International Journal of Systematic and Evolutionary Microbiology (2013), 63, 4735–4743 DOI 10.1099/ijs.0.

048223-0

Bacillus paraflexus sp. nov., isolated from compost

Piyush Chandna,1 Shanmugam Mayilraj2 and Ramesh Chander Kuhad1
Correspondence 1
Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South
Ramesh Chander Kuhad Campus, New Delhi 110 021, India
[email protected] 2
Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH),
Sector 39-A, Chandigarh 160 036, India

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium capable of growing at

15–42 6C (optimum 30 6C) and at pH 5–11 (optimum pH 7) was isolated from compost. Its
taxonomic position was deduced using a polyphasic approach and the strain was designated
RC2T. 16S rRNA gene sequence analysis showed that the isolate belongs to the division
Firmicutes, forming a clade within the cluster containing Bacillus flexus IFO 15715T, and showed
highest similarity to B. flexus IFO 15715T (98.1 %). The cell wall contained meso-diaminopimelic
acid as the diagnostic diamino acid. The major cellular fatty acids of the novel strain were iso-
C15:0 (36.83 %), anteiso-C15:0 (49.19 %) and C16:0 (5.19 %). DNA–DNA hybridization between
strain RC2T and B. flexus DSM 1320T showed a level of relatedness of 54.5 %. The polar lipid
profile of strain RC2T showed the presence of phosphatidylglycerol, diphosphatidylglycerol and
phosphatidylethanolamine. The predominant isoprenoid quinone was MK-7 and the G+C
content of strain RC2T was 37.6 mol%. On the basis of phenotypic characteristics, phylogenetic
analysis and the results of biochemical and physiological tests, strain RC2T was clearly
distinguished from closely related members of the genus, and the strain is assigned to a novel
species, for which the name Bacillus paraflexus sp. nov. is proposed. The type strain is RC2T
(5MTCC 9831T5MCC 2100T5KCTC 13724T5CCM 7754T).

Composting has been defined as an intense microbial standards for describing new taxa of aerobic, endospore-
activity leading to decomposition of most biodegradable forming bacteria (Logan et al., 2009). Because of the close
materials (Weltzien, 1991; Adani et al., 1997). During the phenotypic relatedness observed between members of the
entire composting process, a large variety of mesophilic, genus Bacillus (Lechner et al., 1998), taxonomic revision of
thermotolerant and thermophilic aerobic micro-organisms the genus has been suggested by Ash et al. (1991) and
including bacteria, actinobacteria, yeasts and fungi are Nielsen et al. (1994, 1995). As a consequence, several
involved as decomposers (Doncean et al., 2011). A wide species of the genus Bacillus have been transferred to other
range of thermophilic micro-organisms have been isolated taxa. Broad-range PCR amplification and sequencing of the
from different compost environments, including species of 16S rRNA gene have not only been widely used as a
the genera Pseudomonas, Klebsiella and Bacillus (Falcon taxonomic tool but also recognized as an effective reference
et al., 1987). method for bacterial identification (Schlaberg et al., 2012).
Palys et al. (1997) used the 16S rRNA gene sequence for
The genus Bacillus has undergone considerable taxonomic clustering, phylogenetic tree reconstruction and molecular
changes and is phenotypically heterogeneous, with its discrimination of microbiological families that were very
members exhibiting an extremely wide range of nutritional close to each other. Cellular fatty acid analysis has also been
requirements, growth conditions, metabolic diversity and used to characterize members of the Bacillus subtilis
DNA base composition (Claus & Berkeley, 1986). Fritze subgroup (Palmisano et al., 2001) and strains within B.
(2002) recommended a stepwise approach to the iden- subtilis (Nakamura et al., 1999). Ash et al. (1991) placed 51
tification of aerobic endospore-formers, using the char- species of the genus Bacillus into five phylogenetically
acterization tests of Gordon et al. (1973), and many of distinct groups, and the largest among these groups (RNA
these characters are included in the proposed minimal group 1, Bacillus sensu stricto) contains the type species, B.
subtilis, and 27 other species. Later, within RNA group 1,
Abbreviation: TEM, transmission electron microscopy. species such as Bacillus atrophaeus, B. lautus, B. lentimorbus,
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene B. pumilus, B. amyloliquefaciens, B. licheniformis, B. popilliae
sequence of strain RC2T is FN999943. and B. subtilis were placed in a distinct subgroup. B. lautus
A supplementary table and a supplementary figure are available with the was later reclassified in the genus Paenibacillus, based on
online version of this paper. genotypic and phenotypic analyses (Heyndrickx et al., 1996).
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P. Chandna, S. Mayilraj and R. C. Kuhad

