Ureibacillus suwonensis sp. nov.

, isolated from
cotton waste composts
Byung-Yong Kim,
1
Seon-Young Lee,
1,2
Hang-Yeon Weon,
3
Soon-Wo Kwon,
1
Seung-Joo Go,
1
Yong-Keun Park,
2
Peter Schumann
4
and Dagmar Fritze
4
Correspondence
Soon-Wo Kwon
swkwon@rda.go.kr
1
Korean Agricultural Culture Collection (KACC), Genetic Resources Division, National Institute
of Agricultural Biotechnology, Rural Development Administration (RDA), Suwon 441-707,
Korea
2
School of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea
3
Applied Microbiology Division, National Institute of Agricultural Science and Technology,
RDA, Suwon 441-707, Korea
4
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b,
D-38124 Braunschweig, Germany
The taxonomic position of two spore-forming strains 6T19
T
and 6T29, isolated from cotton
composts for the cultivation of oyster mushroom (Pleurotus ostreatus), was investigated by a
polyphasic approach. Cells of strains 6T19
T
and 6T29 were rod-shaped, Gram-negative and
strictly aerobic. Sequencing and comparative analyses for the 16S rRNA genes of these strains
clearly showed their phylogenetic afliation to the genus Ureibacillus. Their closest relatives
Ureibacillus thermosphaericus and Ureibacillus terrenus have sequence similarity of 96?9 and
97?5%, respectively. The isoprenoid quinones of isolate 6T19
T
were MK-9, MK-8, MK-7, MK-10
and MK-6 (45: 27: 18: 5: 4%), the peptidoglycan type was L-LysrD-Asp and the main cellular
fatty acid was i-C
16: 0
. DNADNA hybridization experiments resulted in relatedness values of 37%
between 6T19
T
and U. thermosphaericus DSM10633
T
and 41%between 6T19
T
and U. terrenus
DSM 12654
T
. Based on the polyphasic data, strains 6T19
T
and 6T29 can be described as
members of a novel species of the genus Ureibacillus, for which the name Ureibacillus
suwonenesis sp. nov. is proposed. The type strain is 6T19
T
(=KACC 11287
T
=DSM 16752
T
).
Ureibacillus was rst described by Fortina et al. (2001) and
it comprises two ureolytic thermophilic bacilli species,
Ureibacillus thermosphaericus and Ureibacillus terrenus.
In Korea, composted cotton wastes are frequently used as
medium for cultivation of oyster mushroom, Pleurotus
ostreatus. During the sterilization and composting of cotton
wastes, the temperature of composts is raised gradually
from room temperature to 65 uC. Two bacterial strains were
obtained from the cotton wastes during the composting
process.
These strains were isolated from trypticase soy agar (TSA) at
pH 7?0 incubated at 50
u
C and maintained in TSA medium.
Colonies were not detected as single entities because of the
smeared growth on both TSA and nutrient agar (Oxoid).
Gram staining was done by using a Gram stain kit (Difco)
according to the manufacturers recommended protocol.
KOH test and L-alanine aminopeptidase assay were also
used (Gregersen, 1978). The presence of agella was sought
according to Heimbrook et al. (1989). Cell morphology
was observed in a phase-contrast microscope after incuba-
tion for 2 days on CASO agar (DSMZ medium no. 220;
http://www.dsmz.de/media/media.htm) supplemented with
10 mg MnSO
4
l
21
. Cells grown on TSA for 24 h were
examined by scanning electron microscopy. An agar block
was cut from the plate, xed in 1 % osmium tetroxide and
observed under a scanning electron microscope (Hitachi
S-2460N).
The following physiological tests were carried out accord-
ing to Gordon et al. (1973) and Claus & Berkeley (1986):
catalase test, anaerobic growth, VogesProskauer (VP) test,
Published online ahead of print on 25 November 2005 as DOI
10.1099/ijs.0.63703-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA
gene sequences of strains 6T19
T
and 6T29 are AY850379 and
AY850380.
Supplementary gure showing the morphological shapes of strain
6T19
T
and a supplementary table showing the cellular fatty-acid
content of Ureibacillus suwonensis and other Ureibacillus species are
available in IJSEM Online.
