International Journal of Systematic and Evolutionary Microbiology (2015), 65, 4315–4322
DOI 10.1099/ijsem.0.000575
Sphaerochaeta associata sp. nov., a spherical
spirochaete isolated from cultures of
Methanosarcina mazei JL01
Olga Troshina,1 Viktoria Oshurkova,1 Natalia Suzina,1 Andrei Machulin,1
Elena Ariskina,1 Natalia Vinokurova,1 Dmitry Kopitsyn,2 Andrei Novikov2
and Viktoria Shcherbakova1
Correspondence
1
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of
Sciences, Prospect Nauki 5, Pushchino, Moscow region 142290, Russia
2
Gubkin Russian State University of Oil and Gas, Leninskiy Prospect 65-1, 119991 Moscow,
Russia
Olga Troshina
[email protected]
An anaerobic, saccharolytic bacterial strain designated GLS2T was isolated from aggregates of
the psychrotolerant archaeon Methanosarcina mazei strain JL01 isolated from arctic permafrost.
Bacterial cells were non-motile, spherical, ovoid and annular with diameter 0.2–4 mm. They
were chemoorganoheterotrophs using a wide range of mono-, di- and trisaccharides as carbon
and energy sources. The novel isolate required yeast extract and vitamins for growth. The
bacteria exhibited resistance to a number of b-lactam antibiotics, rifampicin, streptomycin and
vancomycin. Optimum growth was observed between 30 and 34 8C, at pH 6.8–7.5 and with
1–2 g NaCl l21.Isolate GLS2T was a strict anaerobe but it tolerated oxygen exposure. On the
basis of 16S rRNA gene sequence similarity, strain GLS2T was shown to belong to the genus
Sphaerochaeta within the family Spirochaetaceae. Its closest relatives were Sphaerochaeta
globosa BuddyT (99.3 % 16S rRNA gene sequence similarity) and Sphaerochaeta pleomorpha
GrapesT (95.4 % similarity). The G+C content of DNA was 47.2 mol%. The level of DNA–DNA
hybridization between strains GLS2T and BuddyT was 34.7¡8.8 %. Major polar lipids were
phosphoglycolipids, phospholipids and glycolipids; major fatty acids were C14 : 0, C16 : 0,
C16 : 0 3-OH, C16 : 0 dimethyl acetal (DMA), C16 : 1n8 and C16 : 1 DMA; respiratory quinones
were not detected. The results of DNA–DNA hybridization, physiological and biochemical tests
demonstrated genotypic and phenotypic differentiation of strain GLS2T from the four species of
the genus Sphaerochaeta with validly published names that allowed its separation into a new
lineage at the species level. Strain GLS2T therefore represents a novel species, for which
the name Sphaerochaeta associata sp. nov. is proposed, with the type strain GLS2T
(5DSM 26261T5VKM B-2742T).
One of the first unusual spirochaetes, which had a coccoid
morphology and was not able to demonstrate the characteristic twisting motion, was isolated from the termite
hindgut and named Spirochaeta coccoides SPN1T (Dröge
et al., 2006). Later, the new genus Sphaerochaeta within
the family Spirochaetaceae was proposed by Ritalahti
et al. (2012) to accommodate two newly isolated coccoid
spirochaetes, and at present the genus Sphaerochaeta
comprises four species with validly published names,
Abbreviation: DMA, dimethyl acetal.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA
gene sequence of strain GLS2T is JN944166
Two supplementary figures and two supplementary tables are available
with the online Supplementary Material.
