Journal of Microbiological Methods 77 (2009) 48–57
Contents lists available at ScienceDirect
Journal of Microbiological Methods
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j m i c m e t h
Discovery of marine Bacillus species by 16S rRNA and rpoB comparisons and their
usefulness for species identification
Jang-Seu Ki 1, Wen Zhang 2, Pei-Yuan Qian ⁎
Coastal Marine Laboratory and Department of Biology, Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong
a r t i c l e
i n f o
Article history:
Received 9 September 2008
Received in revised form 11 December 2008
Accepted 3 January 2009
Available online 7 January 2009
Keywords:
Bacillus
Beta subunit of RNA polymerase
Marine
Pathogen
rpoB
16S rRNA
a b s t r a c t
Systematic studies of the Bacillus group have been biased towards terrestrial and pathogenic isolates, and
relatively few studies have examined Bacillus species from marine environments. Here we took twenty Bacillus
strains from diverse marine environments and sequenced their 16S rRNA. Using molecular comparisons, we
separated the strains into thirteen Bacillus genotypes and identified 9 species: B. aquaemaris. B. badius, B. cereus
group, B. firmus, B. halmapalus, B. hwajinpoensis, B. litoralis, B. sporothermodurans, B. vietnamensis, and three
indistinguishable Bacilli. In addition, we sequenced the DNA-directed RNA polymerase beta subunit (rpoB) gene
and assessed its discriminative power in identifying Bacilli. Phylogenetic trees of Bacillus rpoB genes separated
each Bacillus according to their taxonomic positions and were supported statistically. The resolution of Bacillus on
the rpoB phylogenetic tree was approximately 4.5 times greater than on the 16S rRNA phylogenetic tree. These
results demonstrate that the polymorphism of the Bacillus rpoB gene can be used to identify Bacillus species,
providing an improved identification scheme for Bacillus species.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
The genus Bacillus consists of more than 222 recognized species
(http://www.bacterio.cict.fr) distributed widely across many terrestrial and aquatic habitats (Ivanova et al., 1999; Siefert et al., 2000),
including marine sediments (Miranda et al., 2008). Conventionally,
Bacilli have been identified in the laboratory through biochemical
tests and fatty acid methyl ester profiling (Bobbie and White, 1980;
Vaerewijck et al., 2001). These are technically complex and laborintensive procedures, however, and the scarcity of reproducible and
distinguishable phenotypic characteristics for several bacterial species
often makes precise identifications difficult (Khamis et al., 2003). To
date, the development of gene amplification and sequencing,
especially that of the 16S rRNA gene sequences, has simplified the
identification and the detection of specific bacteria (Woese, 1987;
Yamada et al., 1997; Kolbert and Persing, 1999; Shaver et al., 2002;
Wang et al., 2003; Wu et al., 2006), especially those lacking
distinguishable phenotypic characteristics. The 16S rRNA gene is
now used as a framework for the modern classification of bacteria,
including those in the genus Bacillus.
However, 16S rRNA gene sequences sometimes show limited variation
for members of closely related taxa (Fox et al., 1992) due to the conserved
⁎ Corresponding author. Tel.: +852 2358 7331; fax: +852 2358 1559.
E-mail address: [email protected] (P.-Y. Qian).
1
Present address: The Research Institute for Natural Sciences, Hanyang University,
Seoul 133-791, Korea.
2
Present address: China Global Environment Facility (GEF) Office, Foreign Economic
Cooperation Office (FECO) State Environmental Protection Administration (SEPA), P.R.
China.
0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2009.01.003
nature of the gene. In such cases, DNA sequencing of certain housekeeping genes can provide more sensitive DNA-sequencing subtyping
than 16S rRNA sequencing for a number of bacterial species (Mollet et al.,
1997). As alternatives to 16S rRNA sequences, protein-coding genes (e.g.