63703
G
2006 IUMS Printed in Great Britain 663
International Journal of Systematic and Evolutionary Microbiology (2006), 56, 663666 DOI 10.1099/ijs.0.63703-0
temperature range for growth (570
u
C, increments of 5
u
C),
egg-yolk reaction, resistance to lysozyme, growth in the
presence of NaCl (0, 2, 5, 7 and 10 %), growth at pH 5?7,
acids from carbohydrates (D-glucose, L-arabinose, D-xylose
and D-mannitol), formation of gas from glucose, hydrolysis
of starch, utilization of citrate and propionate, nitrate reduc-
tion, production of indole, deamination of phenylalanine,
decomposition of casein and tyrosine and liquefaction of
gelatin. Oxidase test and hydrolysis of aesculin were con-
ducted according to Smibert & Krieg (1994). Motility test
was performed on 1/10 CESP agar (1?5 g casitone, 0?5 g
yeast extract, 0?3 g soytone, 0?2 g peptone, 0?015 g MgSO
4
,
0?007 g FeCl
2
, 0?002 g MnCl
2
, made up to 1 l with distilled
water, pH 7?2) (Fortina et al., 2001).
The 16S rRNA gene sequences were determined by PCR
amplication (Kwon et al., 2003) and direct sequencing
(Hiraishi, 1992). The 16S rRNAgene sequences were aligned
by using the MEGALIGN program of DNASTAR. An evolution-
ary distance matrix was generated as described by Jukes &
Cantor (1969). The evolutionary tree for the datasets was
inferred from the neighbour-joining method of Saitou &
Nei (1987) by using MEGA version 2.1 (Kumar et al., 2001).
The stability of relationships was assessed by performing
bootstrap analyses of the neighbour-joining data based on
1000 resamplings.
DNADNA hybridization was carried out as a lter-
hybridization method described by Seldin & Dubnau
(1985). Probe labelling was conducted by using the non-
radioactive DIG-High prime system(Roche) and hybridized
DNA was visualized by using the DIG luminescent detection
kit (Roche). DNADNA relatedness was quantied by using
a densitometer (Bio-Rad). The DNA G+C content (mol%)
was determined by HPLC analysis of deoxyribonucleosides
as described by Mesbah et al. (1989) using a reverse-phase
column (Supelcosil LC-18-S; Supelco).
Peptidoglycan structure, menaquinones and polar lipids
were analysed according to Fortina et al. (2001). After
growth of cells on TSA for 24 h at 50
u
C, fatty acid methyl
esters were extracted and prepared by the standard protocol
of the Microbial Identication system (MIDI; Microbial
ID).
Supplementary Fig. S1 showing the morphological shapes
of strain 6T19
T
is available in IJSEM Online. Comparisons
of phenotypic properties among Ureibacillus species are
presented in Table 1. In the report of the novel genus Urei-
bacillus, Fortina et al. (2001) characterized the genus as
ureolytic, aerobic bacilli. For the urease test of the Urei-
bacillus species, Fortina et al. (2001) used the method by
Atlas (1993), which relies on the demonstration of alkal-
inity. However, alkalinity can be produced from the use of
peptone or other proteins in the medium, showing false-
positive results (MacFaddin, 2000). In our tests, based on
two different media, the medium used by Fortina et al.
(2001) changed from yellow to pinkred colour, indicating
a positive reaction. However, both control media inoculated
with and without urea also showed a colour change to
pink, indicating it to be an inappropriate test for urease of
Ureibacillus strains. On the other hand, the cultures which
were inoculated in Rustigian and Stuarts urea broth showed
no colour change, indicating a negative reaction in all the
strains tested. The phenotypic comparison among Ureibacil-
lus species is shown in Table 1.
For the phylogenetic analysis of the 16S rRNAgene sequences,
representative sequences retrieved from GenBank were
aligned from position 37 (59) to 1431 (39) (Escherichia coli
numbering; Brosius et al., 1978). Strains 6T19
T
and 6T29
fell within the radiation of cluster comprising the two
Ureibacillus species and Bacillus thermocloacae DSM 5250
T
(Fig. 1). The 16S rRNA gene sequence similarity between
strains 6T19
T
and 6T29 was 99?8 %. The closest relatives of
Table 1. Differentiating phenotypic characteristics of Ureibacillus species
Species: 1, U. suwonenesis sp. nov.; 2, U. thermosphaericus; 3, U. terrenus. Data from Fortina et al.
