Humibacter antri sp. nov., an actinobacterium isolated from a natural cave, and emended description of the genus Humibacter

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1 International Journal of Systematic and Evolutionary Microbiology (2013), 63, DOI /ijs Humibacter antri sp. nov., an actinobacterium isolated from a natural cave, and emended description of the genus Humibacter Soon Dong Lee Correspondence Soon Dong Lee sdlee@jejunu.ac.kr Department of Science Education, Jeju National University, Jeju , Republic of Korea A novel high DNA G+C content bacterium, designated strain D7-27 T, was isolated from clay soils collected inside a natural cave in Jeju, Republic of Korea. The cells of the organism were aerobic, Gram-stain-positive, non-motile rods; its colonies were white, circular and entire in margin. Phylogenetic analyses based on 16S rrna gene sequence comparison showed that strain D7-27 T formed a coherent cluster with Humibacter albus of the family Microbacteriaceae (97.6 % sequence similarity). The cell-wall peptidoglycan contained ornithine and 2,4- diaminobutyric acid as the diagnostic diamino acids. The major menaquinones were MK-12 and MK-11, with MK-10 as a minor component. The polar lipids consisted mainly of phosphatidylglycerol and an unknown glycolipid. Mycolic acids were not present. The predominant fatty acids were anteiso-c 17 : 0 and cyclohexyl-c 17 : 0. The DNA G+C content was 66.3 mol%. DNA DNA relatedness between strain D7-27 T and H. albus DSM T was 28.6 % (17.6 % in a reciprocal test). On the basis of the phenotypic, chemotaxonomic and DNA DNA hybridization data, strain D7-27 T (5KCTC T 5DSM T ) is considered as the type strain of a novel species of the genus Humibacter, for which the name Humibacter antri sp. nov. is proposed. An emended description of the genus Humibacter is also provided. The genus Humibacter was proposed by Vaz-Moreira et al. (2008) for encompassing a member of the family Microbacteriaceae that was isolated from municipal sewage sludge compost. Cells of the type strain of the only known species, Humibacter albus, are Gram-stain-positive, strictly aerobic, motile, short rods. Members of the genus contain ornithine and 2,4-diaminobutyric acid as the diagnostic diamino acids in the cell-wall peptidoglycan with N- acetylated murein, the predominant fatty acids cyclohexyl- C 17 : 0, anteiso-c 17 : 0, anteiso-c 15 : 0 and iso-c 16 : 0 and the major menaquinones MK-11 and MK-12. This paper describes the polyphasic taxonomic characterization of a strain representing the genus Humibacter, which was isolated from a natural cave in the Republic of Korea. During a culture-dependent study on the diversity of cave bacteria, strain D7-27 T was isolated from clay soil samples collected inside a natural cave in Jeju, Republic of Korea. The procedure and medium for bacterial isolation were as described by Lee (2008). After incubation at 30 uc for 14 days, the colonies on the agar plates were subcultured on ISP (International Streptomyces Project) medium 2 (Shirling & Gottlieb, 1966). The pure culture was The GenBank/EMBL/DDBJ accession number for the 16S rrna gene sequence of strain D7-27 T is HF Four supplementary figures are available with the online version of this paper. maintained as 20 % (v/v) glycerol suspensions at 220 uc and 280 uc. For phenotypic comparison and DNA DNA hybridization experiments, Humibacter albus DSM T was grown on ISP medium 2 at 30 uc. Growth of strain D7-27 T was examined on ISP medium 2, trypticase soy yeast extract medium (TSYE; DSM medium 92, Medium92.pdf), trypticase soy agar (TSA; Difco), R2A agar (Difco) and nutrient agar (NA; Difco). Cell morphology and motility were observed by phase-contrast and transmission electron microscopy, with cells grown on ISP 2 agar plates and in ISP 2 liquid broth for 3 days at 30 uc. The presence of flagella was examined using H. albus DSM T as a positive control. Colony appearance and colour were observed after incubation on ISP medium 2 for 5 days at 30 uc. The physiological and biochemical characteristics of strain D7-27 T were, unless otherwise indicated, examined by using ISP 2 medium as the basal medium. Growth was tested at ph (at intervals of 1 ph unit) and different temperatures (4, 10, 20, 28, 30, 32, 35 and 40 uc). The ph of the ISP 2 broth was adjusted with 1 M HCl and 1 M NaOH after sterilization. The temperature and ph ranges for growth were recorded after incubation for 5 days (at 30 uc for ph tests). For growth a different ph, 1 ml of cell suspension (OD 600 adjusted to 0.5) was added into 50 ml ISP 2 broth and the OD 600 of the culture broth G 2013 IUMS Printed in Great Britain 4315

