Cellulomonas aerilata sp. nov., isolated from an air sample

International Journal of Systematic and Evolutionary Microbiology (2008), 58, 2925–2929
DOI 10.1099/ijs.0.2008/002253-0
Cellulomonas aerilata sp. nov., isolated from an air
sample
Chang-Muk Lee,1 Hang-Yeon Weon,2 Seung-Beom Hong,1 YoungAh Jeon,1 Peter Schumann,3 Reiner M. Kroppenstedt,3 Soon-Wo Kwon1
and Erko Stackebrandt3
Correspondence
Soon-Wo Kwon
swkwon@rda.go.kr
1
Korean Agricultural Culture Collection (KACC), Microbial Genetics Division, National Institute of
Agricultural Biotechnology, Rural Development Administration, Suwon 441-707, Republic of Korea
2
Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural
Development Administration, Suwon 441-707, Republic of Korea
3
DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße 7b,
38124 Braunschweig, Germany
A Gram-positive, aerobic, motile, coccoid or short rod-shaped bacterium, 5420S-23T, was
isolated from an air sample collected in the Republic of Korea. According to phylogenetic analysis
based on 16S rRNA gene sequences, strain 5420S-23T revealed 97.5, 97.3, 97.3 and 97.2 %
similarity, respectively, to Cellulomonas biazotea DSM 20112T, Cellulomonas cellasea DSM
20118T, Cellulomonas fimi DSM 20113T and Cellulomonas chitinilytica X.bu-bT. The
peptidoglycan type of strain 5420S-23T was A4b, containing L-ornithine–D-glutamic acid. The
cell-wall sugars were galactose, glucose and xylose. The major fatty acids were anteiso-C15 : 0
(49.7 %) and C16 : 0 (20.0 %). The major menaquinone was MK-9(H4) and major polar lipids were
diphosphatidylglycerol and phosphatidylglycerol. The DNA G+C content was 74 mol%. The
results of DNA–DNA hybridization with strains of closely related Cellulomonas species, in
combination with chemotaxonomic and physiological data, demonstrated that isolate 5420S-23T
represents a novel Cellulomonas species, for which the name Cellulomonas aerilata sp. nov. is
proposed, with strain 5420S-23T (5KACC 20692T 5DSM 18649T) as the type strain.
The genus Cellulomonas was proposed by Bergey et al.
(1923) for some former Bacillus species. In recent years,
Cellulomonas turbata was reclassified as Oerskovia turbata
(Stackebrandt et al., 2002) and Cellulomonas fermentans
was reclassified in a new genus as Actinotalea fermentans
(Yi et al., 2007). At the time of writing, a total of 16
Cellulomonas species, including one species the description
of which was in press (Yoon et al., 2008), were recognized.
In the course of a study of strains from air samples, strain
5420S-23T was isolated from air sampled in the Suwon
region of Korea. The air sample was collected using an
MAS-100 air sampler (Merck) (single-stage multiple-hole
impactor) containing Petri dishes with R2A agar (BBL)
amended with 0.02 % cycloheximide (Sigma). After
sampling, the plates were incubated at 28 uC for 5 days,
and strain 5420S23T was recovered. Routine cultivation
was conducted at 28 uC with R2A medium.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain 5420S-23T is EU560979.
A neighbour-joining tree based on 16S rRNA gene sequences is
available as supplementary material with the online version of this paper.
2008/002253 G 2008 IUMS
Printed in Great Britain
The morphological, cultural, physiological and biochemical characteristics of strain 5420S-23T were investigated
by using routine cultivation on R2A medium at 28 uC.
Cell morphology was examined under phase-contrast
microscopy (Axio; Zeiss). Gram staining, catalase, oxidase
and hydrolysis of CM-cellulose, casein, chitin from crab
shells, DNA, hypoxanthine, pectin, starch, Tween 80,
tyrosine and xanthine were assessed using methods
described by Smibert & Krieg (1994). Growth at various
temperatures (5–40 uC) was measured on R2A agar. The
optimum pH for growth was examined in R2A broth
adjusted to various pH values (pH 4–10 at intervals of 1.0
pH unit). Tolerance of NaCl was tested in R2A broth at
different NaCl concentrations (0, 1, 2, 3 and 5 %, w/v).
