Systematic and Applied Microbiology 42 (2019) 284–290 Contents lists available at ScienceDirect Systematic and Applied Microbiology jou rnal homepage: http://www.elsevier.com/locate/syapm Hymenobacter amundsenii sp. nov. resistant to ultraviolet radiation, ଝ,ଝଝ, isolated from regoliths in Antarctica a,∗,1 b,1 a b Ivo Sedlácekˇ , Roman Pantu˚ cekˇ , Stanislava Králová , Ivana Maslaˇ novᡠ, a a a,b a c Pavla Holochová , Eva Stankovᡠ, Veronika Vrbovská , Pavel Svecˇ , Hans-Jürgen Busse a Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic b Section of Molecular Biology, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic c Institut für Mikrobiologie, Veterinärmedizinische Universität Wien, Veterinärplatz 1, A-1210 Wien, Austria a r a t i b s c l e i n f o t r a c t Article history: A group of thirteen bacterial strains was isolated from rock samples collected in a deglaciated northern Received 28 February 2018 part of James Ross Island, Antarctica. The cells were rod-shaped, Gram-stain-negative, non-motile, cata- Received in revised form lase positive, and produced moderately slimy, ultraviolet light (UVC)-irradiation-resistant and red–pink 27 November 2018 pigmented colonies on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, Accepted 9 December 2018 extensive biotyping, fatty acid profile, chemotaxonomy analyses, and whole genome sequencing were applied in order to clarify the taxonomic position of these isolates. Phylogenetic analysis based on the 16S Keywords: rRNA gene indicated that all isolates constituted a coherent group belonging to the genus Hymenobacter. Hymenobacter amundsenii T T Antarctica The closest relatives to the representative isolate P5136 were Hymenobacter psychrophilus BZ33r and T Hymenobacter rubripertinctus CCM 8852 , exhibiting 97.53% and 97.47% 16S rRNA pairwise similarity, Soil bacteria Regolith respectively. Average nucleotide identity calculated from the whole-genome sequencing data supported T UV-radiation resistance the finding that P5136 represents a distinct Hymenobacter species. The major components in fatty acid Species description profiles were Summed Feature 3 (C16:1 ω7c/C16:1 ω6c), C16:1 ω5c, C15:0 iso and C15:0 anteiso. The cellular quinone content contained unanimously menaquinone MK-6 and MK-7 (ratio 1:5.1). The predominant polar lipid was phosphatidylethanolamine, and moderate to minor amounts of two unknown polar lipids, two unknown aminolipids, one unknown glycolipid and two unknown glycophospholipids were present. The G + C content of genomic DNAs is 60.31 mol%. Based on all the obtained results, we propose a novel T species for which the name Hymenobacter amundsenii sp. nov. is suggested, with the type strain P5136 T T (= CCM 8682 = LMG 29687 ). © 2018 Elsevier GmbH. All rights reserved. Introduction Investigations of the diversity and taxonomy of psychrotrophs have increased over the last two decades, and revealed a high Abbreviations: ANI, average nucleotide identity; FAME, fatty acid methyl esters; complexity within Antarctic microbiota. Numerous novel species, WGS, whole-genome sequencing; MK, menaquinone. mainly from the Cytophaga–Flavobacterium–Bacteroides group, ଝ Subject category: New taxa (Bacteroidetes). have been discovered by cultivation as well as by molecular meth- ଝଝ The GenBank/EMBL/DDBJ accession number for the complete 16S rRNA gene ods [1–4], which has led to rapid changes in the taxonomy of the T T sequence of Hymenobacter amundsenii P5136 = CCM 8682 is MF782439. The Gen- phylum Bacteroidetes [5,6]. The genus Hymenobacter was described Bank/EMBL/DDBJ accession numbers for the partial 16S rRNA genes of other H. amundsenii isolates are MF782440-MF782451. twenty years ago [7] and subsequently the genus description was T The Whole-Genome Shotgun project of H. amundsenii P5136 have been amended [8,9]. At present, the genus Hymenobacter represents a deposited at GenBank/EMBL/DDBJ under the accession NZ NIRR00000000. ∗ member of the family Hymenobacteraceae, phylum Bacteroidetes, Corresponding author at: Masaryk University, Department of Experimental Biol- including 65 species with validly published names (http://www. ogy, Czech Collection of Microorganisms, Building A25, Kamenice 5, 625 00 Brno, bacterio.net/hymenobacter.html; accessed September, 2018) [10]. Czech Republic. E-mail address: [email protected] (I. Sedlácek).