Kai Lou,2 Cheng-Lin Jiang,1 Li-Hua Xu1 and Wen-Jun Li1
1The Key Laboratory for Microbial Resources of the Ministry of Education, PR China, and
Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology,
Yunnan University, Kunming 650091, PR China
2Xinjiang Institute of Microbiology, Xinjiang Academy of Agricultural Science, Urumqi,
Xinjiang 830091, PR China
3DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7b,
D-38124 Braunschweig, Germany
A novel Gram-positive, rod-shaped actinobacterium, designated strain YIM 90718T, was isolated
from a saline soil in Xinjiang province, north-west China, and subjected to polyphasic taxonomy.
The peptidoglycan type was A1c and the cell-wall sugars contained galactose. Phospholipids
were phosphatidylglycerol and diphosphatidylglycerol. The predominant menaquinone was MK-
8(H2). The major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C15 : 0. All of these
chemotaxonomic data assigned the new isolate YIM 90718T consistently to the genus
Brevibacterium. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain
YIM 90718T formed a distinct phyletic lineage in the genus Brevibacterium and showed the
highest sequence similarity (96.2 %) to Brevibacterium samyangense SST-8T and low similarity
(,95.5 %) to other species of the genus Brevibacterium. On the based of the polyphasic
evidence, a novel species, Brevibacterium album sp. nov., is proposed, with the type strain YIM
90718T (5DSM 18261T 5KCTC 19173T 5CCTCC AB 206112T).
The genus Brevibacterium was first proposed by Breed
(1953) for some Gram-positive, non-spore-forming, nonbranching
rods formerly classified as the genus ‘Bacterium’.
The description of the genus was later emended and
restricted only to species that correspond to the type
species Brevibacterium linens on the basis of morphological
and chemotaxonomic characteristics (Collins et al., 1980).
The genus currently contains 16 species with validly
published names isolated from clinical specimens, dairy
products, poultry and terrestrial and marine environments.
While this paper was in preparation, Brevibacterium
samyangense, isolated from beach sediment, was reported
(Lee, 2006). In this article, strain YIM 90718T, isolated
from a saline soil, is described as a novel member of the
genus Brevibacterium based on polyphasic taxonomy.
Strain YIM 90718T was isolated from a saline soil sample
collected from Xinjiang, after 2 weeks incubation at 37 uC
on modified ISP medium 5. This medium contained
(g l21): L-asparagine, 1.0; glycerol, 10.0; yeast extract, 5.0;
K2HPO4, 1.0; KNO3, 5.0; KCl, 150; and agar, 15.0. KCl was
sterilized separately before being added to the medium.
The pH of the medium was adjusted to pH 7.5. The strain
was maintained on ISP 5 slants (Shirling & Gottlieb, 1966)
containing 5% (w/v) NaCl at 4 uC and as glycerol
suspensions (20 %, v/v) at 220 uC. Biomass for chemical
and molecular studies was obtained by cultivation in
shaken flasks (about 150 r.p.m.) using ISP medium 5 [5%
(w/v) NaCl, pH 7.5] at 37 uC for 1 week.
Cell morphology was determined using cultures grown for
6, 12, 16, 24 and 72 h on modified ISP 5 agar medium
supplemented with 5% (w/v) NaCl at 37 uC. Gram
staining was carried out by the standard Gram reaction
and was confirmed by using the KOH lysis test method
(Cerny, 1978). Cell motility was confirmed by the presence
of turbidity throughout tubes of semisolid medium
(Leifson, 1960). Morphological characteristics of the strain
were observed by light microscopy (model BH 2; Olympus)
and by transmission electron microscopy with a model H-
800 transmission electron microscope (Hitachi). Growth
was tested at 4, 10, 20, 28, 37, 45, 55 and 65 uC on ISP
medium 5 containing 5% (w/v) NaCl. For NaCl tolerance
experiments, modified ISP medium 5 was used as the basal
medium. The following NaCl concentrations (w/v) were
used: 0, 1, 3, 5, 10, 15, 20, 25 and 30 %. The pH range for
growth was investigated between pH 4.0 and 10.0 at
intervals of 1 pH unit using the following buffer systems:
pH 4.0–5.0, 0.1 M citric acid/0.1 M sodium citrate;
pH 6.0–8.0, 0.1 M KH2PO4/0.1 M NaOH; pH 9.0–10.0,
0.1 M NaHCO3/0.1 M Na2CO3. Catalase activity was
determined by production of bubbles after the addition
of a drop of 3% H2O2. Oxidase activity was observed by
oxidation of tetramethyl-p-phenylenediamine. Carbon
source utilization tests were carried out using GP2
microplates of the Microlog system (Biolog; 95 substrates).
