Download Allohumibacter endophyticus gen. nov., sp. nov., isolated from the

International Journal of Systematic and Evolutionary Microbiology (2016), 66, 1823–1827
DOI 10.1099/ijsem.0.000948
Allohumibacter endophyticus gen. nov., sp. nov.,
isolated from the root of wild Artemisia princeps
(mugwort)
Yu Ri Kim,1 Tae-Su Kim,1 Ji-Hye Han,1,2 Yochan Joung,1,3 Jisun Park1
and Seung Bum Kim1
Correspondence
Seung Bum Kim
1
Department of Microbiology and Molecular Biology, Chungnam National University,
Daejeon 305-764, Republic of Korea
[email protected]
2
Bacterial Resources Research Team, Freshwater Bioresources Research Division, Nakdonggang
National Institute of Biological Resources, 137, Donam 2-Gil, Sangju, Gyeongsangbuk-Do, 37242,
Republic of Korea
3
Department of Biology, Inha University, Incheon, 402-751, Republic of Korea
A novel actinobacterium designated strain MWE-A11T was isolated from the root of wild
Artemisia princeps (mugwort). The isolate was aerobic, Gram-stain-positive and short rodshaped, and the colonies were yellow and circular with entire margin. Strain MWE-A11T grew
at 15–37 8C and pH 6.0–8.0.The predominant isoprenoid quinones were MK-11 and
MK-10.The predominant fatty acids were anteiso-C15 : 0 and iso-C16 : 0, and the DNA G+C
content was 68.8 mol%. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol
and an unidentified glycolipid. The peptidoglycan contained 2,4-diaminobutyric acid as the
diagnostic diamino acid, and the acyl type was glycolyl. Phylogenetic analyses based on
16S rRNA gene sequence comparisons indicated that strain MWE-A11T was affiliated with the
family Microbacteriaceae, and was most closely related to the type strains of Humibacter antri
(96.4 % 16S rRNA gene sequence similarity), Herbiconiux moechotypicola (96.3 %),
Leifsonia soli (96.3 %), Leifsonia lichenia (96.2 %), Leifsonia xyli subsp. cynodontis (96.1 %),
Microbacterium testaceum (96.0 %) and Humibacter albus (96.0 %). However, the combination
of chemotaxonomic properties clearly distinguished strain MWE-A11T from the related taxa at
genus level. Accordingly, Allohumibacter endophyticus gen. nov., sp. nov. is proposed to
accommodate a new member of the family Microbacteriaceae. The type strain of the type
species is MWE-A11T (5JCM 19371T5KCTC 29232T).
The family Microbacteriaceae was first proposed by Park
et al. (1993) and later emended by Stackebrandt et al.
(1997) and Zhi et al. (2009). At the time of writing, 51
genera are classified as members of the family Microbacteriaceae (http://www.bacterio.net/), including Conyzicola,
Frigoribacterium, Labedella and Okibacterium isolated from
plant roots (Kim et al., 2014; Wang et al., 2015a, b, c).
Members of the family Microbacteriaceae have ornithine,
lysine or 2,4-diaminobutyric acid as diagnostic diamino
acids in their cell-wall peptidoglycan, menaquinones with
7–9 isoprene units as the major respiratory quinones,
Abbreviation: ISP, International Streptomyces Project.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA
gene sequence of strain MWE-A11T is JQ723726.
Two supplementary figures and a supplementary table are available
with the online Supplementary Material.
000948 G 2016 IUMS
anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0 as the main
fatty acids, and DNA G+C contents in the range
59–76 mol% (Evtushenko, 2012; Table 1).
Strain MWE-A11T was isolated from the root of natively
growing Artemisia princeps (mugwort) sampled in the
Daejeon area (geographical location: 36841961.04N,
127853909.66E), by using a standard dilution-plating technique on R2A (BD Difco) plates incubated at 30 8C under
aerobic conditions. The isolate was sub-cultured several
times to obtain a pure culture, and was stored at 280 8C
in 20 % aqueous glycerol solution. The growth of strain
MWE-A11T was observed on other media such as trypticase soy agar (TSA; BD) or nutrient agar (NA; BD) for
2 days at 30 8C.
Cellular morphology was observed using transmission electron microscopy (JEM-1010; JEOL) of cells grown on TSA
for 2 days at 30 8C. A transmission electron micrograph of
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1823
1824
ai-C15 : 0,
i-C16 : 0
67–74
ai-C15 : 0,
i-C16 : 0
58.7
DAB
ND
DAB
Acetyl
C18 : 1v7c/
C15 : 1v6c
64
negatively stained cells is shown as Fig. S1 (available in the
online Supplementary material). Gram staining was performed by using a Gram staining kit (Sigma-Aldrich).