ˇ Based on the phylogenetic clustering of Hymenobacter spp., two 1 These authors contributed equally to this work. https://doi.org/10.1016/j.syapm.2018.12.004 0723-2020/© 2018 Elsevier GmbH. All rights reserved. I. Sedlácekˇ et al. / Systematic and Applied Microbiology 42 (2019) 284–290 285 T new genera Siccationidurans and Parahymenobacter were proposed of type strain H. rubripertinctus CCM 8852 was resequenced in the [11] but these names had not yet been validated. frame of this study (GenBank accession no. MH763858) since the The majority of Hymenobacter species are pigmented brick-red 5 end of sequence MF770258 was not properly corrected. Pairwise or pink with many shades of colour [12], which facilitates their iso- sequence alignment and calculation of similarity values was carried lation from primocultures. Hymenobacter spp. have been retrieved out by the algorithm used in the EzBioCloud (formerly EzTaxon) from various polar environments in recent years, e.g. from sedi- service [24]. 16S rRNA gene sequences of other species of the genus ment in the permafrost [9,13], Antarctic soils [7,14], fragmentary Hymenobacter retrieved from the Gen-Bank/EMBL/DDJB database rocks [15], glacial till [16] or glacier ice [17]. Some hymenobacters were used for phylogenetic analysis performed using the software are unique in being radiation-resistant [18–20], UV-resistant [21] MEGA version 7 [25]. The evolutionary history was inferred using or desiccation-resistant [8,22]. the neighbour-joining (NJ) and maximum-likelihood (ML) meth- Here we describe the novel UV-resistant species of the genus ods, using a bootstrap test based on 500 replications. Hymenobacter, isolated in 2013 from regoliths in Antarctica. A total of several hundred strains were isolated in the frame of the project focused on psychrophilic soil bacteria from Antarctica and analysed Genome analysis and calculating average nucleotide identity by 16S rRNA sequencing. A small group of 13 red–pink pigmented (ANI) values isolates was nearly identical in 16S rRNA sequence analysis and in TM this study these strains were characterized further. WGS sequencing was performed using an Ion Torrent Per- TM sonal Genome Machine (Ion PGM ). The purified genomic DNA T Material and methods of strain P5136 was used for preparing a 400-bp sequencing library with an Ion Plus Fragment Library Kit (Thermo Fisher Isolation, cultivation and reference strains Scientific) as described previously [26]. The sample was loaded onto a 318v2 chip and sequenced using an Ion PGM Hi-Q View Sampling sites were situated in a deglaciated northern part of sequencing kit (Thermo Fisher Scientific). Quality trimming of the James Ross Island, Antarctica (Table 1). Sampling was carried out reads were performed with the Ion Torrent Suite of software (ver- by dispersing 1 g of regolith in 5 ml of sterile saline solution, and sion 5.0.4) with default settings. The assembly computation and 100 ␮l of this suspension was spread by l-loop on the surface of error correction was performed using the Assembler SPAdes 3.9 ◦ an R2A (Oxoid) agar plate and cultivated at 15 C for up to 5 days. with default parameters for Ion Torrent data. The total length of A total of several hundred strains were isolated in the frame of the assembly contained 4,582,236 bp. Assembled contigs larger the project focused on psychrophilic soil bacteria from Antarctica. than 200 bp were used for subsequent analysis. For primal analy- Individual red–pink pigmented colonies were continuously picked sis, the genome was annotated using RAST [27]. To calculate the ◦ out, purified by repeated streaking on R2A medium at 15 C, and the ANI value, the OrthoANI algorithm implemented on the EzBio- ◦ obtained pure cultures were maintained at −70 C until analysed. A Cloud server (http://www.ezbiocloud.net/tools/ani) was used [28]. small group of 13 red–pink pigmented isolates was nearly identical Whole-genome sequences of the related H. psychrophilus CGMCC T in 16S rRNA gene sequence analysis and these strains were charac- 1.8975 (GenBank accession NZ FNOV00000000) and H. actinoscle- T terized further in this study. Reference strains of the phylogenetic rus DSM 15310 (GenBank accession NZ FOHS01000001) were T relatives Hymenobacter psychrophilus CCM 8561 , Hymenobacter obtained from the NCBI database. Whole-genome sequence of H. T T T actinosclerus CCM 8740 , Hymenobacter aerophilus CCM 8584 and rubripertinctus CCM 8852 was determined in this study (GenBank T Hymenobacter rubripertinctus CCM 8852 were retrieved from the accession NZ QYCN00000000). Czech Collection of Microorganisms (http://www.sci.muni.cz/ccm/ ). Further genotypic differentiation of isolates Construction of phylogenetic trees based on 16S rRNA genes The genomic relatedness among isolated strains was studied by TM automated ribotyping with the EcoRI restriction enzyme using a DNA for molecular analyses was extracted with FastPrep RiboPrinter Microbial Characterization System (DuPont Qualicon) Lysing Matrix type B and a FastPrep Homogenizer