Growth under anaerobic conditions was determined after
incubating the strain in a GasPak jar (BBL) at 28 uC for
15 days. Physiological and biochemical properties were
further determined with API ZYM, API 20NE and API ID
32GN test kits (bioMe´rieux). Tests in the commercial
systems were generally performed according to the
manufacturer’s instructions; the API ZYM test strip was
read after 4 h incubation at 37 uC and other API test strips
were examined after 5 days at 28 uC.
2925
C.-M. Lee and others
Biomass for chemical studies was obtained from cultures
grown in R2A broth at 28 uC, checked for purity, harvested
by centrifugation and freeze-dried. Analysis of the
peptidoglycan was performed according to the procedures
described by Schleifer & Kandler (1972). Polar lipids were
extracted, examined using two-dimensional TLC and
identified using published procedures (Minnikin et al.,
1984). Menaquinones were isolated using the methods of
Minnikin et al. (1984) and then separated by HPLC. Sugar
analysis of whole cells and mycolic acid determinations
were carried out as described by Staneck & Roberts (1974)
and Minnikin et al. (1975), respectively. Analyses of the
whole-cell fatty acid pattern followed described methods
using the MIDI system (Microbial ID, Inc.) (Sasser, 1990).
The G+C content of the DNA was determined as
described by Mesbah et al. (1989) using a reversed-phase
column (Supelcosil LC-18 S; Supelco).
Isolation of chromosomal DNA, PCR amplification and
direct sequencing of the purified product were carried out
as described previously (Weon et al., 2006). The resultant
16S rRNA gene sequence (1387 bp) was compared with all
16S rRNA gene sequences available in GenBank, and
alignment of sequences was carried out with the CLUSTAL W
program (Thompson et al., 1994). A phylogenetic tree was
produced using the software package MEGA version 3.1
(Kumar et al., 2004). Distances (using distance options
according to Kimura’s two-parameter model) and clustering using the neighbour-joining and maximum-parsimony
methods were determined by using bootstrap values based
on 1000 replicates. DNA–DNA hybridization was carried
out as described by Seldin & Dubnau (1985). Probe
labelling was conducted by using the non-radioactive DIG
High Prime DNA labelling and detection starter kit II
(Roche Molecular Biochemicals). Reassociation was con-
Table 1. Differential phenotypic characteristics of strain 5420S-23T and type strains of closely related Cellulomonas species
Strains: 1, 5420S-23T; 2, C. biazotea DSM 20112T; 3, C. cellasea DSM 20118T; 4, C. fimi DSM 20113T; 5, C. chitinilytica X.bu-bT. All strains are
positive for catalase and gelatin hydrolysis, but negative for indole production, glucose fermentation and arginine dihydrolase. All strains assimilate
N-acetylglucosamine, but not capric acid, adipic acid, malic acid or trisodium citrate. All strains except C. chitinilytica X.bu-bT (no data available)
test positive for activities of alkaline phosphatase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, a-glucosidase and N-acetyl-bglucosaminidase and negative for activities of esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, b-glucuronidase, amannosidase and a-fucosidase. +, Positive; 2, negative; W, weakly positive; ND, no data available. Data from Stackebrandt & Kandler (1979),
Bagnara et al. (1985), Funke et al. (1995), Collins & Pascual (2000), Elberson et al. (2000), Rivas et al. (2004), An et al. (2005), Yoon et al. (2008)
and this study.
Characteristic
Morphology
Colony colour*
Cell-wall sugarsD
Nitrate reduction
Urease
b-Galactosidase
Assimilation of:
D-Glucose
L-Arabinose
D-Mannose
D-Mannitol
Maltose
Potassium gluconate
Phenylacetic acid
Enzyme activity (API ZYM)
Esterase (C4)
a-Chymotrypsin
Acid phosphatase
a-Galactosidase
b-Galactosidase
b-Glucosidase
G+C content (mol%)
1
2
3
4
5
Coccoid or short
rods
Rods
Rods
Rods
Rods
LO
YW
Y
YW
Y
Gal, Glc, Xyl
2
+
+
Rha, Gal, Man, 6dTal
+
2
+
Rha, Man, 6dTal
+
2
+
GlcN, Rha, Fuc
+
2
+
Gal, Rib, Xyl, Rha
+
2
2
2
2
+
2
2
2
+
+
+
2
+
2
2
+
+
+
+
+
+
+
+
+
+
+
+
2
2
2
2
2
74
2
2
+
+
+
+
71.5–75.6d
W
W
+
+
2
2
+
2
2
+
+
+
+
2
+
75
2
2
+
+
+
+
71.3
W
W
ND
ND
ND
ND
ND
ND
73.6
*LO, Light orange; Y, yellow; YW, yellow–white.
