Antonie van Leeuwenhoek DOI 10.1007/s10482-017-0955-y
ORIGINAL PAPER
Larkinella terrae sp. nov., isolated from soil on Jeju Island, South Korea Leonid N. Ten . Jongguen Jeon . Su-Jin Park . Sangkyu Park . Seung-Yeol Lee . Myung Kyum Kim . Hee-Young Jung
Received: 28 August 2017 / Accepted: 30 September 2017 Ó Springer International Publishing AG 2017
Abstract A Gram-stain negative, non-motile, rodshaped, aerobic bacterium, designated 15J8-8T, was isolated from a soil sample collected on Jeju Island, South Korea, and characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain 15J8-8T belongs to the family Cytophagaceae and is related to Larkinella bovis M2TB15T (95.0%), ‘Larkinella harenae’ 15J9-9 (94.5%), Larkinella arboricola Z0532T (93.2%), and Larkinella insperata LMG 22510T (93.0%). The DNA G?C content of strain 15J8-8T was 50.5 mol%. The detection of phosphatidylethanolamine and two unidentified polar lipids as major polar lipids; menaquinone-7 as the predominant quinone; and C16:1 x5c, C16:0 N alcohol,
Electronic supplementary material The online version of this article (doi:10.1007/s10482-017-0955-y) contains supplementary material, which is available to authorized users. L. N. Ten J. Jeon S.-J. Park S. Park S.-Y. Lee H.-Y. Jung (&) School of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea e-mail: [email protected] M. K. Kim Department of Bio and Environmental Technology, Seoul Women’s University, Seoul 01797, Republic of Korea H.-Y. Jung Institute of Plant Medicine, Kyungpook National University, Daegu 41566, Republic of Korea
and iso-C15:0 as the major fatty acids also supported the affiliation of the isolate to the genus Larkinella. Based on its phenotypic properties and phylogenetic distinctiveness, strain 15J8-8T should be classified in the genus Larkinella as representative of a novel species, for which the name Larkinella terrae sp. nov. is proposed. The type strain is 15J8-8T (= KCTC 52001T = JCM 31990T). Keywords
Larkinella Bacteroidetes Soil bacteria
Introduction The genus Larkinella was originally proposed with Larkinella insperata as the type species (Vancanneyt et al. 2006) and the genus description was later emended by Anandham et al. (2011). At the time of writing, the genus Larkinella contains three species with validly published names (http://www.bacterio. net/larkinella.html). Recently, one other strain, ‘Larkinella harenae’ 15J9-9 (Park et al. 2017), was proposed as a novel Larkinella species and is awaiting validation. The type strains of Larkinella species were recovered from the water of a steam generator in a pharmaceutical company (Vancanneyt et al. 2006), fermented bovine products (Anandham et al. 2011), a humified solution produced by spruce wood decomposition (Kulichevskaya et al. 2009), and soil (Park et al. 2017). Members of the genus Larkinella are
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Gram-negative, non-spore-forming, non-motile or motile by gliding, and pink-pigmented bacteria that are characterized chemotaxonomically as having menaquinone MK-7 as the predominant quinone; isoC15:0, C16:1 x5c, iso-C17:0 3-OH and summed feature 3 (C16:1 x6c and/or C16:1 x7c and/or iso-C15:0 2-OH) as the major fatty acids; and phosphatidylethanolamine, phosphatidylserine, two unidentified aminophospholipids, and two unidentified polar lipids as the major polar lipids. In the course of screening for novel bacteria, strain 15J8-8T was isolated from a soil sample collected on Jeju Island, South Korea. On the basis of 16S rRNA gene sequence analysis, this isolate was considered to be a Larkinella-like strain. Strain 15J8-8T was subjected to a detailed investigation using a polyphasic taxonomic approach that included genotypic, chemotaxonomic, and phenotypic analyses. Based on the results obtained in this study, we propose that strain 15J8-8T be placed in the genus Larkinella as the type strain of a novel species.
Materials and methods Isolation of the bacterial strain and culture conditions Strain 15J8-8T was isolated from a soil sample collected on Jeju Island, South Korea (33°290 5100 N, 126°270 0800 E). The sample was suspended in 10 mL distilled water, vortexed, and serially diluted. One hundred microliters of each dilution was spread onto Reasoner’s 2A (R2A) agar plates (Difco, USA) and incubated at 25 °C for 1 week. On the 107-diluted plate, 30–35 colonies appeared; one pale pink colony, designated 15J8-8T, was purified by transferring it onto a fresh plate and incubating the new plate under the same conditions. The isolate was routinely cultured on R2A agar at 25 °C and was maintained as a glycerol suspension (20%, w/v) at -70 °C. Strain 15J8-8T has been deposited in the Korean Collection for Type Cultures (KCTC) and the Japan Collection of Microorganisms (JCM). Reference strains Larkinella arboricola DSM 21851T was purchased from the German Collection of Microorganisms and
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Cell Cultures, and Larkinella bovis KACC 14040T and L. insperata KACC 11674T were obtained from the Korean Agricultural Culture Collection. Our previously isolated strain, ‘L. harenae’ 15J9-9, was maintained in our laboratory collection. Unless otherwise noted, the morphological, physiological, and biochemical characteristics of strain 15J8-8T and of the four above-mentioned reference strains were investigated by using routine cultivation on R2A agar at 30 °C for 3 days. Phenotypic and biochemical characteristics The Gram reaction of strain 15J8-8T was examined using a standard staining method (Smibert and Krieg 1994). The cell morphology of strain 15J8-8T, grown for 3 days at 25 °C on R2A agar, was observed with an Olympus light microscope (10009 magnification) and a Hitachi HT7700 transmission electron microscope. Motility was investigated on 0.5% (w/v) semisolid R2A agar (Tittsler and Sandholzer 1936) and gliding motility was assessed by the microscopic hanging drop technique (Agarwal et al. 1997). Catalase and oxidase tests were performed as outlined by Cappuccino and Sherman (2010). Growth was assessed on R2A agar, Luria–Bertani (LB) agar, nutrient agar (NA), and trypticase soy agar (TSA), all purchased from Difco. Anaerobic growth was tested as described previously (Liu et al. 2008). The effect of pH on growth was evaluated in R2A broth using three different buffers (final concentration, 100 mM): sodium acetate buffer (for pH 4.0–6.0), potassium phosphate buffer (for pH 7.0–8.0), and Tris buffer (for pH 9.0–10.0). Growth at 4, 8, 15, 20, 25, 30, 37, and 42 °C was assessed after 7 days of incubation on R2A agar. Salt tolerance was tested after 7 days of incubation in R2A broth supplemented with 0.5, 1, 2, 3, 4, 5, and 10% (w/v) NaCl. Enzyme activities, assimilation of carbon sources, acid production from substrates, and other physiological characteristics were determined by inoculating API ZYM, API 20 NE, API ID 32 GN, and API 50 CH strips according to the manufacturer’s instructions (bioMe´rieux).
