J. Microbiol. Biotechnol. (2017), 27(4), 825–833 https://doi.org/10.4014/jmb.1701.01047 Research Article Review jmb Genomic Analysis of a Freshwater Actinobacterium, “Candidatus Limnosphaera aquatica” Strain IMCC26207, Isolated from Lake Soyang Suhyun Kim, Ilnam Kang, and Jang-Cheon Cho* Department of Biological Sciences, Inha University, Incheon 22212, Republic of Korea Received: January 16, 2017 Revised: February 3, 2017 Strain IMCC26207 was isolated from the surface layer of Lake Soyang in Korea by the dilution- Accepted: February 6, 2017 to-extinction culturing method, using a liquid medium prepared with filtered and autoclaved lake water. The strain could neither be maintained in a synthetic medium other than natural freshwater medium nor grown on solid agar plates. Phylogenetic analysis of 16S rRNA gene First published online sequences indicated that strain IMCC26207 formed a distinct lineage in the order February 7, 2017 Acidimicrobiales of the phylum Actinobacteria. The closest relative among the previously *Corresponding author identified bacterial taxa was “Candidatus Microthrix parvicella” with 16S rRNA gene sequence Phone: +82-32-860-7711; similarity of 91.7%. Here, the draft genome sequence of strain IMCC26207, a freshwater Fax: +82-32-232-0541; actinobacterium, is reported with the description of the genome properties and annotation E-mail: [email protected] summary. The draft genome consisted of 10 contigs with a total size of 3,316,799 bp and an average G+C content of 57.3%. The IMCC26207 genome was predicted to contain 2,975 protein-coding genes and 51 non-coding RNA genes, including 45 tRNA genes. Approximately 76.8% of the protein coding genes could be assigned with a specific function. Annotation of the IMCC26207 genome showed several traits of adaptation to living in oligotrophic freshwater environments, such as phosphorus-limited condition. Comparative genomic analysis revealed that the genome of strain IMCC26207 was distinct from that of “Candidatus Microthrix” strains; therefore, we propose the name “Candidatus Limnosphaera aquatica” for this bacterium. pISSN 1017-7825, eISSN 1738-8872 Copyright© 2017 by Keywords: Actinobacteria, “Candidatus Microthrix,” “Candidatus Limnosphaera,” genome, The Korean Society for Microbiology freshwater, dilution-to-extinction culturing and Biotechnology Introduction habitats, where these bacteria can constitute more than 50% of the total epilimnetic bacterial community [4, 5]. Molecular Freshwater ecosystems have unique planktonic bacterial approaches based on sequences of metagenomes and 16S communities that are distinct from bacterial communities rRNA genes have shown the abundance, phylogenetic in other habitats, such as soil and marine environments. diversity, and functional potential of freshwater actinobacteria The freshwater bacterial community is typically dominated [6], revealing that major freshwater actinobacteria are as- by the phyla Actinobacteria, Proteobacteria (mainly the yet-uncultivated in spite of their enormous abundance and classes Alphaproteobacteria and Betaproteobacteria), and diversity. Recent studies using single-cell genomics have Bacteroidetes [1, 2]. Considering the crucial roles of additionally revealed putative ecological niches of the acI freshwater environments in global carbon cycling and clade, one of the most abundant groups among freshwater climate change [3], it is important to study the diversity actinobacteria [7, 8]. However, the physiology and genome and function of the freshwater bacterial communities, properties of many actinobacterial groups that play crucial which mediate diverse biogeochemical reactions. roles in biogeochemical processes in freshwater environments The phylum Actinobacteria is a common and often have mainly remained unknown because the major numerically important component in a variety of freshwater freshwater actinobacterial groups, such as acI and acIV April 2017 ⎪ Vol. 27⎪ No. 4 826 Kim et al. clades, have not been cultivated. 200 µM KCl, 300 µM MgSO4·7H2O, 500 µM CaCl2·2H2O, 300 µM The genus “Candidatus Microthrix” affiliated with the order NaHCO3, 117 nM FeCl3·6H2O, 9 nM MnCl2·4H2O, 0.8 nM ZnSO4·7H2O, Acidimicrobiales of the phylum Actinobacteria currently 0.5 nM CoCl2·6H2O, 0.3 nM Na2MoO4·2H2O, 1 nM Na2SeO3, 1 nM NiCl ·6H O, and 1:5,000 diluted Va vitamin mixture [17] per 1 L of contains two filamentous Candidatus species, retrieved 2 2 from activated sludge of wastewater treatment plants [9- distilled water) amended with the above carbon mixtures were prepared. In addition, the colony-forming ability of strain 11]. Genomic and metagenomic analyses uncovered the IMCC26207 was tested by spotting 10 µl of viable cultures onto metabolic versatility of “Candidatus Microthrix parvicella,” plates