The ISME Journal (2017) 11, 1075–1086 OPEN © 2017 International Society for Microbial Ecology All rights reserved 1751-7362/17 www.nature.com/ismej ORIGINAL ARTICLE Sticking together: inter-species aggregation of bacteria isolated from iron snow is controlled by chemical signaling Jiro F Mori1, Nico Ueberschaar2, Shipeng Lu1,3, Rebecca E Cooper1, Georg Pohnert2 and Kirsten Küsel1,3 1Institute of Ecology, Friedrich Schiller University Jena, Jena, Germany; 2Institute of Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Jena, Germany and 3The German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Leipzig, Germany Marine and lake snow is a continuous shower of mixed organic and inorganic aggregates falling from the upper water where primary production is substantial. These pelagic aggregates provide a niche for microbes that can exploit these physical structures and resources for growth, thus are local hot spots for microbial activity. However, processes underlying their formation remain unknown. Here, we investigated the role of chemical signaling between two co-occurring bacteria that each make up more than 10% of the community in iron-rich lakes aggregates (iron snow). The filamentous iron- oxidizing Acidithrix strain showed increased rates of Fe(II) oxidation when incubated with cell-free supernatant of the heterotrophic iron-reducing Acidiphilium strain. Amendment of Acidithrix supernatant to motile cells of Acidiphilium triggered formation of cell aggregates displaying similar morphology to those of iron snow. Comparative metabolomics enabled the identification of the aggregation-inducing signal, 2-phenethylamine, which also induced faster growth of Acidiphilium.We propose a model that shows rapid iron snow formation, and ultimately energy transfer from the photic zone to deeper water layers, is controlled via a chemically mediated interplay. The ISME Journal (2017) 11, 1075–1086; doi:10.1038/ismej.2016.186; published online 31 January 2017 Introduction of organic matter and its subsequent release into the surrounding water (Grossart and Simon, 1998). Pelagic aggregates form in the water column through Pelagic aggregates are local hot spots for microbial adsorption of inorganic and organic matter, includ- interactions by direct cell contact, feeding activity ing bacteria, phytoplankton, feces, detritus and bio- and also the action of diffusible signals. For example, minerals (Alldredge and Silver, 1988; Simon et al., α-Proteobacteria, including Roseobacter strains, 2002; Thornton, 2002). These aggregates, also called growing on the surface of marine snow produce marine or lake snow, typically range in size from elevated amounts of inhibitory molecules compared millimeters to centimeters (Alldredge and Silver, with planktonic forms in the surrounding aquatic 1988; Passow et al., 2012) and are held together by environment (Long and Azam, 2001). N-acyl homo- extracellular polysaccharides (Thornton, 2002; Giani serine lactones, compounds known to function as et al., 2005; Passow et al., 2012). Marine and lake potential quorum-sensing mediators, are produced snow contribute substantially to the energy transfer by Roseobacter strains (Gram et al., 2002; Zan et al., from the photic zone to deeper water layers (Suess, 1980). During the passage to the sediments, many 2014) and Pantoea sp. (Jatt et al., 2015) in marine motile bacteria and zooplankton colonize these snow communities, suggesting that quorum-sensing organic-rich particles and remain attached as the mechanisms might be at play. Chemical signaling aggregates sink through the water column to the molecules have also been suspected to regulate sediment (Fenchel, 2001; Kiørboe et al., 2003). bacterial colonization, coordinate group behavior, Particle-associated bacteria show higher extracellu- as well as antagonistic activities within pelagic lar enzymatic activities than free-living, planktonic aggregates (Dang and Lovell, 2016). However, the microbes, resulting in rapid and localized turnover exact mechanisms for chemical communication and the identity of the compounds mediating such interactions in pelagic aggregates are poorly Correspondence: K Küsel, Institute of Ecology, Friedrich Schiller understood. University Jena, Dornburger Strasse 159, 07743 Jena, Germany. E-mail: [email protected] The chemical diversity of exuded metabolites Received 19 August 2016; revised 25 October 2016; accepted within such aggregates makes the identification and 15 November 2016; published online 31 January 2017 elucidation of infochemicals highly challenging, due Chemically induced bacterial aggregation JF Mori et al 1076 to the loss of the diluted signals during the purifica- cell observations and bacterial 16S rRNA gene tion process (Prince and