International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3644–3649
DOI 10.1099/ijs.0.068320-0
Lysinibacillus varians sp. nov., an endosporeforming bacterium with a filament-to-rod cell cycle
Chunjie Zhu,1,2,3 Guoping Sun,2,3 Xingjuan Chen,2,3 Jun Guo2,3
and Meiying Xu2,3
Correspondence
Guoping Sun
[email protected]
Meiying Xu
[email protected]
1
School of Bioscience and Bioengineering, South China University of Technology,
Guangzhou Higher Education Mega Centre, Guangzhou 510006, PR China
2
Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong
Institute of Microbiology, 100 Xianlie Road, Yuexiu District, Guangzhou 510070, PR China
3
State Key Laboratory of Applied Microbiology, Southern China (The Ministry–Guangdong Province
Joint Development), 100 Xianlie Road, Yuexiu District, Guangzhou 510070, PR China
Six Gram-stain-positive, motile, filamentous and/or rod-shaped, spherical spore-forming bacteria
(strains GY32T, L31, F01, F03, F06 and F07) showing polybrominated diphenyl ether
transformation were investigated to determine their taxonomic status. After spore germination,
these organisms could grow more than one hundred microns long as intact single cells and then
divide into rod cells and form endospores in 33 h. The cell-wall peptidoglycan of these strains
was type A4a, the predominant menaquinone was MK-7 and the major fatty acids were iso-C16 : 0,
iso-C15 : 0 and C16 : 1v7C. Diphosphatidylglycerol, phosphatidylglycerol and
phosphatidylethanolamine were detected in the polar lipid profile. Analysis of the 16S rRNA gene
sequences indicated that these strains should be placed in the genus Lysinibacillus and they were
most closely related to Lysinibacillus sphaericus DSM 28T (99 % 16S rRNA gene sequence
similarity). The gyrB sequence similarity and DNA–DNA relatedness between strain GY32T and L.
sphaericus JCM 2502T were 81 % and 52 %, respectively. The G+C content of the genomic DNA
of strain GY32T was 43.2 mol%. In addition, strain GY32T showed differences in nitrate reduction,
starch and gelatin hydrolysis, carbon resource utilization and cell morphology. The phylogenetic
distance from its closest relative measured by DNA–DNA relatedness and DNA G+C content, and
its phenotypic properties demonstrated that strain GY32T represents a novel species of the genus
Lysinibacillus, for which the name Lysinibacillus varians sp. nov. is proposed. The type strain is
GY32T (5NBRC 109424T5CGMCC 1.12212T5CCTCC M 2011307T).
The genus Lysinibacillus is typically characterized by rodshaped cells with an A4a cell-wall peptidoglycan type. The
dominant respiratory lipoquinone system is MK-7, and
major polar lipids are diphosphatidylglycerol, phosphatidylglycerol and ninhydrin-positive phosphoglycolipid. At
the time of writing, the genus contains the following
species with validly published names: Lysinibacillus boronitolerans, L. fusiformis, L. sphaericus (Ahmed et al., 2007a),
L. parviboronicapiens (Miwa et al., 2009), L. xylanilyticus
(Lee et al., 2010), L. macroides (Coorevits et al., 2012), L.
mangiferahumi (Yang et al., 2012), L. sinduriensis, L.
massiliensis, L. odysseyi (Jung et al., 2012), L. tabacifolii
Abbreviation: DDH, DNA–DNA hybridization.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene
and gyrB sequence of strain GY32T are JN860068 and JN860069,
respectively.
Three supplementary movies and two supplementary tables are available
with the online version of this paper.
3644
(Duan et al., 2013) and L. chungkukjangi (Kim et al., 2013).
During the selective enrichment experiments of decabrominated diphenyl ether (BDE-209) biotransformation
bacteria from electronic-waste-contaminated river sediment, six Lysinibacillus strains were obtained. They showed
a filament-to-rod cell cycle in either enrichment medium
or LB medium. The cell cycle of these novel isolates is
different from those of other known species of the genus
Lysinibacillus. The phenotypic and genetic properties were
investigated to understand the interesting cell morphology
and the phylogenetic characteristics of these isolates.
For screening of BDE-209 biodegrading bacteria, sediment
samples were collected from an electronic-waste-recycling
site in Guiyu, Guangdong, China. Sediment samples (20 g)
were added to 80 ml sterilized H2O, sediment granule and
microbes were separated by gradient centrifugation at
2000 g and 12 000 g. To enrich the BDE-209 biotransformation bacteria, the microbes were inoculated into a
medium containing 20 mM formate, 20 mM lactate,
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Lysinibacillus varians sp. nov.
