International Journal of Systematic and Evolutionary Microbiology (2015), 65, 4734–4742
DOI 10.1099/ijsem.0.000640
Leucobacter zeae sp. nov., isolated from the
rhizosphere of maize (Zea mays L.)
Wei-An Lai,1 Shih-Yao Lin,2 Asif Hameed,2 Yi-Han Hsu,2
You-Cheng Liu,2 Hsuan-Ru Huang,2 Fo-Ting Shen1,2 and
Chiu-Chung Young1,2
Correspondence
1
Chiu-Chung Young
2
[email protected]
Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan ROC
Department of Soil and Environmental Sciences, College of Agriculture and Natural Resources,
National Chung Hsing University, Taiwan ROC
A novel yellow-pigmented, aerobic, rod-shaped, non-motile bacterium, designated strain CCMF41T, was isolated from rhizosphere soil of maize (Zea mays) collected in Wufeng District,
Taichung, Taiwan. Strain CC-MF41T exhibited 16S rRNA gene sequence similarity of 97.5,
97.3, 97.2 and 97.1 % to Leucobacter chironomi MM2LBT (and ‘Leucobacter kyeonggiensis’
F3-P9 and ‘L. humi’ Re-6, the names of which have not been validly published), Leucobacter
tardus K70/01T, L. komagatae IFO 15245T and ‘Leucobacter margaritiformis’ A23. However,
CC-MF41T and ‘L. margaritiformis’ A23 formed a loosely bound phylogenetic lineage (with a
low bootstrap value) associated with species of the genus Leucobacter. In DNA–DNA
reassociation experiments, the relatedness of strain CC-MF41T to L. chironomi DSM 19883T
was 57.1 % (reciprocal value 29.1 %). The DNA G+C content of strain CC-MF41T was
72.1 mol% and the cell-wall peptidoglycan contained 2,4-diaminobutyric acid, alanine, glycine,
glutamic acid and threonine. The major menaquinone was MK-11 and the predominant fatty
acids were iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. The polar lipid profile of strain CCMF41T contained major amounts of diphosphatidylglycerol followed by an unidentified
glycolipid, phosphatidylglycerol and an unknown phospholipid. Based on its phylogenetic,
phenotypic and chemotaxonomic distinctiveness, strain CC-MF41T represents a novel species
of Leucobacter, for which the name Leucobacter zeae sp. nov. is proposed. The type strain is
CC-MF41T (5BCRC 80515T5LMG 27265T).
The genus Leucobacter (family Microbacteriaceae) was proposed by Takeuchi et al. (1996) and at the time of writing
comprised 14 recognized species: Leucobacter komagatae
(type species; Takeuchi et al., 1996), L. chromiireducens
and L. aridicollis (Morais et al., 2004), L. albus (Lin et al.,
2004), L. alluvii and L. luti (Morais et al., 2006a,b),
L. iarius (Somvanshi et al., 2007), L. tardus (Behrendt
et al., 2008), L. aerolatus (Martin et al., 2010),
L. chironomi (Halpern et al., 2009), L. celer (Shin et al.,
2011), L. exalbidus (Ue, 2011a, b), L. salsicius (Yun et al.,
2011) and L. denitrificans (Weon et al., 2012a,b). The
species L. chromiireducens has subsequently been divided
into the subspecies L. chromiireducens subsp. solipictus
and L. chromiireducens subsp. chromiireducens (Muir &
Tan, 2007). The genus Leucobacter was described for
aerobic, Gram-stain-positive, non-motile, non-sporulating,
rod-shaped bacteria with 2,4-diaminobutyric acid (DAB)
in their cell-wall peptidoglycan. Although all species of
this genus are characterized by the presence of DAB as
the diagnostic diamino acid in the peptidoglycan, the
cell-wall type of the genus is not consistent, as differences
in the amino acid composition have been detected in several species (Behrendt et al., 2008).
Abbreviation: DAB, 2,4-diaminobutyric acid.