During the course of studying the bacterial phylogenetic following concentrations of ethanol in a cold graded
diversity of compost, the aerobic, alkalitolerant, endo- ethanol series (70, 80, 95 and 100 %) for 1–2 min and
spore-forming strain RC2T was isolated from compost and finally washed in Milli-Q water for 10 min. After washing,
appeared to represent a novel species of the genus Bacillus. dehydrated agar blocks were infiltrated with 33 and 66 %
In the present study, the taxonomic position of the strain Möllenhauer’s resin in propylene (1.5 h each). The
was ascertained by using a polyphasic approach. Composting material was embedded in 100 % resin and polymerized
of agricultural residues such as rice bran, wheat bran, rice in an oven at 60 uC for 24 h. Ultrathin sections of 90 nm
husk, ash, mustard oil cake, cow dung, cow urine, molasses were prepared using a microtome, transferred onto a
and other additives like grass clippings and leaves was carried copper grid and stained with uranyl acetate (saturated
out at the University of Delhi South Campus, New Delhi, solution of uranyl acetate in 50 % alcohol) followed by lead
India, in December 2006 to January 2007. The pile citrate. The sample was rinsed again in Milli-Q water and
(1.5060.9060.80 m) was prepared and covered with black dried by wiping with a clean Whatman filter paper no. 1.
polyethylene. Duplicate samples were removed from the pile Sections were viewed in a Morgagni 268D electron
at 30 uC. The samples were mixed thoroughly to homogen- microscope (Fei Electron Optics) equipped with digital
eity and passed through a 10 mm stitch sieve. For bacterial imaging and 35 mm photography system. Furthermore,
isolation, 1 g compost was weighed and serially diluted (1021 the presence of flagella was determined by negative
to 1029) and an aliquot of 100 ml from the 1029 dilution was staining.
plated on sterilized nutrient agar (NA) (M012, HiMedia; Growth at 4, 15, 25, 30, 37, 40, 42, 45, 50, 55, 60 and 65 uC
pH 7.3±0.1) for 18 h at 30 uC. Of different morphotypes was ascertained in trypticase soy agar (TSA) slants, which
examined, a translucent, non-pigmented, oval colony with were incubated for 48 h. Growth with 2, 3, 4, 5, 6, 7, 8,
regular margins and convex elevation was selected and 10 % (w/v) NaCl was ascertained in nutrient broth
characterized. Subcultivation of the isolate was carried out on (originally prepared without NaCl). Similarly, growth at
NA at 30 uC. Stock cultures of the isolate were preserved in pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 was assessed in
nutrient broth with 20 % glycerol at 220 and 280 uC. For trypticase soy broth after sterilization. Growth under
comparison, the reference strain Bacillus flexus DSM 1320T anaerobic conditions was also evaluated after incubating
was obtained from the Deutsche Sammlung von Mikro- in an anaerobic chamber (BBL) on 0.16 TSA supplement-
organismen und Zellkulturen, Braunschweig, Germany. ed with 0.5 % (w/v) glucose or 0.1 % (w/v) potassium
Unless otherwise indicated, morphological, physiological, nitrate. These physiological tests were carried out accord-
molecular and chemotaxonomic studies were performed ing to methods described by Cowan & Steel (1965),