Pohnert, 2010). However, sequencing. comparative metabolomics has emerged as a power- Phylogenetic analyses and identification of the ful tool to overcome the limitations of bioassay- isolates were carried out by genomic DNA extraction guided approaches by allowing the identification and 16S rRNA gene sequencing. Cell colonies were of metabolites produced by microorganisms grown suspended into 20 μl of lysis solution containing in co-culture by comparison with those of single- 0.05 M NaOH and 0.25% SDS and heated for 15 min strain cultures. Notably, upregulated metabolites at 95 °C, followed by centrifugation at 4000 g for are prime-candidates for mediators of microbial 5 min to obtain genomic DNA in the supernatant. A interactions (Gillard et al., 2013; Kuhlisch and 1 μl of the supernatant was subjected to PCR Pohnert, 2015). employing primer set 27F/1492R (Lane, 1991). The In this study, we isolated two predominant PCR product was purified through a spin column bacterial key players from iron-rich lake aggregates (NucleoSpin Gel and PCR Clean-up, Macherey- (iron snow) to investigate chemical communication Nagel, Düren, Germany) and sequenced (Macrogen mechanisms involved in their interactions. Iron Europe, Amsterdam, The Netherlands). Raw snow is characterized by lower microbial and sequences were processed in Geneious 4.6.1 for chemical complexity in comparison with typical trimming and assembling, followed by BLAST organic-rich marine and lake snow aggregates, and homology search (Johnson et al., 2008). Sequences shows high sinking velocities because of its high of the representative isolates were submitted to relative fraction of iron (Reiche et al., 2011). We GenBank database under accession numbers report that an active control by chemical signals LN866581 to LN866603 (Supplementary Table S1). shapes the association of the dominant microbial consortia within the iron snow and their behavior. Comparative metabolomics approach and structure Growth tests of selected bacterial isolates elucidation led to the identification of bacterial Selected bacterial isolates Acidithrix strain C25 extracellular exudates that function as allelopathic and Acidiphilium strain C61 from the central aggregate-inducing signal. These results clearly show basin were transferred into different types of liquid that even during the very limited time of passage media (Supplementary Table S2), and their growth through the water column, interspecies chemical was tested to figure out an appropriate medium interactions between key organisms in iron snow suitable for co-cultivating different bacterial species. enable these microorganisms to adhere to and The artificial pilot-plant water (APPW) medium − 1 − 1 colonize the particles. (pH 2.5, 25 mM FeSO4, 0.022 g l Na2SO4, 0.024 g l − 1 − 1 K2SO4, 3.24 g l MgSO4·7H2O, 0.515 g l CaSO4· − 1 − 1 2H2O, 0.058 g l NaHCO3, 0.010 g l NH4Cl, − 1 − 1 0.014 g l Al2(SO4)3·18H2O, 0.023 g l MnCl2·4H2O, Materials and methods − 1 0.0004 g l ZnCl2) reported by Tischler et al. (2013) − 1 Sampling, bacterial isolation and identification with additional yeast extract (0.2 g l ) was tested in For bacterial isolation from iron snow, lake water addition to iFeo, Feo and YEo. was collected in the water column of the central basin (pH 2.8–3.0) and the northern basin (pH 5.4– 5.6) just below the redoxcline of the acidic lignite Monitoring of cell growth and morphology mine lake (Lake 77) located in the Lusatian mining Bacterial growth was confirmed by microscope, area in Germany, as described in a previous report growth of Fe(II)-oxidizing bacteria was also deter- mined by checking rust-colored Fe(III) oxide precipi- (Mori et al., 2016). Sampling was carried out in tates in the culture and measuring concentrations of September 2012, January and November 2013. The Fe(II) and total dissolved Fe using the phenanthroline lake water was collected and stored at 4 °C and method (Tamura et al., 1974). Bacterial cell morphol- serially diluted (10 − 1 to 10 − 3) with filter-sterilized μ ogy was observed under a fluorescence microscope lake water and 100 l of the dilution was plated onto (Axioplan, Zeiss, Oberkochen, Germany). Cells col- three types double-layer media (Johnson and – lected from the culture were stained with SYTO 13 Hallberg, 2007; Lu et al., 2013; pH 2.5 5.5): iFeo, nucleic acid staining (Thermo Scientific, Waltham, Feo and YEo that contain 25 mM ferrous sulfate and MA, USA) on glass slides and visualized. The 0.25% tryptone soya broth (Feo) or 0.2% yeast fluorescent images were recorded by a mono color extract (YEo), or without carbon source (iFeo). camera (Axio Cam MRm, Zeiss). Total nucleic acids Cycloheximide (Carl Roth, Karlsruhe, Germany) − extraction from bacterial cells and subsequent