40 mM succinate, 5.7 mM Na2HPO4, 3.3 mM KH2PO4,
18 mM NH4Cl, 0.1 % (w/v) yeast extract and a trace
element solution and vitamins (Wolin et al., 1963). BDE209 (1 mM) dissolved in dichloromethane was added to the
culture bottle and evaporated in darkness before adding the
medium above. Enrichment cultures were transferred into
fresh media every month and the concentration of bromine
ions in the systems before and after incubation were
measured by ion chromatography (ICS-1500; DIONEX).
Six months later, the enriched populations were serially
diluted and plated onto Luria–Bertani (LB) agar plates
for single-colony isolation. Colonies displaying different
morphologies were selected and tested for BDE-209 debromination ability after three purifications. Six strains (L31,
GY32T, F01, F03, F06 and F07), producing more than
55.5 mg bromine ion l21 after incubation for 1 month in
the medium containing 1 mM BDE-209, were selected and
maintained on LB slopes at 4 uC or stored at 220 uC in LB
supplemented with 30 % (v/v) glycerol for further studies.
These strains displayed colonies that were yellowish,
circular with jagged margins, opaque on LB plates and
had glossy surfaces at 30 uC after 24 h of incubation.
A light microscope (M165C; Leica), transmission electron
microscope (H-7650; Hitachi) and scanning electron
microscope (S-3000N; Hitachi) were employed for cell
morphological observations. The Invitrogen LIVE/DEAD
Baclight Bacterial Viability kit L7007 was used for nucleic
acid staining according to the manufacturer’s protocols
and nucleoid was immediately analysed by laser scanning
microscopy (Zeiss) and fluorescence microscopy (DMRA2;
Leica). Cell length was measured with a Leica Qwin image
processing and analysis application. All six strains were Gramstain-positive, motile and spore-forming. Interestingly, both
filamentous (10.0–466.160.8–1.3 mm, length6diameter)
and long rod-shaped (5.0–10.060.8–1.3 mm, length6diameter) cells were observed in the field of vision (Fig. 1). The
filamentous cell was not observed in Lysinibacillus sphaericus
JCM 2502T (Movie S1, available in the online Supplementary
Material). Strain GY32T was used as a representative to
observe cell morphology characteristics in detail.
Time-lapse microscopy was used to monitor spore germination, single cell elongation and division, cell growth and
spore formation. For sporulation, 1-week cultures were
(a)
(b)
heat treated at 105 uC for an hour and stored at 24 uC. The
purity of the spores was evaluated by phase-contrast
microscopy and all preparations were free of intact living
vegetative cells. Spore germination experiments were performed according to the method described by Hamoen &
Errington (2003), modified as follows. Spores were heat
shocked for 30 min at 30 uC and quickly cooled on ice.
They were subsequently diluted to 1/2500 and inoculated
into semi-solid LB medium. The inoculated medium was
added to a cell culture dish (35612 mm, 15 mm diameter,
glass bottom; NEST Biotechnology) before medium gelling.
After pre-incubation at 30 uC for 2 h, the culture was
photographed under a laser scanning microscope, by which
images were taken every 20 s for time-lapse experiments.
All photomicrographs were saved with a scale rule. Cell
length was measured with Leica Qwin and calculated as net
size by scaleplate. Time lapse between consecutive images
and cell length measurements were combined for the construction of a length–time diagram. These results showed
that after a spore was pre-incubated at 30 uC for 2 h, the
lifecycle from a spore germination to next-generation
spore-forming, which required approximately 33 h, could
be divided into seven major steps: (i) a spore expands and
germinates in the first 4.5 h; (ii) the bud emerges from the
spore and becomes thicker and longer in the next 2.0 h and
then the rod-shaped bud releases from the cortex and crust
of the spore; (iii) the rod cell elongates to form a filament
in the next 1.5 h (Movie S2); (iv) the filamentous cell
reproduces with a high growth rate and motile rod cells are
observed after a further 3.0 h; (v) in another 5 h, filamentous cells and rod cells co-exist in the medium – some
filaments which could not divide immediately undergo
autolysis; (vi) rod cells become dominant in the following
12 h (Movie S3); and (vii) new endospores subsequently
form after a further 5 h (Fig. 2). These results allow us to
form a conceptual model for the lifecycle of strain GY32T
(Fig. 3).