Bacterial genomic DNA was extracted by using an UltraClean
Microbial DNA Isolation kit (MO BIO) according to the
manufacturer’s instructions. The 16S rRNA gene of strain
CC-LY184T was amplified by using the total genomic DNA
as template (Heiner et al., 1998) by PCR (ABI 9700) with
The GenBank/EMBL/DDBJ accession number for the 16S rRNA
gene sequence of strain CC-MF41T is JX669524.
One supplementary figure and one supplementary table are available in
the online Supplementary Material.
4734
In the present study, strain CC-MF41T was isolated from
the rhizosphere of Zea mays in Wufeng District, Taichung
City, Taiwan, and maintained and subcultivated on marine
agar (MA; HiMedia) at 30 8C for 72 h for analysis of its 16S
rRNA gene sequence and its chemotaxonomic, biochemical
and growth characteristics.
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Printed in Great Britain
Leucobacter zeae sp. nov.
the oligonucleotide primers 1F (59-GAGTTTGATCATGGCTCAG-39) and 9R (59-AAGGAGGTGATCCAACCGCA-39),
which are complementary to conserved regions of the 16S
rRNA gene of Escherichia coli (Brosius et al., 1978; Edwards
et al., 1989). The PCR product was purified by using the
QIAquick Gel Extraction kit (Qiagen) and sequenced as
described by Young et al. (2005).
The 16S rRNA gene sequences of other type strains of the
genus Leucobacter were obtained from NCBI GenBank via
the EzTaxon server (http://www.eztaxon.org/; Chun et al.,
2007). The resultant 16S rRNA gene sequence (1498 nt) of
strain CC-MF41T was compared with available 16S rRNA
gene sequences from GenBank using the BLAST program
(http://www.ncbi.nlm.nih.gov/BLAST/) to determine an
approximate phylogenetic affiliation and was aligned with
sequences of closely related strains using the CLUSTAL _X
1.83 program (Thompson et al., 1997). The software package MEGA 6 (Tamura et al., 2013) was used to reconstruct
phylogenetic trees based on a 1432 nt alignment (E. coli positions 38–1469; Brosius et al., 1978) with neighbour-joining
(Saitou & Nei, 1987), maximum-likelihood (Felsenstein,
1981) and maximum-parsimony (Fitch, 1971) algorithms.
Evolutionary distances were calculated using the method
of Jukes & Cantor (1969). A bootstrap analysis (Felsenstein,
1985) was performed according to the algorithm of
Kimura’s two-parameter model (Kimura, 1980) based on
1000 resamplings (Felsenstein, 1993).
The 16S rRNA gene sequence of strain CC-MF41T
(1432 nt) showed 96.2–97.5 % similarity to those of members of recognized species of the genus Leucobacter. The
highest similarity was found to be with L. chironomi
MM2LBT (97.5 %), followed by L. tardus K70/01T
(97.3 %), L. komagatae IFO 15245T (97.2 %) and other
members of the genus Leucobacter (,97.0 %) (Table S1,
available in the online Supplementary Material). Hence,
we refrained from testing this parameter and consider
strain CC-MF41T to represent a distinct genospecies (see
also Stackebrandt & Ebers, 2006). However, these values
were within the range of similarity of type strains of
other recognized species of Leucobacter (95.5–99.5 %, and
96.2–99.0 % with the type strain of the type species)
(Table S1). However, the similarities of ‘Leucobacter
kyeonggiensis’ F3-P9 (Kim & Lee, 2011), ‘L. humi’ Re-6
(Her & Lee, 2015) and ‘L. margaritiformis’ A23 (Lee &
Lee, 2012) (97.5, 97.5 and 97.1 %, respectively) to CCMF41T were also within the range of interspecies
similarities. In the neighbour-joining tree based on
16S rRNA gene sequences, strain CC-MF41T and
‘L. margaritiformis’A23 formed a loosely bound branch,
as supported by a lower bootstrap value (58 %), within
the radiation encompassing the genus Leucobacter, as evidenced by a higher bootstrap value (98 %) (Fig. 1).
