, proline, and phenylalanine (Table 2).The bacterium further grew on bovine serum albu-min V and lecithin as the sole carbon source.Glycine proved to be cell toxic at the concentrationsused, as the bacterium did not grow in its presence,nor in the presence of both glycine and a secondmetabolizable carbon source. Other non-metabo-lized amino acids did not show any inhibitoryeffects.


DISCUSSION

Previously, only a few studies have been con-ducted to better understand bacteria−diatom inter-actions by using in-depth approaches based on invitro co- cultivations. Paul et al. (2013) conducted co-cultivation studies with Dinoroseobacter shibae andthe diatom Thalassiosira pseudonana, mainly focus-ing on intracellular metabolites, whereas Bruckneret al. (2011) focused on extracellular proteins in co-cultivations of different freshwater diatom specieswith a set of bacterial strains. Amin et al. (2015)have thus far conducted the most comprehensivestudy on bac teria−diatom interactions, combininginformation gained from transcriptomics data ofboth co-cultivated partners with metabolomics data.

Genes and metabolites possibly playing a role during a specific diatom−bacteria interaction wereidentified. A number of environmental meta- transcriptomic and meta bolomic data sets were fur-ther screened for the presence of identified metabo-lites and the expression of genes involved in theirsynthesis pathways.

Interestingly, none of these studies had dealtwith the cytoplasmic proteome of the bacterialinteraction partner. In the current study, we hypo -thesized that a physical interaction of both organ-isms in the phyco sphere of the diatom is notneeded for an exchange of nutrients or signals.Consequently, the 2 interaction partners were sep-arated by dialysis tubing allowing free diffusion ofcompounds.

73

Compound Metabolized by M. adhaerens HP15 Not metabolized by M. adhaerens HP15

Carbohydrates Monosaccharides Fructose L(+)-arabinose, galactose, N-acetyl-D-glucosamine, D(+)-fucose, D-maltose, D(+)-mannose, N-acetyl-D-mannosamine, L(+)-rhamnose, D(+)-xylose

Disaccharides – Lactose (D-glucose & D-galactose; β-1-4), D-maltose (degradation product of starch),D(+)-sucrose (α-D-glucose & β-D-fructose)

Trisaccharides – Affinose (galactose, glucose, fructose)Polysaccharides – Alginic acid, carboxymethyl-cellulose,

carrageenan, cellulose, chitin, dextrin (D-glucose, α(1-4) or α(1-6) linkage), xylan

Amino acids Alanine, L-arginine, L-asparagine, Cystein, glycinea, histidine, methionine, glutamate, glutamine, isoleucine, serine, L-threonine, valineleucine, lysine, proline, phenylalanine

Protein-associated Bovine serum albumin fraction V, –compounds casamino acids

Further compounds Acetate, butyrate, citrate, lecithin, Nicotinic acid, octanoic acid, tested malate, propionate, pyruvate, putrescine, spermidine

succinateaGlycine showed inhibitory effects, as no growth was observed in the presence of a second, growth-supporting carbonsource

Table 2. Compounds tested as potential carbon sources for Marinobacter adhaerens HP15

Organism GH GT PLF CE CBM Sum % of total genes

M. adhaerens HP15 15 41 0 1 7 64 1.43F. johnsoniae UW101 156 64 11 18 30 279 5.41

Table 3. Number of carbohydrate-utilizing gene products of Marinobacter adhaerens HP15 and Flavobacterium johnsoniaeUW101. Numbers were retrieved from the CAZy database. GH: glycoside hydrolase family; GT: glycosyl transferase family;

PLF: polysaccharide lyase family; CE: carbohydrate esterase family; CBM: carbohydrate-binding module family


The experimental set-up included the addition ofglutamate. This compound was primarily added toguarantee sufficient carbon and nitrogen supply forthe synthesis of proteins by Marinobacter adhaerensHP15. This approach aimed to avoid artifacts in theproteomic pattern caused due to e.g. limiting condi-tions instead of the actual response to the diatom ordiatom-borne compounds. As both references andtreatments showed similar bacterial growth, it maybe concluded that glutamate was used as the mainmetabolized carbon source, while diatom exudatessuch as TEP played only a minor role. However,when applying this set-up, we assumed that theinteraction of M. adhaerens HP15 with T. weissflogiiis not an interaction wherein the diatom simply pro-vides carbon to the bacterium, e.g. in form ofreleased TEP. In the latter case, addition of glutamatewould have led to a decoupling of the interaction.Irrespective of this, easy availability of a carbonsource may indeed have impacted the number of dif-ferentially expressed proteins. Furthermore, it can-not be ruled out that the mixotrophic growth of thediatom might have been induced by the presence ofglutamate.