rpoB, gyrB, nifD, recA, atpD) have recently been tested (Palmisano et al.,
2001; Qi et al., 2001; Ko et al., 2003; Blackwood et al., 2004; La Duc et al.,
2004). Of them, the gene encoding the beta subunit of DNA-directed RNA
polymerase, rpoB, has been proposed as a potential biomarker to
overcome identification problems due to the high level of conservation
of the 16S rRNA gene (Mollet et al., 1997; Dahllöf et al., 2000;
Khamis et al.,2003; Walsh et al., 2004; Marianelli et al., 2006). There
are many advantages to using rpoB genes instead of 16S rRNA genes: the
rpoB gene is homogeneous within cells because it is a single-copy gene; it
has relatively long sequences (approximately 3.5 kb in Bacillus); and
many of these rpoB sequences are available in public databases. Many
studies (Dahllöf and Kjelleberg, 2002; Khamis et al., 2003; Walsh et al.,
2004; Marianelli et al., 2006) have demonstrated that rpoB gene
sequences can be used to identify and classify various bacterial species,
including Mycobacterium (Richert et al., 2007), Pseudomonas (Ait Tayeb
et al., 2005), Staphylococcus (Drancourt et al., 2004), Streptomyces (Kim
et al., 2004) and Vibrio (Tarr et al., 2007). So far, in terms of Bacillus
species, the complete rpoB genes of at least ten Bacillus species (Table 1)
have been sequenced by several Bacillus genome sequencing projects
worldwide; also, partial rpoB gene sequences have been determined for
some species to facilitate the detection of some Bacilli pathogens, such as
B. anthracis and B. cereus (Qi et al., 2001; Ko et al., 2003). More recently,
Blackwood et al. (2004) suggested guidelines for the molecular
discrimination of clinical Bacillus based on 16S rRNA, rpoB, vrrA (variable
repeat region A gene), and the 16S-23S rRNA spacer region, and found
J.-S. Ki et al. / Journal of Microbiological Methods 77 (2009) 48–57
49
Table 1
Origins of the Bacillus strains and DNA sequence GenBank accession numbers
Species
B. anthracis
B. aquaemaris
B. badius
B. badius
B. cereus
B. cereus
B. cereus
B. firmus
B. firmus
B. firmus
B. firmus
B. firmus
B. halmapalus
B. hwajinpoensis
B. litoralis
B. sporothermodurans
B. vietnamensis
Bacillus sp. (asahii)
Bacillus sp. (seohaenensis)
Bacillus sp. (lentus)
B. amyloliquefaciens
B. anthracis
B. anthracis
B. anthracis
B. cereus
B. clausii
B. halodurans
B. licheniformis
B. pumilus
B. subtilis
B. weihenstephanensis
B. coagulans
B. coahuilensis
B. arenosi
B. atrophaeus
B. inaquosus
B. massiliensis
B. mojavensis
B. sonorensis
B. tequilensis
B. vallismortis
B. velezensis
G. kaustophilus
a
b
c
Strain
UST2006-BC001
UST040801-016
UST2006-BC002
UST2006-BC003
BU040901-022
BU040901-020
UST2006-BC004
UST981101-006
UST000620-011
UST981101-007
UST2006-BC005
UST991130-006
UST981101-001
UST040801-008
UST2006-BC006
UST2006-BC007
UST040801-005
CU040510-015
UST2006-BC008
UST991130-010
FZB42
ATCC 14578
A0174
A0389
ATCC 14579
KSM-K16
C-125 DNA
ATCC 14580
SAFR-032
str. 168
KBAB4
36D1
m4-4
FSL_F4-143
NRRL BD-622
NRRL B-14697
FSL_h8538
NRRL BD-600
NRRL B-23154
NRRL B-41771
NRRL B-14890
NRRL BD-621
HTA426
Isolation localitya
Length (bp)
Hong Kong: Victoria Harbor
Hong Kong: HKUST pier, Port Shelter
Hong Kong:: Tai O (E4)
Hong Kong: HKUST pier, Port Shelter
Hong Kong: Fish Farm, Long Harbor
Hong Kong: HKUST pier, Port Shelter
Hong Kong: Victoria Harbor
Hong Kong: HKUST pier, Port Shelter
Hong Kong: Victoria Harbor
Hong Kong: Fish Farm, Long Harbor
GenBank access no.