Given several species delimitation thresholds of 16S rRNA
gene sequence similarity, such as 98.65 % (Kim et al.,
2014), 98.7–99.0 % (Stackebrandt & Ebers, 2006) and
98.2–99.0 % (Meier-Kolthoff et al., 2013), the similarity
http://ijs.microbiologyresearch.org
values obtained for CC-MF41T with regard to the reference
strains analysed were well below the reported threshold,
hence strain CC-MF41T represents putative novel species
of the genus Leucobacter. DNA–DNA reassociation was
conducted between strain CC-MF41T and the closely
related strain L. chironomi DSM 19883T. Bacterial genomic
DNA was isolated by using the UltraClean Microbial Genomic DNA Isolation kit (MO BIO) according to the manufacturer’s instructions. DNA samples from strain
CC-MF41T and L. chironomi DSM 19883T were loaded
onto positively charged membranes as described by
Seldin & Dubnau (1985). Genomic DNA of strain
CC-MF41T and L. chironomi DSM 19883T was used to construct hybridization probes by labelling with digoxigenin–
11-dUTP. The experiment was carried out in triplicate
for each sample. DNA–DNA relatedness of strain CCMF41T with L. chironomi DSM 19883T was 57.1¡5.9 %
(the reciprocal value was 29.1¡1.5 %).
Different media including TSA (BBL), R2A (BD) and nutrient agar (HiMedia) were used to test growth of strain CCMF41T at 30 8C. Gram staining of strain CC-MF41T was
performed using an industrial Gram-staining kit (BioStar,
Inc.) according to the manufacturer’s instructions (Murray
et al., 1994). Cell morphology including the presence of flagella was determined by placing cells (1–2 days old) on a
carbon-coated copper grid followed by staining with 0.2 %
uranyl acetate for 5–10 s, brief air-drying and observation
under transmission electron microscope (JEOL JEM-1400).
Strain CC-MF41T formed yellow colonies after 48 h. Microscope examination showed that strain CC-MF41T formed
non-motile, irregular-shaped rods without flagella. Cells
were 0.8–1.0 mm wide and 1.8–3.0 mm long (Fig. S1).
Growth was tested by streaking cells on TSA (BBL), R2A
(BD) and nutrient agar (HiMedia) at 30 8C for 1 week.
The pH range (pH 2–10) and the range of supplemented
NaCl concentration (0–20 %, w/v) in marine broth (MB;
HiMedia) for growth was measured at 30 8C. Growth at
5–50 8C was tested in MB. Thermal tolerance of CCMF41T was measured by incubation of a cell suspension
at 55 8C in MB for 20 min following by testing the survival
of cells in MB at 30 8C. Anaerobic growth was tested using
MA or MA supplemented with 0.1 % (w/v) KNO3 by incubating the culture plates in an anaerobic chamber (COY).
Tolerance of chromium was examined by culturing strain
CC-MF41T in MB supplemented with K2CrO4 at final concentrations of 0–50 mM Cr(VI) for 3 days. Carbon-source
utilization (oxidation) was determined by using the Biolog
GP2 system. Acid production from 49 carbohydrates was
determined by using the API 50CH system (bioMérieux).
Nitrate reduction, indole production, activities of b-galactosidase and urease, hydrolysis of aesculin and gelatin and
assimilation of 12 substrates were tested with API 20NE
strips (bioMérieux). The activities of various enzymes
were detected by using the API ZYM system (bioMérieux)
(Smibert & Krieg, 1994). Catalase activity was determined
by assessing bubble production by cells in 3 % (v/v)
H2O2 and oxidase activity was determined by using 1 %
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4735
W.-A. Lai and others
0.02
Leucobacter albus IAM 14851T (AB012594)
82
Leucobacter komagatae IFO 15245T (AB007419)
Leucobacter aridicollis L9T (AJ781047)
Leucobacter denitrificans M1T8B10T (GQ246672)
Leucobacter iarius 40T (AM040493)
'Leucobacter humi' Re-6 (KC818288)
Leucobacter chromiiresistens JG 31T (GU390657)
T
100 Leucobacter alluvii RB10 (AM072820)