Antonie van Leeuwenhoek
16S rRNA gene sequencing and phylogenetic analysis For phylogenetic analysis, genomic DNA was extracted and purified using DNA extraction and purification kits as described previously (Kwak et al. 2016; Srinivasan et al. 2015). The 16S rRNA gene was amplified from chromosomal DNA using the universal bacterial primers 27F and 1492R (Weisburg et al. 1991), and the purified PCR products were sequenced by Genotech (Daejeon, South Korea). Phylogenetic neighbors were identified, and pairwise 16S rRNA gene sequence similarities were calculated using both the EzBioCloud server (Yoon et al. 2017) and BLAST search program at the National Center for Biotechnology Information (NCBI) website (http://blast.ncbi. nlm.nih.gov/Blast.cgi). The 16S rRNA gene sequences of related taxa were obtained from GenBank and aligned with that of strain 15J8-8T using the program Clustal X (Thompson et al. 1997). Gaps and the 50 and 30 ends of the alignment were edited manually in BioEdit (Hall 1999). Tree topologies were inferred by neighbor–joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (Felsenstein 1981), and maximum-parsimony (MP) (Fitch 1971) methods in the program MEGA7 (Kumar et al. 2016). The NJ tree was constructed using Kimura’s two-parameter model (Kimura 1980). The ML tree was inferred using the nearest neighbor interchange as the maximum-likelihood heuristic search method. The option of complete deletion of gaps was applied for both NJ and ML tree construction. A bootstrap analysis with 1000 replicate data sets was performed to assess support for the clusters. Chemotaxonomic analyses Cellular fatty acids of strain 15J8-8T, ‘L. harenae’ 15J9-9, L. arboricola DSM 21851T, L. bovis KACC 14040T, and L. insperata KACC 11674T were analysed using cells grown on R2A agar for 3 days at 30 °C. Cellular fatty acid saponification, extraction, and methylation were performed according to the Sherlock Microbial Identification System (MIDI) protocol. Fatty acid methyl esters were then analysed by gas chromatography using the Microbial Identification software package (TSBA, version 6.0) (Sasser 1990). Polar lipids were extracted using the procedure described by Minnikin et al. (1984) and identified by
two-dimensional thin layer chromatography, followed by spraying with the appropriate detection reagents (Komagata and Suzuki 1987). The following spray reagents were used for detection: 5% molybdatophosphoric acid (Merck) in ethanol for total lipids; 0.2% ninhydrin in acetone for aminolipids; molybdenum blue (Sigma) for phospholipids; and a-naphtholsulfuric acid reagent for glycolipids. Isoprenoid quinones were extracted with chloroform/methanol (2:1, v/v), evaporated under a vacuum, and reextracted in n-hexane/water (1:1, v/v). The extract was purified using Sep-Pak Silica Vac cartridges (Waters) and then analysed by high performance liquid chromatography (HPLC), as described previously (Hiraishi et al., 1996). DNA G?C content Genomic DNA of strain 15J8-8T was extracted according to the standard CTAB/NaCl protocol (Wilson, 1997). The genomic DNA G?C content of the isolate was determined by a reverse-phase HPLC analysis of individual nucleosides resulting from DNA hydrolysis and dephosphorylation using nuclease P1 and alkaline phosphatase (Mesbah et al. 1989). Singlestranded DNA from salmon testes (D7656; Sigma; DNA G?C content, 41.2 mol%) was used as a standard. DPD taxonnumber and nucleotide sequence accession numbers The Digital Protologue database TaxonNumber for strain 15J8-8T is TA00258. The 16S rRNA gene sequence of strain 15J8-8T obtained in this study has been deposited in NCBI GenBank/EMBL/DDBJ under the accession number LC315195. The accession numbers of the reference strains closely related to strain 15J8-8T are indicated in Fig. 1.
Results and discussion Phylogenetic analysis A nearly complete 16S rRNA gene sequence of strain 15J8-8T (1417 base pairs) was obtained. Sequence similarity calculations using the EzBioCloud server and pairwise sequence comparison of the isolate and
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Antonie van Leeuwenhoek Spirosoma linguale DSM 74T (CP001769) 64 87
Spirosoma aerophilum 5516J-17T (KR024033) Spirosoma fluviale MSd3T (LC019141)
96
Spirosoma pulveris JSH5-14T (KP974819)
68
Spirosoma endophyticum EX36T (GQ342559)
89
Spirosoma luteum SPM-10T (EF451726) Spirosoma rigui WPCB118T (EF507900) Spirosoma oryzae RHs22T (KJ155688)
88
Spirosoma knui 15J8-12T (LC148303)
77 82
Spirosoma soli MIMBbqt12T (KT347096) Spirosoma swuense JBM2-3T (KU865688)
58
Spirosoma aerolatum PR1012kT (JX089325)
97 96
53
Spirosoma panaciterrae Gsoil 1519T (EU370956) Fibrisoma limi BUZ 3T (CAIT01000009) Huanghella arctica R9-9T (JQ303016) Nibrella saemangeumensis GCR0103T (JN607159)
100
Nibrella viscosa GYR3121T (JN607161) 100
57
Larkinella arboricola Z-0532T (FN391025) Larkinella insperata LMG 22510T (AM000022) Larkinella terrae 15J8-8T (LC315195)
100
Larkinella bovis M2T2B15T (GQ246692)
52 62 Rudanella lutea
5715S-11T
‘Larkinella harenae’ 15J9-9 (LC177337) (EF635010) Bacteroides fragilis ATCC 25285T (X83935)
0.050
Fig. 1 Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of strain 15J8-8T among affiliated Larkinella strains and representatives of other members of the family Cytophagaceae. Bootstrap values (based on 1000 replications) greater than 50% are shown at branch points. Filled circles indicate that the corresponding nodes were also recovered in trees generated with
the neighbour–joining and maximum-parsimony algorithms. Open circles indicate that the corresponding nodes were also recovered in the tree generated with the neighbour–joining algorithm. The tree was rooted using Bacteroides fragilis ATCC 25285T (X83935) as an outgroup. Bar, 0.05 substitutions per nucleotide position
‘L. harenae’ 15J8-8T showed that strain 15J8-8T had high sequence similarity to L. bovis M2TB15T (95.0%), ‘L. harenae’ 15J9-9 (94.5%), L. arboricola Z0532T (93.2%), and L. insperata LMG 22510T (93.0%). The 16S rRNA gene sequence similarities between strain 15J8-8T and the type strains of other recognized species within the family Cytophagaceae