of R2A, 1/3 R2A, and 1/10 R2A. which is highly abundant in sludge environments [12]. Several studies in freshwater ecosystems have described Culture Conditions, Electron Microscopy, and Genomic DNA bacterial groups related to the genus “Candidatus Microthrix,” Preparation using cultivation-independent molecular analyses [13, 14]. Strain IMCC26207 was inoculated into 4 L of the liquid medium, To our best knowledge, however, there have been no LNFMC, the same culture medium used for the isolation, and reports on the isolation and genomic characterization of grown aerobically at 15°C. During the cultivation, cell densities freshwater bacteria phylogenetically related to the “Candidatus were monitored using a flow cytometer (Guava EasyCyte Plus; Microthrix” group. Millipore). For morphological characterization, bacterial cells in the stationary growth phase were adsorbed onto a 200-mesh In this study, we report the genome sequence of strain formvar and carbon-coated copper grid (Electron Microscopy IMCC26207, an actinobacterium isolated from an oligotrophic Sciences, USA), stained with uranyl acetate solution (2% (w/v)), freshwater reservoir using dilution-to-extinction culturing and examined with a transmission electron microscope (CM200; and phylogenetically assigned to a novel group related to Philips, The Netherlands). For the genome sequencing, 4 L “Candidatus Microthrix.” Based on morphological, phylogenetic, cultures of strain IMCC26207 were harvested by filtration onto a and genomic differences between strain IMCC26207 and polyethersulfone membrane filter (0.2 µm pore size; Pall “Candidatus Microthrix,” the name “Candidatus Limnosphaera Corporation, USA). The genomic DNA was directly extracted aquatica” is additionally proposed for this bacterium. from the filter using a DNeasy Blood & Tissue Kit (Qiagen, USA) following the protocol for gram-positive bacteria, and was further purified using a PowerClean Pro DNA Clean-Up Kit (Mo Bio Materials and Methods Laboratories, USA). The purity, quality, and concentration of the genomic DNA were determined using 1% (w/v) agarose gel Isolation of Stain IMCC26207 electrophoresis and a Qubit 3.0 fluorometer (Invitrogen). Strain IMCC26207 was isolated from a surface water sample collected from Lake Soyang, an oligotrophic reservoir located in Genome Sequencing, Assembly, and Annotation South Korea, by using the high-throughput dilution-to-extinction A sequencing library was constructed from the genomic DNA culturing (HTC) method [15, 16]. The culture medium (referred to by using Nextera DNA Library Preparation Kits (Illumina, USA) as LNFMC, low nutrient freshwater medium amended with according to the manufacturer’s instructions. The genome of carbon mixtures) used for HTC was prepared by amending strain IMCC26207 was sequenced at ChunLab, Inc. (Korea), using filtered and autoclaved lake water with the following ingredients: the Illumina MiSeq platform, at a read length of 2 × 300 bp. A total 10 µM NH4Cl, 10 µM KH2PO4, 50 µM pyruvic acid, 5 µM D-glucose, of 3,441,153 paired reads obtained from the genome sequencing 5 µM D-ribose, 5 µM N-acetyl-D-glucosamine, 5 µM methyl alcohol, were assembled using SPAdes ver. 3.5.0 [18], generating 10 contigs and 1:10,000 diluted Va vitamin mixture [17]. To determine the (90–836 kbp; N50, 345 kbp) with an average of 63.1× coverage. The cell density for the inoculum, direct cell counting was performed total length of all contigs comprised 3,316,799 bp. Gene calling and microscopically, after staining with 4’,6’-diamidino-2-phenylinole, genome annotation were performed by following the Integrated using an epifluorescence microscope (Nikon 80i; Nikon, Japan). Microbial Genomes-Expert Review (IMG-ER) pipeline [19] Freshwater bacterial cells were diluted to 2 cells/ml with LNFMC, developed by the Joint Genome Institute [20]. tRNA and rRNA and 1 ml of the inoculum was transferred into 48-well plates (BD genes were found using the tRNAscan-SE tool and HMMER, Falcon, USA). Positively grown cells, including strain IMCC26207, respectively [21, 22], and other non-coding RNAs were detected were screened with SYBR Green I (Invitrogen, USA) using an by searching the genome for the corresponding Rfam profiles Easyflow Guava flow cytometer (EasyCyte Plus; Millipore, USA) using INFERNAL [23]. CRISPR repeats were checked by CRT-CLI after 4 weeks of incubation at 15°C in the dark condition. To test and PILER-CR [24]. Protein-coding genes were identified using whether the initial axenic culture of IMCC26207 could grow in Prodigal [25]. The genome map was generated by CGview chemically defined media or synthetic media, R2A broth (BD, software using a merged GenBank file from the RAST server [26]. USA), 1/3 diluted R2A broth, 1/10 diluted R2A broth, and The