Leucobacter luti RF6T (AM072819)
Leucobacter chromiiresistens JG 31T (GU390657)
98
Leucobacter solipictus TAN 31504T (DQ845457)
100
Leucobacter celer NAL101T (GQ504012)
96
'Leucobacter kyeonggiensis' F3-P9 (JQ039895)
Leucobacter chironomi MM2LBT (EU346911)
Leucobacter zeae CC-MF41T (JX669524)
'Leucobacter margaritifomis' A23 (JN038197)
Leucobacter tardus DSM 19811T (AM940158)
Leucobacter exalbidus K-540BT (AB514037)
Leucobacter salsicius M1-8T (GQ352403)
99
T
70 Leucobacter aerolatus Sj10 (FN597581)
Glaciibacter superstes AHU1791T (AB378301)
Microbacterium phyllosphaerae DSM 13468T (AJ277840)
94
90
Microbacterium lacticum DSM 20427T (X77441)
Leifsonia aquaticum DSM 20146T (X77450)
Leifsonia pindariensis PON10T (AM900767)
Escherichia coli KCTC 2441T (EU014689)
Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic relationship of strain CCMF41T and closely related members of the genus Leucobacter. Bootstrap values (.70 %) after 1000 replications are shown
at branching points. Escherichia coli KCTC 2441T was used as an outgroup. Filled triangles, squares and circles indicate
that the corresponding nodes were also recovered in the trees generated using the maximum-likelihood (ML), maximumparsimony (MP), or ML and MP algorithms (with .95 % bootstrap support), respectively. Bar, 0.02 substitutions per nucleotide position.
(w/v) N,N,N9,N9,-tetramethyl 1,4-phenylenediamine (bioMérieux). Antibiotic susceptibility testing was carried out
using ATB STAPH 5 strips (bioMérieux) according to the
manufacturer’s recommendations. For measurement of
hydrolysis of Tweens 20, 40, 60 and 80, a medium consisting of half-strength MB (HiMedia) with 0.4 g CaCl2 l21,
0.15 g NaCl l21 and 15 g agar l21 (pH 7.5) (modified
from Castro et al., 1992) was used with 1.0 % (w/v)
Tween 20, 40, 60 or 80 added individually. Hydrolysis of
these substrates was considered as positive while an
opaque halo of precipitation around the colony was
formed after spot inoculation and 7 days of incubation at
30 8C (Cowan & Steel, 1993).
Differential characteristics of strain CC-MF41T and the three
type strains L. chironomi DSM 19883T, L. tardus DSM 19811T
and L. komagatae DSM 8803T, together with ‘L. kyeonggiensis’
JCM 17539, ‘L. humi’ Re-6 and ‘L. margaritiformis’ A23, are
given in Table 1. The detailed phenotypic characteristics of
strain CC-MF41T are given in the species description.
Cell walls were prepared as described by Rosenthal &
Dziarski (1994) and analysed according to Schleifer &
4736
Kandler (1972). The purified peptidoglycan was hydrolysed
with 6 M HCl at 105 8C for 6 h. The samples were dried
after adding redrying solution mixture (ethanol/water/
triethylamine; 2 : 2 : 1), and derivatization of amino acids
was done by adding ethanol/water/triethylamine/phenylisothiocyanate (7 : 1:1 : 1) and incubating for 20 min, followed by drying and dilution. The amino acid
composition was analysed with a Waters Pico-Tag Amino
Acid Analysis System at the College of Life Science,
National Tsing Hua University (White et al., 1986). The
hydrolysate of the preparation of strain CC-MF41T
contained the amino acids DAB, alanine, glycine, threonine
and glutamic acid in a molar ratio of 1.6 : 2.0 : 1.3 : 1.0 : 1.0,
a composition that confirmed the presence of a B-type
cross-linked peptidoglycan. (Schleifer & Kandler, 1972).
c-Aminobutyric acid, present in some other members of
the genus Leucobacter (L. albus, L. komagatae and
L. chironomi), was not detected (Table 2).
Fatty acid methyl esters were prepared, separated and
identified according to the standard protocol (Paisley,
1996) of the Microbial Identification System (MIDI)
(Sasser, 1990) by gas chromatography (model Agilent
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Leucobacter zeae sp. nov.