were less than 89%. A close relationship between the isolate and the above-mentioned members of the genus Larkinella was also evident in the constructed phylogenetic trees. The maximum-likelihood tree as well as the neighbour-joining and maximum-parsimony trees placed strain 15J8-8T within the genus Larkinella, as shown in Fig. 1. The generally accepted
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criteria for delineating bacterial species state that strains showing 16S rRNA gene sequence dissimilarity above 3% are considered to belong to separate species (Wayne et al. 1987; Stackebrandt and Goebel 1994). Based on this definition, the above data indicate that strain 15J8-8T could not be assigned to any members within the genus Larkinella and should be considered to represent a novel species of the genus Larkinella. Phenotypic characteristics Cells of strain 15J8-8T were Gram-negative, nonmotile, aerobic, pale pink pigment-producing rods without flagella (Supplementary Fig. S1). Growth occurred at 8–30 °C and pH 6–8. Growth was observed on NA and R2A agar, but not on TSA or LB agar. Strain 15J8-8T did not require NaCl for growth but could tolerate up to 1% (w/v) NaCl. Other physiological and biochemical properties of strain 15J8-8T are given in the species description. Phenotypic characteristics that differentiate the isolate from members of the genus Larkinella are listed in Table 1. In particular, strain 15J8-8T could be differentiated from L. bovis, ‘L. harenae’, L. arboricola, and L. insperata based on its ability to reduce nitrate to nitrite, produce a-chymotrypsin, utilize 5-ketogluconate weakly, as well as its inability to utilize Larabinose and to produce acid from amygdalin and Dmelibiose. Chemotaxonomic characteristics The fatty acid profile of strain 15J8-8T (Table 2) was compared with those of members of the genus Larkinella (Table 2). The isolate contained large amounts of C16:1 x5c (30.6%), C16:0 N alcohol (20.1%), and iso-C15:0 (17.6%) and moderate quantities of iso-C17:0 3-OH (7.0%) and C16:0 (6.8%), a profile typical of L. bovis, ‘L. harenae’, L. arboricola, and L. insperata. However, strain 15J8-8T differed from the reference strains in terms of the proportions of the major and some minor fatty acids and could be differentiated from them by its significantly higher amount of C16:0 N alcohol and by its lower content of iso-C17:0 3-OH and C16:0. Strain 15J8-8T contained a large amount of phosphatidylethanolamine (PE) and moderate amounts of two unidentified polar lipids (L1 and L2) (Supplementary Fig. S2), which were
identified as major polar lipids in ‘L. harenae’ 15J99 (Park et al. 2017), L. bovis, and L. insperata (Anandham et al. 2011). In contrast, the absence of phosphatidylserine and an unidentified aminophospholipid distinguished strain 15J8-8T from these three reference strains. In addition, the polar lipid profile of the isolate contained minor quantities of two unidentified aminolipids (AL2 and AL2) and an unidentified polar lipid (L3). The predominant isoprenoid quinone in strain 15J8-8T was menaquinone MK-7, which is also the major respiratory quinone found in other members of the genus Larkinella (Anandham et al. 2011; Park et al. 2017; Vancanneyt et al. 2006). DNA G?C content The DNA G?C content of strain 15J8-8T was 50.5 mol%, which is lower than the values reported for recognized Larkinella species (52.0–53.0%) (Anandham et al. 2011; Kulichevskaya et al. 2009; Vancanneyt et al. 2006). However, the value still lies within the range expected for members of the same genus, and the G?C content range of the genus Larkinella should be extended to take this result into account. Taxonomic conclusion The results of the phylogenetic analysis, phenotypic data, and chemotaxonomic characteristics indicated that strain 15J8-8T belongs to the genus Larkinella. However, based on its phylogenetic distances from other members of the genus Larkinella and its distinctive phenotypic characteristics (Table 1), it is clear that strain 15J8-8T should be classified as a novel species in the genus Larkinella, for which the name Larkinella terrae sp. nov. is proposed. Description of Larkinella terrae sp. nov. Larkinella terrae (ter’rae. L. gen. n. terrae of/from soil) Cells are Gram-negative, non-motile, and short-rod shaped (0.9–1.1 lm in width and 1.7–3.9 lm in length). After 3 days of incubation at 25 °C, colonies on R2A plates are convex, translucent, circular, pale pink, and 0.7–1.5 mm in diameter. Cells grow on NA
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Antonie van Leeuwenhoek Table 1 Differential phenotypic characteristics of strain 15J8-8T and members of the genus Larkinella Characteristic
1
2
3
4
5
Cell shape
Shot rod
Roda
Shot rod
Ring- and horsehoeshapedb
Ring- and horsehoeshapedc
Colony colour
Pale pink
Reddish pink
Pale pink
Pink
Pale pink
Growth at/on LB agar
-
?
-
?
?
TSA agar
-
?
?
-
?
8 °C 37 °C
? -
-a -a
?
?b -b
-c ?c
Gelatin hydrolysis
?
-
-
?
-
Indole production
-
-
-
?
-
Nitrate reduction to nitrite
?
-
-
-
-
Acid phosphatase
w
-d
?
?
-
Alkaline phosphatase
?
?
?
?
-
a-Chymotrypsin
?
-
-
-
-
Cystine arylamidase, esterase (C8), valine arylamidase
?
-
-
?
-
Esterase (C4)
?
-
-
?
?
a-Galactosidase, a-glucosidase, b-glucosidase, amannosidase
?
-
?
?
-
Lipase (C14) Naphthol-AS-BI-phosphohydrolase
-
?
?
we ?
?
Trypsin
?
w
?
-
-
Enzyme activity (API ZYM)
Assimilation of (API 20 NE, API 32 GN) N-Acetyl-D-glucosamine, D-glucose
?
?
?
?
-
L-Arabinose
-
?
w
?
?d
Glycogen
-
?
-
?
?
Gluconate
-
-
w
?
-
5-Ketogluconate
w
-
-
-
D-Mannitol
-
?
-
-e
-
D-Mannose
?
-
?
?
-
D-Melibiose
?
?
?
-
?
D-Ribose
-
-
?
-
-
d
Salicin
-
-
?
?
-
D-Sorbitol
-
-
-
?
-
D-Sucrose Acid production from (API 50 CH)
-
?
?
?
-
N-acetylglucosamine, D-galactose,
-
w
?
-
w
D-Adonitol,
-
-
-
w
-
dulcitol, erythritol, glycerol, inositol, Dmannitol, methyl-b-D-xylopyranoside
Amidon, glycogen, xylitol
-
w
-
w
w
Amygdalin, D-melibiose
-
?
?
?
?
D-Arabitol,
-
w
-
w
-
-
w
-
-
-
w
w
?
-
w
gluconate
L-Arabitol D-Fucose,
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methyl-a-D-glucopyranoside
Antonie van Leeuwenhoek Table 1 continued Characteristic
1
2
3
4
5
D-Glucose
w -
-
? -
-
w ?
Inulin D-Lactose, D-maltose
w
w
?
-
D-Melezitose
-
-
-
w
w
Methyl-a-D-mannopyranoside
w
?
?
-
?
D-Raffinose, D-turanose
-
w
?
-
?
L-Rhamnose
-
-
?