with cells grown on NA (pH 7.3±0.1) at 30 uC. Gordon et al. (1973) and Logan & De Vos (2009).
Colony morphology of 24-h-old cultures was studied using Biochemical characteristics such as activity of oxidase,
a light microscope (DP70; Olympus). Gram staining was catalase, lysine decarboxylase and ornithine decarboxylase,
performed in accordance with the method of Gerhardt et al. nitrate reduction, hydrolysis of aesculin, casein, gelatin, o-
(1981). Motility was examined by phase-contrast micro- nitrophenyl b-D-galactoside (ONPG), starch and Tweens
scopy as described by Cayol et al. (1994) as well as on 20, 40, 60, 80 and 100, carbon source assimilation,
motility agar (Farmer, 1999). For scanning electron production of H2S and indole, as well as sensitivity to 16
microscopy, bacterial cells were grown to mid-exponential antibiotics employing the disc-diffusion method using
phase, harvested by centrifugation at 2952 g for 10 min commercially available discs (Himedia), were determined
and resuspended in PBS (pH 7.4) to yield 108 c.f.u. ml21 following the methods of Smibert & Krieg (1994). Other
(OD60051). After 30 min, cells were centrifuged at 2952 g physiological characteristics including the Voges–Proskauer
for 10 min, again resuspended in PBS (pH 7.4) and then and methyl red tests, utilization of citrate and resistance to
fixed in 2.5 % glutaraldehyde in the same buffer overnight lysozyme were examined as described by Smibert & Krieg
at 4 uC. After fixation, cells were washed three times with (1994). The urease test was performed according to the
0.1 M PBS (pH 7.4), dehydrated through a graded ethanol method prescribed by Kim et al. (2006). Enzyme activities,
series (70, 80 and 95 %) for 1–2 min and finally washed in the assimilation of substrates as sole carbon sources, acid
Milli-Q water for 1 min and dried in a vacuum desiccator. production from substrates and physiological and bio-
An automatic sputter coater was used for coating the chemical characteristics were also determined with the
specimens with 20 nm gold particles. Samples were viewed bioMérieux system and commercially available Biolog GP3
using a digital scanning electron microscope (LEO435VP; Microplate identification system following the manufac-
turers’ specifications.
Carol-Zeiss) with a resolution of 12 and 15 nm. For trans-
mission electron microscopy (TEM), cells were harvested at For cellular fatty acid analyses, strain RC2T and B. flexus
mid-exponential phase, washed and fixed according to the DSM 1320T were grown on TSA plates at 30 uC for 24 h up
method described above. After fixation, the sample was to late exponential growth phase, harvested by centrifu-
post-fixed in 1.0 % (w/v) osmium tetroxide at room gation, washed with distilled water and freeze-dried. Fatty
temperature for 30 min. The fixed cells were embedded in acid methyl esters were obtained from cells by saponifica-
2 % (w/v) agar, which was then cut into 1 mm3 blocks. tion, methylation and extraction and separated in a gas
Agar blocks with fixed cells were dehydrated using the chromatograph (6850 Series II; Agilent). Peaks were
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 Bacillus paraflexus sp. nov.

