Characterization of the first cultured representative ofa Bacteroidetes clade specialized on the scavenging of cyanobacteria Wajdi Ben Hania, Manon Joseph, Boyke Bunk, Cathrin Sproer, Hans-Peter Klenk, Marie-Laure Fardeau, Stefan Spring To cite this version: Wajdi Ben Hania, Manon Joseph, Boyke Bunk, Cathrin Sproer, Hans-Peter Klenk, et al.. Character- ization of the first cultured representative of a Bacteroidetes clade specialized on the scavenging of cyanobacteria. Environmental Microbiology, Society for Applied Microbiology and Wiley-Blackwell, 2017, 19 (3), pp.1134 - 1148. 10.1111/1462-2920.13639. hal-01621708 HAL Id: hal-01621708 https://hal-amu.archives-ouvertes.fr/hal-01621708 Submitted on 3 Jul 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Environmental Microbiology (2017) 19(3), 1134–1148 doi:10.1111/1462-2920.13639 Characterization of the first cultured representative of a Bacteroidetes clade specialized on the scavenging of cyanobacteria Wajdi Ben Hania,1 Manon Joseph,1 Boyke Bunk,2 sequences of the MgMjR-022 clade and cyanobacte- Cathrin Sproer,€ 3 Hans-Peter Klenk,4† ria in the suboxic zone of hypersaline mats points to Marie-Laure Fardeau1 and Stefan Spring4* a specific dependence of members of this clade on 1Laboratoire de Microbiologie IRD, MIO, Aix Marseille decaying cyanobacteria. Based on a comparative Universite, Marseille, France. analysis of phenotypic, genomic and ecological char- 2Department Microbial Ecology and Diversity Research, acteristics we propose to establish the novel taxa Leibniz Institute DSMZ-German Collection of Salinivirga cyanobacteriivorans gen. nov., sp. nov., T Microorganisms and Cell Cultures, Braunschweig, represented by the type strain L21-Spi-D4 , and Sali- Germany. nivirgaceae fam. nov., comprising sequences of the 3Department Central Services, Leibniz Institute MgMjR-022 clade. DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. 4Department Microorganisms, Leibniz Institute Introduction DSMZ-German Collection of Microorganisms and Cell Photosynthetically active laminated microbial mats repre- Cultures, Braunschweig, Germany. sent important model systems for the study of early life on earth and other planets due to a resemblance to distinct Summary fossilized organo-sedimentary structures known as stro- matolites, which represent the first record of life on earth The anaerobic, mesophilic and moderately halophilic and can be traced back to several billion years old rock T strain L21-Spi-D4 was recently isolated from the formations (Grotzinger and Knoll, 1999; Dupraz and suboxic zone of a hypersaline cyanobacterial mat Visscher, 2005). In several hypersaline lakes on the using protein-rich extracts of Arthrospira (formerly Kiritimati Atoll, Central Pacific, thick gelatinous cyanobac- Spirulina) platensis as substrate. Phylogenetic analy- terial mats can be found which are associated with large ses based on 16S rRNA genes indicated an affiliation reticulate microbialites situated in the anoxic zone below of the novel strain with the Bacteroidetes clade the mat. It is assumed that the degradation of extracellular MgMjR-022, which is widely distributed and abundant polymeric substances in the deep anoxic zone of these in hypersaline microbial mats and heretofore com- mats plays an important role in the observed lithification prised only sequences of uncultured bacteria. process (Arp et al., 2012). Members of the Bacteroidetes Analyses of the complete genome sequence of strain phylum are well-known for their ability to degrade polymer- L21-Spi-D4T revealed a possible specialization on the ic compounds (Fernandez-G omez et al., 2013) and have degradation of cyanobacterial biomass. Besides been detected in high abundance and diversity in various genes for enzymes degrading specific cyanobacterial hypersaline mats by using cultivation-independent meth- proteins a conspicuous transport complex for the ods (Farıas et al., 2014; Wong et al., 2016). Therefore, it is polypeptide cyanophycin could be identified that is likely that Bacteroidetes play a key role in the degradation homologous to typical polysaccharide utilization loci and cycling of mat compounds. of Bacteroidetes. A distinct and reproducible co- An analysis of the phylogenetic stratigraphy of the Kiriti- occurrence pattern of environmental 16S rRNA gene mati hypersaline mat revealed that