2 Soon Dong Lee was measured at the time of inoculation and after 3 and 5 days of incubation. A positive reaction was recorded for growth where the increase in OD 600 value was greater than 0.3. Tolerance of NaCl was tested at 1 9 % (w/v) NaCl (at intervals of 1 %) and the agar plates were incubated at 30 uc for 7 days. Gram staining was performed by using the Gram 2 kit (biomérieux) following the instructions of the manufacturer. Oxidase activity was examined with 1 % (w/v) N,N,N9,N9-tetramethyl-p-phenylenediamine and catalase activity was determined by bubble production in 3 % (v/v) H 2 O 2. Other physiological and biochemical properties were examined using API 20NE, API ZYM and API 50CH strips (biomérieux) according to the instructions of the manufacturer. For these tests, the cells of strain D7-27 T and H. albus DSM T were grown on ISP medium 2 for 5daysat30uC and suspended in sterilized distilled water. Strain D7-27 T grew well on ISP medium 2, TSYE medium, TSA and NA but slow growth was observed on R2A agar. The growth rate of strain D7-27 T seemed to be slightly slower than that of H. albus DSM T. Strain D7-27 T showed visible growth after 3 days of incubation on ISP 2 medium and separate colonies appeared after incubation for 5 days, whereas H. albus DSM T showed visible colonies (approx. 0.5 mm in size) after 3 days of incubation. Colonies of strain D7-27 T were white, circular, entire in margin and 0.5 mm in size after incubation on ISP medium 2 for 5 days at 30 uc. Cells of strain D7-27 T were Gram-stain-positive, aerobic, non-motile rods ( mm) and no flagella were observed irrespective of the physical state of media used (Fig. S1, available in IJSEM Online). The results for other physiological and biochemical tests are given in Table 1 and the species description. Genomic DNA isolation and PCR amplification and sequencing of the 16S rrna gene were performed by using previously described methods (Lee, 2008). A preliminary comparison of an almost-complete 16S rrna gene sequence (1412 bp) of strain D7-27 T was performed against 16S rrna gene sequences available in the GenBank database, indicating a close relationship with members of the family Microbacteriaceae. The CLUSTAL X program (Thompson et al., 1997) was used for multiple alignments with the selected sequences. A total of 1376 unambiguously aligned nucleotides were used for phylogenetic analyses by the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods. A neighbour-joining tree was drawn with evolutionary distances calculated by the model of Jukes & Cantor (1969), with the results of bootstrap analysis (Felsenstein, 1985) based on 1000 replications. The 16S rrna gene sequence analysis showed that strain D7-27 T forms a monophyletic cluster with H. albus SC-083 T of the family Microbacteriaceae (a sequence similarity of 97.6 %), being supported by a bootstrap value of 100 % and by the maximum-likelihood and maximum-parsimony treeing methods (Fig. S2). Strain D7-27 T showed 16S rrna gene sequence similarities of less than 95.1 % to representatives of all the other genera in the family Microbacteriaceae. Table 1. Differential characteristics between strain D7-27 T and the type strain of Humibacter albus Taxa: 1, strain D7-27 T ;2,H. albus DSM T. Data were taken from this study. +. Positive; 2, negative; W, weak response. Both strains were positive for nitrate reduction, aesculin degradation and assimilation of D-glucose, N-acetyl-D-glucosamine and DL-malate (weak response) but negative for indole production, glucose fermentation, arginine dihydrolase, gelatin hydrolysis and assimilation of D-arabinose, gluconate, caprate and adipiate (API 20NE). They were positive for esterase (C4) (weak response), esterase lipase (C8) (weak), leucine arylamidase, acid phosphatase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase but negative for alkaline phosphatase, lipase (C14), trypsin, a-chymotrypsin, naphthol-as-bi-phosphohydrolase and b-glucuronidase (API ZYM). Both strains were positive or weakly positive for acid production from D-arabinose, D-glucose, D-mannose, N-acetylglucosamine, arbutin, aesculin, salicin, cellobiose, maltose, sucrose, xylitol and D-fucose, but negative for acid production from glycerol, erythritol, L-xylose, adonitol, L-sorbose, dulcitol, inositol, D-sorbitol, inulin, melezitose, raffinose, starch, glycogen, turanose, D-lyxose, D-tagatose, L-fucose, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate (API 50CH). Characteristic 1 2 Cell size (mm) * Cell motility 2 +* Growth at 35 uc 2 + Growth with 3 % (w/v) NaCl 2 + Enzyme activities Cystine arylamidase W 2 Urease 2 + Valine arylamidase 2 + Assimilation of: Citrate 2 W Maltose W 2 D-Mannitol 2 + D-Mannose W 2 Phenylacetate 2 + Acid produced from: Amygdalin 2 + L-Arabinose 2 + D-Arabitol 2 W D-Fructose 2 + D-Galactose 2 + Gentiobiose 2 + Lactose 2 + D-Mannitol 2 + Melibiose 2 + Methyl a-dmannopyranoside W 2 Methyl a-d-glucopyranoside W 2 Methyl a-d-xylopyranoside 2 + L-Rhamnose 2 + D-Ribose 2 + Trehalose 2 + D-Xylose 2 + DNA G+C content (mol%) * *Data from Vaz-Moreira et al. (2008) International Journal of Systematic and Evolutionary Microbiology 63