(2001) and this study. All strains were negative for urease according to our results. +, Positive; 2,
negative; V, variable among strains.
Characteristic 1 2 3
Spore shape* S/O S S
Growth at/in:
35 uC + + 2
65 uC 2 2 +
5 % NaCl + + V
Aesculin hydrolysis 2 + +
Major menaquinones MK-9, MK-8, MK-7,
MK-10, MK-6
MK-7 MK-9, MK-8, MK-10,
MK-7, MK-11
DNA G+C content (mol%) 41?5 35?739?2 39?641?5
*S, Spherical; O, oval.
664 International Journal of Systematic and Evolutionary Microbiology 56
B.-Y. Kim and others
strain 6T19
T
were U. terrenus (97?5 %) and U. thermo-
sphaericus (96?9 %). On the other hand, strain 6T19
T
was
very distinct from species classied in other genera (<92 %
sequence similarity).
DNADNA hybridization was performed to determine the
genetic relatedness between strains 6T19
T
and 6T29 and
between strain 6T19
T
and the type strains of the two
Ureibacillus species. The reassociation value between 6T19
T
and 6T29 was shown to be 92 %, indicating that the two
strains belong to the same species (Wayne et al., 1987). The
DNADNA reassociation values of 6T19
T
with U. thermo-
sphaericus DSM10633
T
and U. terrenus DSM 12654
T
proved
to be 37 and 41 %, respectively. The DNA G+C content of
strain 6T19
T
was 41?5 mol%.
Strains 6T19
T
and 6T29 contained L-LysrD-Asp type
(variation A4a) (A11.31) as the diagnostic diamino acid in
the cell-wall peptidoglycan. The isoprenoid quinone com-
position of 6T19
T
was MK-9, MK-8, MK-7, MK-10 and
MK-6 (45 : 27 : 18 : 5 : 4 %), and 6T29 contained MK-9, MK-
8, MK-7, MK-6 and MK-10 (36 : 30 : 27 : 5 : 2 %). Strains
6T19
T
and 6T29 contained straight-chain and terminally
saturated fatty acids with a composition of 56 and 7685 %,
respectively (Supplementary Table S1 available in IJSEM
Online). No signicant differences in fatty-acid proles were
found with other Ureibacillus species, except that strains
6T19
T
and 6T29 produced a higher proportion of ai-C
17 : 0
and lower proportions of C
16 : 0
, i-C
15 : 0
and i-C
17 : 0
than the
type strains of the two Ureibacillus species. Polar lipids
consisted of phosphatidylglycerol, diphosphatidylglycerol,
phospholipids and glycolipids of unknown composition.
On the basis of the polyphasic data, strains 6T19
T
and 6T29
can be described as members of a novel species of the genus
Ureibacillus, for which the name Ureibacillus suwonensis
sp. nov. is proposed.
Description of Ureibacillus suwonensis sp. nov.
Ureibacillus suwonensis (su.won.en9sis. N.L. masc. adj.
suwonensis referring to Suwon Region in Korea, where the
bacteria were rst found).
Cells are Gram-negative, spore-forming rods, 0?50?76
1?52?0 mm in size, single or in chains. Cells form spherical
or oval spores, occurring subterminally or terminally in
swollen sporangia. Cells are peritrichously agellated and
motile. Clear colonies are not formed. Colonies grow
smeared. Growth occurs at temperatures ranging from 35 to
60
u
C and in the presence of 5 % NaCl. Positive for catalase,
oxidase and arginine dihydrolase. Weak reaction for phenyl-
alanine deamination. Negative for anaerobic growth, for-
mation of indole and dihydroxyacetone, VP test, nitrate
reduction, acid production from D-glucose, L-arabinose,
D-xylose and D-mannitol and hydrolysis of aesculin, starch,
gelatin, casein and urea. The cross-linkage of peptidoglycan
is L-LysrD-Asp type (variation A4a). The major cellular
fatty acid is i-C
16 : 0
. The major isoprenoid quinones are
MK-9, MK-8 and MK-7. The DNAG+Ccontent of the type
strain 6T19
T
is 41?5 mol%.
The type strain, 6T19
T
(=KACC 11287
T
=DSM 16752
T
),
was isolated from cotton composts in Suwon, Korea.
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