Table 1. Differential characteristics of strain CC-MF41T and the type or proposed type strains of related species of the genus
Leucobacter
Strains: 1, CC-MF41T; 2, L. chironomi DSM 19883T; 3, L. tardus DSM 19811T; 4, L. komagatae DSM 8803T; 5, ‘L. kyeonggiensis’ JCM 17539; 6,
‘L. humi’ Re-6 (data from Her & Lee, 2015); 7, ‘L. margaritiformis’ A23 (Lee & Lee, 2012). Data are from this study unless indicated. All strains
were non-motile and tested positive for catalase, acid phosphatase, naphthol-AS-BI-phosphorylase, butyrate esterase, gelatinase and glucose assimilation. All strains tested negative for nitrite reduction, indole production, glucose fermentation, cytochrome oxidase, a-chymotrypsin, a-galactosidase, b-glucuronidase, a-glucosidase, N-acetyl-b-glucosaminidase, a-fucosidase and assimilation of malate. In API 50 CH tests, all strains were
negative for acid production from all substrates except those indicated in this table. +, Positive; (2, negative; W , weakly positive; NA , no available
data.
Characteristic
1
2
3
4
5
6
7
Pigmentation*
Growth at/with:
Temperature (8C)
NaCl (%, w/v)
pH
Tolerance of 55 8C
Reduction of nitrate
Hydrolysis of:
Tweens 20, 40 and 60 (1 %)
Tween 80 (1 %)
Chromium tolerance (mM)
Enzyme activities
Alkaline phosphatase
Arginine dihydrolase
Caprylate esterase (C8)
Catalase
Cystine arylamidase
b-Galactosidase (PNPG)
a-Glucosidase
b-Glucosidase (aesculin hydrolysis)
Leucine arylamidase
Urease
Valine arylamidase
Assimilation of (API 20NE):
N-Acetylglucosamine
Arabinose
Capric acid
Citric acid
Potassium gluconate
Adipic acid
Phenylacetic acid
Maltose
Mannose
Mannitol
Acid production from (API 50CH):
N-Acetylglucosamine
Adonitol
L -Arabinose
Cellobiose
D -Fructose
Glucose
Glycerol
Glycogen
Melezitose, raffinose, L -rhamnose
D -Ribose
Oxidation of (Biolog GP2):
N-Acetylglutamic acid
Y
Y
Y
WB
C
WC
WC
15–37
0–11
5–11
2
2
30–37
0–9
6–11
+
2
15–37
0–15
5–12
+
+
15–42
0–20
5–11
2
+
4–42
0–10
6–10
+
2
4–42
0–3
6–9
NA
4–42
0–5
7–8
2
+
2
+
0–25
2
2
0–18
2
+
0–45
+
+
0–5
+
+
0–2
NA
NA
NA
NA
NA
NA
+
2
2
+
2
2
2
2
+
2
+
+
+
+
W
W
+
2
+
+
+
2
2
+
+
2
+
http://ijs.microbiologyresearch.org
+
2
W
+
2
+
2
W
W
2
2
+
+
2
2
2
2
+
+
2
+
2
2
+
+
2
2
2
+
2
2
2
2
W
+
W
W
2
2
2
+
2
2
W
2
2
2
2
2
2
+
2
+
2
2
+
+
+
+
+
+
+
+
+
+
+
2
2
2
2
+
2
2
+
2
2
2
+
2
2
2
2
2
2
W
2
2
2
2
+
2
2
+
W
W
2
2
+
2
2
2
2
2
2
+
2
+
2
+
2
W
2
+
W
W
2
W
W
W
W
+
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W
W
2
2
2
W
+
2
2
+
2
2
NA
W
2
+
NA
+
2
+
+
+
2
+
+
2
+
+
2
NA
NA
2
2
2
+
2
+
+
+
2
+
+
+
2
2
+
+
+
+
+
+
2
W
+
+
+
NA
NA
NA
2
NA
NA
NA
+
2
W
2
2
NA
+
NA
NA
NA
W
NA
NA
NA
4737
W.-A. Lai and others
Table 1. cont.