000575 G 2015 IUMS
Spirochaeta coccoides reclassified as Sphaerochaeta coccoides
(Dröge et al., 2006; Abt et al., 2012), Sphaerochaeta globosa
(Ritalahti et al., 2012), Sphaerochaeta pleomorpha (Ritalahti
et al., 2012) and Sphaerochaeta multiformis (Miyazaki et al.,
2014). Furthermore, some other isolates related to the
genus Sphaerochaeta are known, for example strain ACE-P,
isolated from hypolimnion of Antarctic Ace Lake (GenBank 16S rRNA gene accession no. M87055; Franzmann &
Dobson, 1992), bacterial strains obtained from dechlorinating enrichment cultures (GenBank accession nos
DQ833395–DQ833403) and strain MET-E isolated from
an oilfield (AY800103) (Fig. 1). BLAST search revealed
that 16S rRNA gene sequences highly homologous to
Sphaerochaeta 16S rRNA genes were present ubiquitously
both in nature and in anthropogenic environments like,
for example, a lignocellulolytic microbial community
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4315
O. Troshina and others
Sphaerochaeta sp. Jel1-T, DQ833399
Sphaerochaeta sp. Jel1, DQ833402
Sphaerochaeta sp. Grapes TMA14, DQ833395
Sphaerochaeta sp. Jel1-C, DQ833403
0.05
99/100/100
Sphaerochaeta sp. Grapes TMA5, DQ833396
Sphaerochaeta sp. Grapes TMB5, DQ833398
100/100/100
Sphaerochaeta pleomorpha GrapesT, AF357917
Sphaerochaeta sp. TQ1, DQ833400
Sphaerochaeta sp. RCcp2, DQ833401
99/99/100
Spirochaeta sp. MET-E, AY800103
77/98/98
91/99/99
Sphaerochaeta globosa BuddyT, AF357916
Sphaerochaeta associata GLS2T, JN944166
Sphaerochaeta coccoides DSM 17374T, NR_074647
100/100/100
Sphaerochaeta multiformis MO-SPC2T, AB598280
strain ACE-P, M87055
100/100/100
Spirochaeta bajacaliforniensis DSM 16054T, AJ698859
Spirochaeta smaragdinae DSM 11293T, U80597
100/100/100
Spirochaeta alkalica Z-7491T, X93927
99/99/99
100/100/100
94/100/100
Spirochaeta africana Z-7692T, X93928
Spirochaeta asiatica Z-7591T, X93926
Spirochaeta dissipatitropha ASpC2T, AY995150
Fig. 1. Phylogenetic consensus tree of strains of the genera Sphaerochaeta and Spirochaeta based on 16S rRNA gene
sequences demonstrating the position of strain GLS2T. Bootstrap values obtained with the maximum-likelihood/minimum-evolution/neighbour-joining methods were based on 1000 replicates and shown as percentages at branching points. GenBank
accession numbers are presented after the strain names. Bar, 0.05 substitutions per nucleotide sequence position.
(JF834125; Yan et al., 2012), a crude oil reservoir
(JQ088436; Tang et al., 2012), leachate sediment
(HQ183962; Liu et al., 2011), a methanogenic reactor
(JX462552; Kobayashi et al., 2013), dechlorinating
microbial consortia (AB721402; Li et al., 2013), a bioreactor for treatment of wastewater sludge (CU923017;
Rivière et al., 2009), freshwater sediments (Ritalahti et al.,
2012), a hypersaline Antarctic lake (GQ167322; Murray
et al., 2012), a canine oral cavity (JN713550; Dewhirst
et al., 2012) and many other anoxic habitats.
Using molecular techniques, we detected the presence of
contaminating bacteria in a culture of the methanogenic
archaeon Methanosarcina mazei JL01 isolated from arctic
permafrost (Rivkina et al., 2007). The concomitant bacteria
were resistant to ampicillin and streptomycin used for isolation of archaea. These symbiotic bacteria coexisted well
on the medium under conditions optimal for the archaeon.
Establishment of a 16S rRNA gene clone library and
sequencing revealed that these tightly associating bacteria
belonged to genus Sphaerochaeta, a genus within the
family Spirochaetaceae. The genus combines a group of
non-spiral, pleomorphic, non-motile spirochaetes (Ritalahti et al., 2012). Here we describe morphology, physiology, chemotaxonomy and phylogeny of the newly
4316
isolated bacterium designated strain GLS2T. We suggest
that strain GLS2T is a member of a novel species of the
genus Sphaerochaeta.
Enrichment culture of the bacterial contaminant from
M. mazei strain JL01 was obtained on the basal medium
for the archaeon (Rivkina et al., 2007) modified by
adding 1 g glucose l21 and 1 g peptone l21. Cultivation
was performed under N2 headspace at 30 8C. DNA from
the enrichment culture was extracted by the method of
Marmur (1961). The 16S rRNA gene fragments were
amplified with universal bacterial primers (Lane, 1991)
27f (59-AGAGTTTGATCMTGGCTCAG) and 1492r (59TACGGYTACCTTGTTACGACTT) and genomic DNA of
the enrichment culture. PCR mix (25 ml) was composed
of template DNA (up to 10 ng), 2.5 mM MgCl2,
0.25 mM of each dNTP, 1.0 U Taq DNA polymerase,
0.1 mM of each primer in 16 PCR buffer. PCR reagents
and Taq DNA polymerase were purchased from Fermentas.