w
w
L-Sorbose
w
-
-
w
w
50.5
52.0a
48.0
52.1b
53.0c
DNA G?C content (mol%)
Strains: 1, 15J8-8T; 2, Larkinella bovis KACC 14040T; 3, ‘Larkinella harenae’ 15J9-9; 4, Larkinella arboricola DSM 21851T; 5, Larkinella insperata KACC 11674T All data shown were obtained in this study, unless otherwise indicated. All strains were Gram-negative. In API ZYM tests, all strains were positive for N-acetyl-b-glucosaminidase, b-galactosidase, and leucine arylamidase, but negative for a-fucosidase or bglucuronidase. In API 20 NE and API 32 GN tests, all strains were positive for utilization of D-maltose, but were negative for acetate, adipate, L-alanine, caprate, citrate, L-fucose, L-histidine, 3-hydroxybenzoate, 4-hydroxybenzoate, DL-3-hydroxybutyrate, m-inositol, itaconate, 2-ketogluconate, DL-lactate, L-malate, malonate, phenyl acetate, L-proline, propionate, L-rhamnose, L-serine, suberate, and n-valerate. In the API 50 CH system all strains produced acid from aesculin, D-arabinose, L-arabinose, arbutin, D-cellobiose, Dfructose, L-fucose, 5-ketogluconate, D-lyxose, D-mannose, D-ribose, salicin, D-sucrose, D-tagatose, D-xylose, and L-xylose, but not from 2-ketogluconate or D-sorbitol ? Positive reaction, - negative reaction, w weakly positive reaction a
Data from Anandham et al. (2011)
b
Data from Kulichevskaya et al. (2009)
c
Data from Vancanneyt et al. (2006)
d
Anandham et al. (2011) have reported the opposite result
e
Kulichevskaya et al. (2009) have reported the opposite result
and R2A but not on LB or TSA agar. Growth occurs at 8–30 °C and pH 6–8 and in the presence of 0–1% (w/ v) NaCl, with optimum conditions at 25 °C and pH 7 and in the absence of NaCl. In API 20 NE tests, positive for aesculin hydrolysis, b-galactosidase, gelatin hydrolysis, nitrate reduction to nitrite, and weakly positive for glucose fermentation, but negative for arginine dihydrolase, indole production, and urea hydrolysis. In API 32 GN and API 20 NE tests, Nacetyl-D-glucosamine, D-glucose, 5-ketogluconate (weakly, w), D-maltose, D-mannose, and D-melibiose are utilized, but acetate, adipate, L-alanine, L-arabinose, caprate, citrate, L-fucose, gluconate, glycogen, Lhistidine, 3-hydroxybenzoate, 4-hydroxybenzoate, DL3-hydroxybutyrate, itaconate, 2-ketogluconate, DLlactate, L-malate, malonate, D-mannitol, myo-inositol, phenyl acetate, L-proline, propionate, L-rhamnose, Dribose, salicin, L-serine, D-sorbitol, suberate, D-sucrose, and n-valerate are not utilized. In the API ZYM tests, positive for N-acetyl-b-glucosaminidase, acid
phosphatase (w), alkaline phosphatase, a-chymotrypsin, cysteine arylamidase, esterase (C4), esterase (C8), a-galactosidase, b-galactosidase, aglucosidase, b-glucosidase, leucine arylamidase, amannosidase, trypsin, and valine arylamidase, but negative for a-fucosidase, b-glucuronidase, lipase (C14), and naphthol-AS-BI-phosphohydrolase. In the API 50 CH tests, acid is produced from aesculin, Darabinose (w), L-arabinose (w), arbutin, D-cellobiose (w), D-fructose (w), D-fucose (w), L-fucose (w), Dglucose (w), 5-ketogluconate, D-lactose (w), D-lyxose (w), D-maltose (w), D-mannose, methyl-a-D-glucopyranoside (w), methyl-a-D-mannopyranoside (w), Dribose (w), salicin (w), L-sorbose (w), D-sucrose (w), Dtagatose (w), D-xylose (w), and L-xylose, but not from N-acetyl-glucosamine, D-adonitol, amidon, amygdalin, D-arabitol, L-arabitol, dulcitol, erythritol, Dgalactose, gentiobiose, gluconate, glycerol, glycogen, inositol, inulin, 2-ketogluconate, D-mannitol, D-melezitose, D-melibiose, methyl-b-D-xylopyranoside, D-
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Antonie van Leeuwenhoek Table 2 Cellular fatty acid profiles of strain 15J8-8T and members of the genus Larkinella Fatty acids
1
2
3
4
5
Saturated C14:0
1.1
tr
tr
–
1.9
iso-C15:0 anteiso-C15:0
17.6 tr
29.7 1.2
21.0 1.6
20.7 3.3
17.2 2.3
C16:0
6.8
12.2
7.4
9.0
14.2
C17:0
tr
–
–
–
1.1
iso-C17:0
tr
2.0
1.3
–
tr
C18:0
5.1
tr
2.7
–
7.3
Hydroxy iso-C15:0 3-OH
2.4
1.6
1.3
–
1.3
C16:0 3-OH
1.2
1.2
2.2
–
1.6
C16:0 N alcohol
20.1
1.3
3.7
6.1
–
iso-C17:0 3-OH
7.0
12.0
13.6
11.9
8.2
1.3
–
–
C16:1 x5c
30.6
32.1
39.1
43.9
37.8
C18:1 x9c
–
–
–
–
1.2
Summed feature 3(C16:1 x6c and/or C16:1 x7c)a
2.6
tr
5.5
5.1
4.6
Summed feature 4(iso-C17:1 I and/or anteiso-C17:1 B)a
1.4
tr
–
–
–
Unsaturated C14:1 x5c
Strains: 1, 15J8-8T; 2, Larkinella bovis KACC 14040T; 3, ‘Larkinella harenae’ 15J9-9; 4, Larkinella arboricola DSM 21851T; 5, Larkinella insperata KACC 11674T All data are from the present study. Values are percentages, and only fatty acids accounting for more than 1% of the total in at least one of the strains are presented tr trace (\ 1%), – not detected a
Summed feature contained two or three fatty acids that could not be separated by gas–liquid chromatography (GLC) with the MIDI system
raffinose, L-rhamnose, D-sorbitol, D-trehalose, D-turanose, or xylitol. The major cellular fatty acids are C16:1 x5c, C16:0 N alcohol, and iso-C15:0. The predominant isoprenoid quinone is MK-7. The major polar lipids are phosphatidylethanolamine and two unidentified polar lipids. The genomic G?C content is 50.5 mol%. The type strain 15J8-8T (= KCTC 52001T = JCM 31990T) was isolated from a soil sample collected on Jeju Island, South Korea (33°290 5100 N, 126°270 0800 E). Acknowledgements This work was supported by a Cooperative Research Program for Agriculture Science & Technology Development, Rural Development Administration, Republic of Korea and the Brain Pool Program of 2016 through the Korean Federation of Science and Technology Societies (KOFST) funded by the Ministry of Science, ICT and Future Planning, Republic of Korea.
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Conflict of interest The authors declare that they have no conflict of interest.
