Morphogenesis of Ulva Mutabilis (Chlorophyta) Induced by Maribacter Species (Bacteroidetes, Flavobacteriaceae)

Botanica Marina 2017; 60(2): 197–206

Short communication Open Access

Anne Weiss, Rodrigo Costa and Thomas Wichard* Morphogenesis of Ulva mutabilis (Chlorophyta) induced by Maribacter species (Bacteroidetes, Flavobacteriaceae)

DOI 10.1515/bot-2016-0083 related Flavobacteriaceae and a Maribacter strain isolated Received 31 July, 2016; accepted 11 January, 2017; online first from a red alga did not possess any activity. 17 February, 2017 Keywords: bacteroidetes; cell differentiation; green mac- Abstract: Growth and morphogenesis of the sea lettuce Ulva roalga; morphogens; thallusin. (Chlorophyta) depends on the combination of regulative morphogenetic compounds released by specific associated bacteria. Axenic Ulva gametes develop parthenogenetically The green macroalga Ulva mutabilis Føyn (Chlorophyta) is into callus-like colonies consisting of undifferentiated cells not able to develop and differentiate into blade, stem and without normal cell walls. In Ulva mutabilis Føyn, two bac- rhizoid cells under axenic conditions, or when its microbi- terial strains, Maribacter sp. strain MS6 and Roseovarius ome is not appropriate. Instead, the alga forms callus-like, strain MS2, can restore the complete algal morphogenesis slow growing structures with colourless protrusions from forming a tripartite symbiotic community. Morphogenetic the exterior cell wall (Spoerner et al. 2012, Wichard 2015). compounds ( = morphogens) released by the MS6-strain Early experiments of Provasoli (1958) have already pointed induce rhizoid formation and cell wall development in U. out that treatment of Ulva with antibiotics results in abnor- mutabilis, while several bacteria of the Roseobacter clade, mal growth. Further experiments examined the role of including the MS2-strain, promote blade cell division and isolated bacteria in activating developmental and growth thallus elongation. In this study, 12 type strains of the Fla- promoting traits in Ulva and revealed species-specific inter- vobacteriaceae family, including six Maribacter strains, actions, as no combination of bacteria showed the com- were examined for their morphogenetic activity in compari- plete recovery of the normal morphotypes (Provasoli and son to the original MS6-strain isolated from U. mutabilis. Pintner 1980, Marshall et al. 2006, Spoerner et al. 2012, The bioassay is based on the functional complementation Wichard 2015). Two bacterial isolates from non-axenic lab- of the tested Flavobacteriaceae strain with the Roseovarius oratory cultures of U. mutabilis were later found to induce MS2-strain. If the test-strain possesses morphogenetic the complete morphogenesis of U. mutabilis , forming a tri- activity complementary to the factor of the MS2-strain, the partite community (Spoerner et al. 2012, Wichard 2015). The complete morphogenesis of U. mutabilis can be restored. bacterial strains were originally described as Roseobacter This bioassay revealed not only the stand-alone activity of sp. strain MS2 and Cytophaga sp. MS6. The strain MS2 alone certain bacteria, but also their essential capability to take induces cell division and elongation of the blades but not part in the orchestrated bacteria-induced morphogenesis of of the rhizoid, whereas the strain MS6 promotes rhizoid U. mutabilis. All Maribacter type strains isolated from Ulva formation and proper cell wall synthesis without cell-wall could phenocopy the MS6-strain, whereas some distantly protrusions (Figure 1; Spoerner et al. 2012). Overall these morphogenesis-inducing bacteria secreted a bouquet (i.e. MS6- and MS2-factors) of still uncharacterised morpho- *Corresponding author: Thomas Wichard, Institute for Inorganic and Analytical Chemistry, Jena School of Microbial Communication, genesis-inducing factors ( = morphogens) into the culture Friedrich Schiller University Jena, Jena, Lessingstr. 8, 07743 Jena, medium of Ulva (Spoerner et al. 2012). However, each of Germany, e-mail: [email protected] these bacteria can be replaced with partly purified com- Anne Weiss: Institute for Inorganic and Analytical Chemistry, Jena pounds extracted from the corresponding bacterial culture School of Microbial Communication, Friedrich Schiller University medium (Spoerner et al. 2012, Wichard, unpublished Jena, Jena, Lessingstr. 8, 07743 Jena, Germany results). Morphogens with similar activity to the com- Rodrigo Costa: Institute for Bioengineering and Biosciences (IBB), Department of Bioengineering, Instituto Superior Técnico, pounds released by the MS6-strain were also determined in Universidade de Lisboa, Lisbon, Portugal sterile-filtered lagoon water of the Ria Formosa (Portugal) at