16Sb
rpoB
16S
rpoB
1131
1460
1154
1201
1428
1198
1086
1453
1303
1344
1180
1212
1135
1332
1064
1165
1346
1452
1108
245
1555 (rrnA)
1305
1509 (rrnA)
1509 (rrnA)
1481 (rrnA)
1553 (rrnA)
1552
1545 (rrnA)
1551
1553 (rrnA)
1545
1180
832
1178
1164
1137
1204
1206
1103
1168
1101
1173
1160
1096
1126
1193
1073
1128
815
1169
1133
3591
3534
3534
3534
3534
3544
3543
3582
3591
3582
3534
3420
3558
717
964
964
697
964
964
964
964
964
3573
FJ188293
FJ188294
FJ188295
FJ188296
FJ188297
FJ188298
FJ188299
FJ188300
FJ188301
FJ188302
FJ188303
FJ188304
FJ188305
FJ188306
FJ188325
FJ188308
FJ188309
FJ188310
FJ188311
FJ188312
NC_009725c
AB190217
NZ_ABLT00000000c
NZ_ABLB00000000c
AF290547
NC_006582c
BA000004c
AE017333c
NC_009848c
Z99104c
NC_010184c
NZ_AAWV00000000c
NZ_ABFU01000005c
FJ188313
FJ188314
FJ188315
FJ188316
FJ188317
FJ188318
FJ188319
FJ188320
FJ188321
FJ188322
FJ188323
FJ188324
FJ188325
FJ188326
FJ188327
FJ188328
FJ188329
FJ188330
FJ188331
FJ188332
1553
AE016879
AE016877
EF156913
EU138858
EU138804
EU147236
EU138851
EU138818
EU138832
EU138808
EU138857
NC_006510c
HKUST, Hong Kong University of Science and Technology.
Parenthesis represents the 16S rRNA operons (e.g. rrnA, rrnB, rrnC etc.) used in this study.
Genome sequence from GenBank.
that the rpoB gene proved to be the best alternative target, with a
conserved 4-nucleotide difference between B. cereus and B. anthracis
(Blackwood et al., 2004). However, apart from the rpoB genes of some
Bacillus species, the rpoB gene sequences of only very few members of
the diverse Bacillus species have been documented so far.
In this study, we determined the 16S rRNA gene sequences of Bacillus
strains isolated from various marine environments. By using molecular
comparisons (e.g. GenBank, phylogenetic analyses), we identified and
separated marine isolates into individual Bacillus species. In addition, we
sequenced the rpoB genes of the strains as an alternative biomarker to
determine the value of this approach in underpinning phylogenetic
relationships and to see whether it might serve for routine identification
of closely related marine bacilli. Finally, we discuss the usefulness of
rpoB sequencing in differentiating and identifying marine Bacilli, taking
into consideration the available Bacillus genome data.
and Technology (HKUST; Table 1). All of the strains had been isolated
from a variety of marine environments, including intertidal areas, fish
farms, and marine biofilms, water column, and sediments. The strains
had previously been identified on the basis of their morphological
characteristics and partial 16S rRNA gene sequences. We took bacterial
cells from the bacterial stocks kept in liquid nitrogen, re-grew them on
nutrient agar (0.3% yeast extract, 0.5% peptone, and 1.5% agar in 0.22µm-filtered seawater), and incubated them for 48 h at 30 °C under
aerobic conditions. After incubation, the cells on the plates were
harvested with a spatula, transferred to a 1.5 mL microcentrifuge tube,
resuspended in 100 µL of 1 × TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM
EDTA), and immediately stored at − 20 °C until DNA extraction.
The genomic DNA of each pure Bacillus strain was extracted from
the bacterial cells using the TaKaRa MiniBEST Bacterial Genomic DNA
Extraction Kit ver. 2.0 (TaKaRa, Dalian, China), according to the
manufacturer's instructions.
2. Materials and methods
2.2. PCR amplification and sequencing of 16S rRNA and rpoB genes
2.1. Bacillus strains and genomic DNA extraction
Twenty marine Bacillus strains were obtained from the Marine
Bacterial Culture Collection at the Hong Kong University of Science
The 16S rRNA gene in the DNA samples was amplified by PCR using
combinations (27F + 1492R, 27F+ 1389R, 519F+ 1492R) of primer pairs
(27F, 5′-AGA GTT TGA TCM TGG CTC AG-3′; 519F, 5′-CAG CMG CCG
50
J.-S. Ki et al. / Journal of Microbiological Methods 77 (2009) 48–57
CGG TAA TAC-3′; 1389R, 5′-TGA CGG GCG GTG TGT ACA AG-3′; 1492R, 5′GGT TAC CTT GTT ACG ACT T-3′), which are specific for the domain
Bacteria. For amplification of the rpoB gene, we used the primers
rpoB1206 (5′-ATC GAA ACG CCT GAA GGT CCA AAC AT-3′) and
rpoBR3202 (5′-ACA CCC TTG TTA CCG TGA CGA CC-3′). The primers
were designed by a comparison of Firmicute rpoB gene sequences.
The two genes were amplified under the same PCR conditions.