M. adhaerens HP15 is not a carbohydrate degrader

In individual cases, up to 80% of algal exudates aremade up of carbohydrates (Myklestad et al. 1989,Myklestad 1995, Biddanda & Benner 1997) that areoften regarded as classical bacterial attractants (Sey-mour et al. 2008). Common sugars identified in suchextracellular matrices may be specific for a givenalgal species and its respective growth state (Bruck-ner et al. 2011). Arabinose, fucose, glucose, galac-tose, mannose, rhamnose, and xylose were identifiedas such sugars (Biersmith & Benner 1998). Conse-quently, the majority of these carbohydrates wereconsidered experimentally in the present study. Cer-tain bacterial groups, such as members of the Bac-teroidetes, are known for their ability to degrade car-bohydrate compounds resulting from algae exudates(Teeling et al. 2012, Hahnke et al. 2013, Mann et al.2013). According to our proteomics and carbon uti-lization results, none of the tested carbohydrates wasused as a carbon source by M. adhaerens HP15except for fructose. In support of this, the proteomicsapproach did not show any carbohydrate uptake systems or carbohydrate-utilizing proteins being up-regulated in M. adhaerens HP15 in the presence ofthe diatom. The findings were further substantiatedby the remarkably low number of carbohydrate uti-

lization-associated proteins encoded by the genomeof M. adhaerens HP15, as compared to those of clas-sical degraders of algae exudates, such as Flavobac-terium johnsoniae UW101 (McBride et al. 2009) orother members of the Flavobacteria (Mann et al.2013).

Shifts in protein patterns suggest a favorablenutrient state during co-cultivation

Expression of 1 aldehyde and 2 quinoprotein alcohol dehydrogenases was apparently down-regu-lated in M. adhaerens HP15 during co-cultivation.Such enzymes usually facilitate the oxidation of alco-hols via aldehydes towards alkanoic acids. Interest-ingly, the respective down-regulated dehydrogenasegenes are located in a genomic region that alsoencodes a number of proteins, associated with theutilization of nitrogen-rich molecules such as urea,amino acids, spermidine, and putrescine. We testedwhether these proteins are organized in a gene clus-ter. However, this hypothesis was not confirmed,since the genomic region of concern is divided intodifferent clusters, from which a functional relation-ship could not be easily retrieved. A functional hint,however, becomes apparent when looking at homol-ogous genes in another marine bacterium: the γ-pro-teobacterium Alteromonas sp. KE10 expresses thealdehyde dehydrogenase OlgA (BAA24014.1) duringlow-nutrient stress. During high-nutrient conditions,this dehydrogenase was found to be down-regulated(Maeda et al. 2000). The aldehyde dehydrogenaseADP98908.1 of M. adhaerens HP15 that was down-regulated in the presence of the diatom shows a 74%protein identity to OlgA of Alteromonas sp. KE10.Therefore, diatom presence might represent a favor-able nutrient status for the bacterium.

Isocitrate lyase is a central enzyme in the glyoxy-late cycle that was also down-regulated in the bac-terium during co-cultivation with the diatom. In thecase of a suppression of the tricarboxylic acid (TCA)cycle due to e.g. unfavorable nutrient supply or lim-ited availability of specific carbon compounds, theglyoxylate cycle may short-cut the reaction fromisocitrate via α-ketoglutarate and succinyl-CoA tosuccinate (as it would happen in the TCA cycle) in asingle-step reaction. Isocitrate lyase represents abranching point in this cycle due to the reaction ofisocitrate to succinate and glyoxylate (Betts et al.2002). While glyoxylate continues fueling the glyoxy-late cycle, succinate replenishes the TCA cycle(Vanni et al. 1990). The relevance of isocitrate lyase,

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Stahl & Ullrich: Bacterial response to diatoms analyzed by proteomics 75

and consequently the importance of the glyoxylatecycle in surviving nutrient-depleted conditions, hadbeen demonstrated previously (Idnurm & Howlett2002, Gengenbacher et al. 2010). The down-regula-tion of a central enzyme of the glyoxylate cycle thusindicates a favorable substrate supply to the TCAcycle. Consequently, the bacterial cell metabolismmight be provided with a richer nutrient source, or asignal representing such, during co-cultivation withthe diatom as compared to the growth in the refer-ence medium. Contradictory to this hypothesis is theobservation that cell numbers of M. adhaerens HP15did not change during sampling. The glyoxylatecycle is also activated during ex clusive supply ofacetate or fatty acids and will be shut down uponaddition of other carbon sources (Cronan & Laporte2006). However, the reference treatment was sup-plied with glutamate as the sole carbon source,which does not directly explain the presence of isoc-itrate lyase and induction of the glyoxylate cycle. Ananalysis of metabolites available in both the culturemedium and intracellularly (as e.g. done by Paul etal. 2013) may add valuable information to the under-standing of this finding.