D6dTal, 6-Deoxytalose; Fuc, fucose; Gal, galactose; Glc, glucose; GlcN, glucosamine; Man, mannose; Rha, rhamnose; Rib, ribose; Xyl, xylose.
dRange of four independent determinations quoted by Stackebrandt & Kandler (1979).
2926
International Journal of Systematic and Evolutionary Microbiology 58
Cellulomonas aerilata sp. nov.
ducted at 65 uC. The hybridized DNA was visualized using
the DIG luminescent detection kit (Roche). DNA–DNA
relatedness was quantified by using a densitometer (BioRad).
Cells of strain 5420S-23T were Gram-positive, motile, lightorange-coloured coccoids or short rods, 0.961.3 mm.
Strain 5420S-23T grew on R2A, nutrient agar (Difco) and
trypticase soy agar (Difco), but not on MacConkey agar
(Difco). The phenotypic characteristics that differentiate
strain 5420S-23T from related species are listed in Table 1.
The 16S rRNA gene sequence of strain 5420S-23T showed
the highest similarity to members of the genus
Cellulomonas; Cellulomonas biazotea DSM 20112T, C.
cellasea DSM 20118T, C. fimi DSM 20113T and C.
chitinilytica X.bu-bT showed 97.5, 97.3, 97.3 and 97.2 %
sequence similarity, respectively, to strain 5420S-23T.
Sequences from the other species of the genus
Cellulomonas showed less than 97 % similarity to strain
5420S-23T. In the phylogenetic tree constructed by using
the maximum-parsimony algorithm (Fig. 1), strain 5420S23T formed a cluster with C. biazotea, C. cellasea, C. fimi
and C. chitinilytica, which was also supported by the
neighbour-joining algorithm (see Supplementary Fig. S1,
available in IJSEM Online). DNA–DNA relatedness values
between strain 5420S-23T and C. biazotea DSM 20112T, C.
cellasea DSM 20118T and C. fimi DSM 20113T were 28, 26
and 16 %, respectively.
The peptidoglycan composition of strain 5420S-23T
corresponded to type A4b, containing L-ornithine–Dglutamic acid. This peptidoglycan type is present in most
members of the genus Cellulomonas (Stackebrandt &
Schumann, 2000). The cell-wall sugars were galactose,
glucose and xylose. The major fatty acids were anteisoC15 : 0 (49.7 %), C16 : 0 (20.0 %), anteiso-C15 : 1 A (9.2 %) and
iso-C16 : 0 (6.7 %) (Table 2). The major menaquinone was
MK-9(H4). Polar lipids detected were diphosphatidylglycerol and phosphatidylglycerol as major components and
three unknown phospholipids as minor components. The
DNA G+C content of strain 5420S-23T was 74 mol%.
Strain 5420S-23T can be differentiated from its close
phylogenetic relatives C. biazotea, C. cellasea, C. fimi and C.
chitinilytica as follows. Strain 5420S-23T displayed coccoid
or short rod-shaped cells and light-orange-coloured
colonies and, furthermore, was unique in its inability to
reduce nitrate and to assimilate D-glucose, L-arabinose, Dmannose and maltose and its ability to hydrolyse urea.
Chemotaxonomically, strain 5420S-23T had different cellwall sugars (galactose, glucose and xylose) and showed a
larger amount of C16 : 0 in its fatty acid composition (Table
2).
In conclusion, the 16S rRNA gene sequence, physiological
characteristics and chemotaxonomic properties of strain
5420S-23T differed from those of its phylogenetic neighbours. Furthermore, the low levels of DNA–DNA relatedness supported the novelty of the isolate. On the basis of
the data presented, therefore, strain 5420S-23T represents a
novel species of the genus Cellulomonas, for which the
name Cellulomonas aerilata sp. nov. is proposed.
Description of Cellulomonas aerilata sp. nov.
Cellulomonas aerilata (ae.ri.la9ta. L. n. aer air; L. part. adj.
latus -a -um carried; N.L. fem. part. adj. aerilata airborne).