integrated automatically and fatty acid names and relatedness was quantified by using a scintillation counter
percentages were determined using the MIDI Sherlock (1450 LSC Luminescence counter Wallac Microbeta Trilux;
MIS system (MIDI; Microbial ID). Cells grown on TSA Perkin Elmer). Reciprocal reactions (A6B and B6A) were
plates at 30 uC for 3 days under aerobic conditions were performed, and their variation was within the limits of this
extracted using procedures described by Minnikin et al. method (Goris et al., 1998).
(1984) and analysed for polar lipids by two-dimensional
Cells of strain RC2T were Gram-stain-positive, motile and
TLC followed by spraying with the appropriate detection
rod-shaped, with rounded ends, occurring singly or in
reagents (Komagata & Suzuki, 1987). Respiratory quinones
short chains or filaments, with peritrichous flagella. Single
were extracted from dried cells of RC2T with chloroform/
cells were 1–2 mm long, with rounded ends, and flagella
methanol (2 : 1) and purified on TLC as described by
were lost during slide preparation. TEM revealed that
Collins & Jones (1981). Preparation of the cell wall and
determination of the peptidoglycan composition were spores of RC2T were oval, occurring centrally in swollen
performed by the method described by Schleifer & sporangia. TEM of spores of RC2T showed a diagnostic
Kandler (1972) with slight modifications. The peptidogly- feature of an outer fibrillar coat with a dense outer layer of
can was detected by TLC on cellulose sheets (art. no. 5577; the thinner cell wall (exospores) and the electronlucent
Merck) instead of paper chromatography. DNA of strain inner layer (endospore), separated by a space of 96.06 nm,
RC2T was extracted by using a standard method as encasing almost oval entities and measuring 137.6 nm in
described by Marmur (1961) and the G+C content of diameter (Fig. 1). The endospore core appeared swollen,
the genomic DNA determined using the thermal dena- and the rest of the spore structure was found to be intact.
turation method (Marmur & Doty, 1962). Whole-cell The nucleus might have been lost during sample prepara-
proteins were extracted from strain RC2T and reference tion. Colonies on NA plates with 2 to 10 % (w/v) NaCl
strain B. flexus DSM 1320T by employing the method became yellow and circular after cultivation at 30 uC for
described by Morris & Park (1973) and visualized using 24 h. Growth occurred at pH 4.0–11.0, 2–10 % (w/v) NaCl
Coomassie blue on SDS-PAGE, in accordance with and 15–42 uC.
procedure of Laemmli (1970). Strain RC2T was positive for catalase, but negative for
The almost-complete 16S rRNA gene sequence (1409 oxidase and H2S production. The novel isolate could be
bases) of RC2T was amplified by PCR using the universal differentiated from recognized species of the genus Bacillus
primers 8f, 342f, 519r and 1542r. The sequence of the based on different physiological and biochemical char-
amplified 16S rRNA gene was determined using a DNA acteristics. Characteristics that differentiate strain RC2T
sequencer (ABI 310 sequencer; Applied Biosystems). 16S and B. flexus DSM 1320T are presented in Table 1. All
rRNA gene sequences were aligned using CLUSTAL X 2.1 phenotypic tests were carried out at least in triplicate with
software (Thompson et al., 1997). Terminal nucleotides strain RC2T and B. flexus DSM 1320T under similar
not present in all sequences were removed manually. conditions. The carbon substrate utilization profile of
Common gaps from all selected sequences were removed RC2T, as measured by the bioMérieux system, showed an
and the alignment was then corrected manually by deleting identification match for the genus Bacillus (Table S1,
gaps and missing data. Phylogenetic trees were recon-
structed by the neighbour-joining (Saitou & Nei, 1987) and
maximum-parsimony (Nei & Kumar, 2000) methods using
PHYLIP version 3.5 (Felsenstein, 1993) and TREECON (Van de
Peer & De Wachter, 1994). Calculation of bootstrap values
was based on 1000 resamplings. Phylogenetic trees were Length: 492.04 nm
also reconstructed using the maximum-likelihood method
(Guindon et al., 2005) and neighbour-joining method Length: 137.64 nm
(Saitou & Nei, 1987) using MEGA version 5 (Tamura et al.,
2011) and the tree topologies were evaluated by bootstrap Length: 96.06 nm
analysis based on 1500 replicates. Strain RC2T clustered
with B. flexus IFO 15715T and was more distantly related to
other members of the genus Bacillus.
The relationship between strain RC2T and B. flexus DSM
1320T was further examined using DNA–DNA hybridiza-
tion. DNA–DNA hybridization between strain RC2T and B.
flexus DSM 1320T, the most closely related strain based on
16S rRNA gene sequencing, was performed using the
membrane filter method (Tourova & Antonov, 1987). The
probe DNA was labelled with [a-32P]ATP with a mean Fig. 1. Transmission electron micrograph of an endospore of strain
specific activity of 3000 Ci mmol21 (Board of Radiation RC2T, showing a core, 137.6 nm long; from core to extraneous
and Isotope Technology, Hyderabad, India). DNA–DNA layer, 96.06 nm.
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P. Chandna, S. Mayilraj and R. C. Kuhad

