Jiangella Anatolica Sp. Nov. Isolated from Coastal Lake Soil

1 Jiangella anatolica sp. nov. isolated from coastal lake soil 2 Hilal Ay1*, Imen Nouioui2, Lorena Carro2¥, Hans-Peter Klenk2, Demet Cetin3, José M. 3 Igual4, Nevzat Sahin1, Kamil Isik5 4 1H. Ay, N. Sahin 5 Department of Molecular Biology and Genetics, Faculty of Science and Arts, Ondokuz Mayis 6 University, Samsun, Turkey 7 *Corresponding author: [email protected], Tel: +90 362 312 1919, Fax: +90 362 457 6081 8 2I. Nouioui, L. Carro, H.-P. Klenk 9 School of Natural and Environmental Sciences, Newcastle University, Ridley Building 2, 10 Newcastle upon Tyne, NE1 7RU, UK 11 ¥Present address: Dpto. de Microbiología y Genética. Universidad de Salamanca, Salamanca, 12 37007, Spain. 13 3D. Cetin 14 Science Teaching Programme, Gazi Faculty of Education, Gazi University, 06500, Ankara, 15 Turkey 16 4J. M. Igual 17 Instituto de Recursos Naturales y Agrobiologia de Salamanca, Consejo Superior de 18 Investigaciones Cientificas (IRNASA-CSIC), Salamanca, Spain 19 5K. Isik 20 Department of Biology, Faculty of Science and Arts, Ondokuz Mayis University, Samsun, 21 Turkey 22 23 24 Abstract 25 A novel actinobacterial strain, designated GTF31T, was isolated from a coastal soil sample of 26 Gölcük Lake, a crater lake in southwest Anatolia, Turkey. The taxonomic position of the strain 27 was established using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene 28 sequences and showed that the strain is closely related to Jiangella gansuensis DSM 44835T 29 (99.4 %), Jiangella alba DSM 45237T (99.3 %) and Jiangella muralis DSM 45357T (99.2 %). 30 Optimal growth was observed at 28 °C and pH 7 – 8. Whole cell hydrolysates were found to 31 contain LL-DAP, glucose, mannose, rhamnose and ribose. The predominant menaquinone was 32 identified as MK-9(H4). The polar lipid profile was found to contain diphosphatidylglycerol, 33 phosphatidylglycerol, phosphatidylinositol, glycophospholipids and unidentified phospholipids. 34 The major fatty acids were identified as anteiso-C15:0, iso-C16:0 and anteiso-C17:0. The G+C 35 content of the type strain was determined to be 72.5 % and the size of the draft genome is 7.0 36 Mb. The calculated digital DDH values between strain GTF31T and the type strains of J. 37 gansuensis, J. alba, J. muralis and Jiangella alkaliphila ranged from 24.4 to 34.4% and ANI 38 values ranged between 81.0 – 87.9 %. Based upon the consensus of phenotypic and 39 phylogenetic analyses as well as whole genome comparisons, strain GTF31T (= DSM 100984T = 40 CECT 9378T) is proposed to represent the type strain of a novel species, Jiangella anatolica sp. 41 nov. 42 Keywords: Phylogenomic, Gölcük Lake, Jiangella, Polyphasic taxonomy 43 Introduction 44 The genus Jiangella was first proposed by Song et al. (2005) within the family 45 Nocardioidaceae. Subsequently, Tang et al. (2011) established the family Jiangellaceae to 46 accommodate the genera Jiangella (Song et al. 2005) and Haloactinopolyspora (Tang et al. 47 2011), mainly based on phylogenetic analysis of the phylum Actinobacteria. Currently, the 48 family Jiangellaceae includes the genera Jiangella, Haloactinopolyspora and the recently 49 described Phytoactinopolyspora (Li et al. 2015). The type genus Jiangella encompasses 50 aerobic, Gram-positive, filamentous actinomycetes with substrate mycelium which fragment 51 into short and elongated rods. Members of the genus are characterised by the presence of LL- 52 diaminopimelic acid in the cell wall peptidoglycan and MK-9(H4) as the major menaquinone. 53 None of the species described so far contain mycolic acids. The genus accommodates Jiangella 54 gansuensis YIM 002T (Song et al. 2005), Jiangella alba YIM 61503T (Qin et al. 2009), 55 Jiangella alkaliphila D8-87T (Lee 2008), Jiangella muralis 15-Je-017T (Kämpfer et al. 2011) 56 and Jiangella mangrovi 3SM4-07T (Suksaard et al. 2015) isolated from desert soil, surface 57 sterilised stem of Maytenus austroyunnanensis, cave soil, cellar wall and mangrove soil, 58 respectively. 59 Members of the phylum Actinobacteria are well-known for being prolific producers of many 60 bioactive secondary metabolites (Goodfellow and Fiedler 2010). Thus, selective isolation of 61 novel actinobacteria from unexplored habitats such as marine sediments (Carro et al. 2018) or 62 lakes (Ay et al. 2017; Ay et al. 2018) holds considerable importance for the discovery of novel 63 bioactive compounds. In a continuation of our investigations on actinobacterial diversity in 64 unexplored habitats a novel strain, GTF31T, was isolated from a coastal soil sample of Gölcük 65 Lake, a crater lake in southwest Anatolia (Turkey). Polyphasic characterisation of strain 66 GTF31T showed that the strain is a novel species of the genus Jiangella, for which the name 67 Jiangella anatolica sp nov is proposed. 