References Agarwal S, Hunnicutt DW, McBride MJ (1997) Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA. Proc Natl Acad Sci USA 94:12139–12144 Anandham R, Kwon S, Weon H, Kim S, Kim Y, Gandhi PI, Kim Y, Jee H et al (2011) Larkinella bovis sp. nov., isolated from fermented bovine products, and emended descriptions of the genus Larkinella and of Larkinella insperata Vancanneyt et al. 2006. Int J Syst Evol Microbiol 61:30–34 Cappuccino JG, Sherman N (2010) Microbiology: a laboratory manual, 9th edn. Benjamin Cummings, San Francisco
Antonie van Leeuwenhoek Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376 Fitch WM (1971) Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20:406–416 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucl Acids Symp Ser 41:95–98 Hiraishi A, Ueda Y, Ishihara J, Mori T (1996) Comparative lipoquinone analysis of influent sewage and activated sludge by highperformance liquid chromatography and photodiode array detection. J Gen Appl Microbiol 42:457–469 Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120 Komagata K, Suzuki KI (1987) Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19:161–205 Kulichevskaya IS, Zaichikova MV, Detkova EN, Dedysh SN, Zavarzin GA (2009) Larkinella arboricola sp. nov., a new spiral-shaped bacterium of the phylum Bacteroidetes isolated from the microbial community of decomposing wood. Microbiol 78:741–746 Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874 Kwak Y, Li QX, Shin JH (2016) Draft genome sequence of Mycobacterium rufum JS14T, a polycyclic-aromatic hydrocarbon-degrading bacterium from petroleum-contaminated soil in Hawaii. Stand Genomic Sci 11:47 Liu QM, Ten LN, Yu HS, Jin FX, Im WT, Lee ST (2008) Emticicia ginsengisoli sp. nov., a species of the family ‘Flexibacteraceae’ isolated from soil of a ginseng field. Int J Syst Evol Microbiol 58:1100–1105 Mesbah M, Premachandran U, Whitman WB (1989) Precise measurement of the G?C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39:159–167 Minnikin DE, O’Donnell AG, Goodfellow M, Alderson G, Athalye M, Schaal A, Parlett JH (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2:233–241 Park SJ, Lee JJ, Lee SY, Lee DS, Kim MK, Ten LN, Jung JY (2017) Larkinella harenae sp. nov., isolated from Korean beach soil. Curr Microbiol 74:798–802 Saitou N, Nei M (1987) The neighbor–joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. Newark, DE: MIDI Inc Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC, pp 607–654 Srinivasan S, Kim M, Joo E, Lee SY, Lee DS, Jung HY (2015) Complete genome sequence of Rufibacter sp. DG31D, a bacterium resistant to gamma and UV radiation toxicity. Mol Cell Toxicol 11:415–421 Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25:4876–4882 Tittsler RP, Sandholzer LA (1936) The use of semi-solid agar for the detection of bacterial motility. J Bacteriol 31:575–580 Vancanneyt M, Nedashkovskaya OI, Snauwaert C, Mortier S, Vandemeulebroecke K, Hoste B, Dawyndt P, Frolova GM, Janssens D, Swings J (2006) Larkinella insperata gen. nov., sp. nov., a bacterium of the phylum ‘Bacteroidetes’ isolated from water of a steam generator. Int J Syst Evol Microbiol 56:237–241 Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Stackebrandt E, Starr MP, Tru¨per HG (1987) Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37:463–464 Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703 Wilson K (1997) Preparation of genomic DNA from bacteria. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology. Wiley, New York, pp 2.4.1–2.4.5 Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017) Introducing EzBioCloud: a taxonomically united database of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol 67:1613–1617
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ORIGINAL PAPER
Larkinella terrae sp. nov., isolated from soil on Jeju Island, South Korea Leonid N. Ten . Jongguen Jeon . Su-Jin Park . Sangkyu Park . Seung-Yeol Lee . Myung Kyum Kim . Hee-Young Jung
Received: 28 August 2017 / Accepted: 30 September 2017 Ó Springer International Publishing AG 2017
Abstract A Gram-stain negative, non-motile, rodshaped, aerobic bacterium, designated 15J8-8T, was isolated from a soil sample collected on Jeju Island, South Korea, and characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain 15J8-8T belongs to the family Cytophagaceae and is related to Larkinella bovis M2TB15T (95.0%), ‘Larkinella harenae’ 15J9-9 (94.5%), Larkinella arboricola Z0532T (93.2%), and Larkinella insperata LMG 22510T (93.0%). The DNA G?C content of strain 15J8-8T was 50.5 mol%. The detection of phosphatidylethanolamine and two unidentified polar lipids as major polar lipids; menaquinone-7 as the predominant quinone; and C16:1 x5c, C16:0 N alcohol,
Electronic supplementary material The online version of this article (doi:10.1007/s10482-017-0955-y) contains supplementary material, which is available to authorized users. L. N. Ten J. Jeon S.-J. Park S. Park S.-Y. Lee H.-Y. Jung (&) School of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea e-mail: [email protected] M. K. Kim Department of Bio and Environmental Technology, Seoul Women’s University, Seoul 01797, Republic of Korea H.-Y. Jung Institute of Plant Medicine, Kyungpook National University, Daegu 41566, Republic of Korea
and iso-C15:0 as the major fatty acids also supported the affiliation of the isolate to the genus Larkinella. Based on its phenotypic properties and phylogenetic distinctiveness, strain 15J8-8T should be classified in the genus Larkinella as representative of a novel species, for which the name Larkinella terrae sp. nov. is proposed. The type strain is 15J8-8T (= KCTC 52001T = JCM 31990T). Keywords
Larkinella Bacteroidetes Soil bacteria
Introduction The genus Larkinella was originally proposed with Larkinella insperata as the type species (Vancanneyt et al. 2006) and the genus description was later emended by Anandham et al. (2011). At the time of writing, the genus Larkinella contains three species with validly published names (http://www.bacterio. net/larkinella.html). Recently, one other strain, ‘Larkinella harenae’ 15J9-9 (Park et al. 2017), was proposed as a novel Larkinella species and is awaiting validation. The type strains of Larkinella species were recovered from the water of a steam generator in a pharmaceutical company (Vancanneyt et al. 2006), fermented bovine products (Anandham et al. 2011), a humified solution produced by spruce wood decomposition (Kulichevskaya et al. 2009), and soil (Park et al. 2017). Members of the genus Larkinella are
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Gram-negative, non-spore-forming, non-motile or motile by gliding, and pink-pigmented bacteria that are characterized chemotaxonomically as having menaquinone MK-7 as the predominant quinone; isoC15:0, C16:1 x5c, iso-C17:0 3-OH and summed feature 3 (C16:1 x6c and/or C16:1 x7c and/or iso-C15:0 2-OH) as the major fatty acids; and phosphatidylethanolamine, phosphatidylserine, two unidentified aminophospholipids, and two unidentified polar lipids as the major polar lipids. In the course of screening for novel bacteria, strain 15J8-8T was isolated from a soil sample collected on Jeju Island, South Korea. On the basis of 16S rRNA gene sequence analysis, this isolate was considered to be a Larkinella-like strain. Strain 15J8-8T was subjected to a detailed investigation using a polyphasic taxonomic approach that included genotypic, chemotaxonomic, and phenotypic analyses. Based on the results obtained in this study, we propose that strain 15J8-8T be placed in the genus Larkinella as the type strain of a novel species.
Materials and methods Isolation of the bacterial strain and culture conditions Strain 15J8-8T was isolated from a soil sample collected on Jeju Island, South Korea (33°290 5100 N, 126°270 0800 E). The sample was suspended in 10 mL distilled water, vortexed, and serially diluted. One hundred microliters of each dilution was spread onto Reasoner’s 2A (R2A) agar plates (Difco, USA) and incubated at 25 °C for 1 week. On the 107-diluted plate, 30–35 colonies appeared; one pale pink colony, designated 15J8-8T, was purified by transferring it onto a fresh plate and incubating the new plate under the same conditions. The isolate was routinely cultured on R2A agar at 25 °C and was maintained as a glycerol suspension (20%, w/v) at -70 °C. Strain 15J8-8T has been deposited in the Korean Collection for Type Cultures (KCTC) and the Japan Collection of Microorganisms (JCM). Reference strains Larkinella arboricola DSM 21851T was purchased from the German Collection of Microorganisms and
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Cell Cultures, and Larkinella bovis KACC 14040T and L. insperata KACC 11674T were obtained from the Korean Agricultural Culture Collection. Our previously isolated strain, ‘L. harenae’ 15J9-9, was maintained in our laboratory collection. Unless otherwise noted, the morphological, physiological, and biochemical characteristics of strain 15J8-8T and of the four above-mentioned reference strains were investigated by using routine cultivation on R2A agar at 30 °C for 3 days. Phenotypic and biochemical characteristics The Gram reaction of strain 15J8-8T was examined using a standard staining method (Smibert and Krieg 1994). The cell morphology of strain 15J8-8T, grown for 3 days at 25 °C on R2A agar, was observed with an Olympus light microscope (10009 magnification) and a Hitachi HT7700 transmission electron microscope. Motility was investigated on 0.5% (w/v) semisolid R2A agar (Tittsler and Sandholzer 1936) and gliding motility was assessed by the microscopic hanging drop technique (Agarwal et al. 1997). Catalase and oxidase tests were performed as outlined by Cappuccino and Sherman (2010). Growth was assessed on R2A agar, Luria–Bertani (LB) agar, nutrient agar (NA), and trypticase soy agar (TSA), all purchased from Difco. Anaerobic growth was tested as described previously (Liu et al. 2008). The effect of pH on growth was evaluated in R2A broth using three different buffers (final concentration, 100 mM): sodium acetate buffer (for pH 4.0–6.0), potassium phosphate buffer (for pH 7.0–8.0), and Tris buffer (for pH 9.0–10.0). Growth at 4, 8, 15, 20, 25, 30, 37, and 42 °C was assessed after 7 days of incubation on R2A agar. Salt tolerance was tested after 7 days of incubation in R2A broth supplemented with 0.5, 1, 2, 3, 4, 5, and 10% (w/v) NaCl. Enzyme activities, assimilation of carbon sources, acid production from substrates, and other physiological characteristics were determined by inoculating API ZYM, API 20 NE, API ID 32 GN, and API 50 CH strips according to the manufacturer’s instructions (bioMe´rieux).