Table 1. Phenotypic characteristics that differentiate strain The major cellular fatty acids of strain RC2T were iso-C15:0
RC2T from its closest phylogenetic neighbour in the genus (36.83 %), anteiso-C15:0 (49.19 %) and C16:0 (5.19 %). A
Bacillus comparison of the fatty acid profiles of strain RC2T and its
closest phylogenetic neighbour B. flexus DSM 1320T is
Data were obtained in this study. Both strains display the following
given in Table 2. The major polar lipids detected in strain
properties. Gram-stain-positive, rod-shaped, motile, endospore-
RC2T were diphosphatidylglycerol, phosphatidylglycerol,
forming with swollen sporangia and catalase-positive. Growth in 2–
phosphatidylethanolamine and four unknown lipids (Fig.
10 % (w/v) NaCl and at 15–42 uC. Positive for acid production from
glucose, fructose, ONPG, urease, mannitol, salicin and melibiose.
S1). The predominant isoprenoid quinone was MK-7,
Negative for fluorescence (UV), growth in the presence of lysozyme
which supports the affiliation of strain RC2T to the genus
(0.001 %), hydrolysis of urea, aesculin and Tweens 20, 40, 60 and 80,
Bacillus. Strain RC2T contained meso-diaminopimelic acid
production of indole and H2S, utilization of citrate, Voges–Proskauer
as the diagnostic diamino acid in the cell-wall peptidogly-
test and acid production from lactose, mannose, malonate, rhamnose, can. The genomic DNA G+C content of strain RC2T was
melezitose, xylitol, methyl a-D-mannoside, methyl a-D-glucoside, D- 37.6 mol%. In comparison to the reference strain B. flexus
arabinose, xylose, adonitol, sorbitol, inulin, glycerol, glucosamine, DSM 1320T, the protein profile showed that strain RC2T
dulcitol and ribose, b-glucuronidase activity and deamination of exhibited a significant degree of overall genus similarity.
phenylalanine. No growth on MacConkey agar. Susceptible to However, a few dissimilar faint bands were also observed in
streptomycin, tetracycline, clindamycin, lincomycin, erythromycin, strain RC2T (Fig. 2). The whole-cell protein profile
vancomycin, levofloxacin, cefotaxime and resistant to chlorampheni- reiterates the phylogenetic relationship with the genus
col. +, Positive; 2, negative; w, weakly positive; S, susceptible; R, Bacillus. The chemotaxonomic features of strain RC2T were
resistant. typical of those found in members of the genus Bacillus.
For phylogenetic analysis of the 16S rRNA gene sequence,
Characteristic RC2T B. flexus representative sequences retrieved from GenBank were
DSM 1320T
aligned using the CLUSTAL X program version 2.1. Com-
Cell shape Short rods Long rods parative 16S rRNA gene sequencing analyses revealed that
Endospore position Central Terminal strain RC2T was closely related phylogenetically to B. flexus
Optimum NaCl concentration for 2–4.0 2–5.0 IFO 15715T (sequence similarity 98.1 %), B. nealsonii FO-
growth (%, w/v) 92T (95 %), B. niabensis 4T19T (95 %) and B. azotoformans
Optimum temperature for growth (uC) 30 35 LMG 9581T (94 %). Strain RC2T exhibited relatively low
pH for growth levels of 16S rRNA gene sequence similarity with respect to
Range 5–11 5–8 other species of the genus Bacillus. The phylogenetic tree
Optimum 7.0 7.5
Methyl red test + 2
Oxidase 2 +
Table 2. Fatty acid profiles of strain RC2T and B. flexus DSM
Hydrolysis of:
1320T
Casein + 2
Gelatin + 2 Strains: 1, RC2T; 2, B. flexus DSM 1320T. Data were obtained in this
Starch W 2 study. Values are percentages of total fatty acids; 2, not found. DMA,
Tween 100 + 2 Dimethylacetal; EPA, eicosapentaenoic acid.
Nitrate reduction 2 +
Acid production from: Fatty acid 1 2
Glucose 2 +
Galactose 2 + iso-C11 : 0 2 1.67
Cellobiose 2 + C12 : 0 0.23 2
Sorbose 2 + iso-C13 : 0 2 2.27
Lysine + 2 C14 : 0 2.52 4.92
Ornithine + 2 iso-C14 : 0 2 7.41
myo-Inositol 2 + anteiso-C14 : 0 0.17 2
Raffinose + 2 iso-C15 : 0 36.83 9.69
Trehalose + 2 anteiso-C15 : 0 49.19 33.72
Antibiotic susceptibility iso-C15 : 0 3-OH 0.17 2
Cefradine S R C16 : 0 5.19 14.96
Fusidic acid S R iso-C16 : 0 2 8.69
Kanamycin R S iso-C17 : 0 1.41 2
anteiso-C17 : 0 3.20 2
iso-C17 : 1v5c 0.40 2
C18 : 0 0.29 7.02
available in IJSEM Online). Phenotypically, as measured by C18 : 1v9c 0.24 2
the Biolog system, the strain showed a resemblance to the C20 : 0 0.16 2
genus Bacillus.
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 Bacillus paraflexus sp. nov.

