Laceyella sediminis sp. nov., a thermophilic
bacterium isolated from a hot spring
Jiu-Jiu Chen,1,2Lian-Bing Lin,1Lan-Lan Zhang,1Jin Zhang,2
Shu-Kun Tang,1Yun-Lin Wei1and Wen-Jun Li2
1Biotechnology Research Center, Faculty of Life Science and Technology,
Kunming University of Science and Technology (KUST), Kunming 650093, PR China
2Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, PR China, and
Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Institute of Microbiology,
Yunnan University, Kunming 650091, PR China
A thermophilic bacterium, designated strain RHA1T, was isolated from a sediment sample
collected from a hot spring in Tengchong county, Yunnan province, south-west China, and was
characterized by using a polyphasic approach. Based on its phenotypic and phylogenetic
characteristics, strain RHA1Twas affiliated to the genus Laceyella. The strain formed white aerial
and yellow–white substrate mycelia, bearing single endospores on short sporophores. The
cell-wall peptidoglycan contained meso-diaminopimelic acid. Whole-cell hydrolysates contained
ribose and glucose. The major fatty acids were iso-C15:0(62.39%) and anteiso-C15:0(17.55%).
The predominant menaquinone was MK-9. The G+C content of the genomic DNA of strain
RHA1Twas 47.9 mol%. Based on DNA–DNA hybridization data, chemotaxonomic characteristics
and differential physiological properties, strain RHA1Tis considered to represent a novel species
of the genus Laceyella, for which the name Laceyella sediminis sp. nov. is proposed; the type
strain is RHA1T(5DSM 45263T5CCTCC AA 208058T).
The genus Laceyella within the family Bacillaceae was
proposed by Yoon et al. (2005), but was subsequently
placed in the family Thermoactinomycetaceae by Matsuo
et al. (2006) based on data from a polyphasic taxonomic
study. Members of the genus Laceyella are aerobic, chemo-
organotrophic, Gram-positive and thermophilic filamen-
tous bacteria. Substrate and aerial mycelia are formed, and
the aerial mycelium is white. Sessile endospores may be
produced on sporophores. Greyish-yellow or yellow–
brown diffusible pigment may be produced. The predom-
inant menaquinone is MK-9. The cell-wall peptidoglycan
contains meso-diaminopimelic acid, but no characteristic
sugars. The major fatty acids are iso-C15:0and anteiso-
C15:0, and the DNA G+C content of the type strains of
recognized Laceyella species is in the range 48–49 mol%
(Yoon et al., 2005). At the time of writing, the genus
comprised three recognized species: Laceyella sacchari
(basonym Thermoactinomyces sacchari; Lacey & Vince,
1971; Yoon et al., 2000, 2005), Laceyella putida (basonym
Thermoactinomyces putidus; Lacey & Cross, 1989; Yoon
et al., 2005) and Laceyella tengchongensis (Zhang et al.,
During long-term investigations on the diversity of micro-
organisms from particular habitats, a thermophilic strain,
designated RHA1T, was isolated from a sediment sample of
a hot spring (55 uC, pH 6.5). In this study, 1 g sediment
was suspended in 50 ml liquid DSM 88 medium and
incubated at 55 uC with shaking (140 r.p.m.) for 24 h.
Samples of 100 ml were then spread onto the surface of
isolation plates containing soluble starch (1.0%, w/v) and
incubated at 55 uC for 24 h. After three purification steps,
one strain, designated RHA1T, was picked from the
isolation plate and maintained on DSM 88 medium agar
slants at 4 uC and as glycerol suspensions (20%, v/v) at
Cultures grown on International Streptomyces Project (ISP)
3 agar medium (Shirling & Gottlieb, 1966) for 1–7 days at
55 uC were observed by light microscopy (Olympus BH-2).
The colours of substrate and aerial mycelia and any soluble
pigments produced were determined with reference to
Kelly (1964). Aerial and substrate mycelia were abundant,
well developed, non-fragmented and white; endospores
were formed (Fig. 1). Scanning electron micrographs
(Philip XL30 ESEM-TMP) were taken of mature spores
in aerial mycelia of strain RHA1Tgrown on ISP 3 agar
medium for 5 days at 55 uC.
The physiological features and biochemical characteris-
tics of strain RHA1Twere examined as described by
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain RHA1Tis FJ422144.
