International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3804–3809
DOI 10.1099/ijs.0.068205-0
Genome-based reclassification of Bacillus cibi as a
later heterotypic synonym of Bacillus indicus and
emended description of Bacillus indicus
Samantha J. Stropko,3 Shannon E. Pipes3 and Jeffrey D. Newman
Correspondence
Jeffrey D. Newman
[email protected]
Biology Department, Lycoming College, Williamsport, PA 17701, USA
While characterizing a related strain, it was noted that there was little difference between the 16S
rRNA gene sequences of Bacillus indicus LMG 22858T and Bacillus cibi DSM 16189T.
Phenotypic characterization revealed differences only in the utilization of mannose and galactose
and slight variation in pigmentation. Whole genome shotgun sequencing and comparative
genomics were used to calculate established phylogenomic metrics and explain phenotypic
differences. The full, genome-derived 16S rRNA gene sequences were 99.74 % similar. The
average nucleotide identity (ANI) of the two strains was 98.0 %, the average amino acid identity
(AAI) was 98.3 %, and the estimated DNA–DNA hybridization determined by the genome–
genome distance calculator was 80.3 %. These values are higher than the species thresholds for
these metrics, which are 95 %, 95 % and 70 %, respectively, suggesting that these two strains
should be classified as members of the same species. We propose reclassification of Bacillus
cibi as a later heterotypic synonym of Bacillus indicus and an emended description of Bacillus
indicus.
Bacillus indicus Sd/3T was isolated from arsenic-contaminated aquifer sand and described by Suresh et al. (2004).
Bacillus cibi JG-30T was isolated from the Korean fermented
seafood dish jeotgal, and a description was published by
Yoon et al. (2005). While Yoon et al. (2005) used information about reference strains and cited Suresh et al. (2004),
a comparison with the B. indicus Sd/3T 16S rRNA gene sequence was not reported, and B. indicus Sd/3T was not used
as a reference strain despite tight clustering of the original
DNA sequences on the neighbour-joining tree (Fig. 1).
While investigating unknown environmental organisms
isolated from a freshwater creek in the undergraduate microbiology course at Lycoming College (Newman, 2000; Strahan
et al., 2011; Kirk et al., 2013), strain SJS was found to be
closely related to B. indicus and B. cibi. The type strains B.
indicus LMG 22858T and B. cibi DSM 16189T were obtained
from BCCM and DSMZ, respectively, characterized phenotypically and their genomes were sequenced.
Genomic DNA was isolated from B. indicus LMG 22858T
and B. cibi DSM 16189T using the Qiagen DNeasy Blood
3These authors contributed equally to this work.
The GenBank/EMBL/DDBJ accession numbers for the Whole Genome
Shotgun projects of Bacillus indicus LMG 22858T and Bacillus cibi DSM
16189T are JGVU00000000 and JNVC00000000, respectively. The
versions described in this paper are versions JGVU02000000 and
JNVC02000000.
Two supplementary figures and two supplementary tables are available
with the online Supplementary Material.
3804
and Tissue kit according to the manufacturer’s instructions
for Gram-positive bacteria. Libraries were prepared and
then sequenced on an Illumina MiSeq (V3 26300 base) by
the Indiana University Center for Genome Studies as part
of a Genome Consortium for Active Teaching NextGen
Sequencing Group (GCAT-SEEK) shared run (Buonaccorsi
et al., 2011, 2014). Sequencing reads were filtered (median
phred score .20), trimmed (phred score .16), and assembled using the paired-end de novo assembly option in
NextGENe V2.3.4.2 (SoftGenetics). The assembled genomes were uploaded to the Rapid Annotation with Subsystem Technology web service (Aziz et al., 2008; Overbeek
et al., 2014) for analysis, guided contig reordering and
assembly improvement.
Approximately 0.9 million reads from the B. indicus LMG
22858T genome were assembled into 22 contigs with a total
genome length of 4 129 127 bp, a mean coverage of 526
and a DNA G+C content of 44.4 mol%, significantly
higher than the 41.2 mol% reported by Suresh et al.,
(2004). Automated annotation identified 4285 potential
protein-coding sequences and 83 RNAs.