Characteristic
Adenosine
L -Alaninamide
L -Alanine, L -alanyl glycine
L -Arabinose
L -Asparagine
2,3-Butanediol
b-Cyclodextrin
29-Deoxyadenosine
Dextrin
D -Fructose
D -Fructose 6-phosphate
D -Galactose, D -galacturonic acid
Gluconic acid
Glucose
D -Glucose 1-phosphate
D -Glucose 6-phosphate
L -Glutamic acid
Glycerol
DL -a-Glycerol phosphate
Glycyl L -glutamic acid
a-Hydroxybutyric acid
p-Hydroxyphenylacetic acid
a-Ketoglutaric acid
a-Ketovaleric acid
Lactamide
L -Lactic acid
L -Malic acid
Mannan
Mannitol
Melezitose
Methyl b-D -galactoside
3-Methyl glucose
Methyl D -lactic acid
Methyl pyruvate
Methylsuccinic acid, D -psicose
Putrescine
Pyruvic acid
L -Rhamnose
D -Ribose
L -Serine
D -Succinamic acid, D -sorbitol, succinic acid, thymidine
Tween 40
Tween 80
Uridine
Uridine 5-phosphate
D -Xylose
*C , Cream;
WB ,
whitish brown;
WC ,
1
2
3
4
5
2
+
+
2
+
2
+
2
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
2
2
2
2
2
2
+
2
2
2
2
2
2
+
2
+
2
2
2
+
2
2
2
2
2
2
2
2
2
2
2
2
2
+
2
2
2
2
2
W
W
2
W
W
2
2
2
2
2
2
+
W
W
2
2
2
W
2
W
W
W
2
2
W
2
2
W
2
2
W
2
2
2
2
W
2
2
+
W
2
W
W
W
2
W
W
2
W
+
2
W
2
2
2
2
2
2
2
2
W
W
2
W
2
2
2
W
W
W
W
2
2
+
+
2
+
2
+
+
2
+
+
2
+
W
2
+
2
2
W
2
+
+
2
+
2
+
+
2
+
+
+
2
2
+
2
+
W
W
W
2
2
2
W
W
2
2
2
2
2
2
2
2
2
2
2
2
2
W
W
W
2
W
W
W
2
W
W
W
W
2
W
W
W
W
W
2
+
+
2
2
2
2
2
2
2
2
+
W
2
2
2
7
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
white, creamy; Y , yellow.
7890) fitted with a flame-ionization detector. For this purpose, a culture was grown on trypticase soy agar (BBL) at
30 8C for 3 days. One loop of cells on the plate was scraped
and was subjected to saponification, methylation and
extraction (Miller, 1982). Identification and comparison
4738
2
2
2
W
6
were made by using the Aerobe (RTSBA6) database of
the MIDI System. The major cellular fatty acids of strain
CC-MF41T were C16 : 0 (8.3 %), iso-C16 : 0 (21.8 %), anteiso-C17 : 0 (22.5 %) and anteiso-C15 : 0 (39.9 %). Minor
amounts of iso-C15 : 0 (1.8 %) and C16 : 1v5c (2.0 %) were
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Leucobacter zeae sp. nov.
Table 2. Differential chemotaxonomic and genotypic characteristics of strain CC-MF41T and the type or proposed type strains of
related species of the genus Leucobacter
Strains: 1, CC-MF41T; 2, L. chironomi DSM 19883T; 3, L. tardus DSM 19811T; 4, L. komagatae DSM 8803T; 5, ‘L. kyeonggiensis’ JCM 17539 (data
from Kim & Lee, 2011); 6, ‘L. humi’ Re-6 (Her & Lee, 2015); 7, ‘L. margaritiformis’ A23 (Lee & Lee, 2012). Data are from this study unless indicated
otherwise. Values given for cell-wall amino acids are molar ratios; other data for cell-wall amino acids indicate presence or absence only. Fatty acid
amounts are shown as means¡SD (n54), and values above 5 % are shown in bold. (2, Not detectable or ,1 %; ND , no data available.
