International Journal of Systematic and Evolutionary Microbiology (2012), 62, 342–346 DOI 10.1099/ijs.0.031880-0 Knoellia locipacati sp. nov., from soil of the Demilitarized Zone in South Korea Na-Ri Shin, Seong Woon Roh, Min-Soo Kim, Mi-Ja Jung, Tae Woong Whon and Jin-Woo Bae Correspondence Department of Life and Nanopharmaceutical Sciences and Department of Biology, Jin-Woo Bae Kyung Hee University, Seoul 130-701, Republic of Korea [email protected] A Gram-positive, aerobic, rod- or coccus-shaped, non-motile bacterium, designated DMZ1T, was isolated from soil of the Demilitarized Zone, South Korea. Strain DMZ1T grew optimally at 30 6C, at pH 7–8 and with 1 % (w/v) NaCl. The isolate showed high 16S rRNA gene sequence similarity with Knoellia aerolata 5317S-21T (98.2 %). The cell-wall sugars were glucose and ribose. The peptidoglycan amino acids were meso-diaminopimelic acid, glutamic acid and glycine. The major cellular fatty acids were iso-C16 : 0, iso-C15 : 0 and iso-C14 : 0. The polar lipids were phosphatidylethanolamine, phosphatidylinositol, diphosphatidylglycerol, phosphatidylglycerol and five unknown phospholipids. The isolate did not contain mycolic acids. The DNA G+C content was 72.6 mol%. The isolate showed ,28 % DNA–DNA relatedness with members of the genus Knoellia. Phylogenetic, phenotypic and genotypic analysis indicated that strain DMZ1T represents a novel species of the genus Knoellia, for which the name Knoellia locipacati sp. nov. is proposed. The type strain is DMZ1T (5KACC 15114T 5JCM 17313T). The Demilitarized Zone (DMZ) separating North and South soy broth (TSB; Bacto) at 4, 10, 15, 25, 30, 37, 45 and 55 uC, Korea is an area where weapons are prohibited. After the at pH 4–12 (at intervals of one pH unit) by adjusting the pH Korean War, the Military Demarcation Line was established. of TSB with 10 mM MES (pH 4–6), 10 mM N-tris(hydroxy- The 4 km-wide DMZ was formed along this line, with 2 km methyl)methyl-3-aminopropanesulfonic acid (pH 8–10) or located on each side of the divide. Since the DMZ has been 10 mM Na2HPO4 (pH11–12)andwith0,1,2,3,4,5,8,10, rigidly off limits to civilians for over 50 years, the natural 12, 15 and 20 % (w/v) NaCl. The turbidity of each culture was ecology has been highly preserved; therefore, it has attracted determined by measuring the optical density at 600 nm much attention from ecologists worldwide. (Synergy Mx; BioTek) after 24 h, 48 h and 7 days of incu- bation. Growth under anaerobic conditions was assessed Members of the genus Knoellia (Groth et al., 2002) are in an anaerobic chamber under a N2/H2/CO2 (90:5:5) Gram-positive, aerobic, non-motile microbes that possess atmosphere at 37 uC for 7 days. The isolate grew at 10–37 uC, an A1c-type peptidoglycan moiety and MK-8(H4) as the at pH 6–9 and with 0–5 % (w/v) NaCl. Optimal growth major menaquinone. Currently, three species have been occurred at 30 uC, at pH 7–8 and with 1 % (w/v) NaCl. isolated from soil and air: Knoellia sinensis, K. subterranea Anaerobic growth was not observed. Gram-staining was (Groth et al., 2002) and K. aerolata (Weon et al., 2007). In performed using a Gram-staining kit (bioMe´rieux), accord- this paper, we describe a novel species to accommodate ing to the manufacturer’s instructions. Cell morphology and T strain DMZ1 , which was isolated from soil during an Gram-staining were observed by light microscopy (Eclipse investigation of the microbial ecology of the DMZ. 50i; Nikon). The motility of the isolate was examined using A soil sample was diluted with filtered PBS and incubated semi-solid agar (Tittsler & Sandholzer, 1936). Cells had an at 25 uC on trypticase soy agar (TSA; Bacto). The isolate was irregular rod or coccoid form and were Gram-positive and purified by subcultivation. All investigations were per- non-motile. formed in triplicate. Physiological and biochemical experi- For the phylogenetic analysis, the 16S rRNA gene of the ments were performed at 25 uC for 48 h unless otherwise isolate was amplified by colony PCR using PCR Pre- stated. Conditions for growth were determined in trypticase Mix (iNtRON Biotechnology) and four universal bacterial primers: 8F, 518R, 785F and 1492R (Baker et al., 2003; Abbreviation: DAP, diaminopimelic acid. Kumar et al., 2003). After purification (QIAquick PCR The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene Purification kit; Qiagen), sequencing of the amplification sequence of strain DMZ1T is HQ171909. products was performed using the BigDye Terminator Two supplementary figures are available with the online version of this Cycle Sequencing Ready Reaction kit (Applied Biosystems), paper. according to the manufacturer’s instructions. The reaction 342 031880 G 2012 IUMS Printed in Great Britain Knoellia locipacati sp. nov. mixtures were analysed using an automated DNA analyser by bubble production with 3 % (v/v) hydrogen peroxide and (ABI Prism 3730 