Motility was examined by light microscopy. Catalase and
oxidase activity, and hydrolysis of casein, starch and
DNA were determined following the methods of Han
et al. (2003). Biochemical characteristics were tested by
using API 20NE and API ZYM kits (bioMérieux), and
acid production was tested using the API 50 CH system
(bioMérieux) according to the instructions of the manufacturer. Growth at different temperatures, pH and NaCl concentrations were determined on agar plates after incubation
for 7 days. Growth on different media was examined using
NA, TSA, R2A and International Streptomyces Project (ISP)
2 (0.4 % glucose, 0.4 % yeast extract, 1 % malt extract and
20 % agar) media at 30 8C. Growth at 4, 10, 15, 20, 25, 30,
35, 37 and 42 8C was examined on TSA over 7 days.
Growth at different pH (pH 4–10 at 1 unit intervals) and
at different salt concentrations (0–4 % at 0.5 % intervals
and 5–10 % at 1 % intervals, w/v) was tested using TSA
as the basal medium. Sodium acetate (for pH 4 and 5),
potassium phosphate (for pH 6, 7 and 8) or sodium
bicarbonate (for pH 9 and 10) buffer was used for pH
adjustment of the medium.
DAB
Acetyl
ai-C15 : 0, ai-C17 : 0,
i-C16 : 0
69–73
ND
DAB
Acetyl
ai-C15 : 0, ai-C17 : 0,
i-C16 : 0
62–73
Colonies of strain MWE-A11T on TSA were pale yellow,
round and convex. The cells were aerobic, non-motile and
short rod-shaped. Strain MWE-A11T grew at 15–37 8C
(optimum 30 8C) and also at pH 6.0–8.0, and could tolerate
up to 5 % (w/v) NaCl (optimum 0 %). The biochemical
and physiological properties are listed in the species
description.
Diamino acids
Murein Acyl type
Major fatty acidsD
DAB
Glycolyl
ai-C15 : 0,
i-C16 : 0
DNA G+C content (mol%)
68.8
Orn, DAB
Acetyl
ai-C17 : 0,
ch-C17 : 0
62.8–68
DAB
Acetyl*
ai-C15 : 0,
ai-C17 : 0
66–71.1
Extraction of genomic DNA, and PCR amplification and
sequence analysis of the 16S rRNA gene sequence followed
previously described procedures (Kim et al., 2014). The
16S rRNA gene sequence of the isolate was compared
with other sequences in the EzTaxon-e database
(http://eztaxon-e.ezbiocloud.net; Kim et al., 2012b) based
on the pairwise alignment method. The 16S rRNA gene
sequences of the isolate and other closely related species
were aligned, and phylogenetic trees were reconstructed
using MEGA software version 6 (Tamura et al., 2013). Phylogenetic trees were inferred with the neighbour-joining
(Saitou & Nei, 1987), maximum-parsimony (Kluge &
Farris, 1969) and maximum-likelihood (Felsenstein, 1981)
algorithms. Tree topologies were evaluated by bootstrap
analysis (Felsenstein, 1985) based on 1000 replicates.
*Determined from this study.
Dai, anteiso; i, iso; ch, cyclohexyl.
Rods
2
MK-13, 12
Rods
2
MK-12(H2),
11(H2)
Lys
Fragmenting hyphae/rods
Rods
2
2
MK-12 (11, 13)
MK-11, 10
Rods/filaments
+/2
MK-11 (10, 12)
Rods
Rods
Rods
2
+/2
2
MK-11, 10 MK-11, 12 MK-11 (10)
Morphology
Motility
Major quinones
8
7
6
5
4
3
2
1
Characteristic
Taxa: 1, MWE-A11T; 2, Humibacter (data from Kim et al., 2015; Lee, 2013; Vaz-Moreira et al., 2008); 3, Herbiconiux (Behrendt et al., 2011; Hamada et al., 2012; Kim et al., 2012a) ; 4, Leifsonia
(Evtushenko, 2012); 5, Agromyces (Evtushenko, 2012); 6, Schumannella (An et al., 2008); 7, Lysinimonas (Jang et al., 2013); 8, Rudaibacter (Kim et al., 2013). DAB, 2,4-diaminobytyric acid;
Orn, ornithine; Lys, lysine; +, positive; 2, negative; ND , not determined.