The physiological and biochemical characteristics of all
isolates (GY32T, L31, F01, F03, F06 and F07) were
determined along with those of L. sphaericus JCM 2502T.
Growth at various temperatures (10–50 uC with 5 uC
intervals), NaCl concentrations (0, 0.5, 1.0, 2.0, 3.0, 4.0,
5.0, 6.0 and 7.0 %, w/v) and pH values (pH 4.0210.0) was
measured in the LB liquid medium. Optimal growth was
(c)
Fig. 1. Filamentous cells of strain GY32T in LB medium. (a) 5 h culture under fluorescence microscope; (b, c) 10 h culture
under scanning electronic microscope. Bars, 5 mm (a), 3 mm (b) and 300 nm (c).
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C. Zhu and others
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 2. Spore germination and filament formation observed by laser scanning microscopy. (a) Spore swelling; (b) spore
germination and outgrowth; (c) vegetative cell release from cortex and crust of the spore; (d) cell elongation; (e) cell division; (f)
endospore formation. Bars, 2 mm (a) and 5 mm (b–f).
observed at 35 uC and pH 7.0 in the LB medium without
NaCl. Use of carbon sources was determined by using the
Biolog GP2 characterization system according to the
manufacturer’s protocol. Oxidase and catalase activity,
nitrate reduction and hydrolysis of starch and gelatin, were
assessed as previously described (Dong & Cai, 2001). Except
for the difference of delayed positive oxidase activity of
strains L31 and F01, all novel isolates gave identical results in
these tests (Tables S1 and S2). Such characteristics were
compared in strain GY32T and in other species of the genus
Lysinibacillus (Table 1); the results of the nitrate reduction
test and hydrolysis of gelatin and starch revealed that strain
GY32T was different from its relatives.
Spore
For peptidoglycan analysis, strain GY32T and L. sphaericus
JCM 2502T were grown in shaking flasks containing liquid
4.5 h
Germination and outgrowth
5.0 h
2.0 h
Rod-shaped cells
Vegetative cell release
1.5 h
12.0 h
Filaments and rods
coexistence
Filamentous cell
3.0 h
5.0 h
Filaments division
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Fatty acid methyl esters of 24 h cultures were extracted and
prepared by the method described by Xu et al. (2005).
Extracts were analysed using a Hewlett Packard model
HP6890 GC equipped with a flame-ionization detector
(HP CHEMSTATION version A 5.01) and an Ultra-2
column (0.2 mm internal diameter; 25 m long). The major
fatty acids of strain GY32T were iso-C16 : 0, iso-C15 : 0 and
C16 : 1v7c. Strain GY32T had larger relative amounts of isoC16 : 0 and iso-C14 : 0 than other species of the genus
Lysinibacillus.
Fig. 3. A conceptual model for the lifecycle of
strain GY32T in LB culture at 30 6C. After
spore germination, a rod cell outgrows and
elongates into a filament. Immediately, the
filament asymmetrically divides into long rodshaped cells. Subsequently, rod cells become
predominant and spores are formed in the
medium. The lifecycle from spore germination
to next-generation spore formation requires
approximately 33 h. The length of rod cells are
5.0–10.0 mm and filaments are 10.0–
466.1 mm. The diameter of cells maintained
at 0.8–1.3 mm.
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Lysinibacillus varians sp. nov.
Table 1. Phenotypic characteristics of various species of the genus Lysinibacillus
Strains: 1, GY32T (data from this study); 2, L. sphaericus JCM 2502T (this study); 3, L. fusiformis DSM 2898T (previously Bacillus fusiformis; Priest
et al., 1988); 4, L. macroides LMG 18474T (Coorevits et al., 2012); 5, L. boronitolerans DSM 10aT (Ahmed et al., 2007a). +, Positive; 2, negative; ND,
no data available; B, Bulging; C, central; O, oval or slightly oval; R, round; T, terminal.
Characteristic
Cell size (length6diameter) (mm)
Spore shape and position
Growth temperature (uC)
pH range for growth
Growth in 7 % (w/v) NaCl
Voges–Proskauer test
Nitrate reduction
Oxidase
Catalase
Hydrolysis of:
Gelatin
Starch
1
2
3
4
5
5.0–466.160.8–1.3
R/O, T, B
15–45
6.0–9.0
–
–
+
+
+
3.0–5.061.2–1.3
R/O, T, B
15–40
6.0–9.0
–
–
–
+
+
3.0–5.061.0–1.3
R, C/T, B
17–40
6.0–9.5
+
–
–
+
3.0–5.060.9–1.1
ND
10–45
7.0–9.0
–
+
–
–
+
3.0–5.060.8–1.5
R/O, T, B
16–45
5.5–9.5
–
+
–
+
–
–
+
+
+
–
ND
ND
–
–
LB on a rotary shaker for 24 h at 30 uC. Quantitative
analysis of cell-wall peptidoglycan showed that strain
GY32T contained lysine and aspartic acid, representing
the type A4a (Schleifer & Kandler, 1972). This result is a
key marker used to discriminate the genus Lysinibacillus or
the genus Kurthia from other members of Bacillus group 2
(Rheims et al., 1999; Lee et al., 2010).