Characteristic
Cell-wall amino acids
DAB
Alanine
Glycine
Glutamic acid
c-Aminobutyric acid
Threonine
Menaquinones
Major amounts
Minor amounts
Fatty acids (%)
C14 : 0
C16 : 0
iso-C14 : 0
iso-C15 : 0
iso-C16 : 0
anteiso-C15 : 0
anteiso-C17 : 0
C14 : 1v5c
C15 : 1v5c
C16 : 1v5c
iso-C17 : 1v10c
DNA G+C content (mol%)
1
2
3
4
5
1.6
2.0
1.3
1.0
2
1.0
0.5
2.5
1.2
1.0
+a*
0.9
0.5
3.2
2.1
1.0
2
2
0.8
1.9
0.9
1.0
0.7
2
11
10, 12
11
10, 12
2
8.3¡0.2
2
1.8¡0.1
21.8¡0.3
39.9¡0.3
22.5¡0.2
2
2
2.0¡0.2
2
72.1
2.9¡0.2
3.4¡0.9
2.1¡0.0
1.2¡0.1
27.3¡0.4
38.8¡1.6
18.1¡0.4
2
2
3.9¡0.7
1.6¡0.5
70.7a
10, 11
9
11
10, 12
2
3.4¡0.1
2
1.8¡0.2
20.2¡1.0
36.4¡0.1
23.4¡0.7
2
2
2
2
ND
2
1.9¡0.2
5.2¡0.2
6.8¡0.1
17.6¡0.4
49.0¡1.5
13.4¡0.4
1.1¡0.0
1.3¡0.1
2.8¡0.7
2
66.2b
6
7
+
+
+
+
2
2
+
+
+
+
2
2
+
+
2
+
+
2
11
9, 10
11
10
11
10
2
2.2
2
1.6
17.1
45.2
32.6
2
2
2
2
66.6
2
3.2
2.0
4.8
31.5
43.2
13.9
2
2
2
2
67.0
2
2
2
9.9
14.5
48.5
22.7
2
2
2
2
67.5
*Data from: a, Halpern et al. (2009); b, Takeuchi et al. (1996).
also found. The detailed fatty acid profile of strain CCMF41T is compared with those of the type strains of related
species of the genus Leucobacter in Table 2.
Polar lipids were extracted and analysed by two-dimensional
TLC, and isoprenoid quinones were purified according to
Minnikin et al. (1984) and analysed by HPLC as described
by Collins (1985). The polar lipids in CC-MF41T were
diphosphatidylglycerol, phosphatidylglycerol, an unknown
glycolipid and an unknown phospholipid (Fig. 2). It is
known that the polar lipid profiles of many other representative species of Leucobacter contain diphosphatidylglycerol,
phosphatidylglycerol and an unknown glycolipid (Muir &
Tan, 2007; Yun et al., 2011; Ue, 2011a; Kim & Lee, 2011;
Lee & Lee, 2012; Weon et al., 2012a). Strain CC-MF41T contained respiratory quinone MK-11 as a major component
and MK-9 and MK-10 in minor amounts (Table 2). The
quinone system supported our assignment of strain CCMF41T to the genus Leucobacter.
For analysis of DNA G+C content, a DNA sample was
prepared and degraded enzymically into nucleosides as
http://ijs.microbiologyresearch.org
described by Mesbah et al. (1989). The nucleoside mixture
obtained was then separated by HPLC. The genomic DNA
G+C content of CC-MF41T was determined as 72.1 mol%
(Table 2). It is known that the genomic DNA G+C content of L. komagatae (the type species) is 66.2 mol%
(Takeuchi et al., 1996). However, the DNA G+C content
of CC-MF41T is within the range of values known for
related species of Leucobacter, such as L. chironomi
(70.7 mol%), L. exalbidus (64.9 mol%), L. salsicius
(62.8 mol%) and L. celer (68.8 mol%) (Halpern et al.,
2009; Ue, 2011a; Yun et al., 2011; Shin et al., 2011).
On the basis of our phenotypic, phylogenetic and chemotaxonomic analyses, we suggest that strain CC-MF41T represents a novel species of the genus Leucobacter, for which
the name Leucobacter zeae sp. nov. is proposed.
Description of Leucobacter zeae sp. nov.
Leucobacter zeae [L. gen. n. ze9ae of spelt, of Zea mays,
referring to its isolation from rhizosphere soil of corn
(Zea mays)].