Figure 1: Control experiment for complementary activity of morphogenesis-inducing bacteria (reference strains). Axenic Ulva mutabilis sl G mt( + ) germlings (A) were inoculated with either Roseovarius sp. MS2 (B) or Maribacter sp. MS6 (C) or both bacteria (D). Gametophytes were propagated for gamete production and release under laboratory conditions (Wichard and Oertel 2010). Afterwards axenic gametes were prepared according to Wichard (2015). Purified gametes [mating type ( + )] were inoculated with selected bacteria (final concen- − 6 tration OD600 = 1 × 10 ) in 10 ml Ulva culture medium and kept in the dark for 24 h to let gametes settle on culture tissue flask. After 14 days growth at 18°C and 90–120 μmol photons s − 1 m − 2 for 17 h light and 7 h dark, thallus development of 50–70 germlings derived from triplicate experiments was examined with an inverted Leica DMIL LED microscope (Leica, Solms, Germany) equipped with a digital camera (Nikon, Düs- seldorf, Germany). The four morphotypes are colour-coded. Black arrows indicate protrusions from the exterior cell wall. Scale bars = 100 μm. sampling sites where Ulva sp. was usually abundant. These metabolism is aerobic or facultatively anaerobic (Nedashk- results indicated a tight interaction of the MS6-factor-pro- ovskaya et al. 2004a, 2010, Barbeyron et al. 2008, Lo et al. ducing bacterium with Ulva. In contrast, the MS2-like bioac- 2013, Weerawongwiwat et al. 2013, Hu et al. 2015, Jackson tivity factor was abundant all over the lagoon regardless of et al. 2015). They were found in various marine sources and the presence or absence of Ulva species (Grueneberg et al. climatic zones (Cho et al. 2008, Zhang et al. 2009, Tang 2016). In this context further phylogenetic analysis classi- et al. 2015) and could be isolated from the water column fied the MS6-strain to the genus Maribacter rather than to (Yoon et al. 2005, Barbeyron et al. 2008) and the sea sedi- Cytophaga, while the MS2-strain was reclassified to Roseo- ment (Nedashkovskaya et al. 2004a, Cho et al. 2008). varius (Grueneberg et al. 2016). The same study has also Maribacter strains are often associated with the surface shown that cultivable bacteria isolated from the surface of of macroalgae such as Ulva fenestra Postels & Ruprecht Ulva rigida C. Agardh could not replace the eco-physiologi- (Nedashkovskaya et al. 2004a, 2010, Weerawongwiwat cal functions of the Maribacter sp. MS6. The uniqueness of et al. 2013), U. mutabilis (Spoerner et al. 2012) and Polysi- the MS6-factors seems to be produced by difficult-to-culture phonia (Nedashkovskaya et al. 2007), where they harbour bacteria, which may accidentally get lost in the laboratory various enzyme activities involved in degradation of, for (Spoerner et al. 2012, Grueneberg et al. 2016). example, algal polysaccharides (Bakunina et al. 2012). Maribacter is a bacterial genus within the Flavobac- In this study, we tested whether the Maribacter genus, teriaceae comprising 20 documented species at present. and possibly closely related genera, could phenocopy Mar- Maribacter are gram-negative, rod shaped cells, which ibacter sp. MS6 in our tripartite model system (Table 1). produce non-diffusible yellow to orange pigments. The To have morphogenesis-inducing strains available for A. Weiss et al.: Morphogenesis of Ulva 199 Spoerner et al. 2012 Spoerner et al. Spoerner et al. 2012 Spoerner et al. Reference Weerawongwiwat et al. 2013 et al. Weerawongwiwat 2008 emend, et al. Cho 2013 et al. Weerawongwiwat 2004a et al. Nedashkovskaya 2010 et al. Nedashkovskaya 2004a et al. Nedashkovskaya 2007 et al. Nedashkovskaya 2004c et al. Nedashkovskaya 2004b et al. Nedashkovskaya et al. 2006 emend, Fukui et al. Yoon 2013 2009 et al. Nedashkovskaya 2006 et al. Nedashkovskaya 2013 et al. Yang