Approximately 1.4 million B. cibi DSM 16189T reads were
assembled into 24 contigs with a total genome length of
4 072 175 bp, a mean coverage of 716 and a DNA G+C
content of 44.4 mol%, in good agreement with the
45 mol% reported by Yoon et al. (2005). Automated annotation identified 4253 potential protein-coding sequences
and 85 RNAs. Contigs were ordered and oriented to
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Printed in Great Britain
Reclassification of Bacillus cibi as Bacillus indicus
100 Bacillus cibi JG-30T (AY550276)
96
Bacillus indicus Sd/3T (AJ583158)
Bacillus idriensis SMC 4352-2T (AY904033)
Bacillus altitudinis 41KF2bT (AJ831842)
97
100
Bacillus stratosphericus 41KF2aT (AJ831841)
Bacillus aerophilus 28KT (AJ831844)
Bacillus pumilus ATCC 7061T (ABRX01000007)
97 Bacillus safensis FO-36bT (AF234854)
Bacillus aerius 24KT (AJ831843)
96
100 Bacillus sonorensis NBRC 101234T (AYTN01000016)
Bacillus licheniformis ATCC 14580T (AE017333)
Bacillus atrophaeus JCM 9070T (AB021181)
75
Bacillus mojavensis RO-H-1T (JH600280)
97
Bacillus subtilis subsp. inaquosorum KCTC 13429T (AMXN01000021)
Bacillus subtilis subsp. spizizenii NRRL B-23049T (CP002905)
0.005
Bacillus tequilensis 10bT (HQ223107)
Bacillus vallismortis DV1-F-3T (JH600273)
Bacillus amyloliquefaciens subsp. plantarum FZB42T (CP000560)
Bacillus amyloliquefaciens subsp. amyloliquefaciens DSM 7T (FN597644)
Bacillus siamensis KCTC 13613T (AJVF01000043)
Bacillus methylotrophicus CBMB205T (EU194897)
Bacillus subtilis subsp. subtilis NCIB 3610T (ABQL01000001)
Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic relationship between B. indicus
LMG 22858T, B. cibi DSM 16189T and the most closely related species of the genus Bacillus. The neighbour-joining tree
(Saitou & Nei, 1987) was reconstructed with the Kimura two-parameter method (Kimura, 1980) in MEGA6 (Tamura et al., 2013)
from 208 16S rRNA gene sequences (997 positions) of species of the genus Bacillus with validly published names. Bootstrap
values (expressed as percentages of 1000 replications) greater than 70 % are given at the nodes (Felsenstein, 1985). Bar,
0.005 substitutions per nucleotide position.
maximize synteny of the 3964 shared coding sequences
(Fig. S1, available in the online Supplementary Material).
The 16S rRNA gene sequences obtained from the sequenced genomes were compared to the partial sequences
reported at the time of initial publication using the
pairwise alignment (Myers & Miller, 1988) feature implemented on the EzTaxon-e web-based service (Kim et al.,
2012). The Bacillus indicus LMG 22858T genome-derived
16S rRNA gene sequence was identical to the Bacillus
indicus Sd/3T 16S rRNA gene partial sequence (AJ583158)
(Suresh et al., 2004) except for two differences and an
insertion within the first 14 bases of the partial rRNA gene
sequence, which apparently did not have the amplification
primer sequences trimmed. Because the genome-derived
sequence is present within a large contig as part of a
complete rRNA operon with the 23S and 5S rRNA genes, it
is very likely to be the correct sequence. There was one
difference between the B. cibi DSM 16189T genome-derived
16S rRNA gene sequence and the B. cibi JG-30T partial
sequence (AY550276) (Yoon et al., 2005). The two
genome-derived sequences differed at four of 1533 bases
http://ijs.sgmjournals.org
giving a similarity of 99.74 %. The four differences
corresponded to two pairs of substitutions that maintained
base-pairing within stems.