1 2 3 kDa reconstructed using the neighbour-joining method showed

that strain RC2T is a member of rRNA group 6 of the genus
Bacillus, which includes B. flexus, and formed a compact
175 cluster with 100 % bootstrap support. Strain RC2T formed
a clade with B. flexus IFO 15715T with bootstrap value of
116 100 % (Fig. 3). The resulting gene sequences were aligned
again using the MEGA version 5 software package and
phylogenetic trees were reconstructed using the maximum-
likelihood method and neighbour-joining method. Boot-
66.4 strap analysis with 1500 replicates was performed to assess
45
the support of the clusters. The phylogenetic tree based on
16S rRNA gene sequences (Fig. 4) revealed a monophyletic
cluster supported by a high bootstrap value (100 %).
35 Phylogenetic tree reconstruction employing both max-
imum-likelihood and neighbour-joining methods revealed
that the branches are conserved in the two trees.
25
18.4
DNA–DNA relatedness between strain RC2T and B. flexus
DSM 1320T was 54.5 %, calculated on the basis of the mean
of four replicates. The pooled standard deviation of all the
hybridization experiments was 1 %. Since the hybridization
values were ,70 %, as recommended for the delineation of
bacterial species (Wayne et al., 1987; Stackebrandt &
Goebel, 1994), the results confirmed that strain RC2T
Fig. 2. Comparative whole-cell protein profile of strain RC2T with represents a novel species of the genus Bacillus.
reference strain B. flexus DSM 1320T. Lanes: 1, 15 ml sample from
B. flexus DSM 1320T plus 5 ml tracking dye; 2, 15 ml sample from The phenotypic properties of strain RC2T, such as the
strain RC2T plus 5 ml tracking dye; 3, 10 ml size markers (ranging ability to hydrolyse Tween 100, casein and gelatin but the
from 175 to 18.4 kDa). inability to reduce nitrate, also supported the view that