Briefly, each PCR mixture contained 1 µL of extracted DNA, 1.25 U of
rTaq DNA polymerase (TaKaRa, Dalian, China), 0.25 mM dNTPs, 0.1 µM
of each primer, and PCR buffer in a total volume of 25 µL. PCR was
performed on a PTC-100™ programmable thermal controller with a
heated lid (MJ Research, USA) with the following cycling program:
95 °C for 3 min, 35 cycles at 95 °C for 20 s, 55 °C for 30 s, and 72 °C for
1.5 min, with a final extension at 72 °C for 5 min. Subsequently, 2 µL of
each PCR product was subjected to electrophoresis on a 1% agarose gel
in 1 X TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA [pH
8.3]). The PCR products on the gel were visualized by UV illumination
after staining with ethidium bromide. The desired PCR products were
purified with the PCR Purification Mini Kit (Watson Biotechnologies
Inc., China), according to the manufacturer's instructions.
DNA sequencing reactions were performed directly with the PCR
products and a DYEnamic™ ET terminator sequencing premix
(Amersham Biosciences), according to the manufacturer's instructions. The above PCR and internal walking primers (e.g. 805R, 5′-GAC
TAC CAG GGT ATC TAA TCC-3′; 1055R, 5′-CAC GAG CTG ACG ACA GCC
AT-3′ for 16S rRNA; rpoBF1721: 5'-AAC ATC GGT CTG ATC AAC TC-3';
rpoBF2063: 5'-ATG GGT GCG AAC ATG CAA CGT CA-3'; rpoBR2410: 5'TGA CGT TGC ATG TTC GCA CCC AT-3' for rpoB gene) were used for the
sequencing reactions of the PCR amplicons. The sequencing reactions
were carried out using the following cycling program: 30 cycles of
denaturation at 94 °C for 10 s, primer annealing at 50 °C for 10 s, and
extension at 60 °C for 2 min. The DNA fragments were separated on an
automated sequencer (MegaBASE™ 500; Amersham Biosciences).
Editing and contig assembly of the DNA sequences were performed
using Sequencher 4.1.4 (Gene Codes). For additional verification, all the
rpoB and 16S rRNA gene sequences were compared with nucleotide
sequences in the GenBank database (http://blast.ncbi.nlm.nih.gov/
Blast.cgi) using the Basic Local Alignment Search Tool (BLAST) search
algorithm (Altschul et al., 1990). All DNA sequences determined here
have been deposited in the GenBank database (Table 1).
2.3. Neighbor-joining tree analyses of Bacillus 16S rRNA gene
For sequence comparisons, we constructed a sequence data matrix of
the 16S rRNA genes (a total of 157 rRNA gene sequences), including 125
Bacillus type strains and 11 genome data, and then added the nucleotide
sequences determined in this study to the dataset. These sequences
were aligned using Clustal W 1.8 (Thompson et al., 1994). The variable
and incomplete sites at both the 5′ and 3′ ends of the 16S rRNA gene
sequences were excluded from the alignment. Various regions were
further aligned using BioEdit 5.0.6 (North Carolina State University, NC).
Regions that could not be aligned unambiguously were excluded from
the analysis. Finally, the remaining alignment sites (989 bp), which
included regions V3–V8, were selected for the subsequent analyses.
Phylogenetic trees were inferred using the neighbor-joining (NJ)
algorithm with the Kimura two-parameter model in MEGA 4.0 (Tamura
et al., 2007). The strengths of the internal branches of the resultant trees
were statistically evaluated by bootstrap analysis with 1000 bootstrap
replications. Geobacillus kaustophilus (GenBank accession no.
NC_006510) was used as the outgroup.
Table 2
Similarity scores between marine isolates and the highly matched type strain identified
by NJ analysis (Fig. 1)
Strain
nt.
compared
UST2006-BC001
NC_006510UST040801016
UST2006-BC002
BU040901-020
UST991130-006
UST981101-001
UST040801-008
UST2006-BC006
UST2006-BC007
UST040801-005
CU040510-015
UST2006-BC008
1089
1460
UST991130-010
%
Similarity
GenBank
nos.
Species
99.9
99.0
AB190217
AF483625
B. anthracis
B. aquaemaris
1151
1198
1212
1135
1331
1063
1165
1249
1453
1077
99.8
100
100
98.6
99.7
99.9
100
99.6
95.9
96.3
X77790
AF290547
AB271750
X76447
AF541966
AY608605
U49078
AB099708
AB109209
AY667495
245
96.7
AB271746
B. badius
B. cereus
B. firmus
B. halmapalus
B. hwajinpoensis
B. litoralis
B. sporothermodurans
B. vietnamensis
Bacillus sp. (asahii)
Bacillus sp.