Some physiological properties were tested by using the API
CORYNE and API ZYM strips (bioMe´rieux) according to
the manufacturer’s instructions. The morphological, cultural
and physiological properties of strain YIM 90718T are
given in Table 1 and in the species description.
Peptidoglycan was purified and the cell-wall amino acids
and peptides in cell-wall hydrolysates were analysed by twodimensional
ascending TLC on cellulose plates using the
solvent system of Schleifer & Kandler (1972). The cell-wall
sugars were analysed according to the procedures developed
by Hasegawa et al. (1983). Polar lipids were extracted,
examined by two-dimensional TLC and identified using
procedures described previously (Minnikin et al., 1984).
Menaquinones were isolated according to Minnikin et al.
(1984) and separated by HPLC (Kroppenstedt, 1982).
Cellular fatty acid analysis was performed as described by
Sasser (1990) using the Microbial Identification System
(MIDI). The peptidoglycan type of YIM 90718T was A1c and
galactose was the cell-wall sugar. Phospholipids contained
phosphatidylglycerol and diphosphatidylglycerol. The predominant
menaquinone was MK-8(H2). The major fatty
acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C15 : 0. All of
these features are consistent with the chemotaxonomic
description of the genus Brevibacterium (Collins et al., 1980).
Details of the phospholipids and menaquinones and the
cellular fatty acid profile are reported in the species
description
Extraction of genomic DNA and PCR amplification of the
16S rRNA gene were done as described by Li et al. (2007).
Multiple alignments with sequences of the most closely
related members of Brevibacterium and calculations of
levels of sequence similarity were carried out using
CLUSTAL_X (Thompson et al., 1997). Phylogenetic analyses
were performed using three tree-making algorithms: the
neighbour-joining (Saitou & Nei, 1987), maximumlikelihood
(Felsenstein, 1981) and maximum-parsimony
(Fitch, 1971) methods. A phylogenetic tree was constructed
using the neighbour-joining method from Knuc values
(Kimura, 1980) using MEGA version 2.1 (Kumar et al.,
2001). The topology of the phylogenetic tree was evaluated
by the bootstrap resampling method of Felsenstein (1985)
with 1000 replicates. Genomic DNA of strain YIM 90718T
for the determination of G+C content was prepared
according to the method of Marmur (1961). The G+C
content of the DNA was determined by reversed-phase
HPLC of nucleosides according to Mesbah et al. (1989).
A neighbour-joining phylogenetic tree (Fig. 1) based on
16S rRNA gene sequence comparison clearly showed that
strain YIM 90718T belongs to the genus of Brevibacterium
and forms a distinct subclade with B. samyangense. The 16S
rRNA gene sequence similarity between strain YIM 90718T
and its nearest neighbour, B. samyangense SST-8T, was
96.2 %, and low 16S rRNA gene sequence similarity
(,95.5 %) was revealed with other species of the genus
Brevibacterium. The G+C content of the DNA was
70.0 mol%.
On the basis of the phenotypic, chemotaxonomic and
phylogenetic data, strain YIM 90718T merits recognition
within a novel species of the genus Brevibacterium, for
which we propose the name Brevibacterium album sp. nov
Description of Brevibacterium album sp. nov.
Brevibacterium album (al9bum. L. neut. adj. album white).
Cells are aerobic, Gram-positive, motile, catalase-positive,
oxidase-negative, non-spore-forming rods, 1.864.0–
5.0 mm. Colonies are white on most media tested.