Table 1. Differential characteristics between strain MWE-A11T and related genera of the family Microbacteriaceae
Y. R. Kim and others
The nearly complete 16S rRNA gene sequence (1450 bp) of
strain MWE-A11T was obtained, and the comparative analysis
based on the EzTaxon-e database indicated that strain
MWE-A11T displayed the highest 16S rRNA gene sequence
similarity with Humibacter antri D7-27T (96.4 %) (Lee,
2013). Herbiconiux moechotypicola RB-62T (96.3 %), Leifsonia
soli TG-S248T (96.3 %), Leifsonia lichenia 2SbT (96.2 %),
Leifsonia xyli subsp. cynodontis JCM 9733T (96.1 %), Microbacterium testaceum DSM 20166T (96.0 %) and Humibacter
albus DSM 18994T (96.0 %) were also found as neighbouring
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Allohumibacter endophyticus gen. nov., sp. nov.
taxa. However, strain MWE-A11T formed an independent
lineage in the maximum-likelihood, neighbour-joining and
maximum-parsimony trees (Fig. 1). The 16S rRNA gene
sequence of strain MWE-A11T contained the signature
nucleotides defined for the family Microbacteriaceae
(Stackebrandt et al., 1997; Zhi et al., 2009), which supported
the affiliation of the isolate to the family.
The genomic DNA G+C content was determined by
the Tm method of Gonzalez & Saiz-Jimenez (2002).
Microbacterium lacticum DSM 20427T (X77441)
0.01
Microbacterium ginsengiterrae DCY37T (EU873314)
100
Microbacterium testaceum DSM 20166T (X77445)
92
Microbacterium paraoxydans CF36T (AJ491806)
66
Microbacterium luteolum NBRC 15074T (AB004718)
92
77
Microbacterium liquefaciens DSM 20638T (X77444)
T
70 Microbacterium maritypicum DSM 12512 (AJ853910)
68 Microbacterium oxydans DSM 20578T (Y17227)
Lysinimonas soli SGM3-12T (JN378395)
99
Lysinimonas kribbensis MSL-13T (EF466129)
Leifsonia kafniensis KFC-22T (AM889135)
Herbiconiux moechotypicola RB-62T (FJ828659)
Herbiconiux solani K134/01T (FN432340)
70
Herbiconiux ginsengi wged11T (DQ473536)
88
Leifsonia xyli subsp. cynodontis JCM 9733T (AB016985)
52
98
Leifsonia naganoensis JCM 10592T (DQ232612)
99
Leifsonia aquatica ATCC 14665T (KI271991)
54
Leifsonia soli TG-S248T (EU912483)
98
Leifsonia shinshuensis JCM 10591T (DQ232614)
Leifsonia lichenia 2SbT (AB278552)
59
Leifsonia poae VKM Ac-1401T (AF116342)
Rudaibacter terrae 5GHs34-4T (JQ639054)
Schumannella luteola KHIAT (AB362159)
68
Humibacter ginsengisoli DCY90T (KF915800)
98
Humibacter antri D7-27T (HF570028)
81
100
Humibacter ginsengiterrae DCY60T (JQ010859)
Humibacter albus DSM 18994T (ATXT01000006)
Allohumibacter endophyticus MWE-A11T (JQ723726)
T
100 Agromyces cerinus subsp. cerinus DSM 8595 (X77448)
Agromyces cerinus subsp. nitratus ATCC 51763T (AY277619)
100
Agromyces ramosus DSM 43045T (X77447)
Gulosibacter chungangensis CAU 9625T (HQ268603)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences (1353 bp) showing the relationship
between strain MWE-A11T and related taxa of the family Microbacteriaceae. Bootstrap values based on 1000 replications
are given at branch points; only values .50 % are shown. Filled circles indicate branches that were also recovered in
the maximum-likelihood and maximum-parsimony trees. Gulosibacter chungangensis CAU 9625T was used as the
outgroup. Bar, 0.01 substitutions per nucleotide position.
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Y. R. Kim and others
The isoprenoid quinones were extracted with chloroform/
methanol (2 : 1, v/v), purified using Sep-Pak vac silica cartridges (Waters) and analysed by HPLC as described by
Collins et al. (1982). For the analysis of cell-wall amino
acid and sugars, the cell wall was prepared as described
by Schleifer & Kandler (1972). The amino acid composition of the peptidoglycan was determined by one-dimensional TLC as described by Harper & Davis (1979). The
sugars of the peptidoglycan were also separated and identified by TLC using cellulose plates as described by Staneck &
Roberts (1974). The murein acyl type of the cell wall was
analysed according to the method of Uchida et al.
(1999). Polar lipids were extracted from dried cell biomass
and analysed using two-dimensional TLC as described by
Minnikin et al. (1984). Fatty acid methyl esters were analysed by gas chromatography with Sherlock MIDI software
(version 6.0) and a TSBA database (version 6.0).