Several phylogenomic metrics have been developed to
measure genome similarity for taxonomic determinations.
The traditional metric of DNA–DNA hybridization (DDH)
(Tindall et al., 2010) has been criticized due to technical
difficulty, lack of reproducibility, and inability to incorporate
into databases. Meier-Kolthoff et al. (2013) developed the
Genome–Genome Distance Calculator to estimate the DNA–
DNA hybridization value based on high-scoring segment
pairs from genome sequence comparisons. The estimated
DDH between the B. cibi DSM 16189T and B. indicus LMG
22858T genomes was 80.3 % (95 % confidence interval 78.3–
82.3 %), which exceeds the 70 % DDH species boundary.
Average nucleotide identity (ANI) reflects the similarity of
1 kb sequence fragments using the algorithm described by
Goris et al., (2007). The implementation on the ExGenome
Web service (http://www.ezbiocloud.net/ezgenome/ani)
(Kim et al., 2012) yielded a value of 97.8 %, while the
Kostas Lab implementation (http://enve-omics.ce.gatech.
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3805
S. J. Stropko and others
edu/ani/) calculated the value as 98.24 %. In both cases,
this is higher than the 94–96 % threshold that correlates to
the demarcation of separate species (Konstantinidis &
Tiedje, 2005a; Richter & Rosselló-Móra, 2009).
Average amino acid identity (AAI) reflects the similarity of
orthologous proteins. Because amino acid sequences change
more slowly than nucleotide sequences and have greater
complexity, AAI values are more sensitive over greater
evolutionary distances, yet can still be used to distinguish
organisms at the species level (Konstantinidis & Tiedje,
2005b). Based on the bidirectional best hits (3964 coding
sequences) identified by the sequence-based comparison
tool of the SEED viewer (Overbeek et al., 2005), AAI was
calculated as 98.33 % with the web-based NewmanLab AAI
calculator (http://lycofs01.lycoming.edu/~newman/aai/). Again,
this is greater than the 95 % threshold that correlates to
separate species.
During phenotypic characterization as described by Kirk
et al. (2013), several discrepancies with published results
were noted. Motility during wet mounts was observed for
both strains, which each have more than 80 genes for
motility and chemotaxis; however, B. indicus was originally
described as non-motile (Suresh et al., 2004). Both organisms grew with 4 %, 6 % and 8 % (w/v) NaCl on TSA plates
and with 4 % and 8 % (w/v) NaCl in Biolog GenIII plates,
although B. indicus was originally reported as negative for
growth under these moderately high NaCl concentrations.
Growth with 8 % NaCl and motility were also observed for
both strains by Khaneja et al. (2010). Neither organism
grew at 44 uC, in contrast to the positive growth result
reported by Yoon et al. (2005) at this temperature. Khaneja
et al. (2010) also observed that both strains showed resistance
to arsenic and arsenate, although B. cibi DSM 16189T
exhibited slightly lower maximum tolerated concentrations.
While examining the utilization of carbon sources on the
Biolog GenIII plates, both organisms yielded a strong
positive response in the negative control well when using
Biolog Inoculating Fluid A, suggesting that they contained
stored carbon sources. Consistent with this hypothesis,
both organisms contained a glycogen synthesis and utilization operon. A weaker response in the negative control well
was observed with inoculating fluid B, which allowed the
assessment of exogenous carbohydrate utilization abilities.
The primary nutritional differences between the two strains
were that B. indicus LMG 22858T utilized mannose, but
not galactose, while B. cibi DSM 16189T utilized galactose
but not mannose. Examination of the genome sequences
revealed the existence of a 55 kb region with a mannose
utilization operon that was present in B. indicus LMG
22858T but absent in B. cibi DSM 16189T (Table S1). A
likely explanation for the lack of galactose utilization by B.
indicus LMG 22858T is a 1 base deletion near the 39 end of
the galactose-1-phosphate uridylyltransferase gene that
results in an apparently non-functional in-frame fusion
with the adjacent aldose-1-epimerase gene. While these
organisms shared approximately 93 % of their genomes,
3806
B. indicus LMG 22858T contained 321 unique coding sequences,
including a 34 kb prophage (Table S1). B. cibi DSM 16189T
contained 282 unique coding sequences (Table S2).