The PCR conditions were 94 8C for 5 min; 30 cycles at
94 8C for 30 s, 55 8C for 30 s, and 72 8C for 2 min; and
final elongation at 72 8C for 10 min. The PCR products
were purified after agarose gel electrophoresis using a
PCR gel purification kit with SiO2-coated magnetic particles (Sileks). The purified fragments were ligated into
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Sphaerochaeta associata sp. nov.
the pAL-TA plasmid (Evrogen JSC) and subsequently
transformed into competent cells of Escherichia coli
DH10B according to standard guidelines (Sambrook
et al., 1989). Sequencing was carried out by the dideoxy
method using an ABI PRISM BigDye Terminator v.3.1
sequencing reaction kit on an Applied Biosystems 3730
DNA analyser. The sequence data obtained were submitted
to GenBank to search for similar sequences using the
BLAST algorithm (Altschul et al., 1990, 1997). Phylogenetic
trees were reconstructed using the neighbour-joining,
maximum-likelihood and minimum-evolution methods
implemented in MEGA6 (Tamura et al., 2013). The evolutionary distances were computed using the Tamura–Nei
model. The rate variation among sites was modelled with
a gamma distribution (shape parameter55). The phylogenetic trees were evaluated by bootstrap analysis based on
1000 replications. The analysis involved 22 nt sequences.
There were a total of 1372 positions in the final dataset.
Cell morphology was observed using a Nikon Eclipse Ci
microscope with Jenoptic ProgRes SpeedXTcore5 camera.
Gram-stain reaction was performed using the Hucker
staining method (Murray et al., 1994) and KOH technique
(Buck, 1982). For ultrathin sectioning, cells were harvested
by centrifugation and then fixed in a solution of 1.5 %
(w/v) glutaraldehyde in 0.05 M cacodylate buffer
(pH 7.2) at 4 8C for 1 h. The cells were washed three
times in the same buffer and post-fixed in 1 % OsO4 in
0.05 M cacodylate buffer (pH 7.2) for 3 h at 20 8C. The
preparation was dehydrated in a series of ethanol and
embedded in Epon 812 epoxy resin. Ultrathin sections
were mounted on grids and post-stained in 3 % (w/v)
uranyl acetate in 70 % (v/v) ethanol for 30 min and afterwards they were additionally stained in lead citrate according to the method of Reynolds (1963). Thin sections were
examined in a JEOL JEM-100B transmission electron
microscope at an acceleration voltage of 80 kV.
Amplification of 16S rRNA gene fragments followed by
cloning and sequencing allowed identification of a contaminating bacterium in the culture of M. mazei JL01 as
a representative of the coccoid spirochaetes of the genus
Sphaerochaeta. On the basis of information obtained
from molecular experiments, a modified basal salt
medium (SM) (Leadbetter & Breznak, 1996) and antibiotics (Ritalahti et al., 2012) were chosen for isolation of
bacterial satellites by serial dilutions in roll-tubes (Hungate,
1969). The strain isolated was named GLS2T. A nearly fulllength 16S rRNA gene sequence (1504 bp) of GLS2T was
deposited in GenBank (JN944166). The isolate shared
85.6–99.3 % 16S rRNA gene sequence similarity with
type strains of species of the genus Sphaerochaeta: Sphaerochaeta globosa BuddyT, with the highest degree of
sequence similarity (99.3 %), and Sphaerochaeta multiformis MO-SPC2T (85.6 %). The phylogenetic tree (Fig. 1)
demonstrates the position of strain GLS2T among other
isolates of the genus Sphaerochaeta.
Isolate GLS2T formed round, loose, pale colonies with
diameter about 2–3 mm. Under the phase-contrast microscope, cells of GLS2T were non-motile and had spherical or
ovoid shape with sizes of 0.2 to 1–2 mm (Fig. 2a). Many
cells were associated in bubbles of various dimensions, a
feature described by Ritalahti et al. (2012) for strain
BuddyT. Such bubble-like structures filled with one to
dozens of cells were particularly apparent in exponentially
growing GLS2T culture. Solitary enlarged bright spherical
bodies (about 4 mm) were also observed. In the stationary
growth phase, the cells occurred mostly as single separate
spheres. Transmission electron microscopy (TEM) images
showed a Gram-negative cell wall structure (Fig. 2b–d)
thereby supporting Gram staining tests (Buck, 1982;
Murray et al., 1994). In addition, TEM revealed another
type of cell morphology, namely, annular cells (Fig. 2c).