We further observed the down-regulation of theperiplasmic substrate-binding compound UrtA of theurea uptake system (Beckers et al. 2004). Urea servesas a nitrogen source for a wide range of bacteria(Mobley & Hausinger 1989). The operon encoding forthe urea uptake system (made up of 5 genes, urt -ABCDE) is induced by nitrogen-limiting conditionsin Corynebacterium glutamicum (Beckers et al.2004). Due to the down-regulation of 1 protein of theurea uptake system, the nitrogen supply seems to bemore optimal in the treatment protocol as comparedto the reference protocol in which the amino acidglutamate is the only nitrogen source provided.

The enzyme 3-hydroxyisobutyrase dehydrogenase(EC 1.1.1.31), less expressed in the presence ofthe diatom, is part of the valine degradation path -way and oxidizes 3-hydroxyisobutyrate to methyl-malonate semialdehyde (Robinson & Coon 1957,Bannerjee et al. 1970). Reduced expression of arespective gene might suggest that the bacteriumwas not in need of valine utilization as a carbon ornitrogen source during co-cultivation. However,since the corresponding protein was present whenonly glutamate was provided (Débarbouillé et al.1991) it is assumed that in the reference protocol,probably nitrogen and carbon became more limiting.Alternatively, nitrogen supply might be balancedduring co-cultivation of M. adhaerens HP15 with thediatom. This hypothesis is in line with the down-

regulation of a urease uptake system, and leads tothe conclusion that the diatom might provide anadditional source of nitrogen to the bacterium.

Compounds gained by M. adhaerens HP15 duringco-cultivation with the diatom T. weissflogii

According to the current results, proteins, peptides,or amino acids might be the major carbon sources ofthe bacterium. Their presence in exopolymeric sub-stances has been shown in previous studies (Myk-lestad et al. 1989, Myklestad 1995, Biddanda & Ben-ner 1997).

During exposure to the diatom, a possible benefitfor M. adhaerens HP15 might be the release of spe-cific amino acids by the diatom. Two bacterial pro-teins up-regulated while being co-cultivated with T.weissflogii can be associated with amino acid uptakeand facilitate the binding of amino acids in theperiplasm. LivK is part of a branched amino acid uptake system transporting leucine, isoleucine, andvaline (Adams et al. 1990, Ribardo & Hendrixson2011). AotJ, another amino acid-binding protein,belongs to an arginine/ornithine uptake system (Wissen bach et al. 1995, Nishijyo et al. 1998). A thirdup-regulated transport-associated protein, a DctPsubunit, could not be assigned to a substrate, but issuspected to be part of the above mentioned argi-nine/ornithine transporter due to the close proximityof the corresponding genes. Amino acids other thanglutamate might serve as possible additional carbonand nitrogen sources when diatoms are present, sug-gesting that several of such amino acids might besecreted by the diatom cells. This finding is sup-ported by the down-regulation of a urea uptaketransporter element during co-cultivation with thediatom, a process usually associated with growthunder nitrogen-rich conditions (Beckers et al. 2004).Only 1 protein of a whole transporter unit, whichcomprises several proteins, was found to be altered(Beckers et al. 2004). This observation may be partlyexplained by our experimental approach that did notisolate and identify membrane-associated, hydro -phobic proteins (e.g. UrtB and UrtC, LivH and LivK,and AotQ and AotM), which thus were not repre-sented in the cytoplasmic protein fraction that weanalyzed.