Antonie van Leeuwenhoek
16S rRNA gene sequencing and phylogenetic analysis For phylogenetic analysis, genomic DNA was extracted and purified using DNA extraction and purification kits as described previously (Kwak et al. 2016; Srinivasan et al. 2015). The 16S rRNA gene was amplified from chromosomal DNA using the universal bacterial primers 27F and 1492R (Weisburg et al. 1991), and the purified PCR products were sequenced by Genotech (Daejeon, South Korea). Phylogenetic neighbors were identified, and pairwise 16S rRNA gene sequence similarities were calculated using both the EzBioCloud server (Yoon et al. 2017) and BLAST search program at the National Center for Biotechnology Information (NCBI) website (http://blast.ncbi. nlm.nih.gov/Blast.cgi). The 16S rRNA gene sequences of related taxa were obtained from GenBank and aligned with that of strain 15J8-8T using the program Clustal X (Thompson et al. 1997). Gaps and the 50 and 30 ends of the alignment were edited manually in BioEdit (Hall 1999). Tree topologies were inferred by neighbor–joining (NJ) (Saitou and Nei 1987), maximum-likelihood (ML) (Felsenstein 1981), and maximum-parsimony (MP) (Fitch 1971) methods in the program MEGA7 (Kumar et al. 2016). The NJ tree was constructed using Kimura’s two-parameter model (Kimura 1980). The ML tree was inferred using the nearest neighbor interchange as the maximum-likelihood heuristic search method. The option of complete deletion of gaps was applied for both NJ and ML tree construction. A bootstrap analysis with 1000 replicate data sets was performed to assess support for the clusters. Chemotaxonomic analyses Cellular fatty acids of strain 15J8-8T, ‘L. harenae’ 15J9-9, L. arboricola DSM 21851T, L. bovis KACC 14040T, and L. insperata KACC 11674T were analysed using cells grown on R2A agar for 3 days at 30 °C. Cellular fatty acid saponification, extraction, and methylation were performed according to the Sherlock Microbial Identification System (MIDI) protocol. Fatty acid methyl esters were then analysed by gas chromatography using the Microbial Identification software package (TSBA, version 6.0) (Sasser 1990). Polar lipids were extracted using the procedure described by Minnikin et al. (1984) and identified by
two-dimensional thin layer chromatography, followed by spraying with the appropriate detection reagents (Komagata and Suzuki 1987). The following spray reagents were used for detection: 5% molybdatophosphoric acid (Merck) in ethanol for total lipids; 0.2% ninhydrin in acetone for aminolipids; molybdenum blue (Sigma) for phospholipids; and a-naphtholsulfuric acid reagent for glycolipids. Isoprenoid quinones were extracted with chloroform/methanol (2:1, v/v), evaporated under a vacuum, and reextracted in n-hexane/water (1:1, v/v). The extract was purified using Sep-Pak Silica Vac cartridges (Waters) and then analysed by high performance liquid chromatography (HPLC), as described previously (Hiraishi et al., 1996). DNA G?C content Genomic DNA of strain 15J8-8T was extracted according to the standard CTAB/NaCl protocol (Wilson, 1997). The genomic DNA G?C content of the isolate was determined by a reverse-phase HPLC analysis of individual nucleosides resulting from DNA hydrolysis and dephosphorylation using nuclease P1 and alkaline phosphatase (Mesbah et al. 1989). Singlestranded DNA from salmon testes (D7656; Sigma; DNA G?C content, 41.2 mol%) was used as a standard. DPD taxonnumber and nucleotide sequence accession numbers The Digital Protologue database TaxonNumber for strain 15J8-8T is TA00258. The 16S rRNA gene sequence of strain 15J8-8T obtained in this study has been deposited in NCBI GenBank/EMBL/DDBJ under the accession number LC315195. The accession numbers of the reference strains closely related to strain 15J8-8T are indicated in Fig. 1.
Results and discussion Phylogenetic analysis A nearly complete 16S rRNA gene sequence of strain 15J8-8T (1417 base pairs) was obtained. Sequence similarity calculations using the EzBioCloud server and pairwise sequence comparison of the isolate and
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Antonie van Leeuwenhoek Spirosoma linguale DSM 74T (CP001769) 64 87
Spirosoma aerophilum 5516J-17T (KR024033) Spirosoma fluviale MSd3T (LC019141)
96
Spirosoma pulveris JSH5-14T (KP974819)
68
Spirosoma endophyticum EX36T (GQ342559)
89
Spirosoma luteum SPM-10T (EF451726) Spirosoma rigui WPCB118T (EF507900) Spirosoma oryzae RHs22T (KJ155688)
88
Spirosoma knui 15J8-12T (LC148303)
77 82
Spirosoma soli MIMBbqt12T (KT347096) Spirosoma swuense JBM2-3T (KU865688)
58
Spirosoma aerolatum PR1012kT (JX089325)
97 96
53
Spirosoma panaciterrae Gsoil 1519T (EU370956) Fibrisoma limi BUZ 3T (CAIT01000009) Huanghella arctica R9-9T (JQ303016) Nibrella saemangeumensis GCR0103T (JN607159)
100
Nibrella viscosa GYR3121T (JN607161) 100
57
Larkinella arboricola Z-0532T (FN391025) Larkinella insperata LMG 22510T (AM000022) Larkinella terrae 15J8-8T (LC315195)
100
Larkinella bovis M2T2B15T (GQ246692)
52 62 Rudanella lutea
5715S-11T
‘Larkinella harenae’ 15J9-9 (LC177337) (EF635010) Bacteroides fragilis ATCC 25285T (X83935)
0.050
Fig. 1 Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of strain 15J8-8T among affiliated Larkinella strains and representatives of other members of the family Cytophagaceae. Bootstrap values (based on 1000 replications) greater than 50% are shown at branch points. Filled circles indicate that the corresponding nodes were also recovered in trees generated with
the neighbour–joining and maximum-parsimony algorithms. Open circles indicate that the corresponding nodes were also recovered in the tree generated with the neighbour–joining algorithm. The tree was rooted using Bacteroides fragilis ATCC 25285T (X83935) as an outgroup. Bar, 0.05 substitutions per nucleotide position
‘L. harenae’ 15J8-8T showed that strain 15J8-8T had high sequence similarity to L. bovis M2TB15T (95.0%), ‘L. harenae’ 15J9-9 (94.5%), L. arboricola Z0532T (93.2%), and L. insperata LMG 22510T (93.0%). The 16S rRNA gene sequence similarities between strain 15J8-8T and the type strains of other recognized species within the family Cytophagaceae