(seohaenensis)
Bacillus sp. (lentus)
2.4. Phylogenetic analyses of Bacillus rpoB gene DNA and protein sequences
For phylogenetic inferences of Bacillus rpoB genes, we constructed a
data set of the rpoB gene sequences of the genus Bacillus, including both
the data from this study and sequences from public sources. A total of 24
rpoB gene DNA sequences that were from selected specie were aligned
using the procedure employed in 16S rRNA gene analysis. The aligned
sequences were trimmed to the same length, and the remaining 676
alignment sites were used for the comparative rpoB gene analysis.
Phylogenetic analysis of the data set was performed using the Bayesian
method with MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001). The
general time reversible (GTR) model (–lnL = 7427.2) was selected from
MrModeltest v2 (Nylander, 2004) as the best-fit model for this data set.
A Bayesian tree of the rpoB data set was constructed with the GTR model,
in which the among–site rate variation was modeled with a proportion
of sites being invariant; the rates for variable sites were drawn from an
invgamma distribution, in MrBayes 3.1.2. The Markov chain Monte Carlo
(MCMC) process was set to chains and applied with 1,000,000
generations. The sampling frequency was every 100 generations. After
analysis, the first 2000 trees were deleted as burn–in processes and the
consensus tree was constructed from the remaining trees. The
phylogenetic trees were visualized using TreeView 1.6.6 (Page, 1996).
Additional phylogenetic analysis of deduced amino acid sequences
of the above rpoB gene data was carried out with the same manner
used in the previous DNA analysis. We aligned 24 rpoB amino acid
sequences, and used only unambiguous positions of 328 out of 450
alignment positions. The best-fitting model of the protein sequence
evolution was selected as the RtREV model (–lnL = 3027.9), by using
PROTTEST 1.4 (Abascal et al., 2005) among a set of 95 candidate
models (e.g. Dayhoff, Blosum62, JTT, WAG, and VT). Bayesian analysis
was conducted using MrBayes 3.1.2. Two independent runs of four
incrementally heated MCMC chains were simultaneously run for
1,000,000 generations under the RtREV model. The convergence of
MCMC was monitored by examining the values of the marginal
likelihood. Posterior Probabilities (PP) of each clade were obtained
from the 0.5 majority rule consensus of all the trees sampled every
100 generations on both independent runs after removing the 2000
first trees as a conservative burn-in. Phylogenetic tree was visualized
with TreeView 1.6.6.
After phylogenetic sequence comparisons were made with NJ,
Bayesian trees and genetic distances, we selected 13 different
Fig. 1. NJ trees inferred from Bacillus 16S rRNA sequences (Bacillus type strains and marine isolates). The tree on the left was done by 157 16S rDNA sequences, containing 20 marine
isolates, 11 genomic sequences, as 125 type strains, and 1 outgroup. The tree on the right was predicted with all sequences (71 selected Bacillus sequences) boxed in color in the lefthand-side tree. The boxes represent clades containing marine Bacillus. The two NJ trees were constructed from each data set using the Kimura two-parameter model in MEGA 4.0.
Branch lengths are proportional to the scale given. The numbers at the nodes are bootstrap values greater than 50% with 1000 bootstrap replications. The Firmicute Geobacillus
kaustophilus (GenBank no. NC_006510) was used as the outgroup. Bold letters represent the isolate names of sequences determined in this study. Asterisks represent genomesequences data.
J.-S. Ki et al. / Journal of Microbiological Methods 77 (2009) 48–57
51
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J.-S. Ki et al. / Journal of Microbiological Methods 77 (2009) 48–57
species from the 20 marine strains of Bacillus for further parsimony
analyses. Intraspecific differences among B. cereus and B. firmus
were investigated by comparing DNA similarities and genetic
distances of both 16S rRNA and rpoB gene sequences. Genetic
variations among the 13-selected marine Bacillus were calculated
according to 16S rRNA and rpoB gene sequences. The corrected
pairwise (p-) distances were calculated by investigating nucleotide
substitutions. Overall genetic characteristics, such as parsimony
informative sites and conserved and variable sites, were calculated
using MEGA 4.0.