Respiratory quinones were extracted and purified from
500 mg dried cells and were separated by HPLC and
individually identified by MS (Nishijima et al., 1997). Polar
lipids were extracted and purified from 200 mg dried cells
using the protocol described by Minnikin et al. (1984) and
were identified by two-dimensional TLC (Merck). The
predominant menaquinone of strain GY32T was MK-7
(95.7 %) and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine.
Ninhydrin-positive phosphoglycolipid was not observed in
strain GY32T as in all other species of the genus
Lysinibacillus.
The 16S rRNA gene was amplified by universal primers 27f
and 1492r (Dunbar et al., 2001). The gyrB gene was
amplified using primers UP-1 and UP-2r (Yamamoto &
0.01
ND
ND
Harayama, 1995). The 16S rRNA gene sequence of strain
GY32T, consisting of 1456 bp, was used to search the
GenBank database. Sequence searches showed that strain
GY32T was most closely related to L. sphaericus DSM 28T
(99 % 16S rRNA gene sequence similarity) (Fig. 4). The
gyrB sequence similarity between strain GY32T (GenBank
accession no. JN860069) and its most closely related strain
L. sphaericus JCM 2502T was 81 % (Fig. 5).
DNA G+C content of strain GY32T was determined by
HPLC using a procedure previously described (Ahmed
et al., 2007b). The DNA–DNA renaturation rate was
determined using a Lambda 35 UV/VIS spectrometer
equipped with a PTP-1 Peltier temperature programmer
(PerkinElmer) and the experiment was carried out by the
service of the Institute of Microbiology, Chinese Academy
of Sciences. The DNA G+C content of strain GY32T was
43.2 mol% compared with 38.4 mol% for L. sphaericus
JCM 2502T. The mean DNA–DNA hybridization (DDH)
value between strain GY32T and L. sphaericus JCM 2502T
was 52±0.1 % (SD). A value of 70 % DDH was proposed by
Wayne et al. (1987) as a recommended standard for
delineating species. According to the equation described by
T
99 L. sphaericus DSM 28 (AJ310084)
Lysinibacillus varians GY32T (JN860068)
L. fusiformis DSM 2898T (AJ310083)
L. parviboronicapiens BAM-582T (AB300598)
L. xylanilyticus KCTC 13423T (FJ477040)
83
95 L. macroides LMG 18474T (AJ628749)
L. boronitolerans DSM 17140T (AB199591)
Bacillus subtilis ‘subsp. spizizenii’ DSM 618 (DQ529249)
40
85
Fig. 4. Phylogenetic tree of the genus Lysinibacillus based on 16S rRNA gene sequences. The tree was reconstructed by
using the neighbour-joining method. GenBank accession numbers are given in parentheses; the sequence determined in this
study is in bold type. Bar, 0.01 substitutions per nucleotide position.
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C. Zhu and others
0.1
99
100 Lysinibacillus varians GY32T (JN860069)
Lysinibacillus sphaericus JCM 2502T (KM090846)
Lysinibacillus parviboronicapiens KCTC 13154T (KM090847)
Bacillus cereus ATCC14579T (AB014101)
Bacillus subtilis ATCC 21332 (HQ844067)
Escherichia coli ATCC 25922 (AB083953)
100
Fig. 5. Phylogenetic tree of strain GY32T and representatives of some other taxa based on gyrB gene sequences. The tree was
reconstructed by using the neighbour-joining method. GenBank accession numbers are given in parentheses; the sequence
determined in this study is in bold type. Bar, 0.1 substitutions per nucleotide position.
Goris et al. (2007), the 52 % DDH corresponds to 89 %
average nucleotide identity of common genes and 46 %
conserved DNA between a pair of strains.