were less than 89%. A close relationship between the isolate and the above-mentioned members of the genus Larkinella was also evident in the constructed phylogenetic trees. The maximum-likelihood tree as well as the neighbour-joining and maximum-parsimony trees placed strain 15J8-8T within the genus Larkinella, as shown in Fig. 1. The generally accepted
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Antonie van Leeuwenhoek
criteria for delineating bacterial species state that strains showing 16S rRNA gene sequence dissimilarity above 3% are considered to belong to separate species (Wayne et al. 1987; Stackebrandt and Goebel 1994). Based on this definition, the above data indicate that strain 15J8-8T could not be assigned to any members within the genus Larkinella and should be considered to represent a novel species of the genus Larkinella. Phenotypic characteristics Cells of strain 15J8-8T were Gram-negative, nonmotile, aerobic, pale pink pigment-producing rods without flagella (Supplementary Fig. S1). Growth occurred at 8–30 °C and pH 6–8. Growth was observed on NA and R2A agar, but not on TSA or LB agar. Strain 15J8-8T did not require NaCl for growth but could tolerate up to 1% (w/v) NaCl. Other physiological and biochemical properties of strain 15J8-8T are given in the species description. Phenotypic characteristics that differentiate the isolate from members of the genus Larkinella are listed in Table 1. In particular, strain 15J8-8T could be differentiated from L. bovis, ‘L. harenae’, L. arboricola, and L. insperata based on its ability to reduce nitrate to nitrite, produce a-chymotrypsin, utilize 5-ketogluconate weakly, as well as its inability to utilize Larabinose and to produce acid from amygdalin and Dmelibiose. Chemotaxonomic characteristics The fatty acid profile of strain 15J8-8T (Table 2) was compared with those of members of the genus Larkinella (Table 2). The isolate contained large amounts of C16:1 x5c (30.6%), C16:0 N alcohol (20.1%), and iso-C15:0 (17.6%) and moderate quantities of iso-C17:0 3-OH (7.0%) and C16:0 (6.8%), a profile typical of L. bovis, ‘L. harenae’, L. arboricola, and L. insperata. However, strain 15J8-8T differed from the reference strains in terms of the proportions of the major and some minor fatty acids and could be differentiated from them by its significantly higher amount of C16:0 N alcohol and by its lower content of iso-C17:0 3-OH and C16:0. Strain 15J8-8T contained a large amount of phosphatidylethanolamine (PE) and moderate amounts of two unidentified polar lipids (L1 and L2) (Supplementary Fig. S2), which were
identified as major polar lipids in ‘L. harenae’ 15J99 (Park et al. 2017), L. bovis, and L. insperata (Anandham et al. 2011). In contrast, the absence of phosphatidylserine and an unidentified aminophospholipid distinguished strain 15J8-8T from these three reference strains. In addition, the polar lipid profile of the isolate contained minor quantities of two unidentified aminolipids (AL2 and AL2) and an unidentified polar lipid (L3). The predominant isoprenoid quinone in strain 15J8-8T was menaquinone MK-7, which is also the major respiratory quinone found in other members of the genus Larkinella (Anandham et al. 2011; Park et al. 2017; Vancanneyt et al. 2006). DNA G?C content The DNA G?C content of strain 15J8-8T was 50.5 mol%, which is lower than the values reported for recognized Larkinella species (52.0–53.0%) (Anandham et al. 2011; Kulichevskaya et al. 2009; Vancanneyt et al. 2006). However, the value still lies within the range expected for members of the same genus, and the G?C content range of the genus Larkinella should be extended to take this result into account. Taxonomic conclusion The results of the phylogenetic analysis, phenotypic data, and chemotaxonomic characteristics indicated that strain 15J8-8T belongs to the genus Larkinella. However, based on its phylogenetic distances from other members of the genus Larkinella and its distinctive phenotypic characteristics (Table 1), it is clear that strain 15J8-8T should be classified as a novel species in the genus Larkinella, for which the name Larkinella terrae sp. nov. is proposed. Description of Larkinella terrae sp. nov. Larkinella terrae (ter’rae. L. gen. n. terrae of/from soil) Cells are Gram-negative, non-motile, and short-rod shaped (0.9–1.1 lm in width and 1.7–3.9 lm in length). After 3 days of incubation at 25 °C, colonies on R2A plates are convex, translucent, circular, pale pink, and 0.7–1.5 mm in diameter. Cells grow on NA
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Antonie van Leeuwenhoek Table 1 Differential phenotypic characteristics of strain 15J8-8T and members of the genus Larkinella Characteristic
1
2
3
4
5
Cell shape
Shot rod
Roda
Shot rod
Ring- and horsehoeshapedb
Ring- and horsehoeshapedc
Colony colour
Pale pink
Reddish pink
Pale pink
Pink
Pale pink
Growth at/on LB agar
-
?
-
?
?
TSA agar
-
?
?
-
?
8 °C 37 °C
? -
-a -a
?
?b -b
-c ?c
Gelatin hydrolysis
?
-
-
?
-
Indole production
-
-
-
?
-
Nitrate reduction to nitrite
?
-
-
-
-
Acid phosphatase
w
-d
?
?
-
Alkaline phosphatase
?
?
?
?
-
a-Chymotrypsin
?
-
-
-
-
Cystine arylamidase, esterase (C8), valine arylamidase
?
-
-
?
-
Esterase (C4)
?
-
-
?
?
a-Galactosidase, a-glucosidase, b-glucosidase, amannosidase
?
-
?
?
-
Lipase (C14) Naphthol-AS-BI-phosphohydrolase
-
?
?
we ?
?
Trypsin
?
w
?
-
-
Enzyme activity (API ZYM)
Assimilation of (API 20 NE, API 32 GN) N-Acetyl-D-glucosamine, D-glucose
?
?
?
?
-
L-Arabinose
-
?
w
?
?d
Glycogen
-
?
-
?
?
Gluconate
-
-
w
?
-
5-Ketogluconate
w
-
-
-
D-Mannitol
-
?
-
-e
-
D-Mannose
?
-
?
?
-
D-Melibiose
?
?
?
-
?
D-Ribose
-
-
?
-
-
d
Salicin
-
-
?
?
-
D-Sorbitol
-
-
-
?
-
D-Sucrose Acid production from (API 50 CH)
-
?
?
?
-
N-acetylglucosamine, D-galactose,
-
w
?
-
w
D-Adonitol,
-
-
-
w
-
dulcitol, erythritol, glycerol, inositol, Dmannitol, methyl-b-D-xylopyranoside
Amidon, glycogen, xylitol
-
w
-
w
w
Amygdalin, D-melibiose
-
?
?
?
?
D-Arabitol,
-
w
-
w
-
-
w
-
-
-
w
w
?
-
w
gluconate
L-Arabitol D-Fucose,
123
methyl-a-D-glucopyranoside
Antonie van Leeuwenhoek Table 1 continued Characteristic
1
2
3
4
5
D-Glucose
w -
-
? -
-
w ?