3. Results
3.1. 16S rRNA and rpoB gene sequences
In the present study, we determined the 16S rRNA gene sequences,
including V3 to V8 sites, as well as the rpoB gene sequences, of the
twenty marine strains we investigated (Table 1). Using BLAST-N or
BLAST-X searches, we found that all the strains belonged to the genus
Bacillus. The identities of marine Bacillus isolates were determined by
comparing them with the available 125 sequences among 222 Bacillus
type strains described so far (see http://www.bacterio.cict.fr/number.
html) and with high-scored rRNA sequences in BLAST searches. After
judging similarity scores, we separated the 20 included marine Bacillus
into 13 species or genotypes. For example, there was a high degree of
similarity among species that were the closest matched, such as B.
cereus group (99.9%), B. aquaemaris (99.0%), B. badius (99.8%), B.
cereus (100%), and B. firmus (100%). By making further similarity
comparisons with each closest-matched species of type strains, we
could separate the 20 marine Bacillus isolates into 13 species (Table 2),
including 9 identified species: B. aquaemaris. B. badius, B. cereus group,
B. firmus, B. halmapalus, B. hwajinpoensis, B. litoralis, B. sporothermodurans, B. vietnamensis. In some cases, we, however, observed relatively
low levels of similarity between each marine isolate and its closest
neighbor, as identified on the branches of our phylogenetic trees
(described later). These included Bacillus sp. #CU040510-015 (the
closest neighbor in our analysis was B. asahii with 95.9% similarity of
16S rRNA gene), Bacillus sp. #UST2006-BC008 (B. seohaenensis with
96.3%), and Bacillus sp. #UST991130-010 (B. lentus with 96.3%),
respectively. In further BLAST-N searches, we could not determine
their identities exactly. Hence, here we have tentatively assigned the
three unidentifiable marine isolates to Bacillus sp. (asahii) for
CU040510-015, Bacillus sp. (seohaenensis) for UST2006-BC008, and
Bacillus sp. (lentus) for UST991130-010, respectively.
In addition, we found that some isolates belonged to the same
species: B. badius (UST2006-BC002, UST2006-BC003), B. cereus
(BU040901-022, BU040901-020, UST2006-BC004), and B. firmus
(UST981101-006, UST000620-011, UST981101-007, UST2006-BC005,
UST991130-006), as determined by comparing 16S rRNA gene
sequences. Intraspecific variations in 16S rRNA genes were measured
by comparing 16S rRNA gene sequences among the identical marine
Bacillus species (e.g. B. cereus, B. firmus), and these comparisons
showed that the overall similarities of 16S rRNA gene sequences were
nearly identical (more than 99.5% similarity).
3.2. Phylogenetic NJ tree of Bacillus 16S rRNA
A NJ tree of Bacillus 16S rRNA sequences (157), including 125 type
strains of Bacillus species, clustered all of the isolates belonging to the
previously identified species to the corresponding species together
(Fig. 1A). We found that the 20 marine Bacillus isolates were
distributed among five clades of the NJ tree (Fig. 1A). In addition,
marine Bacillus species revealed in the present study were separated
into each known type strain of Bacillus (Fig. 1B). The NJ tree shows
that, of 20 marine isolates, 13 Bacillus species, including the B. cereus
group, could be discriminated phylogenetically from the other marine
isolates. Of them, a total of 17 Bacillus isolates (consisting of 13 Bacillus
species) were distinguishable clustering with the given Bacillus
species; however, 3 un-identifiable isolates (CU040510-015,
UST2006-BC008, and UST991130-010) were present independent
from the other Bacillus species.
3.3. rpoB genes of marine Bacillus isolates
Fig. 2 shows two separate Bayesian phylograms inferred from the
Bacillus rpoB gene DNA and protein sequences of the 23 selected
species, including the 13 marine Bacillus species. Additional NJ
bootstrap proportions (BP) were incorporated into the Bayesian
trees to support the strength of each branch. Comparing the two
inference algorithms, the Bayesian method separated each Bacillus
species better than the NJ algorithm did, as judged by PP and BP
values. In many cases, NJ methods generated low BP supported (less
than 50% BP) and polytomic clades (more than 2). Comparisons of the
two Bayesian phylograms showed that the branch topologies of the
rpoB gene DNA-based tree (Fig. 2A) were generally congruent with
those of rpoB protein-based tree (Fig. 2B), with two differences in B.
badius and B. licheniformis, including some slight differences. Based on
the sum of branch lengths (SBL), higher phylogenetic resolution was
recorded at rpoB DNA (SBL = 2.398) rather than at rpoB protein
(SBL = 0.914).
Taking into consideration the above genetic and phylogenetic
information, we reconstructed a phylogenetic tree with Bacillus rpoB
gene DNA sequences, comprising data from 20 marine isolates, 11
other strains and 12 genomes, using the Bayesian algorithm (Fig. 3).