Portugal, Ria Formosa Ria Portugal, Portugal, Ria Formosa Ria Portugal, Geographical origin Geographical Korea, Jeju Island Korea, Ny-Ålesund Spitsbergen, Norway, the Peter of Gulf Japan, of Sea Great Peter of Gulf Japan, of Sea Russia, Bay Troitsa the Great, Russia Peter of Gulf Japan, of Sea Russia, Bay Troitsa the Great, Russia Peter of Gulf Japan, of Sea Russia, Bay Troitsa the Great, Korea South Japan of Sea Bay, Posiet Russia, Bay Pallada Japan, of Sea Fugong province, Fujian China, Area Reservation Nature Mangrove

U. mutabilis U. U. mutabilis U. Source Undaria pinnatifida Undaria sediment Marine sample sediment Bottom fenestrata Ulva fenestrata U. japonica Polysiphonia fenestrata U. fenestrata U. water Sea fenestrata U. fenestrata U. sediment Mangrove

EU359909 EU359911 GenBank GenBank # accession JN036550 AY771762 AY271623 EU246691 AY271626 AM497875 AY187689 AY243096 DQ004686 AB084261 AJ575643 JN426849

T T T T T T T T T T T T CCUG 61948 CCUG MS2 MS6 DSM 23546 DSM 19840 DSM 19891 DSM 15366 DSM 23514 DSM 15365 DSM 16195 DSM 17204 DSM 19858 DSM 17539 DSM 25030 DSM Strain

Maribacter chungangensis Maribacter sp.) Roseobacter y (formerl sp. Roseovarius sp.) Cytophaga (formerly sp. Maribacter Maribacter arcticus Maribacter sedimenticola Maribacter stanieri Maribacter ulvicola Maribacter polysiphoniae Maribacter lectus Algibacter litoralis Ulvibacter dokdonensis Polaribacter thermophila Pseudozobellia palladensis Arenibacter zhangzhouensis Muricauda . mutabilis on Ulva activities on morphogenetic screening bioassay for selected control-strains and type- 1: List other Flavobacteria of and Maribacter Table Name 200 A. Weiss et al.: Morphogenesis of Ulva

Figure 2: Bioassay screening for morphogenetic activity among selected Maribacter strains (rows). Two-week old germlings are shown. Maribacter strains were tested with axenic Ulva gametes alone (left column: A–F) and in combination with Roseovarius sp. MS2 (middle column: G–L) or with Maribacter sp. MS6 (right column: M–R). Yellow framing (F) highlights an axenic-like development and morphotype with protrusions from the exterior cell wall (black arrow). Purple framing (L) shows cell divisions and blade formation with malformed cell walls (black arrow) indicating an MS2-like morphotype. Red framing (A–E, M–R) shows longitudinal growth and normal cell wall formation similar to the MS6-like morphotype. If the tested strain harboured an MS6-like bioactivity and was inoculated with Roseovarius sp. MS2, the complete morphogenesis was observed (green framing, G–K). Scale bars = 100 μm. A. Weiss et al.: Morphogenesis of Ulva 201