Two supplementary figures are available with the online version of this
International Journal of Systematic and Evolutionary Microbiology (2012), 62, 38–42
38 028282G2012 IUMSPrinted in Great Britain
Goodfellow (1971) and Williams et al. (1983) and are
detailed in Table 1 and in the species description. Growth
of strain RHA1Toccurred at 28–65 uC (optimum 55 uC), at
pH 5.0–9.0 (optimum pH 7.0) and in the presence of 0–
1% (w/v) NaCl (optimum 0%). The phenotypic prop-
erties of strain RHA1Tclearly distinguished it from
recognized Laceyella species (Tables 1 and 2).
For chemical and molecular systematic studies, biomass of
strain RHA1Tand the type strains of L. sacchari, L. putida
and L. tengchongensis was obtained after incubation in
DSM 88 broth at 55 uC for 3 days in shaken flasks (about
150 r.p.m.). Amino acids and sugars of whole-cell hydro-
lysates were analysed according to the procedures described
by Hasegawa et al. (1983). Polar lipids were extracted and
examined by two-dimensional TLC and were identified by
using the procedures described by Minnikin et al. (1984)
(see Supplementary Fig. S1 in IJSEM Online). Menaquin-
ones were extracted according to Minnikin et al. (1984)
and were analysed by HPLC (Kroppenstedt, 1982). Cellular
fatty acid assays were performed according to the standard
protocol of the Microbial Identification System (MIDI)
(Sasser, 1990) (Sherlock version 6.1; MIDI database
TSBA6), according to the manufacturer’s instructions.
Whole-cell hydrolysates of strain RHA1Tcontained meso-
diaminopimelic acid, ribose and glucose. The phospholipids
comprised disphosphatidylglycerol, phosphatidylethanola-
mine, phosphatidylglycerol, phosphatidylinositol, phospha-
tidylinositolmannosides and one unknown phospholipid.
The predominant menaquinone was MK-9; MK-8 was
detected as a minor component. The cellular fatty acids
included branched, straight-chain and unsaturated compo-
nents (Kroppenstedt, 1985). The major fatty acids were iso-
C15:0 (62.39%) and anteiso-C15:0 (17.55%). A detailed
fatty acid profile of strain RHA1Tis given in Table 2; it was
similar to those of L. sacchari DSM 43356T, L. putida DSM
44608Tand L. tengchongensis YIM 10002T.
Extraction of genomic DNA, and PCR amplification and
sequencing of the 16S rRNA gene of strain RHA1Twere
carried out according to Li et al. (2007). The almost-
complete 16S rRNA gene sequence (1467 bp) of strain
RHA1Twas determined. The 16S rRNA gene sequence of
strain RHA1Twas compared with available sequences from
GenBank by using the program BLAST (http://blast.ncbi.nlm.
nih.gov/Blast.cgi) to determine an approximate phylo-
genetic affiliation. The program CLUSTAL X (Thompson
et al., 1997) was used for multiple alignment and three algo-
rithms, neighbour-joining (Saitou & Nei, 1987), maximum-
parsimony (Fitch, 1971) and maximum-likelihood (Guindon
& Gascuel, 2003), were used to construct phylogenetic trees;
Bacillus subtilis NCDO 1769Twas used as an outgroup. The
topology of the trees generated with these methods was
evaluated by the bootstrap resampling method of Felsenstein
(1985) based on 1000 resamplings.
A BLAST analysis of the 16S rRNA gene sequence of
strain RHA1Tshowed that it was affiliated to the family
Thermoactinomycetaceae. Fig. 2 shows the phylogenetic
position of strain RHA1Twithin the radiation of species of
the genus Laceyella. Strain RHA1Toccupied a distinct
branch with L. sacchari DSM 43356T, L. tengchongensis YIM
10002Tand L. putida DSM 44608T, with which it shared
99.8, 99.6 and 97.8% 16S rRNA gene sequence similarity,
respectively. This position was also supported in the tree
generated with the maximum-likelihood algorithm with a
bootstrap value of 100% (Supplementary Fig. S2, available
in IJSEM Online).
For determination of the G+C content of strain RHA1T,
DNA was obtained according to the method described by
Marmur (1961) and the value was determined by the
HPLC method (Mesbah et al., 1989). The DNA G+C
content of strain RHA1Twas 47.9 mol%. DNA–DNA
hybridization was carried out by the optical renaturation
method (De Ley et al., 1970; Huß et al., 1983; Jahnke,
1992). Levels of DNA–DNA relatedness between strain
RHA1Tand its closest phylogenetic neighbours, L. sacchari
DSM 43356T, L. tengchongensis YIM 10002Tand L. putida
DSM 44608T, were 60, 47 and 44%, respectively, values
below the 70% threshold for the delineation of genomic
species (Stackebrandt & Goebel, 1994).