The Bayesian tree of rpoB gene sequences (n = 44), including the
outgroup, showed clearer relationships among the Bacillus species
than did the NJ tree of rpoB gene (low BP supportings in most nodes)
or 16S rRNA gene sequences (Fig. 1). Overall, the 13 previously
identified Bacillus species and other species were separated from one
another, and identical species (e.g. B. anthracis, B. badius, B. cereus, and
B. firmus) of different isolates were clustered near one other according
to their taxonomic names. Upon comparison, the branch topologies of
the 16S rRNA- and rpoB-based trees were generally congruent.
However, the Bayesian tree of Bacillus rpoB gene sequences was
more strongly supported statistically, as judged by posterior probabilities, and species were separated more clearly at the taxonomic
level of species with higher resolution than that achieved with 16S
rRNA gene sequences. While species within the B. cereus-group clade
(including B. anthracis, B. thuringiensis, B. cereus, and B. mycoides)
were separated according to the Bayesian tree (1.00 PP, 97% BP), they
had nearly identical genotypes, judging by the extremely low genetic
distances of rpoB genes (Table 3), and therefore their relationship was
not resolved clearly by our rpoB gene analysis.
3.4. Significance of rpoB gene compared with 16S rRNA gene
In the present study, we measured genetic divergences of both
rpoB and 16S rRNA genes, precisely comparing the 13 selected marine
Bacillus species we identified. Variations in the 16S rRNA gene
sequences of the selected Bacillus species were high (Table 3). In the
present study, we found that, of the 13 species, the levels of similarity
ranged from 90.3% (B. hwajinpoensis and B. sporothermodurans) to
99.8% (B. anthracis and B. cereus). Excluding the B. cereus group (e.g.
B. anthracis, B. cereus, B. mycoides, B. thuringiensis, and B. weihenstephanensis), in most cases these degrees of relatedness were
supported phylogenetically as well (Figs. 1–3). For example, B.
hwajinpoensis and B. sporothermodurans were separate and distantly
related species, but B. anthracis and B. cereus, including B. mycoides,
B. thuringiensis and B. weihenstephanensis, were clustered together
rather than shown as separate species. Excluding this group, a total of
20 marine Bacillus strains were identified by 16S rRNA gene
sequences.
J.-S. Ki et al. / Journal of Microbiological Methods 77 (2009) 48–57
53
Fig. 2. Bayesian trees inferred from Bacillus rpoB gene DNA (A) and deduced amino acid (B) sequences, including 13 sequences determined in this study and 10 genomic sequences
(see Table 1). Additional NJ trees were constructed with the same data matrixes, using the Kimura two-parameter model for DNA sequences and the Jones, Taylor, Thornton (JTT)
model for protein, respectively, in MEGA 4.0. The strengths of the internal branches of the resultant trees were statistically evaluated by bootstrap analysis with 1000 bootstrap
replications. Overall, NJ analyses generated similar topologies of the trees when compared with those of the Bayesian prediction. The first numbers at the nodes are posterior
probabilities (PP), in which PP above 0.50 are indicated at each node; the second numbers provide bootstrap proportions (BP) of more than 50% in the NJ analysis. Asterisks represent
incompatible branches between NJ and Bayesian trees. Dotted lines and species outlined by a box represent unexpected placements between DNA and protein trees.
On the other hand, similarity analysis based on rpoB gene
sequences, which were determined from the identical strains used
in 16S rRNA gene analysis, recorded high dissimilarities among the
marine Bacillus isolates (Table 3). In a comparison of the rpoB gene
DNA sequences, similarity scores were highly discriminative, e.g. we
found a 74.4% similarity between B. hwajinpoensis and Bacillus sp.
(lentus); however, in some cases, similarities were high (e.g. 98.3%
between B. anthracis and B. cereus). In comparison with the deduced
amino acid sequences, similarity scores were generally higher than
those of DNA sequences. For example, amino acid similarity (99.5%)
was measured between B. anthracis and B. cereus (vs. 98.3% DNA) as
well as between B. hwajinpoensis and Bacillus sp. (lentus) (83.5% vs.
74.4% DNA). When comparing analyses using the sequences of 16S
rRNA, rpoB gene DNA and rpoB gene protein (Fig. 4), similarities in
rpoB gene DNA sequences were significantly lower than those of 16S
rRNA gene (one-factor ANOVA, post-hoc Scheffe, p b 0.01); however,
they were not significantly different from rpoB gene protein comparisons (p N 0.05).