0.1 Bacillus bataviensis LMG 21833T (AJ542508)

1000 Bacillus novalis LMG 21837T (AJ542512)
Bacillus soli LMG 21838T (AJ542513)
Bacillus asahii MA 001T (AB109209)
697
993 Bacillus psychrosaccharolyticus ATCC 23296T (X60635/AB021195)
Bacillus muralis LMG 20238T (AJ628748)
Bacillus niabensis 4T 19T (AY998119)
Bacillus nealsonii FO-092T (AF234863)
T
1000 Bacillus flexus IFO 15715 (AB021185)
998 Bacillus paraflexus RC2T (FN999943)
Bacillus amyloliquefaciens DSM 7T (X60605)
997
Bacillus azotoformans LMG 9581T (X60609)
Bacillus psychrophilus ATCC 23304T (X60634)
768
1000 1000 Bacillus fusiformis ATCC 7055T (L14013)
Bacillus sphaericus ATCC 14577T (L14010)
Bacillus alvei DSM 29T (X57304)
1000
Bacillus macerans ATCC 8244T (X57306)
704
717 Bacillus gordonae ATCC 29948T (X60617)
Bacillus pulvifaciens NCDO 1141T (X60636)
Alicyclobacillus acidocaldarius DSM 446T (AJ496806)

Fig. 3. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, indicating the position of strain RC2T among
related members of the genus Bacillus. Bootstrap values (from 1000 replications) greater than 500 are shown at branch nodes.
Alicyclobacillus acidocaldarius DSM 446T was used as an outgroup. Bar, 0.1 substitutions per nucleotide position.

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P. Chandna, S. Mayilraj and R. C. Kuhad

(a) 100 * Bacillus paraflexus RC2T (FN999943)

0.02
Bacillus flexus IFO 15715T (AB021185)

Bacillus amyloliquefaciens DSM 7T (X60605)

Bacillus nealsonii FO-092T (AF234863)
Bacillus azotoformans LMG 9581T (X60609)
Bacillus niabensis 4T 19T (AY998119)
Bacillus asahii MA 001T (AB109209)
83
56 Bacillus psychrosaccharolyticus ATCC 23296T (X60635/AB021195)
89 Bacillus muralis LMG 20238T (AJ628748)
Bacillus soli LMG 21838T (AJ542513)
96
86 Bacillus bataviensis LMG 21833T (AJ542508)
60 Bacillus novalis LMG 21837T (AJ542512)

Bacillus psychrophilus ATCC 23304T (X60634)

65 Bacillus sphaericus ATCC 14577T (L14010)
100 Bacillus fusiformis ATCC 7055T (L14013)

88 Bacillus pulvifaciens NCDO 1141T (X60636)

Bacillus gordonae ATCC 29948T (X60617)
99 Bacillus macerans ATCC 8244T (X57306)
74 Bacillus alvei DSM 29T (X57304)
Alicyclobacillus acidocaldarius DSM 446T (AJ496806)

(b) 91 Bacillus psychrosaccharolyticus ATCC 23296T (X60635/AB021195)

0.02
79 Bacillus muralis LMG 20238T (AJ628748)
Bacillus asahii MA 001T (AB109209)
Bacillus niabensis 4T 19T (AY998119)
Bacillus soli LMG 21838T (AJ542513)
98 Bacillus bataviensis LMG 21833T (AJ542508)
55 Bacillus novalis LMG 21837T (AJ542512)

Bacillus azotoformans LMG 9581T (X60609)

Bacillus nealsonii FO-092T (AF234863)
92
Bacillus paraflexus RC2T (FN999943)
*
100 Bacillus flexus IFO 15715T (AB021185)
99
Bacillus amyloliquefaciens DSM 7T (X60605)
Bacillus psychrophilus ATCC 23304T (X60634)

60 Bacillus sphaericus ATCC 14577T (L14010)

100 Bacillus fusiformis ATCC 7055T (L14013)

85 Bacillus pulvifaciens NCDO 1141T (X60636)

Bacillus gordonae ATCC 29948T (X60617)
100 Bacillus macerans ATCC 8244T (X57306)
62 Bacillus alvei DSM 29T (X57304)
Alicyclobacillus acidocaldarius DSM 446T (AJ496806)

Fig. 4. Maximum-likelihood (a) and neighbour-joining (b) phylogenetic trees based on 16S rRNA gene sequences, showing
relationships between strain RC2T and representatives of the family Bacillaceae. Bootstrap values (.50 %) based on 1500
replicates are shown as percentages at branch nodes. Alicyclobacillus acidocaldarius DSM 446T was used as an outgroup.
Bars, 0.02 substitutions per nucleotide position.