The A4a cell-wall peptidoglycan type and the 16S rRNA
gene sequence analysis clearly placed strain GY32T in the
genus Lysinibacillus. However, strain GY32T differs from all
other previously described species of the genus Lysinibacillus in several aspects, such as DNA G+C content, gyrB
sequence similarity, biochemical and physiological features,
cell cycle and DNA–DNA relatedness. Based on these
results, strains GY32T, L31, F01, F03, F06 and F07 were
assigned to a novel species of the genus Lysinibacillus, for
which the name Lysinibacillus varians sp. nov. is proposed.
Description of Lysinibacillus varians sp. nov.
Lysinibacillus varians (va9ri.ans. L. part. adj. varians
varying, referring to the change of cell shape).
Cells are motile, Gram-stain-positive, filamentous (10.0–
466.1 mm60.8–1.3 mm, length6diameter), and/or rodshaped (5.0–10.0 mm60.8–1.3 mm, length6diameter),
spore-forming with cell length changes during the growth
cycle. Colonies are yellowish, circular with jagged margins,
opaque on LB agar and have glossy surfaces at 30 uC in
24 h. Growth is observed at 15–45 uC and pH 6.0–9.0. The
type strain could tolerate 150 mM NaBO3 and 4 % (w/v)
NaCl in the LB broth. The cell-wall peptidoglycan type is
A4a (Lys–Asp) and menaquinone MK-7 is the predominant quinone. Phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol are the major polar
lipids. The major fatty acids are iso-C16 : 0, iso-C15 : 0 and
C16 : 1v7c. Oxidase, catalase and nitrate reduction tests are
positive, whereas gelatin and starch hydrolysis and Voges–
Proskauer tests are negative. The following compounds can
be utilized as sole sources of carbon: mannan, L-arabinose,
D-ribose, D-tagatose, D-xylose, acetic acid, a-hydroxybutyric acid, a-ketovaleric acid, L-lactic acid, L-malic acid, Lasparagine, L-glutamic acid, L-serine, 29-deoxyadenosine,
propionic acid, thymidine and uridine. The following
compounds are not utilized as sole sources of carbon: acyclodextrin, â-cyclodextrin, dextrin, glycogen, nulin,
Tween 40, Tween 80, N-acetyl-D-galactosamine, N-acetylD-glucosamine, amygdalin, D-arabitol, arbutin, D-cellobiose, D-fructose, L-fucose, D-galactose, D-galacturonic
3648
acid, gentiobiose, D-gluconic acid, a-D-glucose, m-inositol,
a-D-lactose, lactulose, maltose, maltotriose, D-mannitol,
D-mannose, D-melezitose, D-melibiose, a-Methyl-D-Galactoside, b-methyl-D-galactoside, 3-methyl-D-glucose, a-methylD-glucoside, b-methyl-D-glucoside, a-methyl-D-mannoside,
palatinose, D-psicose, D-raffinose, L-rhamnose, salicin,
sedoheptulosan, D-sorbitol, stachyose, sucrose, D-trehalose,
turanose, xylitol, b-hydroxy butyric acid, c-hydroxy butyric
acid, p-hydroxy- phenylacetic acid, a-ketoglutaric acid, lactamide, D-lactic acid methyl ester, D-malic acid, pyruvic acid
methyl ester, succinic acid mono-methyl ester, pyruvic
acid, succinamic acid, succinic acid, N-acetyl-L-lactamide
glutamic acid, L-alaninamide, D-alanine, L-alanine, L-alanylglycine, glycyl-L-glutamic acid, L-pyroglutamic acid,
putrescine, 2,3-butanediol, glycerol, adenosine, inosine,
adenosine-59- monophosphate, thymidine-59- monophosphate, uridine-59-monophosphate, D-fructose-6-phosphate,
a-D-glucose-1-phosphate, D-glucose- 6-phosphate, D-L-aglycerol phosphate.
The type strain is GY32T (5NBRC 109424T5CGMCC
1.12212T5CCTCC M 2011307T), isolated from an electronic-waste-contaminated river sediment from Guiyu,
Guangdong, China. The DNA G+C content of the type
strain is 43.2 mol%.
Acknowledgements
The authors thank Professor Zhili He for improving the English
language. This work was supported by the National Basic Research
Program of China (973 Program) (2012CB22307), the National
Natural Science Foundation of China (21207019), the International
Cooperation Projects of Guangdong Province (2011B050400005),
Guangdong Province-Chinese Academy of Science strategic cooperative project (2012B091100257), Guangdong Provincial Programs for
Science and Technology Development (2012A061100009) and
Guangdong Provincial Innovative development of marine economy
regional demonstration projects (GD2012-D01-002).
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