XL; Applied Biosystems). To obtain the indophenol blue production with 1 % (w/v) tetramethyl-p- complete 16S rRNA gene sequence, fragments were phenylenediamine (bioMe´rieux), respectively. The results of assembled with SeqMan software (DNASTAR). A comparison the biochemical analysis are given in Table 1 and the species of the 16S rRNA gene sequence of the isolate with those in description. The isolate was catalase-positive and oxidase- the EzTaxon database (Chun et al., 2007) revealed that negative and could be distinguished from the reference strain DMZ1T belonged to the genus Knoellia in the class strains by assimilation of D-xylose and N-acetylglucosamine Actinobacteria. The isolate had the highest levels of 16S and activities for trypsin and b-galactosidase. T rRNA gene sequence similarity with K. aerolata 5317S-21 Chemotaxonomic analysis was performed using cell biomass (98.2 %), K. sinensis HKI 0119T (98.1 %) and K. subterranea T prepared at 25 uC for 72 h on R2A agar (Difco). Cell-wall HKI 0120 (98.1 %). The multiple sequence alignment sugars were extracted as described by Schleifer & Kandler program CLUSTAL W (Thompson et al., 1994) was used to (1972) and hydrolysed using 0.5 M H2SO4 at 95 uC for 2 h. align the 16S rRNA gene sequence of the isolate with The cell-wall sugars and standard sugars (Scho¨n & Groth, sequences exhibiting high pairwise similarity. The trimmed 2006) were separated on a cellulose TLC plate (Merck) and alignment was converted to MEGA and PHYLIP formats for developed twice with ethyl acetate/pyridine/water (100 : 35 : analysis. The phylogenetic relationship between the isolate 25, v/v). Sugars were detected by spraying with aniline- and closely related strains was investigated using the phthalate spray reagent. For analysis of the cell-wall neighbour-joining (Saitou & Nei, 1987), maximum-par- peptidoglycan amino acids, purified peptidoglycan hydroly- simony (Kluge & Farris, 1969) and maximum-likelihood sates (6 M HCl, 121 uC, 15 min) were analysed using (Felsenstein, 1981) algorithms and bootstrap analysis was one-dimensional TLC on a cellulose TLC plate, accord- based on 1000, 1000 and 300 randomly selected replicates, ing to the method of Bousfield et al. (1985). LL-and respectively. The phylogenetic consensus tree was recon- meso-diaminopimelic acid (DAP), 2,4-diaminobutyric structed using MEGA version 4 (Tamura et al., 2007) for the acid, glutamic acid, glycine, alanine, lysine, threonine and neighbour-joining and maximum-parsimony methods ornithine were used as standards. The whole cell-wall and PHYLIP (Felsenstein, 1996) for the maximum-likelihood hydrolysates of strain DMZ1T possessed glucose and ribose method. The phylogenetic analysis indicated that the isolate (Supplementary Fig. S1, available in IJSEM Online), meso- formed a monophyletic clade in a cluster containing the DAP, glutamic acid and glycine. Diagnostic cell-wall sugars members of the genus Knoellia (Fig. 1). According to the have rarely been detected previously in the reference strains; T branch lengths of the phylogenetic tree, strain DMZ1 had however, two or more sugar components of the cell wall the highest levels of 16S rRNA gene sequence similarity were detected in the isolate and the reference strains in this T T with K. sinensis HKI 0119 (98.6 %), K. aerolata 5317S-21 study. (98.3 %) and K. subterranea HKI 0120T (98.3 %). Cellular fatty acids were obtained as described by the Type strains of the three described species of the genus Sherlock Microbial Identification System (MIDI). The Knoellia were obtained from the DSMZ for use as reference cellular fatty acids were analysed by GC (Hewlett Packard strains. Assimilation of sole carbon sources and acid 6890) and identified using the TSBA 40 database in the production from carbohydrates were examined using the Microbial Identification software package (Sasser, 1990). API 50 CH system (bioMe´rieux) with AUX and API 50 The major cellular fatty acids of the isolate were iso-C16 : 0 CHB/E media, respectively, according to the manufacturer’s (32.6 %), iso-C15 : 0 (12.6 %) and iso-C14 : 0 (10.6 %). The instructions. Results were recorded after incubation at 30 uC cellular fatty acid compositions of the isolate and the for 72 h. The API ZYM system (bioMe´rieux) was used to reference strains are compared in Table 2. Polar lipids were screen enzyme activities, according to the manufacturer’s extracted by the method described by Xin et al. (2000), instructions. Catalase and oxidase activities were determined separated using two-dimensional TLC on a silica gel plate Fig. 1. Phylogenetic consensus tree based on 16S rRNA gene sequences showing the position of strain DMZ1T with its closest neighbours. Bootstrap values (.70 %) based on 1000, 1000 and 300 replicates for the neighbour-joining, maximum-parsimony and maximum-likelihood algorithms, respectively, are shown at branch nodes.