Cells are Gram-positive, aerobic, motile and coccoid or
short rod-shaped. Colonies on R2A are circular, lightorange-coloured and convex with clear margins. Spores
and mycelia are not formed. The temperature range for
growth is 5–35 uC, with optimum growth at 28 uC. The pH
Fig. 1. Maximum-parsimony tree constructed
from a comparative analysis of 16S rRNA gene
sequences showing the relationships of
5420S-23T with related taxa. Bootstrap values
(expressed as percentages of 1000 replications) greater than 40 % are shown at branch
points. Dots indicate that the corresponding
nodes were also recovered in a tree generated
with
the
neighbour-joining
algorithm
(Supplementary Fig. S1). Bar, 10 expected
changes per site.
http://ijs.sgmjournals.org
2927
C.-M. Lee and others
Table 2. Cellular fatty acid compositions of strain 5420S-23T
and type strains of closely related Cellulomonas species
T
T
Strains: 1, 5420S-23 ; 2, C. biazotea DSM 20112 ; 3, C. cellasea DSM
20118T; 4, C. fimi DSM 20113T; 5, C. chitinilytica X.bu-bT (data from
Yoon et al., 2008). Data for all strains except C. chitinilytica X.bu-bT
were obtained in this study from biomass harvested after growth on
R2A medium for 2 days. Values are percentages of total fatty acids; 2,
,1 % or not detected.
Fatty acid
anteiso-C13 : 0
C14 : 0
iso-C14 : 0
anteiso-C15 : 0
iso-C15 : 0
anteiso-C15 : 1 A
C16 : 0
iso-C16 : 0
C17 : 0
anteiso-C17 : 0
iso-C17 : 0
C18 : 0
1
2
3
4
5
2
3.9
5.1
49.7
2
9.2
20.0
6.7
2
4.3
2
2
1.3
4.0
8.9
41.2
7.4
2.2
9.6
8.8
3.7
8.4
1.2
1.9
1.3
3.4
11.0
42.8
3.2
9.2
7.9
16.6
2
3.6
2
2
2
7.3
4.6
44.9
2
2.1
12.7
7.6
2
12.9
2
5.4
2
2
3.0
61.3
3.0
2
2.9
1.0
2.4
15.9
5.4
3.0
range for growth is pH 7.0–9.0, with optimum growth at
pH 7–8. Growth occurs in the presence of 0–1 % NaCl.
Hydrolyses CM-cellulose and starch but not casein, chitin,
DNA, hypoxanthine, pectin, tyrosine, Tween 80 or
xanthine. Positive for catalase, hydrolysis of urea, aesculin
and gelatin and activity of b-galactosidase. Negative for
oxidase, nitrate reduction, indole production, glucose
fermentation and arginine dihydrolase. Assimilates Dmannitol and N-acetylglucosamine. Does not assimilate
D-glucose, L-arabinose, D-mannose, maltose, potassium
gluconate, capric acid, adipic acid, malic acid, trisodium
citrate, phenylacetic acid, L-rhamnose, D-ribose, inositol,
sucrose, itaconic acid, suberic acid, sodium malonate,
sodium acetate, lactic acid, L-alanine, potassium 2ketogluconate, potassium 5-ketogluconate, glycogen, 3hydroxybenzoic acid, 4-hydroxybenzoic acid, L-serine,
salicin, melibiose, L-fucose, D-sorbitol, propionic acid,
valeric acid, L-histidine, 3-hydroxybutyric acid or Lproline. Positive for activities of alkaline phosphatase,
esterase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, a-glucosidase and N-acetyl-b-glucosaminidase.
Negative for activities of esterase lipase, lipase, valine
arylamidase, cystine arylamidase, trypsin, a-chymotrypsin,
acid phosphatase, a-galactosidase, b-glucuronidase,
b-glucosidase,
a-mannosidase
and
a-fucosidase.
Peptidoglycan contains L-ornithine–L-glutamic acid (type
A4b). Major fatty acids are anteiso-C15 : 0 and C16 : 0. Major
isoprenoid quinone is MK-9(H4). Cell-wall sugars are
galactose, glucose and xylose. Major polar lipids are
diphosphatidylglycerol and phosphatidylglycerol.
Acknowledgements
This work was supported by a grant (Grant No. 20080401034028)
from the BioGreen 21 Program, Rural Development Administration,
Republic of Korea.
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