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bioRxiv preprint doi: https://doi.org/10.1101/682252; this version posted December 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 EXPERIMENTAL ARTICLES 2 3 Biodiversity of Magnetotactic Bacteria in the Freshwater Lake Beloe Bordukovskoe, Russia 4 V. V. Koziaevaa, *, L. M. Alekseevaa, b, M. M. Uzuna, b, P. Leãoc, M. V. Sukhachevaa, E. O. 5 Patutinaa, T. V. Kolganovaa, and D. S. Grouzdeva 6 7 a Institute of Bioengineering, Research Center of Biotechnology, Russian Academy of Sciences, 8 Moscow, 119071, Russia 9 b Moscow State University, Moscow, 199991, Russia, 10 c Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio 11 de Janeiro, RJ, 21941-902, Brazil 12 * E-mail: [email protected] 13 Received 14 15 Abstract---According to 16S rRNA gene- or genome-based phylogeny, magnetotactic bacteria 16 (MTB) belong to diverse taxonomic groups. Here we analyzed the diversity of MTB in a sample 17 taken from the freshwater lake Beloe Bordukovskoe near Moscow, Russia, by using molecular 18 identification based on sequencing of the 16S rRNA gene and mamK, a specific marker gene for 19 these bacteria. A protein encoded by the mamK gene is involved in magnetosome chain 20 arrangement inside the cell. As a result, six operational taxonomic units (OTUs) of MTB were 21 identified. Among them, OTUs affiliated with the phylum Nitrospirae were predominant. ‘Ca. 22 Etaproteobacteria’ and Alphaproteobacteria represented the minor groups of MTB. We also 23 identified a novel MTB belonging to the family Syntrophaceae of the Deltaproteobacteria class. 24 Using a combination of fluorescence and transmission electron microscopy, the bacteria belonging 25 to these new MTB groups were visualized. Electron microscopy revealed that Syntrophaceae MTB 26 were rod-shaped and synthesized elongated magnetosomes, arranged as a disorganized cluster. 27 Among the Nitrospirae group, two groups with vibrioid cell shape and one group of ovoid-shaped 28 bacteria were identified, all of which had elongated magnetosome crystals consisting of magnetite. 29 Keywords: magnetotactic bacteria, magnetosomes, magnetotaxis, 16S rRNA gene, phylogenetic 30 analysis, Nitrospirae, Syntrophaceae 1 bioRxiv preprint doi: https://doi.org/10.1101/682252; this version posted December 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 31 Prokaryotes capable of directed active movement that is guided by geomagnetism are 32 collectively called magnetotactic bacteria (MTB) (Blakemore, 1975). MTB are physiologically, 33 morphologically and phylogenetically diverse, sharing only the ability to synthesize special 34 organelles termed magnetosomes. Magnetosomes consist of nanometer-size magnetite (Fe3O4) or 35 greigite (Fe3S4) crystals surrounded by a lipid bilayer membrane containing proteins specific to the 36 organelle. Magnetosomes are often assembled into chains inside the cell. MTB evolved the ability 37 to conduct a special type of movement called magnetotaxis, which is based on orientation relative 38 to magnetic field lines (Faivre and Schuler, 2008). Specific genes involved in magnetosome 39 formation were found in the genomes of all MTB. There are currently nine highly conserved 40 primary genes in magnetite- and greigite-producing MTB (mamA, mamB, mamM, mamQ, mamO, 41 mamI, mamP, mamK, and mamE) (Lefèvre and Bazylinski, 2013). One of them, the mamK gene, 42 encodes the actin-like protein MamK, which is responsible for the ordered arrangement of the 43 magnetosome chain in the cell (Uebe and Schüler, 2016). 44 MTB are found within the phyla Proteobacteria, Nitrospirae, Planctomycetes, the candidate 45 phyla ‘Omnitrophica’ and ‘Latescibacteria’ (Dziuba et al., 2016; Lefèvre and Bazylinski, 2013; Lin 46 et al., 2017a; Lin and Pan, 2015). ‘Ca. Etaproteobacteria’ and Alphaproteobacteria are the most 47 commonly observed and extensively characterized types of MTB (Lefèvre and Bazylinski, 2013; 48 Monteil et al., 2018). Many of cultured marine and freshwater MTB belong to the class 49 Alphaproteobacteria, including various vibrios and spirilla affiliated to the genera 50 Magnetospirillum, Magnetovibrio, Magnetospira, and Terasakiella (Lefèvre and Bazylinski, 2013; 51 Monteil et al., 2018). Numerous magnetotactic cocci and rods are affiliated to the order 52 Magnetococcales of the class ‘Ca. Etaproteobacteria’ (Koziaeva et al., 2019). The two 53 Deltaproteobacteria orders, Desulfovibrionales and the Desulfobacteriales, also contain 54 magnetotactic members. Cultured strains Desulfovibrio magneticus RS-1, FH-1, ML-1, AV-1, ZZ- 55 1, and an uncultured bacterium WYHR-1 belong to the order Desulfovibrionales (Lefèvre and 56 Bazylinski, 2013; Li et al., 2019). The order Desulfobacteriales contains the strains Desulfamplus 57 magnetovallimortis BW-1T and SS-2, as well as several uncultured multicellular magnetotactic 58 prokaryotes (MMP) (Abreu et al., 2007; Leão et al., 2017; Lefèvre and Bazylinski, 2013). 