Colonies are smooth, circular, opaque and approximately
1–2 mm in diameter after 48 h incubation at 37 uC on ISP
medium 5 containing 5% (w/v) NaCl. Growth occurs in
the temperature, pH and salt ranges of 28–45 uC, pH 6–8,
0–10% NaCl (w/v), 0–20% KCl (w/v) and 0–30%
MgCl2 . 6H2O (w/v). Good growth occurs at 37 uC,
pH 7.5, 0–5% (w/v) NaCl, 0–10% KCl (w/v) and 0–
15% MgCl2 . 6H2O (w/v). The following substrates are
utilized as sole carbon sources for growth in Biolog GP2
microplates: b-cyclodextrin, Tween 80, N-acetyl-D-glucosamine,
L-fucose, D-gluconic acid, myo-inositol, lactulose,
D-mannitol, methyl b-D-galactoside, methyl a-D-glucoside,
palatinose, L-mannose, salicin, trehalose, turanose, D-malic
acid, succinic acid, D-alanine, L-alanyl glycine, putrescine,
uridine 59-monophosphate and fructose 6-phosphate. The
following substrates are not utilized in the Biolog GP2
system: a-cyclodextrin, dextrin, glycogen, inulin, mannan,
Tween 40, N-acetyl-b-D-mannosamine, amygdalin, Larabinose,
D-arabitol, arbutin, cellobiose, D-fructose, Dgalactose,
D-galacturonic acid, gentiobiose, a-D-glucose,
a-D-lactose, maltose, maltotriose, D-mannose, melezitose,
melibiose, methyl a-D-galactoside, 3-methyl glucose,
methyl b-D-glucoside, methyl a-D-mannoside, D-psicose,
raffinose, D-ribose, sedoheptulosan, D-sorbitol, stachyose,
sucrose, D-tagatose, xylitol, D-xylose, acetic acid, a-, b- and
c-hydroxybutyric acid, p-hydroxyphenylacetic acid, aketoglutaric
acid, a-ketovaleric acid, lactamide, D-lactic
acid methyl ester, L-lactic acid, L-malic acid, methyl
pyruvate, monomethyl succinate, propionic acid, pyruvic
acid, succinamic acid, N-acetyl-L-glutamic acid, L-alaninamide,
L-alanine, L-asparagine, L-glutamic acid, glycyl Lglutamic
acid, L-pyroglutamic acid, L-serine, 2,3-butanediol,
glycerol, adenosine, 29-deoxyadenosine, inosine,
thymidine, uridine, adenosine 59-monophosphate, thymidine
59-monophosphate, glucose 1-phosphate, glucose 6-
phosphate and DL-a-glycerol phosphate. In the API
CORYNE system, tests for pyrazinamidase, pyrrolidonyl
arylamidase, alkaline phosphatase, gelatin hydrolysis and
acid production from D-ribose are positive. Tests for
nitrate reduction, b-glucuronidase, b-galactosidase, aglucosidase,
N-acetyl-b-glucosaminidase, b-glucosidase
(aesculin hydrolysis), urease and acid production from Dglucose,
D-xylose, D-mannitol, maltose, D-lactose, sucrose
and glycogen are negative. Using the API ZYM system, acid
and alkaline phosphatase, esterase (C4), esterase lipase
(C8), leucine arylamidase, valine arylamidase, cystine
arylamidase, trypsin, a-chymotrypsin and naphthol-ASBI-
phosphohydrolase tests are positive. Lipase (C14),
a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase,
b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase
and a-fucosidase tests are negative. The
peptidoglycan is A1c, meso-diaminopimelic acid directly
cross-linked. Cell-wall sugar contains galactose. The
phospholipids are phosphatidylglycerol and diphosphatidylglycerol.
The menaquinones are MK-8(H2), MK-7(H2),
MK-6(H2), MK-8, MK-6 and MK-9(H2) (ratio of peak
areas, 76 : 10 : 5 : 3 : 2 : 1). The fatty acid profile contains
anteiso-C15 : 0 (58.71 %), anteiso-C17 : 0 (19.0 %), iso-C15 : 0
(12.29 %) and iso-C16 : 0 (7.24 %). The G+C content of the
DNA of the type strain is 70.0 mol%.
The type strain is YIM 90718T (5DSM 18261T 5KCTC
19173T 5CCTCC AB 206112T), isolated from a saline soil
in the north-west of China.
Acknowledgements
This research was supported by the National Basic Research Program
of China (no. 2004CB719601), the National Natural Science
Foundation of China (nos 30560001 and 30600001), the Yunnan
Provincial International Cooperative Program (no. 2005GH21), the
Ministry of Science of Technology, PR China (2006DFA33550), and
the Open Project Program of the Extremophiles Lab of Xinjiang (no.
XJYS0203-2005-01). W.-J. L. was supported by the Program for New
Century Excellent Talents in University.
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Table 1. Differential phenotypic characteristics between strain
YIM 90718T and its nearest phylogenetic neighbour, B.
samyangense SST-8T
Data for strain SST-8T were taken from Lee (2006).
Characteristic Strain YIM 90718T B. samyangense SST-8T
Colony colour White Bright yellow
Rod–coccus cycle - +
Temperature range 28-45 10-45
(C)
pH range 6–8 6.1–10.1
Gelatin hydrolysis + -
Utilization of:
Tween 40 - +
Sucrose - +
a-D-Lactose - +
D-Malic acid + -
a-Glucosidase - +
b-Galactosidase - +
a-Chymotrypsin + -
Polar lipids* DPG,PG PG
*DPG, Diphosphatidylglycerol; PG, phosphatidylglycerol.
Fig. 1. Phylogenetic dendrogram obtained by distance matrix
analysis of 16S rRNA gene sequences, showing the position of
strain YIM 90718T and its phylogenetic neighbours. Numbers at
branch nodes are bootstrap values (1000 resamplings; only values
over 50% are given). The sequence of Micrococcus luteus ATCC
381T was used as an outgroup. Bar, 1% sequence divergence.