Previous studies support our hypothesis of aminoacids playing a major role during bacteria–diatominteractions: Sulfitobacter sp. SA11 received trypto-phan from its diatom partner and provided IAA tothe host organism (Amin et al. 2015). It was


assumed that tryptophan was even directly used forthe synthesis of IAA in the bacterium, underliningthe closely coupled trade-off between the partners.Paul et al. (2013) found that intracellular free aminoacid concentrations were increased in T. pseudo-nana during exposure to D. shibae. Alterations inthe concentration of extracellular free amino acidswere also observed in co-cultivations of mainly ben-thic freshwater diatoms towards different bacterialstrains (Bruckner et al. 2011). Gärdes et al. (2012)confirmed for T. weissflogii cultures that the quan-tity and quality of both dissolved free and dissolvedcombined amino acids is highly dependent on thenutrient status of the culture. Further, the axenic orxenic status (being co-cultivated with M. adhaerensHP15) of the culture impacted the net concentrationof free and combined amino acids (Gärdes et al.2012). A shift in amino acid composition duringphosphate-depleted conditions was observed, irre-spective of the culture being xenic or axenic. Theproportions of the branched amino acids leucineand isoleucine — but not of valine — in the totalamino acid pool increased during phosphate limita-tion. Specifically, the ratio of leucine and isoleucineas part of total dissolved free amino acids increasedfrom ~5% in both nutrient-balanced and nitrogen-depleted scenarios to ~30%. Indeed, indications fora phosphate limitation were identified in the presentstudy: an ABC type transporter component, a phos-phonate substrate binding unit (PhnD), was up-reg-ulated in M. adhaerens HP15 in the presence of thediatom. The phnD gene was previously shown to beinduced during phosphate-limiting conditions (Met-calf & Wanner 1991, Gebhard et al. 2006). This find-ing would further explain and support the up-regu-lation of the LivK subunit as part of an uptaketransporter for branched amino acids in the pres-ence of diatom cells in the current study. Inaddition, an enzyme as part of the valine degrada-tion pathway is down-regulated, which would be inline with the low presence of valine during phos-phate limitation in comparison to other branchedamino acids. However, whether the release of suchamino acids by the diatom is indeed due to phos-phate limitation, as suggested by Gärdes et al.(2012), remains speculative and deserves furtherinvestigation.

An obvious and unsolved question is why diatomsactively release high-value metabolites, such asamino acids, when coming into contact with certainbacteria. One possible explanation is a potentialtrade-off: the diatom provides a high nutrient invest-ment in order to trigger attachment of certain bacte-

ria. In turn, these bacteria might be favorable for thediatom by providing a metabolite to which thediatom is auxotrophic. Such a metabolite remains tobe identified in further experiments. Amin et al.(2015) already demonstrated such a metabolite: theyobserved a supply of tryptophan by the diatom,whereas the bacterium provided the diatom with theplant hormone IAA.

Our results suggest that M. adhaerens HP15 mightbenefit from diatom-borne amino acids. A potentialbenefit of this interaction for the diatom could be thesupply of vitamins by the bacterium. Cobalamin (vit-amin B12) has been postulated as such a potentialcandidate (Cole 1982, Croft et al. 2005). However, thegenome analysis of M. adhaerens HP15 revealed thatthis organism does not synthesize cobalamin, butmight produce a set of other vitamins that may deter-mine synergistic bacteria−diatom interactions (Croftet al. 2006, Wagner-Döbler et al. 2010), whichdeserves future investigations.

CONCLUSIONS

Herein, a proteomics approach combined with ascreening for carbon substrate preferences was cho-sen to investigate processes taking place in a bacter-ial cell during its interaction with a diatom. Theapproach allowed the interpretation of characteris-tics during the interaction. Our results underline theimportance of amino acids as a ‘traded’ componentgroup serving as a nitrogen and carbon source forbacteria. The role of Marinobacter adhaerens HP15was further defined as being involved in amino acid-,peptide-, or protein-associated turn-over processes.A potential role of algal TEP as a source of aminoacids or peptides remains to be investigated in futurestudies. The actual benefits for the diatom are so farunknown for this model. Therefore, future studiesshould also aim to include proteome analysis of thediatom to better understand adaptions taking placein this organism during interaction with bacteria.Integrating the analysis of further protein fractionslike the membrane or extracellular fraction will im -prove and substantiate our results. Correspondingstudies should also be extended to a metabolic ana -lysis of bacteria−diatom interactions in order to de -velop a mechanistic understanding of the process.

Acknowledgements. This project was funded by theHelmholtz Graduate School for Polar and Marine Research(POLMAR) and the Deutsche Forschungsgemeinschaft(UL169/6-1).

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Editorial responsibility: Ilana Berman-Frank, Ramat Gan, Israel

Submitted: July 7, 2016; Accepted: October 28, 2016Proofs received from author(s): December 5, 2016

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proteomics analysis of the response of the marine ...bertani agar medium. bacterial cell numbers...

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