68 Materials and methods 69 Isolation, maintenance and cultural conditions 70 Strain GTF31T was obtained from a coastal soil sample of Gölcük Lake, a freshwater lake, (37° 71 44’ 03.90’’ N; 30° 29’ 39.24’’ E), collected in September 2013. The strain was isolated on 72 International Streptomyces Project Medium 2 (ISP 2 medium) (Shirling and Gottlieb 1966) 73 supplemented with nystatin (50 µg/ml) and rifampicin (5 µg/ml) using a standard dilution-plate 74 technique. Briefly, the soil sample was suspended in ¼ strength Ringer’s solution (Oxoid) and 75 pretreated with wet heat at 60 ºC for 20 minutes. Then, the diluted suspensions (10-2 and 10-3) 76 were spread on ISP 2 medium. Inoculated plates were incubated at 28 ºC for two weeks. The 77 Jiangella isolate was purified and maintained on slopes of tryptone-yeast extract agar (DSM 78 medium No. 680) at 4 ºC and as glycerol stock solutions (25 % w/v) at – 20 ºC. 79 J. gansuensis JCM 13367T, J.alba JCM 16901T and J.muralis JCM 17970T were obtained from 80 Japan Collection of Microorganisms (JCM). These reference strains for phenotypic comparison 81 were grown and tested under identical conditions. 82 Cultural properties and phenotypic tests 83 Cultural and morphological characteristics were observed on ISP media 1 – 7 (International 84 Streptomyces Project, Shirling and Gottlieb (1966)), nutrient agar and tryptic soy agar (TSA; 85 Difco) after incubation at 28 °C for 14 days. The ISCC-NBS Colour Charts Standard Samples 86 No. 2106 (Kelly 1964) was used to determine the colours of aerial mycelium, substrate 87 mycelium and diffusible pigments. Micromorphological characteristics were observed by 88 scanning electron microscopy (JEOL JSM 6060) using cultures grown on glucose-yeast extract- 89 malt extract (DSMZ Medium No. 65) at 28 °C for 14 days. The temperature (4, 10, 20, 28, 37, 90 50 ºC) and pH range (7 – 12, at intervals of 1.0 pH unit) for growth were determined on ISP 2 91 medium (Shirling and Gottlieb 1966). Enzyme activity profile of the strain was established 92 using the commercial kit API ZYM (BioMerieux) following the manufacturer’s instructions. 93 Carbon source utilisation and chemical sensitivity properties were determined using GEN III 94 Microplates in an Omnilog device (BIOLOG Inc., Hayward, CA, USA). The GEN III 95 Microplates were inoculated with cells suspended in a viscous inoculation fluid (IF C) provided 96 by the manufacturer at 85–87 % of transmittance for strain GTF31T and the closely related type 97 strains. The strains were studied in two independent technical replicates. Data were exported 98 and analysed using the opm package for R (Vaas et al. 2013; Vaas et al. 2012). 99 100 101 Chemotaxonomy 102 Biomass for chemotaxonomic analyses was obtained by cultivation in shake flasks at 28 °C for 103 14 days on a rotary shaker (150 rpm) using glucose-yeast extract-malt extract (DSMZ Medium 104 No. 65). Biomass was harvested by centrifugation at 9000 rpm, washed several times with 105 sterile distilled water and then freeze-dried. Whole cell amino acids and sugars were extracted 106 following a standard protocol described by Lechevalier and Lechevalier (1970) and analysed by 107 thin layer chromatography (TLC) according to Staneck and Roberts (1974). Polar lipids were 108 extracted and analysed by two-dimensional TLC following Minnikin et al. (1984) using 109 modifications of Kroppenstedt and Goodfellow (2006). Menaquinones were extracted from 110 freeze-dried biomass and purified according to Collins (1985). The extracted menaquinones 111 were analysed using a high performance liquid chromatography (HPLC; LC-10AS, Shimadzu 112 63 Co.) fitted with a reverse-phase C18 column (150 x 4.6 mm, 5 µm particle size, RP-18- 113 Lichrosorb column, Penomenex UK) at 270 nm. Fatty acid methyl esters were extracted from 114 fresh cultures grown on TSA at 28 °C for 10 days and analysed using Microbial Identification 115 System (MIDI) Sherlock software version 6.1 and the library RTSBA6 according to the 116 technical instructions provided by this system (Sasser 1990). 117 Phylogeny 118 Genomic DNA was extracted as described by Chun and Goodfellow (1995) and PCR 119 amplification of the 16S rRNA gene achieved using the universal primers 27F (5′– 120 AGAGTTTGATC(AC)TGGCTCAG–3′) and 1492R (5′– 121 ACGG(CT)TACCTTGTTACGACTT–3′) (Weisburg et al. 1991). The purified PCR products 122 were sequenced using an ABI PRISM 3730 XL automatic sequencer and an almost complete 123 16S rRNA gene sequence (1491 bp) was deposited in the GenBank data library and 124 corresponding sequences of the type strains of the genus Jiangella were retrieved by using the 125 EzBioCloud server (Yoon et al. 2017). A multiple sequence alignment was generated with 126 MUSCLE program (Edgar 2004). A Neighbour Joining tree (Saitou and Nei 1987) was 127 constructed in MEGA 6 (Tamura et al. 2013) using the model of Jukes and Cantor (Jukes and 128 Cantor 1969). Phylogenetic analyses for maximum likelihood (ML) and maximum parsimony 129 (MP) methods were conducted by the GGDC web server using the DSMZ phylogenomics 130 pipeline (Meier-Kolthoff et al.

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