3 Humibacter antri sp. nov. Biomass for chemotaxonomy was prepared by cultivation of strain D7-27 T and H. albus DSM T in ISP 2 broth for 5 days at 30 uc. Purified cell wall was obtained by using the method of Hancock (1994) and the amino acid composition of hydrolysed cell walls (6 M HCl, 16 h, 120 uc) was determined by HPLC as described by Lee (2007). The acyl type of the peptidoglycan was analysed according to the method of Uchida & Aida (1984). Menaquinones were extracted and purified by the method of Collins (1985) and subsequently analysed by HPLC (Kroppenstedt, 1985). Polar lipids were extracted and analysed by using two-dimensional TLC (Minnikin et al., 1977). Mycolic acids were analysed by the method of Minnikin et al. (1980). The base composition of the DNA was determined by HPLC (Mesbah et al., 1989). For determination of cellular fatty acids, strain D7-27 T and H. albus DSM T were grown on ISP medium 2 and TSA for 3 days at 30 uc and the identities of both strains were checked by 16S rrna gene sequencing before analysis. Fatty acid methyl esters were prepared and analysed according to the instructions of the Sherlock Microbial Identification System (version 6.1; MIDI), with the ACTIN6 database for identification of fatty acids. The main peak, identified as a summed feature which could not be separated by GLC using the MIDI system, was further analysed by using a GCMS-QP2010 Ultra (Shimazu) with a RTX-5MS capillary column (30 m, 0.25 mm i.d. and 0.25 mm film thickness; RESTEK) and GCMS solution software version The results of the 16S rrna gene sequence analysis were consistent with most of the chemotaxonomic characteristics. The diagnostic diamino acids of the cell-wall peptidoglycan of strain D7-27 T were ornithine and 2,4- diaminobutyric acid, being typical for members of the genus Humibacter (Vaz-Moreira et al., 2008). The molar ratio of the amino acids ornithine, 2,4-diaminobutyric acid, alanine, glycine and glutamic acid was found to be 0.4 : 0.6 : 0.7 : 1.8 : 1.0. The acyl type of cell-wall muramic acid was acetyl type. The polar lipid profile of strain D7-27 T consisted of phosphatidylglycerol, an unknown glycolipid, an unknown phospholipid and three unknown lipids. The phospholipid composition of H. albus DSM T was not determined in a previous study (Vaz- Moreira et al., 2008). In this analysis, H. albus DSM T showed a similar profile to that of strain D7-27 T (Fig. S3). Diphosphatidylglycerol, which is found in most of members of the family Microbacteriaceae, was not detected in either strain tested. Thus, the absence of diphosphatidylglycerol can be a key characteristic that differentiates the genus Humibacter from the other genera of the family Microbacteriaceae. The menaquinone profile of strain D7-27 T contained menaquinones MK-12, MK-11 and MK-10 (79, 17 and 4 %, respectively), whereas H. albus DSM T, in this study, revealed a similar profile (MK- 12, MK-11 and MK-10 in the ratio 33 : 58 : 9) to that reported previously (Vaz-Moreira et al., 2008). The DNA G+C content of strain D7-27 T was 66.3 mol%, slightly lower than the 68 % of H. albus DSM T. The cellular fatty acid profiles of strain D7-27 T and H. albus DSM T are given in Table 2. Both strains showed consistent patterns according to the culture media used and contained considerable amounts of iso- and anteiso-methyl-branched acids together with cylcohexyl fatty acids, with anteiso-c 17 : 0 and cylcohexyl-c 17 : 0 predominating. The fatty acids iso-c 16 : 0 and C 16 : 0, as main components in both strains, were variable depending on culture media. The main peaks identified as summed feature 7 (C 18 : 1 v9t/v6t/v11c) in fatty acid methyl esters of both strains using the MIDI system were further analysed by GC-MS, confirming this fatty acid as cylcohexyl-c 17 : 0 (Fig. S4). The two mass spectra were identical to that from Curtobacterium pusillum (Suzuki et al., 1981). The fatty acid cyclohexyl-c 17 : 0 has been reported as a distinctive feature that distinguishes the genus Humibacter from the other genera in the family Microbacteriaceae (Vaz-Moreira et al., 2008) but was not included in the genus description. The presence of cyclohexyl-c 17 : 0 in the cells of strain D7-27 