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4739
W.-A. Lai and others
DPG
GL
PG
PL
Fig. 2. Polar lipids isolated from strain CC-MF41T as separated
by two-dimensional TLC. Total polar lipids were visualized by
spraying the TLC plates with 10 % ethanolic molybdatophosphoric acid. DPG, Diphosphatidylglycerol; PG, phosphatidylglycerol;
PL, unidentified phospholipid; GL, unidentified glycolipid.
Cells are rod shaped, 0.8–1.0 mm in diameter and 1.8–
3.0 mm long, non-motile and Gram-stain-positive. Good
growth on TSA (BBL), R2A (BD) and nutrient agar
(HiMedia) at 30 uC. Colonies grown on MA (HiMedia)
are opaque, yellow and circular, with entire margins. The
conditions for growth are 15–37 uC, pH 5–11 and 0–8%
(w/v) NaCl. Cells are positive for catalase, aesculin hydrolysis (b-glucosidase), gelatinase, alkaline phosphatase, acid
phosphatase, naphthol-AS-BI-phosphorylase, butyrate
(C4) esterase, caprylate (C8) esterase, leucine arylamidase
and cystine arylamidase, but are negative for oxidase,
nitrate reduction, glucose fermentation, arginine dihydrolase, urease, indole production from L -tryptophan,
myristate (C14) lipase, valine arylamidase, trypsin, a-chymotrypsin, a-galactosidase, b-galactosidase, a-glucosidase,
b-glucuronidase, N-acetylglucosaminidase, a-mannosidase
and a-fucosidase (API ZYM and API 20NE systems). Glucose, mannose, N-acetylglucosamine, potassium gluconate
and citric acid are assimilated, but arabinose, mannitol,
capric acid, adipic acid, maltose, malate and phenylacetic
acid are not (API 20NE system). Acids are produced
from D -ribose and N-acetylglucosamine, and produced
weakly from erythritol, D - and L -arabinose, D - and L xylose, D -adonitol, methyl b-D -xylopyranoside,
D -galactose, D -glucose, D -fructose, D -mannose, L -sorbose,
L -rhamnose, dulcitol, inositol, D -mannitol, D -sorbitol and
4740
potassium 5-ketogluconate, but are not produced from
the following: glycerol, methyl a-mannopyranoside,
methyl a-glucopyranoside, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose,
D -lyxose, D -tagatose, D - and L -fucose, D - and L -arabitol,
potassium gluconate and potassium 2-ketogluconate (API
50CH system). Dextrin and putrescine are utilized strongly
(oxidized) as sole carbon sources, and lactamide, L -alaninamide, D -lactic acid methyl ester, uridine, D -fructose, L -glutamic acid, mannan, D -ribose, Tween 40, uridine 5monophosphate, pyruvic acid, D -fructose 6-phosphate,
p-hydroxyphenylacetic acid, a-D -glucose 1-phosphate, Nacetyl L -glutamic acid, glycerol and DL -a-glycerol phosphate are oxidized weakly (Biolog GP2 system). The
major cellular fatty acids are C16:0, iso-C16:0, anteiso-C15:0
and anteiso-C17:0. The major isoprenoid quinone is
MK-11. The polar lipids are diphosphatidylglycerol, phosphatidylglycerol, an unknown glycolipid and an unknown
phospholipid. The type strain is sensitive to cotrimoxazole,
gentamicin, erythromycin, vancomycin, teicoplanin and
levofloxacin, but resistant to penicillin, clindamycin, tetracycline, rifampicin, norfloxacine, nitrofurantoin, quinupristin-dalfopristin, coag(–)oxacillin and oxacillin, and
shows intermediate resistance to minocycline and fusidic
acid (ATB STAPH system).
The type strain, CC-MF41T (5BCRC 80515T5LMG
27265T), was isolated from rhizosphere soil of maize (Zea
mays L.) from Wufeng, Taichung, Taiwan. The genomic
DNA G+C content of the type strain is 72.1 mol%.
Acknowledgements
We would like to thank Fen-Syun Wu for technical assistance. Our
research work was kindly supported by grants from the Ministry of
Science and Technology, the Council of Agriculture, Executive
Yuan, and in part by the Ministry of Education, Taiwan, ROC,
under the ATU plan.
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