2,4-diminobutyric acid, and the diagnostic cell-wall
sugars are rhamnose and xylose. The murein is of glycolyl
type. The polar lipids include diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The major
fatty acids are anteiso-C15 : 0 and iso-C16 : 0. Phylogenetically, the genus belongs to the family Microbacteriaceae.
The genomic DNA G+C content of strain MWE-A11T was
68.8¡0.5 mol%. The isoprenoid quinones consisted of
MK-10 (30.4 %), MK-11 (62.2 %) and MK-12 (7.4 %).
The diagnostic diamino acid in the cell wall peptidoglycan
was 2,4-diaminobutyric acid. The diagnostic cell-wall
sugars were rhamnose and xylose. The murein was of glycolyl type. The major polar lipids of strain MWE-A11T
were diphosphatidylglycerol, phosphatidylglycerol and an
unidentified glycolipid, and minor amounts of unidentified
lipids were also present (Fig. S2). The major fatty acid
of strain MWE-A11T was anteiso-C15 : 0 (52.8 %), and
iso-C16 : 0 (15.7 %), anteiso-C17 : 0 (8.1 %) and a
summed feature consisting of C16 : 1v7c and/or C16 : 1v6c
(6.3 %) were also present. The detailed fatty acid profiles
of strain MWE-A11T and related taxa are listed in Table S1.
The species shows the following properties in addition to
those given for the genus. Cells are short rods, approximately
0.5–1 mm long and 0.5–0.7 mm wide. Colonies are glistening
and soft yellow after incubation for 2 days at 30 uC. Growth
occurs at 15–37 uC, at pH 6.0–8.0, and in the presence of
0–5 % (w/v) NaCl; optimum growth is observed at 30 uC,
pH 7 and with 0 % NaCl on TSA. Tweens 20 and 80,
starch, casein, cellulose and DNA are hydrolysed. According
to API ZYM tests, gelatin and aesculin are hydrolysed.
b-Glucosidase and b-galactosidase activities are present,
but arginine dihydrolase and urease activities are absent.
According to API 20NE tests, nitrate is reduced, but indole
is not produced. D -Glucose is not utilized. The following
substrates are assimilated: D -glucose, D -mannitol, D -maltose, potassium gluconate and trisodium citrate. According
to the API 50CH test, acids are produced from glycerol,
L -arabinose, D -galactose, D -glucose, D -fructose, D -mannose,
D -mannitol, amygdalin, arbutin, aesculin, salicin, cellobiose
and maltose. The main fatty acids (w5 %) are anteiso-C15 : 0,
iso-C16 : 0, anteiso-C17 : 0, and a summed feature consisting
of C16 : 1v7c and/or C16 : 1v6c.
The chemotaxonomic properties clearly distinguished strain
MWE-A11T from other related genera, as the major isoprenoid quinones of strain MWE-A11T differed from those of
the genera Humibacter and Herbiconiux, and the diamino
acids and cell-wall sugars also differed from those of the
genus Humibacter (Table 1). In addition, species of the
genera Humibacter and Herbiconiux are known to contain
acetyl type murein, but that of strain MWE-A11T was glycolyl type. Moreover, fatty acid profiles also differed substantially (Tables 1 and S1). Hence, based on phylogenetic and
chemotaxonomic analysis, strain MWE-A11T evidently
represents a novel species of a new genus in the family
Microbacteriaceae, for which the name Allohumibacter
endophyticus gen. nov., sp. nov. is proposed.
Description of Allohumibacter gen. nov.
Allohumibacter (Gr. adj. allos another, the other, different;
N.L. masc. n. Humibacter a bacterial genus name; N.L.
masc. n. Allohumibacter the other Humibacter, a genus
different from, but phylogenetically related to Humibacter).
Cells are Gram-positive, aerobic and non-motile.
The major isoprenoid quinones are MK-10 and MK-11.
The diamino acid in the cell-wall peptidoglycan is
1826
The type species is Allohumibacter endophyticus.
Description of Allohumibacter endophyticus
sp. nov.
Allohumibacter endophyticus (Gr. pref. endo within; Gr. n.
phuton plant; L. masc. suff. -icus adjectival suffix used
with the sense of belonging to; N.L. masc. adj. endophyticus
within plant, endophytic, pertaining to the original
isolation from plant tissues).
The type strain is MWE-A11T (5JCM 19371T5KCTC
29232T), isolated from the root of Artemisia princeps (mugwort). The genomic DNA G+C content of the type strain
is 68.8 mol%.
Acknowledgements
The authors acknowledge support from the Project on Survey and
Excavation of Korean Indigenous Species of the National Institute
of Biological Resources (NIBR) under the Ministry of Environment,
Korean Government, and also from the CNU Research Grant (grant
no. 2015-1417-01) of Chungnam National University.
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