T suggests that the description of the genus Humibacter needs to be emended. DNA DNA hybridization experiments between strain D7-27 T and H. albus DSM T were conducted by using photobiotin-labelled DNA probes in microdilution wells as described by Ezaki et al. (1989). DNA DNA relatedness between strain D7-27 T and H. albus DSM T was 28.6 % (17.6 % in a reciprocal hybridization with DNA of H. albus DSM T as a probe). These relatedness values were lower than the threshold value of 70 % that is considered for definition of bacterial genomic species Table 2. Cellular fatty acid profiles of strain D7-27 T and the type strain of Humibacter albus Strains: 1, D7-27 T ;2,H. albus DSM T. Both strains were grown on ISP medium 2 and TSA for 3 days at 30 uc., Not detected; TR, trace (,1.0 % of the total); TBSA, tuberculostearic acid. Fatty acid 1 2 ISP 2 TSA ISP 2 TSA Unknown TR 1.4 C 14 : TR TR iso-c 15 : anteiso-c 15 : iso-c 16 : C 16 : iso-c 17 : anteiso-c 17 : C 18 : Methyl C 18 : TR (TBSA) Summed feature 7* *Summed features represent groups of two or more fatty acids which could not be separated by GLC using the MIDI system. Summed feature 7 contained C 18 : 1 v9t/v6t/v11c

4 Soon Dong Lee (Wayne et al., 1987), thus revealing that strain D7-27 T represents a different species from H. albus. Furthermore, strain D7-27 T can be readily differentiated from H. albus DSM T by physiological features such as growth temperature, utilization of carbon sources and enzyme activities (Table 1) and cellular fatty acid composition (Table 2). The combination of the morphological, physiological and chemotaxonomic data as well as DNA DNA relatedness suggested that strain D7-27 T represents a novel species of the genus Humibacter, for which the name Humibacter antri sp. nov. is proposed. Emended description of the genus Humibacter Vaz-Moreira et al Humibacter (Hu.mi.bac9ter. L. masc. n. humus earth, soil and, in earth sciences or agriculture, humus; N.L. masc. n. bacter rod; N.L. masc. n. Humibacter rod living in humus). The description is as given by Vaz-Moreira et al. (2008) with the following modifications. Cells are motile or non-motile depending on species. The polar lipids consist mainly of phosphatidylglycerol and an unknown glycolipid. The predominant fatty acids are anteiso-c 17 : 0 and cylcohexyl- C 17 : 0. The DNA G+C content is mol%. Description of Humibacter antri sp. nov. Humibacter antri (an9tri. L. gen. n. antri of a cave, referring to the sample from which the type strain was isolated). Cells are Gram-stain-positive, aerobic, oxidase-negative, catalase-positive and non-motile rods ( mm). Colonies are white, circular and entire in margin and 0.5 mm in size after incubation on ISP medium 2 for 5 days at 30 uc. Growth occurs at uc (optimum at uc) and ph (optimum ph ) and in the presence of 1.0 % (w/v) NaCl. Growth does not occur at 32 uc, ph 9.0 or with 2.0 % (w/v) NaCl. Nitrate reduction and aesculin degradation are observed. D-Glucose, N-acetyl- D-glucosamine, D-mannose (weakly), maltose (weakly) and DL-malate (weakly) are assimilated. Enzyme activities such as esterase (C4) (weak response), esterase lipase (C8) (weak), leucine arylamidase, acid phosphatase, cystine arylamidase (weak), a-galactosidase, b-galactosidase, a-glucosidase, b- glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase are present. Acid is produced from D-arabinose (weakly), D-glucose (weakly), D-mannose (weakly), N-acetylglucosamine (weakly), arbutin, aesculin, salicin, cellobiose, maltose, sucrose (weakly), xylitol (weakly), methyl a-d-mannoside (weakly), methyl a-dglucoside (weakly) and D-fucose (weakly). Other physiological and biochemical properties are given in Table 1. The cell-wall peptidoglycan contains ornithine and 2,4-diaminobutyric acid as the diagnostic diamino acids. The major menaquinones are MK-12 and MK-11. The polar lipids comprise phosphatidylglycerol, an unknown glycolipid, an unknown phospholipid and three unknown lipids. Mycolic acids are not present. The predominant cellular fatty acids are anteiso-c 17 : 0 and cylcohexyl-c 17 : 0. The type strain, D7-27 T (5KCTC T 5DSM T ), was isolated from clay soils collected inside a natural cave in Jeju, Republic of Korea. The DNA G+C content of the type strain is 66.3 mol%. Acknowledgements This work was supported by a research grant from the Chuongbong Academic Research Fund of Jeju National University in The author is thankful to Dr R. Pukall for providing the type strain of Humibacter albus. References Collins, M. D. (1985). Analysis of isoprenoid quinones. Methods Microbiol 18, Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. 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