Based on the combination of morphological, physiological,
chemotaxonomic and phylogenetic data discussed here, it
is evident that strain RHA1Tshould be affiliated to the
genus Laceyella. Differences in several phenotypic char-
acteristics can be used to distinguish the isolate from
recognized Laceyella species. For example, strain RHA1T
Fig. 1. Scanning electron micrographs of mature spores in aerial
mycelia of strain RHA1Tgrown on ISP 3 agar medium for 5 days at
55 6C. Representative measurements are given in (b). Bars: (a)
5 mm; (b) 1 mm.
Laceyella sediminis sp. nov.
and the type strains of L. sacchari, L. tengchongensis and L.
putida showed different results for gelatin liquefaction,
starch hydrolysis, nitrate reduction and melanin produc-
tion. They could also be distinguished based on cell-wall
sugars and DNA G+C content. The above data, together
with low levels of DNA–DNA relatedness (,70%) between
strain RHA1Tand its closest phylogenetic neighbours,
demonstrate that strain RHA1Trepresents a novel species
of the genus Laceyella, for which the name Laceyella
sediminis sp. nov. is proposed.
Description of Laceyella sediminis sp. nov.
Laceyella sediminis (se.di9mi.nis. L. gen. n. sediminis of
Cells are Gram-positive, aerobic, thermophilic and fila-
mentous. White aerial and yellow–white substrate mycelia
are produced, bearing single endospores on short sporo-
phores. No soluble pigments are produced on any of
the media tested. Growth occurs at 28–65 uC (opti-
mum 55 uC), at pH 5.0–9.0 (optimum pH 7.0) and in the
Table 1. Differential characteristics between strain RHA1Tand the type strains of recognized species of the genus Laceyella
Strains: 1, RHA1T; 2, L. sacchari DSM 43356T; 3, L. tengchongensis YIM 10002T; 4, L. putida DSM 44608T. All data are from this study. V, Variable.
Temperature range (uC)
DPG, PE, PG,
PI, PIM, PL
Xyl, Ara, Glu
DPG, PE, PME,
PI, PIM, PL
MK-9, MK-8, MK-10
Rib, Xyl, Glu
DPG, PE, PG,
PI, PIM, PL
Xyl, Ara, Glu
DPG, PE, PG,
PI, PIM, PL
DNA G+C content (mol%)
*Ara, Arabinose; Glu, glucose; Rib, ribose; Xyl, xylose.
DDPG, Disphosphatidylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIM, phosphatidylinositol-
mannosides; PME, phosphatidylmonomethylethanolamine; PL, unknown phospholipid.
Table 2. Cellular fatty acid profiles of strain RHA1Tand type
strains of recognized species of the genus Laceyella
Strains: 1, RHA1T; 2, L. sacchari DSM 43356T; 3, L. tengchongensis
DSM 45262T; 4, L. putida DSM 44608T. All data are from this study;
cultures were grown under identical conditions. Values are percen-
tages of the total fatty acids.
J.-J. Chen and others
40International Journal of Systematic and Evolutionary Microbiology 62
presence of 0–1% (w/v) NaCl (optimum 0%). Positive
for gelatin liquefaction, degradation of starch, and milk
peptonization and coagulation, but negative for nitrate
reduction, and H2S and melanin production. Utilizes
lactose, trehalose, maltose and gelatin as carbon sources,
but not D-mannitol, L-rhamnose, raffinose, D-mannose,
L-valine, adenine, glycine, L-asparagine and L-arginine,
but not L-lysine, L-cysteine, L-threonine, L-serine or L-
hydroxyproline. The diagnostic amino acid in the peptido-
glycan is meso-diaminopimelic acid. Hydrolysates of the
cell-wall contain ribose and glucose. The predominant
menaquinone is MK-9. The major cellular fatty acids are
The type strain is RHA1T(5DSM 45263T5CCTCC AA
208058T), isolated from a sediment sample of a hot
spring collected from Tengchong county, Yunnan province,
south-westChina.TheDNA G+C contentof thetypestrain
is 47.9 mol%.
We are grateful to Professor Hans-Peter Klenk for the reference type
strains. This research was supported by the National Basic Research
Program of China (no. 2010CB833801) and National Natural Science
Foundation of China (nos 31070007 and 30960022).
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of strain RHA1Tamong its closest neighbours. Numbers at nodes are bootstrap values (percentages of 1000 replications); only
values .50% are shown. The sequence of Bacillus subtilis NCDO 1769Twas used as an outgroup. Bar, 0.01 inferred
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42International Journal of Systematic and Evolutionary Microbiology 62