Such forms were also observed for Sphaerochaeta multiformis MO-SPC2T (Miyazaki et al., 2014). Like its closest
relatives Sphaerochaeta globosa BuddyT and Sphaerochaeta
pleomorpha GrapesT, strain GLS2T was able to grow in
the presence of ampicillin. Genome sequencing of
BuddyT and GrapesT showed the absence of several genes
coding for penicillin-binding proteins involved in transglycosylation and transpeptidation processes during the final
steps of peptidoglycan synthesis (Caro-Quintero et al.,
2012). Apparently, BuddyT and GrapesT as well as strain
GLS2T synthesize a non-rigid defective cell wall resulting
in spheric and sometimes pleomorphic morphology.
These strains might be considered therefore as cell-wall
deficient organisms of the spheroplast type. Electron
microscopy of GLS2T cells grown in SM medium with
ampicillin (175 mg ml21) showed cells without evident
cell walls (Fig. S1 available in the online Supplementary
Material). Some of these cells had very small sizes (about
100 nm). They resembled, in relation to morphology and
sizes described previously, cell-wall-less mycoplasma in
aggregates of Methanosarcina sp. (Zhilina & Zavarzin,
1979). Mycoplasma in methanosarcina aggregates were
identified by electron microscopy. They were seen on
The SM medium for cultivation of GLS2T contained
(per 1000 ml) NaCl, 1.0 g; KCl, 0.5 g; MgCl2 . 6H2O,
0.4 g; CaCl2 . 2H2O, 0.1 g; NH4Cl, 0.3 g; KH2PO4, 0.2 g;
Na2SO4, 0.15 g; xylose, 20 mmol; yeast extract, 1.0–3.0 g;
NaHCO3, 0.5 g; trace element solution SL7 (Widdel &
Pfennig, 1981), 1 ml; seven vitamin solution (DSMZ
medium 503), 1 ml. The medium was prepared according
to the Hungate anaerobic technique (Hungate, 1969).
Cysteine hydrochloride at a final concentration of
0.2 mM was added. Nitrogen-purged reduced medium
was then dispensed anaerobically into 15 ml Hungate
tubes or into 125 ml serum bottles. Sodium hydrogen carbonate, vitamins, xylose and antibiotics (when necessary)
were added to the autoclaved medium from sterile anaerobic stock solutions. The medium was adjusted to pH 7.2–
7.3 with 10 % (w/v) NaHCO3 before inoculation. Cultivation was carried out at 30 8C under an atmosphere of N2
without shaking. For comparative studies of strain GLS2T
and reference strain Sphaerochaeta globosa BuddyT, both
strains were cultivated on SM medium as described above.
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4317
O. Troshina and others
(a)
(c)
(b)
(d)
OM
OM
OM
CM
CM
Fig. 2. Phase-contrast micrograph of cells of strain GLS2T (a). Transmission electron micrographs of negatively stained thin
sections of GLS2T: image of coccoid cell (b), annular cell (c) and magnified cell-wall area (d). CM, Cytoplasmic membrane;
OM, outer membrane. Bars, 10 mm (a), 1 mm (b), 0.5 mm (c), 0.1 mm (d).
ultrathin sections as spheroid bodies of various sizes
between 0.15 and 1 mm and without cell walls. Mycoplasma
were located in places of depolymerization of the methanochondroitin matrix of Methanosarcina sp. As can be seen
from Fig. S1a, these extremely small cells were possibly
generated by a mechanism of membrane extrusion or
blebbing. Such a mechanism of cell propagation was
suggested for L-forms of the Gram-positive bacterium
Bacillus subtilis (Leaver et al., 2009). It is suggested as a
mode of cell division of early cells before cell-wall evolution. GLS2T cells grown in the presence of ampicillin
were able to pass through a 0.22 mm-pore-sized sterile membrane and to induce growth. Phase-contrast microscopy of
cultures grown after filtration indicated typical GLS2T cell
morphology (data not presented).
Temperature, pH and NaCl ranges for growth were determined by monitoring the OD600. The effect of temperature
was checked within a temperature range from 4 to 55 8C.