Figure 3: Bioassay screening for morphogenetic activity among selected Flavobacteriaceae (rows). Two-week old germlings are shown. Selected strains were tested with axenic Ulva gametes alone (left column: A–F) and in combination with Roseovarius sp. MS2 (middle column: G–L) or with Maribacter sp. MS6 (right column: M–R). Black arrows indicate protrusions from the exterior cell wall. Different colours of frames indicate different morphotypes, as explained in Figure 2 and shown for the control experiments in Figure 1. Scale bars = 100 μm. 202 A. Weiss et al.: Morphogenesis of Ulva bioassays in the long term, cultivable type strains of the no effect on the morphogenesis of U. mutabilis, was iso- cluster Maribacter/Arenibacter/Muricauda and more dis- lated from the red alga Polysiphonia japonica (Figure 2F, tantly related flavobacterial type strains were purchased L). It is thus tempting to assume that mutualistic interac- from the German Collection of Microorganisms and Cell tions have evolved between Ulva and specific Maribacter Cultures (DSMZ). Twelve different type strains were tested strains releasing the MS6-factor or factors similar to the for their MS6-like bioactivity on the morphogenesis of MS6-bioactivity. It is noteworthy that the morphogenetic U. mutabilis (Figures 2 and 3). As the same bacteria (MS2- activity of the MS6-factor from Maribacter sp. MS6, induces and MS6-strain) induce in combination the predisposed the morphogenesis of different Ulva strains and species in morphotype (Spoerner et al. 2012), either wildtype or the same way (Spoerner et al. 2012, Vesty et al. 2015), but it “slender”, the simply organised ribbon-shaped develop- remains to be proven whether the newly-identified bioactive mental mutant “slender”, with a shorter developmental Maribacter strains have a widespread activity on morpho- cycle, was preferred for the bioassays. genesis within the order Ulvales. Interestingly, a general Experiments were started with axenic gametes pre- bioactivity was suggested for thallusin, the first isolated pared and treated under strictly controlled conditions morphogen, retrieved from an epiphytic bacterium associ- (Spoerner et al. 2012). Control experiments have proven ated with Monostroma oxyspermum (Matsuo et al. 2005). that U. mutabilis grown in the absence of symbiotic bacteria In order to assess the distribution of morphogenetic developed into callus-like structures with no cell differen- activities similar to Maribacter sp. in the Flavobacte- tiation and a disturbed cell wall synthesis (Figure 1A). By riaceae, a broader screening of different type strains adding Roseovarius sp. MS2 to axenic cultures, cell division isolated from Ulva spp., seawater samples and from a leads to blade formation, but cell wall protrusions are still mangrove sediment sample was performed (Figure 3). visible (Figure 1B). Under the influence of Maribacter sp., Muricauda zhangzhouensis (Yang et al. 2013), Pseudo- axenic gametes of the “slender” mutant develop into minute zobellia thermophila (Nedashkovskaya et al. 2009) and short rows of degenerated blade cells with normal cell walls Arenibacter palladensis (Nedashkovskaya et al. 2006) and rhizoid formation (Figure 1C). Maribacter sp. MS6 in combination with Roseovarius sp. MS2 results in the morphogenesis being completely restored (Figure 1D). Using the “slender” mutant of U. mutabilis the bioassay was thus used to determine the bacterial ability which induced proper cell wall formation and completed the intrinsic morphogenesis in combination with Roseovarius sp. MS2 (Figures 2 and 3). Cross-testing experiments with bacteria from U. mutabilis on Ulva linza L. have already shown that the combination of MS2- and MS6-strains can also induce the morphogenesis of U. linza. However, bacterial morphogens might have an Ulva-species-specific component, for example, to control the accurate formation of algal hold- fasts (Vesty et al. 2015). The Maribacter strains tested in this study were equally able to induce the morphogenesis of U. mutabilis (slender) regardless of their origin. Indeed, Maribacter chungangensis (Weerawongwiwat et al. 2013), Maribacter Figure 4: Relative morphogenetic activity of the tested stanieri (Nedashkovskaya et al. 2010) and Maribacter ulvi- Flavobacteriaceae. Algae with normal cell wall formation (i.e. without any protrusions) cola (Nedashkovskaya et al. 2004a), isolated from green were counted after 14 days of co-cultivation with following bacte- seaweed, and Maribacter arcticus (Cho et al. 2008) and rial type strains: Maribacter chungangensis, Maribacter arcticus, Maribacter sedimenticola (Nedashkovskaya et al. 2004a), Maribacter sedimenticola, Maribacter stanieri, Maribacter ulvicola, isolated from marine sediment, all displayed similar Maribacter polysiphoniae, Algibacter lectus, Ulvibacter litoralis, morphogenetic activities (Figure 2A–E). Importantly, in Polaribacter dokdonensis, Pseudozobellia thermophila, Arenibacter combination with Roseovarius sp. MS2, U. mutabilis cell palladensis, Muricauda zhangzhouensis and the isolated strains Roseovarius sp. MS2 (formerly Roseobacter sp. MS2) and Maribacter division was enhanced and the complete morphotype was sp. MS6 (formerly Cytophaga sp. MS6). Error bars represent standard developed (Figure 2G–K). The only exception, Maribacter deviation (n = 50–70 individual algae). Roseovarius sp. MS2 and polysiphoniae (Nedashkovskaya et al. 2007), which had Maribacter sp. MS6 were used as control strains for comparison. A. Weiss et al.: Morphogenesis of Ulva 203 showed the same morphogenetic activity on U. mutabilis able to suppress the formation of cell wall protrusions of as the Maribacter strains (Figure 3D–F). However, Algibac- the inspected germlings in the tested population of Ulva. ter lectus (Nedashkovskaya et al. 2004c) and Ulvibacter However, in case of M. ulvicola, half of the investigated litoralis (Nedashkovskaya et al. 2004b), isolated from U. individuals still possessed malformed cell walls (Figure 4). fenestra, and Polaribacter dokdonensis (Yoon et al. 2006), This indicates that the concentration of the morphogen isolated from a seawater sample, had no effect on U. muta- released by the bacterium was close to the threshold con- bilis (Figure 3A–C). Algae inoculated with these strains centration that is required to induce a proper cell wall for- developed into calli with colourless protrusions from mation of all individuals in the bioassay. the exterior cell wall (Figure 1A). Moreover, a detailed Phylogenetic analysis revealed that the morphogenesis- analysis of the degree of formation of cell wall protrusions inducing genera Maribacter, Muricauda, Pseudozobellia and (Figure 4) as a result of a lack of MS6-morphogens con- Arenibacter are more closely related to each other than to the firmed the microscopic observations of the development other genera tested within the Flavobacteriaceae that were of Ulva juveniles (Figures 2 and 3). All active strains were not active (Figure 5). In fact, the inactive M. polysiphoniae