Inulin D-Lactose, D-maltose
w
w
?
-
D-Melezitose
-
-
-
w
w
Methyl-a-D-mannopyranoside
w
?
?
-
?
D-Raffinose, D-turanose
-
w
?
-
?
L-Rhamnose
-
-
?
w
w
L-Sorbose
w
-
-
w
w
50.5
52.0a
48.0
52.1b
53.0c
DNA G?C content (mol%)
Strains: 1, 15J8-8T; 2, Larkinella bovis KACC 14040T; 3, ‘Larkinella harenae’ 15J9-9; 4, Larkinella arboricola DSM 21851T; 5, Larkinella insperata KACC 11674T All data shown were obtained in this study, unless otherwise indicated. All strains were Gram-negative. In API ZYM tests, all strains were positive for N-acetyl-b-glucosaminidase, b-galactosidase, and leucine arylamidase, but negative for a-fucosidase or bglucuronidase. In API 20 NE and API 32 GN tests, all strains were positive for utilization of D-maltose, but were negative for acetate, adipate, L-alanine, caprate, citrate, L-fucose, L-histidine, 3-hydroxybenzoate, 4-hydroxybenzoate, DL-3-hydroxybutyrate, m-inositol, itaconate, 2-ketogluconate, DL-lactate, L-malate, malonate, phenyl acetate, L-proline, propionate, L-rhamnose, L-serine, suberate, and n-valerate. In the API 50 CH system all strains produced acid from aesculin, D-arabinose, L-arabinose, arbutin, D-cellobiose, Dfructose, L-fucose, 5-ketogluconate, D-lyxose, D-mannose, D-ribose, salicin, D-sucrose, D-tagatose, D-xylose, and L-xylose, but not from 2-ketogluconate or D-sorbitol ? Positive reaction, - negative reaction, w weakly positive reaction a
Data from Anandham et al. (2011)
b
Data from Kulichevskaya et al. (2009)
c
Data from Vancanneyt et al. (2006)
d
Anandham et al. (2011) have reported the opposite result
e
Kulichevskaya et al. (2009) have reported the opposite result
and R2A but not on LB or TSA agar. Growth occurs at 8–30 °C and pH 6–8 and in the presence of 0–1% (w/ v) NaCl, with optimum conditions at 25 °C and pH 7 and in the absence of NaCl. In API 20 NE tests, positive for aesculin hydrolysis, b-galactosidase, gelatin hydrolysis, nitrate reduction to nitrite, and weakly positive for glucose fermentation, but negative for arginine dihydrolase, indole production, and urea hydrolysis. In API 32 GN and API 20 NE tests, Nacetyl-D-glucosamine, D-glucose, 5-ketogluconate (weakly, w), D-maltose, D-mannose, and D-melibiose are utilized, but acetate, adipate, L-alanine, L-arabinose, caprate, citrate, L-fucose, gluconate, glycogen, Lhistidine, 3-hydroxybenzoate, 4-hydroxybenzoate, DL3-hydroxybutyrate, itaconate, 2-ketogluconate, DLlactate, L-malate, malonate, D-mannitol, myo-inositol, phenyl acetate, L-proline, propionate, L-rhamnose, Dribose, salicin, L-serine, D-sorbitol, suberate, D-sucrose, and n-valerate are not utilized. In the API ZYM tests, positive for N-acetyl-b-glucosaminidase, acid
phosphatase (w), alkaline phosphatase, a-chymotrypsin, cysteine arylamidase, esterase (C4), esterase (C8), a-galactosidase, b-galactosidase, aglucosidase, b-glucosidase, leucine arylamidase, amannosidase, trypsin, and valine arylamidase, but negative for a-fucosidase, b-glucuronidase, lipase (C14), and naphthol-AS-BI-phosphohydrolase. In the API 50 CH tests, acid is produced from aesculin, Darabinose (w), L-arabinose (w), arbutin, D-cellobiose (w), D-fructose (w), D-fucose (w), L-fucose (w), Dglucose (w), 5-ketogluconate, D-lactose (w), D-lyxose (w), D-maltose (w), D-mannose, methyl-a-D-glucopyranoside (w), methyl-a-D-mannopyranoside (w), Dribose (w), salicin (w), L-sorbose (w), D-sucrose (w), Dtagatose (w), D-xylose (w), and L-xylose, but not from N-acetyl-glucosamine, D-adonitol, amidon, amygdalin, D-arabitol, L-arabitol, dulcitol, erythritol, Dgalactose, gentiobiose, gluconate, glycerol, glycogen, inositol, inulin, 2-ketogluconate, D-mannitol, D-melezitose, D-melibiose, methyl-b-D-xylopyranoside, D-
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Antonie van Leeuwenhoek Table 2 Cellular fatty acid profiles of strain 15J8-8T and members of the genus Larkinella Fatty acids
1
2
3
4
5
Saturated C14:0
1.1
tr
tr
–
1.9
iso-C15:0 anteiso-C15:0
17.6 tr
29.7 1.2
21.0 1.6
20.7 3.3
17.2 2.3
C16:0
6.8
12.2
7.4
9.0
14.2
C17:0
tr
–
–
–
1.1
iso-C17:0
tr
2.0
1.3
–
tr
C18:0
5.1
tr
2.7
–
7.3
Hydroxy iso-C15:0 3-OH
2.4
1.6
1.3
–
1.3
C16:0 3-OH
1.2
1.2
2.2
–
1.6
C16:0 N alcohol
20.1
1.3
3.7
6.1
–
iso-C17:0 3-OH
7.0
12.0
13.6
11.9
8.2
1.3
–
–
C16:1 x5c
30.6
32.1
39.1
43.9
37.8
C18:1 x9c
–
–
–
–
1.2
Summed feature 3(C16:1 x6c and/or C16:1 x7c)a
2.6
tr
5.5
5.1
4.6
Summed feature 4(iso-C17:1 I and/or anteiso-C17:1 B)a
1.4
tr
–
–
–
Unsaturated C14:1 x5c
Strains: 1, 15J8-8T; 2, Larkinella bovis KACC 14040T; 3, ‘Larkinella harenae’ 15J9-9; 4, Larkinella arboricola DSM 21851T; 5, Larkinella insperata KACC 11674T All data are from the present study. Values are percentages, and only fatty acids accounting for more than 1% of the total in at least one of the strains are presented tr trace (\ 1%), – not detected a
Summed feature contained two or three fatty acids that could not be separated by gas–liquid chromatography (GLC) with the MIDI system
raffinose, L-rhamnose, D-sorbitol, D-trehalose, D-turanose, or xylitol. The major cellular fatty acids are C16:1 x5c, C16:0 N alcohol, and iso-C15:0. The predominant isoprenoid quinone is MK-7. The major polar lipids are phosphatidylethanolamine and two unidentified polar lipids. The genomic G?C content is 50.5 mol%. The type strain 15J8-8T (= KCTC 52001T = JCM 31990T) was isolated from a soil sample collected on Jeju Island, South Korea (33°290 5100 N, 126°270 0800 E). Acknowledgements This work was supported by a Cooperative Research Program for Agriculture Science & Technology Development, Rural Development Administration, Republic of Korea and the Brain Pool Program of 2016 through the Korean Federation of Science and Technology Societies (KOFST) funded by the Ministry of Science, ICT and Future Planning, Republic of Korea.
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Conflict of interest The authors declare that they have no conflict of interest.
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