All other standard phenotypes tested and listed in the
emended species description below were identical in the two
strains and to previously reported results except for bglucosidase activity on the API ZYM test strip (bioMérieux),
which was present in B. indicus LMG 22858T but not in B.
cibi DSM 16189T.
Fatty acid profiles were generated by GLC (MIDI Sherlock
version 6.1, method RTSBA6) using cells grown on tryptic
soy broth agar (TSBA) for 24 h at 30 uC and extracted
using the standard method (Sasser, 1990). The fatty acid
composition (Table 1) was essentially identical in the two
strains and the most abundant fatty acids were iso-C15 : 0
and anteiso-C15 : 0.
While colonies of B. indicus LMG 22858T and B. cibi DSM
16189T grown on TSBA at 30 uC for 24 h appear similar,
the pigmentation of B. cibi DSM 16189T colonies is darker
by 72 h (Fig S2a). To extract the pigments, 5–10 mg of cell
mass was scraped from a plate, spread around the inside
of a 1.5 ml microcentrifuge tube and 1 ml was acetone
added. The tube was then incubated in a shaker-incubator at
250 r.p.m. and 28 uC for 1 h. The acetone extract was
decanted into a 2 ml sample vial and analysed on an Agilent
1200 HPLC with an Agilent Zorbax Eclipse XDB C18
column (4.6 mm6250 mm) at 40 uC and a diode array
detector. Separation was achieved using 50 mM NaH2PO4
(pH 4.5) as solvent A; methanol containing 0.l % (v/v)
glacial acetic acid as solvent B; a flow rate of 1 ml min21; and
Table 1. Fatty acid contents (percentages) of B. indicus LMG
22858T and B. cibi DSM 16189T grown on TSBA at 30 6C for
24 h
Strains: 1, B. indicus LMG 22858T; 2, B. cibi DSM 16189T. Fatty acids
amounting to ,1 % of the total fatty acids in the both strains are not
shown. All data from this study.
Fatty Acid
iso-C14 : 0
C14 : 0
iso-C15 : 0
anteiso-C15 : 0
C16 : 1 v7c alcohol
iso-C16 : 0
C16 : 1v11c
C16 : 0
iso-C17 : 1v10c
Summed feature 4*
iso-C17 : 0
anteiso-C17 : 0
1
7.2
2.8
46.4
15.7
4.6
3.4
5.6
4.4
2.5
2.0
1.7
2.3
2
5.9
2.1
48.9
14.9
4.9
3.7
5.7
3.2
3.3
2.2
1.6
2.4
*Summed feature 4 comprises iso-C17 : 1 I and/or anteiso-C17 : 1 B,
which could not be separated by the MIDI system.
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Reclassification of Bacillus cibi as Bacillus indicus
the following gradient: 0–5 min at 0 %; increase to 75 %
solvent B at 10 min; increase to 100 % solvent B at 30 min;
remain at 100 % solvent B until 45 min. The elution profiles
revealed a variety of compounds with different spectra that
absorbed light in the visible range (Fig. S2b, c). The optimal
wavelength to detect all of the peaks was 452 nm (Fig. 2a).
There were three clusters of peaks that could be distinguished based on absorbance spectrum (Fig. 2b); the 29–
32 min peaks had spectra consistent with 4,4’-diaponeurosporene, the 36–40 min peaks had spectra consistent with
4,4’-diaponeurosporenoate, and the 33–36 min peaks had
spectra consistent with staphyloxanthin. Analysis of the two
strains’ genomes revealed several operons with homologues
of the Staphylococcus aureus crtOPQMN genes that are
responsible for the synthesis of the golden pigment staphyloxanthin (Pelz et al., 2005). Some of the different peaks in
each region are very likely to be different geometric isomers
(Melendez-Martinez et al., 2013). Both organisms contain
all of the genes necessary to produce staphyloxanthin, but B.
indicus LMG 22858T contains less of this pigment (Fig. 2b),
which is apparent in the more yellow, less golden colour
(Fig. S2a).