Figure 5: Phylogenetic tree of strains of Flavobacteriaceae tested for morphogenetic activities. Maximum Likelihood (ML) phylogenetic inference of 16S rRNA gene sequences of the genus Maribacter and related genera in the family Flavobacteriaceae (phylum Bacteroidetes). Maribacter sp. strain MS6 (bold) and all type strains found to be MS6 substitutes in Ulva morphogenesis bioassays are marked in green. Test type strains which did not elicit the MS6 morphotype are marked in dark-red. For taxonomic robustness, the tree was constructed only with high-quality, ≥ 1300 bp 16S rRNA gene sequences, mostly from type strains. The gene sequence of strain MBIC04683 (349 bp) was added to the tree a posteriori using the ARB parsimony function. Numbers at tree nodes are bootstrap values calculated in ML analysis, and values ≥ 70% are shown. The unrooted tree is drawn to scale, and the scale bar represents the number of nucleotide substitutions per site (Costa et al. 2013, Keller-Costa et al. 2014). Arrows indicate strains which were identified as morphogenesis-inducing bacteria in previous studies (Matsuo et al. 2003, Spoerner et al. 2012). 204 A. Weiss et al.: Morphogenesis of Ulva displayed somewhat moderate levels of 16S rRNA gene Acknowledgements: This work was supported by the resemblance (94%–96%) with most Maribacter type strains, Deutsche Forschungsgemeinschaft (CRC 1127 ChemBio- being placed next to the Maribacter clade in our phylogenetic Sys to AW and TW), the Jena School for Microbial Com- assessment (Figure 5). However, more exploratory studies of munication (to AW and TW), the European Union Seventh the Flavobacteriaceae and its 100 genera are necessary for a Framework Programme Research Infrastructure Initiative better understanding of the phylogenetic breadth of bacteria (ASSEMBLE-227799 to TW) and the H2020-Marie Sklo- releasing MS6-factors. Attempts to isolate and cultivate Flavo- dowska-Curie Actions (MSCA)-ITN-2014 (grant agreement bacteriaceae species with alternative and specific procedures no. 642575 to TW). The authors would like to acknowledge (Hahnke and Harder 2013) will increase the number of culti- networking support by the COST Action “Phycomorph” vable Flavobacteriaceae available in culture collections and (European Cooperation in Science and Technology, Grant better reflect the distribution and abundance of this family in No.: FA1406). Prof. Dr. Georg Pohnert (University Jena, various marine habitats. In this context, research on specific Germany) is acknowledged for his great support and help- nutrient requirements holds promise for broadening our bio- ful discussion during the preparation of the manuscript. assay-based surveys of Flavobacteriaceae in the future. We thank Dr. Jörn Petersen (DSMZ, German Collection Matsuo et al. (2003) have isolated about 1000 bac- of Microorganisms and Cell Cultures) for essential type teria from algae and sponges. Among these isolates, only strains. We gratefully acknowledge the critical reviews by eight, mainly retrieved from the green algae Ulva and two anonymous reviewers. Monostroma, showed morphogenesis-inducing activity on M. oxyspermum. The main activity of these isolates was attributed to the first identified morphogen, thallusin, References which was isolated from the epiphytic bacterium YM2-23 (deposition No. MBIC 04683) associated with M. oxysper- Bakunina, I.Y., O.I. Nedashkovskaya, S.B. Kim, T.N. Zvyagintseva mum. The compound was also effective in Ulva (Matsuo and V.V. Mikhailov. 2012. Diversity of glycosidase activities in et al. 2005). Moreover, strain UP7 (deposition No. MBIC the bacteria of the phylum Bacteroidetes isolated from marine 01484) induced growth acceleration and the development algae. 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Bull. 114: obtained her Diploma in Biology in the Molecular Botany group (Uni- 375–384. versity Jena) working on natural products in freshwater microalgae. Provasoli, L. and I.J. Pintner. 1980. Bacteria induced polymorphism Her current research interests are the chemical communication of in an axenic laboratory strain of Ulva lactuca (Chlorophyceae). bacteria-macroalgae interactions, bacterial dependent development J. Phycol. 16: 196–201. of Ulva sp. and aquaculture of macroalgae. 206 A. Weiss et al.: Morphogenesis of Ulva