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The pH range was tested from pH 5.5 to 8.5 and, when
necessary, MES, PIPES, HEPES and MOPS buffers at a
final concentration of 30 mM were used. NaCl ranged
from 0 to 20 g l21 in experiments to determine salt requirements. Substrate utilization studies were performed in
basal SM medium without xylose and containing one of
the following substrates: sugars (10 mM), organic acids at
a final concentration of 1 and 2 g l21, galactosamine (0.5
and 1 g l21), glucuronic acid (0.5 and 1 g l21), Casamino
acids (2 g l21), ethanol (17 mM), methanol (120 mM), trimethylamine (17 mM), xylan (1 g l21), cellulose (1 g l21)
and H2 + CO2 (80 : 20, v/v). Ability to use various electron acceptors was tested in SM medium containing yeast
extract (2 g l21) and xylose (10 mM). The following acceptors were tested: nitrate (20 mM), sulfate (20 mM), thiosulfate (15 mM), sulfite (2 mM), ferric citrate (3 mM)
and ferric EDTA (3 mM). Reduction of Fe(III) was determined as described by Lovley & Phillips (1986), sulfide was
measured by the Pachmayr method (Cline, 1969) and
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Sphaerochaeta associata sp. nov.
nitrite was determined using Griess reagent. All experiments were carried out in triplicate and with two consequent transfers.
GLS2T was an anaerobic chemoorganoheterotroph with
fermentative metabolism, and used mono-, di- and trisaccharides as sources of carbon and energy for growth. It
grew well on arabinose, xylose, maltose, galactose, raffinose
and cellobiose, but weaker growth was observed on glucose,
lactose, sucrose and starch and it did not grow on fructose,
cellulose, xylan or yeast extract as the only carbon source
(Table 1). Strain GLS2T grew on lactate and glucuronic
acid but it could not grow on a number of other organic
acids like malate, citrate, pyruvate, benzoate, fumarate,
propionate, butyrate or acetate as well as on ethanol,
methanol or trimethylamine. Strain GLS2T did not grow
on H2 + CO2. The isolate could use for growth the components of methanochondroitin of methanosarcina,
namely glucuronic acid and glucose but not galactosamine.
Casamino acids did not support growth of GLS2T bacteria.
Yeast extract was required for growth on all substrates and
it could not be replaced by vitamin mixture. Similar to
strain BuddyT, isolate GLS2T was able to reduce Fe (III)
citrate and Fe (III) EDTA to Fe (II) during cultivation
with xylose. Strain GLS2T could not reduce the following
electron acceptors: sulfate, thiosulfate, sulfite and nitrate,
when cells were grown with xylose. Sulfite inhibited
growth of GLS2T completely.
Isolate GLS2T was a strict anaerobe but tolerated exposure
to air. It was able to grow anaerobically after 4 days of
exposure to air. The optimal growth temperature was 30–
34 8C and the temperature range for growth was 20–
40 8C. Optimal pH for growth was pH 6.8–7.5, with
growth occurring between pH 5.7 and 8.2. The bacterium
required NaCl at a concentration of 1–2 g l21 for optimal
growth. The growth was inhibited by 5 g NaCl l21.
T
The effect of antibiotics on strain GLS2 was determined
on SM medium using antibiotics at the following concentrations (l21): ampicillin (200 mg), carbenicillin (250 mg),
rifampicin (30 mg), vancomycin (50 mg), cefepime (1 g),
streptomycin (100 mg), kanamycin (150 mg), erythromycin, (12.5 mg) and tetracycline (10 mg). As for other bacteria of the genus Sphaerochaeta, strain GLS2T was resistant
to ampicillin, cefepime, rifampicin and vancomycin and it
was sensitive to kanamycin, erythromycin and tetracycline.
GLS2T grew in the presence of streptomycin.
Catalase activity was determined by bubble formation in
3 % (v/v) H2O2 mixed with the cell suspension. Oxidase
activity was determined by using an oxidase reagent (bioMérieux). An API ZYM test (bioMérieux) was used according to manufacturer’s protocol for characterizing the
enzymic profiles of isolate GLS2T and the close relative
Sphaerochaeta globosa BuddyT. Strain GLS2T was catalaseand oxidase-negative. As determined with the API ZYM
test, both GLS2T and Sphaerochaeta globosa BuddyT had
high activities of acid and alkaline phosphatases,
naphthol-AS-BI-phosphohydrolase and a-galactosidase as
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well as weaker activity of b-galactosidase. The two strains
differed by the presence of esterase and esterase lipase
activities in BuddyT while GLS2T showed highly active
valine arylamidase (Table S1).