On the basis of the minor phenotypic and genomic
differences, it is proposed that B. cibi Yoon et al. 2005
should be considered as a later heterotypic synonym of B.
indicus Suresh et al. 2004.
Emended description of Bacillus indicus Suresh
et al. 2004
The description is the same as that given by Suresh et al.
(2004) except for the following traits. Cells are motile.
Growth is observed on R2A and tryptic soy agars, but not
on phenylethanol or mannitol-salt agars. Yellow–golden,
non-diffusible, carotenoid-type pigments are produced
that are consistent with staphyloxanthin and biosynthetic
precursors. The pH range for growth is pH 6–9, and cells
grow with up to 8 % (w/v) NaCl. Cells exhibit oxidase,
(a)
20
18
16
B. cibi
B. indicus
A452 (mAU)
14
12
10
8
6
4
2
0
20
25
30
35
40
45
Time (min)
Absorbance (mAU)
(b)
10
B. cibi 38.85 min
8
B. cibi 30.65 min
6
B. cibi 33.77 min
4
2
0
200
250
300
350
400
450
500
550
600
650
700
Wavelength (nm)
Fig. 2. Pigment analysis. (a) Acetone extracts of B. indicus LMG 22858T and B. cibi DSM 16189T cell mass were separated by
reversed-phase HPLC. Elution profile at 452 nm is shown. (b) UV–visible absorbance spectra of representative B. cibi DSM
16189T pigment peaks. mAU, milli absorption units.
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3807
S. J. Stropko and others
catalase, amylase and caseinase activities, but do not liquefy
gelatin. On the API ZYM panel, positive for C4 esterase, C8
esterase lipase, leucine arylamidase (weakly positive), achymotrypsin, acid phosphatase (weakly positive), naphtholAS-BI-phosphohydrolase, b-galactosidase and a-glucosidase,
and negative for alkaline phosphatase, C14 lipase, valine and
cystine arylamidases, trypsin, a-galactosidase, b-glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase and afucosidase; b-glucosidase activity varies. When using inoculating fluid B and the Biolog GenIII plate (27 uC), positive for
utilization of dextrin, maltose, trehalose, cellobiose, gentiobiose, sucrose, turanose, stachyose, raffinose, melibiose,
methyl b-D-glucoside, D-salicin, N-acetyl-D-glucosamine, aD-glucose, D-fructose, glycerol, gelatin, glycyl-L-proline,
L-alanine, L-aspartic acid, L-glutamic acid, L-histidine, Lpyroglutamic acid, L-serine, pectin, D-gluconic acid, Dglucuronic acid, methyl pyruvate, D-lactic acid methyl ester,
L-lactic acid, a-ketoglutaric acid, L-malic acid, Tween-40,
hydroxy-b-DL-butyric acid, acetoacetic acid and acetic acid;
utilization of D-mannose and galactose varies; negative for
the remaining utilization tests. Also on the GenIII plate,
tolerant of pH 6, NaCl at 1, 4 and 8 % (w/v), 1 % (w/v)
sodium lactate, LiCl, K-tellurite, aztreonam and sodium
butyrate; sensitive to the remaining inhibitory conditions.
T
T
T
The type strain is Sd/3 (5MTCC 4374 5DSM 15820 5
LMG 22858T); the former type strain of B. cibi, JG-30T
(5KCTC 3880T5DSM 16189T), is a second strain of B.
indicus. The DNA G+C content of strain DSM 16189T
LMG 22858T is 44.4 mol%.
Acknowledgements
This work was funded by a Lycoming College Professional
Development Grant to J. D. N and a Summer Student Research
Grant to S. J. S. GCAT-SEEK has been supported by US National
Science Foundation award DBI-1248096: RCN-UBE - GCAT-SEEK:
The Genome Consortium for Active Undergraduate Research and
Teaching Using Next-Generation Sequencing.
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