different clades of Bacteroidetes prevail in distinct layers of the mat Received 9 September, 2016; revised 26 November, 2016; (Schneider et al., 2013) reflecting a profound niche separa- accepted 1 December, 2016. *For correspondence. E-mail ssp@ tion within this group of bacteria. In the upper photic-oxic dsmz.de; Tel. (10049) 531 2616 233; Fax (10049) 531 2616 418. †Present address: School of Biology, Newcastle University, Newcastle zone bacteria affiliated with the genera Salinibacter and Upon Tyne, UK Salisaeta were clearly dominating. Representatives of VC 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Cyanobacteria foraging in hypersaline mats 1135 these genera are extremely halophilic and probably can observed phylogenetic divergence within the MgMjR-022 use light as additional energy source for mixotrophic clade matches most closely the taxonomic rank of a family. growth, which explains their high abundance in association The closest related cultured strains were found within the with endoevaporitic deposits on the surface of microbial Marinilabiliaceae. Based on a comparison with 16S rRNA mats (e.g., Sahl et al., 2008). Bacteria representing this sequences of type strains the most closely related type spe- clade have traditionally been classified within the family cies were Marinilabilia salmonicolor NBRC 15948T (86.2%), Rhodothermaceae of the Bacteroidetes, but according to a Anaerophaga thermohalophila Fru22T (85.8%), Alkaliflexus recent proposal both genera should be assigned to the imshenetskii Z-7010T (85.8%), Alkalitalea saponilacus SC/ family Salinibacteraceae and transferred from the Bacteroi- BZ-SP2T (85.6%) and Natronoflexus pectinivorans AP1T detes to the novel sister phylum Rhodothermaeota (Munoz (85.6). These 16S rRNA sequence identity values were et al., 2016). On the other hand, in the deeper layers of the below the threshold of 86.5% proposed for assigning Kiritimati hypersaline mat phylotypes related to members sequences to a family (Yarza et al., 2014), therefore con- of the Marinilabiaceae became dominating and reached firming the placement of strain L21-Spi-D4T in a distinct proportions of up to 25% of the total number of retrieved novel family within the order Marinilabiliales. bacterial 16S rRNA gene sequences. The family Marinila- biaceae and related phylogenetic groups were recently transferred to the order Marinilabiliales (Wu et al., 2016), Phenotypic characteristics which represents mainly facultatively aerobic or anaerobic Morphology. Cells of strain L21-Spi-D4T were Gram- bacteria with a fermentative metabolism that have been negative straight to slightly curved slender rods, often with frequently isolated from marine and hypersaline anoxic a hook at one end. They had an average size of 0.4–0.5 sediments (e.g., Shalley et al., 2013; Iino et al., 2015; Wu mm 3 5–8 mm, were non motile and occurred single or in et al., 2016). pairs. An active bending or flexing of cells as described for In a previous study extracts of the cyanobacterium most representatives of the Marinilabiliaceae was not Arthrospira (formerly Spirulina) platensis containing around detected. Formation of cell aggregates was occasionally 60% single-cell protein were used as substrate to isolate observed in liquid media, but no visible colonies were strain L21-Spi-D4T representing phylotypes affiliated with a formed on solid agar media, so that gliding motility on sur- novel family-level clade of the class Bacteroidia that have faces could not be determined. In older cultures bulged been frequently detected in the suboxic zone of hypersa- cells and spherical bodies were found (Fig. 2A and B). line mats (Spring et al., 2015). In this study, we present a Thin cuts viewed by transmission electron microscopy detailed analysis of the spatial distribution and characteris- revealed a cell wall structure typical of Gram-negative bac- tics of the novel strain, which revealed specific adaptations teria comprising an outer membrane, periplasmic space to the degradation of cyanobacterial biomass. and cytoplasmic membrane. Occasionally, protrusions of the outer membrane could be detected (Fig. 2C and D). Results and discussion Pigments were not formed and no flagella, endospores or Phylogenetic placement within the Bacteroidia other intracellular inclusion bodies became apparent by microscopy. Phylogenetic