59 Axenic cultures of MTB of the Nitrospirae phylum have not yet been obtained. According 60 to Parks et al., the most well-known members of this group form the candidate family 61 ‘Magnetobacteraceae’. The family ‘Ca. Magnetobacteriaceae’ currently includes several candidate 62 genera, which accommodate only MTB: ‘Ca. Magnetobacterium, ‘Ca. Magnetoovum’, and ‘Ca. 63 Magnetominusculus’ (Lefèvre and Bazylinski, 2013; Lin et al., 2011, 2017b). Most of MTB 64 belonging to the Nitrospirae phylum were found in mesophilic freshwater habitats, with the 65 exception of two representatives, WGC and BGC, the recently described giant cocci with multiple 2 bioRxiv preprint doi: https://doi.org/10.1101/682252; this version posted December 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 66 chains of magnetosomes (Qian et al., 2019), and multiple sequences retrieved from the Yelow Sea 67 in China (Xu et al., 2018). A thermophilic MTB named ‘Ca. Thermomagnetovibrio paiutensis’ 68 HSMV-1 was also described. It is closely related to cultured Thermodesulfovibrio spp. 69 Despite the high diversity of MTB found in environmental samples, they are difficult to 70 isolate in axenic culture. Therefore, culture-independent techniques are indispensable in research on 71 these bacteria. Members of the phyla Proteobacteria and Nitrospirae have been studied extensively. 72 However, analyses of metagenomic datasets revealed the presence of MTB within the phyla 73 Planctomycetes and ‘Ca. Latescibacteria’ (Lin and Pan, 2015; Lin et al., 2017a). This finding 74 indicates that the biodiversity of MTB is much broader than is currently known. Therefore, 75 modifying and developing the strategies for investigation offers great promise towards identifying 76 MTB groups. Another challenge in MTB biodiversity investigation is detailed characterization of 77 cell morphology and crystallographic properties of their magnetosomes. A recently developed 78 method of coordinated fluorescence in situ hybridization and electron microscopy (FISH-TEM) 79 successfully linked the phylogeny with cell ultrastructure and magnetosome morphology of several 80 uncultured MTB strains, including the Gammaproteobacteria strain SHHR-1, Deltaproteobacteria 81 strain WYHR-1 and ‘Ca. Magnetaquicoccus inordinatus’ UR-1 (Li et al., 2017, 2019; Koziaeva et 82 al., 2019). 83 Here we describe a diversity of MTB in the lake Beloe Bordukovskoe based on a novel 84 isolation approach that does not depend on cell motility. For phylogenetic identification of collected 85 cells, we used a universal primer system on the marker gene mamK and analysis of the 16S rRNA 86 gene sequences. To characterize the morphology of cells and magnetosomes of bacteria of retrieved 87 OTUs, the FISH-TEM method was applied. 88 MATERIALS AND METHODS 89 Sampling and MTB enrichment. Water and sediment samples were collected at the 90 freshwater lake Beloye Bordukovskoe, Shatura District, Russia (55°37'56"N, 39°44'38"E). Samples 91 with a 1 : 2 sediment : water ratio were used to create a microcosm (3 L) and incubated in the dark 92 at room temperature for one year. The pH value and salinity were measured using an Acvilon pH 93 meter (Russia) and a handheld portable refractometer, respectively. The elemental composition of 94 organic carbon (С%) in the sediment was measured using a Flash 1112 elemental analyzer coupled 95 with a Thermo-Finnigan Delta V Plus isotope mass spectrometer (Thermo Fisher Scientific, United 96 States) at the Core Facility of the Severtsov Institute of Ecology and Evolution, Russian Academy 97 of Sciences. 98 Magnetic collection of MTB was performed using the MTB-CoSe (magnetotactic bacteria 99 column separation) method developed in our laboratory (Fig. 1). Initially, sediment cores (15 mm in 3 bioRxiv preprint doi: https://doi.org/10.1101/682252; this version posted December 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 100 diameter) were taken from three different sites of the microcosm and mixed. To desorb the cells 101 from large clay particles, PBS buffer (final concentration 1.25×) and glass microbeads (2 g; 150– 102 200 µm diameter;

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