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 Bacillus paraflexus sp. nov.

isolate was distinguishable from closely related Bacillus (30), bacitracin (10) and polymyxin B (30). The major
species (e.g. B. flexus DSM 1320T) (Table 1). Therefore, on cellular fatty acids are iso-C15:0, anteiso-C15:0 and C16:0.
the basis of its physiological, biochemical and phylogenetic MK-7 is the predominant isoprenoid quinone. The polar
properties, strain RC2T represents a novel species within lipids include phosphatidylglycerol, diphosphatidylglycerol
the genus Bacillus, for which the name Bacillus paraflexus and phosphatidylethanolamine, as well as some uniden-
sp. nov. is proposed. tified phospholipids and unidentified aminolipids. The
diagnostic diamino acid in the cell-wall peptidoglycan is
Description of Bacillus paraflexus sp. nov. meso-diaminopimelic acid.
Bacillus paraflexus (pa.ra.fle9xus. Gr. prep. para beside, The type strain, RC2T (5MTCC 9831T5MCC 2100T5
alongside, near, like; L. masc. adj. flexus flexible, and also a KCTC 13724T5CCM 7754T), was isolated from compost
specific epithet; N.L. masc. adj. paraflexus near Bacillus at the University of Delhi South Campus, New Delhi,
flexus). India. The DNA G+C content of the type strain is
37.6 mol%.
Colonies grown at 30 uC on NA are translucent with
regular margins and convex elevation, non-pigmented and
do not display UV fluorescence. The colony surface is oval Acknowledgements
in appearance. Cells are short, Gram-strain-positive rods,
We are grateful to Professor Rup Lal, Department of Zoology,
1–2 mm long, with rounded ends, occurring singly or in University of Delhi, Delhi, India, for providing the dot blot facility.
pairs, straight, motile and aerobic. Endospores are oval and The authors are also grateful to Dr J. P. Euzéby for etymological
make the sporangia swollen, and the spores are located advice during preparation of the manuscript. The authors also
centrally. Grows at 15–42 uC (optimal 30 uC) with a broad gratefully acknowledge timely help from Dr Jung-Sook Lee (Curator,
pH range (pH 5–11; optimum pH 7.0), but does not grow KCTC, Korea), Dr Sanjay Kumar Gupta (University of Delhi, Delhi)
in medium supplemented with lysozyme (0.001 %). and Professor T. Satyanarayana (University of Delhi South Campus,
New Delhi). The authors wish to express their gratitude to Ms
Halotolerant; capable of growing at 2–10 % (w/v) NaCl. Urvashi Kuhad, Department of Modern Indian Languages and
Casein, Tween 100, gelatin and starch are hydrolysed, but Literary Studies, University of Delhi, Delhi, for editing the
Tweens 20, 40, 60 and 80, aesculin and urea are not manuscript. We are extremely grateful to Professor J. P. Khurana,
hydrolysed. Positive for catalase and urease, but negative Department of Plant Molecular Biology and Biotechnology,
for oxidase. The Voges–Proskauer test is negative and University of Delhi South Campus, New Delhi, for reviewing and
methyl red test is positive. Nitrate is not reduced to nitrite editing the manuscript.
and H2S and indole are not produced. Produces acid from
D-glucose, sucrose, fructose, raffinose, ornithine, salicin,
trehalose, melibiose, ONPG and lysine but not from References
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