Rodrigo Costa Thomas Wichard Institute for Bioengineering and Biosciences Institute for Inorganic and Analytical (IBB), Department of Bioengineering, Chemistry, Jena School of Microbial Instituto Superior Técnico, Universidade de Communication, Friedrich Schiller University Lisboa, Lisbon, Portugal Jena, Jena, Lessingstr. 8, 07743 Jena, Germany, [email protected]

Rodrigo Costa acquired his PhD degree in Life Sciences from the Thomas Wichard is a Research Group Leader at the Institute for Technical University of Braunschweig, Germany (2006), and cur- Inorganic and Analytical Chemistry of the Friedrich Schiller Univer- rently is an Assistant Professor at the Department of Bioengineering sity Jena. After he had been awarded a PhD in Biochemistry for his of Instituto Superior Técnico (IST), University of Lisbon. His research studies at the Max Planck Institute for Chemical Ecology in Jena, addresses the diversity and function of microorganisms in natural he began investigating the metal recruitment of nitrogen fixers at and fabricated biomes – with emphasis on Eukaryote-Prokaryote the Princeton Environmental Institute (USA). Now the main focus symbioses –, their implications to host/ecosystem health and of his research group is to elucidate the mutualistic interactions climate regulation, and their potential use as renewable sources of between bacteria and the marine macroalga Ulva (“cross-kingdom- innovative biotechnological appliances. cross-talk”). The group applies various methodologies in analytical chemistry, chemical ecology and molecular biology to understand the basis of eco-physiological processes.