The G+C content of the DNA was determined as
described by Mesbah et al. (2011) and Owen & Pitcher
(1985) on a DU800 spectrophotometer (Beckman Coulter)
using as a control the DNA of the reference strain Sphaerochaeta globosa BuddyT. DNA hybridization was carried
out with DNA of Sphaerochaeta globosa BuddyT according
to the method of Rosselló-Móra et al. (2011). Despite the
high degree of 16S rRNA gene sequence similarity, strains
GLS2T and BuddyT were characterized by a low level of
DNA–DNA hybridization, only 34.7¡8.8% (SD ).
Polar lipids and isoprenoid quinones were extracted from
lyophilized cells (approx. 100 mg) according to procedures
described by Minnikin et al. (1979, 1984). The separation
of lipids was carried out by two-dimensional TLC on
Silica Gel 60F TLC-plates (Merck) using the following solvent systems: chloroform/methanol/water (65 : 25 : 4, by
vol.) in the horizontal dimension and chloroform/acetic
acid/methanol/water (80 : 15 : 12 : 4, by vol.) in the vertical dimension. A 5 % (w/v) solution of phosphomolybdic
acid in ethanol was used for detection of all lipids. Phospholipids were detected by molybdenum blue and glycolipids were detected by l-naphthol (Minnikin et al.,
1984). The polar lipid analysis showed that isolate GLS2T
had glycolipids, phospholipids and phosphoglycolipids,
typical for members of the genus Sphaerochaeta. However,
the polar lipid profile of strain GLS2T differed significantly
in both qualitative and quantitative terms from other
described species of the genus Sphaerochaeta (Miyazaki
et al., 2014) (Fig. S2). Isoprenoid quinones were absent.
Cellular fatty acids profiles were determined by GC-MS as
described previously (Slobodkina et al., 2013). Fatty acid
content was determined as the percentage of the total ion
current peak area.
The major fatty acids for the novel strain were C14 : 0,
C16 : 0, C16 : 0 3-OH and C16 : 1n8. Dimethyl acetals
(DMAs) C16 : 0 DMA and C16 : 1n8 DMA were also detected
(Tables 1 and S2). Interestingly, C16 : 0 DMA accounted for
about 20 % of the fatty acids content in both GLS2T and
BuddyT. Another major component was C16 : 0 3OH
(16 %). The fatty acid profile of strain GLS2T differed
from that of strain BuddyT, its closest recognized relative,
in terms of the absence or low content of different C18
fatty acids components like C18 : 0, C18 : 1n9 and C18 : 1
DMA as well as the absence of C20 : 4n6 (Table S2).
It was found that the physiological ranges of strain GLS2T
and M. mazei JL01 were the same. This fact along with the
ability of strain GLS2T to grow on components of methanochondroitin such as glucose and glucuronic acid as well as its
resistance to a wide variety of antibiotics and its properties of
cell-wall deficiency characteristic to many pathogens explain
the selection and tight association of both GLS2T and
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4319
4320
ND
Fresh water sediments
2
+
+
+
2
2
2
+
+
+
ND
2
+
+
+
2
+
+
+
+
+
+
(+)
+
2
+
(+)
+
+
(+)
(+)
2
M. mazei JL01
2
Fresh water sediments
2
46.2
C14 : 0; C16 : 0; C16 : 1; br-C17 : 1
C14 : 0; C16 : 0; C16 : 1;
C18 : 1; br-C17 : 1
48.9
2
C14 : 0; C16 : 0; C16 : 0 3-OH; C16 : 0
DMA; C16 : 1; C16 : 1 DMA
47.2
ND
6.5–7.5
1
ND
5.7–8.2
6.8–7.5
1–2
15–30
20–25
Pleomorphic spherical, elongated
or crescent shapes)
2
3
6.5–7.5
1
20–37
30
20–40
30–34
2
2
2
Coccus
1
Pleomorphic (spherical, ovoid, annular)
*Data from Miyazaki et al. (2014).
DNA G+C
content (mol%)
Cytochrome
oxidase
Carbon substrates
D -Arabinose
D -Glucose
D -Galactose
D -Fructose
Cellobiose
Lactose
Maltose
Raffinose
Sucrose
Starch
Yeast extract
Isolation source
Motility
Temperature for
growth (8C)
Range
Optimum
pH
Range
Optimum
NaCl optimum
(g l21)
Major fatty acids
Morphology
Feature
+
2
2
2
+
2
+
2
2
2
+
Hindgut of termite
Neotermes castaneus
2
50.6
2
+
2
2
+
2
+
2
2
2
+
Marine subsurface sediments
(+)
32.3
C14 : 0; C16 : 0; C16 : 1
6–8
6.8–7.2
20–30
5.5–9.5
7.4
1
C14 : 0; C16 : 0; iso-C16 : 0*
0–17
9
Pleomorphic (spherical, annular,
curved rod, helical)
+ (Only for helical cells)
5
15–40
30
2
Coccus
4
Strains: 1, GLS2T (data from this study); 2, Sphaerochaeta globosa BuddyT (Ritalahti et al., 2012); 3, Sphaerochaeta pleomorpha GrapesT (Ritalahti et al., 2012); 4, Sphaerochaeta coccoides SPN1T
(Dröge et al., 2006; Abt et al., 2012; Miyazaki et al., 2014); 5, Sphaerochaeta multiformis MO-SPC2T (Miyazaki et al., 2014). +, Positive; 2, negative; (+), weakly positive; ND , no data available;
DMA, dimethyl acetal. All strains were Gram-stain-negative. All strains were positive for assimilation of D -xylose. All were catalase-negative.
Table 1. Differential characteristics of strain GLS2T and the type strains of species of the genus Sphaerochaeta
O. Troshina and others
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Sphaerochaeta associata sp. nov.
M. mazei JL01. The characteristics differentiating strain
GLS2T from species of the genus Sphaerochaeta with
validly published names are detailed in Table 1.
REFERENCES
On the basis of morphological, physiological, genotypic
and phylogenetic traits, strain GLS2T is considered to
represent a novel species within the genus Sphaerochaeta.
We propose the name Sphaerochaeta associata since the
novel bacterium was isolated from a binary culture with
M. mazei, coexisting together in symbiotic relationship
for a long time under conditions of substrate limitation
for growth.
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Description of Sphaerochaeta associata sp. nov
Sphaerochaeta associata (as.so.ci.a9ta. N.L. fem. part. adj.
associata united with, occurring together, referring to isolation from binary cultures with M. mazei).
Cells are non-motile, coccoid, ovoid and annular with
sizes of 0.2–4 mm. Gram-stain-negative, catalase- and
oxidase-negative, anaerobic chemoorganoheterotroph.
Saccharolytic fermenting metabolism. Good growth occurs
on mono-, di- and trisaccharides. Growth occurs in the
presence of arabinose, xylose, galactose, maltose, cellobiose,
raffinose, glucose, lactose, sucrose, starch, lactate and
glucuronic acid, but not on fructose, cellulose, xylan, yeast
extract, galactosamine, methanol, ethanol, glycerol, acetate,
formate, pyruvate, propionate, butyrate, benzoate, fumarate, trimethylamine, Casamino acids or H2 + CO2 alone.
Yeast extract is required for growth on all substrates.
Requires 1–2 g NaCl l21 for optimal growth. Positive for
the following in the API ZYM test: acid and alkaline phosphatases, naphthol-AS-BI-phosphohydrolase, a-galactosidase and valine arylamidase. Mesophile with optimum
growth at 30–34 uC and a growth temperature range of
20–40 uC. Neutrophile with optimum pH 6.8–7.5 and
growth within the range pH 5.7–8.2. Resistant to ampicillin,
carbenicillin, cefepime, vancomycin, rifampicin and streptomycin but sensitive to kanamycin, erythromycin and tetracycline. Fatty acid profile is mostly composed of C14 : 0,
C16 : 0, C16 : 0 3-OH, C16 : 0 DMA, C16 : 1n8 and C16 : 1 DMA.
Major polar lipids are phosphoglycolipids, phospholipids
and glycolipids. Isoprenoid quinones are absent.
Type strain is GLS2T (5DSM 26261T5VKM B-2742T).
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We are grateful for help with analysis of isoprenoid quinones to Boris
Baskunov, Skryabin Institute of Biochemistry and Physiology of
Microorganisms, Russian Academy of Science. This work was supported by a grant of the Russian Foundation of Basic Research, no.
15-04-08612.The fatty acids analysis was supported by President of
Russia (grant MK-4530.2015.4).
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