By parsimonious analysis, the corrected p-distances of the 13
selected Bacillus, based on their pairwise DNA genetic distance scores
for 16S rRNA and rpoB genes, were 0.043 and 0.248, respectively
(Table 4). In general, the Bacillus rpoB gene had higher genetic distance
(e.g. lower similarity) values than did 16S rRNA genes. Higher
parsimony-informative (PI) sites were recorded for the protein-coding
rpoB gene (35.4%) than for the 16S-rRNA-coding gene (8.0%). The rpoB
gene sequence is considerably more polymorphic than the corresponding 16S rRNA gene sequence. This high degree of polymorphism
is particularly evident for species that are not well differentiated by 16S
rRNA gene sequence analysis (Tables 3 and 4).
4. Discussion
The current classification of species within the genus Bacillus and
related genera is well established and is based on a combination of
numerous experimental approaches (Xu and Côté, 2003). In addition,
many phylogenetic studies of the Bacillus (Goto et al., 2000;
Blackwood et al., 2004) have been done, most of which are biased
towards terrestrial isolates (e.g. B. subtilus), particularly because of
clinical concerns about certain pathogens such as B. cereus,
B. thuringiensis, and B. anthracis (Shangkuan et al., 2000). However,
relatively few taxonomic works on marine Bacillus have been
attempted so far. In the present study, we have taken a total of 20
marine Bacillus isolates and determined their 16S rRNA and rpoB
gene sequences for phylogenetic comparisons (Figs. 1–3). According
to the assessment of 16S rRNA gene sequence-based targets for the
identification of Bacillus species (Goto et al., 2000; Blackwood et al.,
2004) and the present rpoB gene comparisons, we discriminated
thirteen species against the others. Of them, we could identify nine
54
J.-S. Ki et al. / Journal of Microbiological Methods 77 (2009) 48–57
Fig. 3. Phylogenetic tree of Bacillus rpoB genes. The Bayesian tree was inferred from a data set of rpoB gene DNA sequences (676 alignment sites, after trimming at the 5′ and 3′ ends)
and a GTR model. Additional NJ analysis was performed with the same DNA data with 1000 bootstrap replications in MEGA 4.0. The first numbers at the nodes are posterior
probabilities (PP), in which PP above 0.50 are indicated at each node; the second numbers provide the NJ analysis bootstrap proportions (BP) of more than 50%. Asterisks represent
incompatible branches between NJ and Bayesian trees. Branch lengths are proportional to the scale given. The Firmicute Geobacillus kaustophilus (GenBank no. NC_006510) was used
as the outgroup. The rpoB gene sequences are listed in Table 1.
Bacillus species and three indistinguishable species. In addition, to
verify their identity, we further analyzed these species phylogenetically and also searched them with BLAST-N algorithm in The National
Center for Biotechnology Information (NCBI) database. All the
analyses showed the same results and supported our present species
assignments for the 13 genotypes or species from 20 marine isolates
(Table 1). In one case, however, we could not separate the B. cereus
group (three B. cereus-like strains and a B. anthracis-like strain) and
identify its members to the species level using the present rpoB gene
sequence comparisons (more than 1100 bp) or the 16S rRNA gene
sequences. Excluding the B. cereus group, the Bacillus species
compared in the present study had significantly higher phylogenetic
divergence in the rpoB gene than in the 16S rRNA gene (p b 0.01).
Most bacilli of marine origins belong to the species Bacillus subtilis,
according to their phenotypic characteristics, antibiotic susceptibility
profiles, and fatty acids patterns (Ivanova et al., 1999). In addition,
B. horti, B. pumilus, B. licheniformis, and two indistinguishable Bacilli were
identified from marine invertebrates and sea water from different areas
of the Pacific Ocean. Also, Siefert et al. (2000) studied marine Bacillus
from the Gulf of Mexico using a phylogenetic analysis of 16S rRNA, and
J.-S. Ki et al. / Journal of Microbiological Methods 77 (2009) 48–57
55
Table 3
Pairwise similarity scores (%) calculated from 13 aligned sequences of the partial 16S rRNA sequences (above the diagonal) and the rpoB sequences (below the diagonal) from the
marine Bacillus species
No
Species #strain
Similarity (%)
[1]
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
B. cereus group (anthracis)
#UST2006-BC001
B. aquaemaris #UST040801-016
79.8
(91.1)⁎
B. badius #UST2006-BC002
76.4
(89.7)
B. cereus group (cereus)
98.3
#BU040901-020
(99.5)
B. firmus #UST991130-006
81.3
(91.5)
B. halmapalus #UST981101-001
82.8
(91.1)
B. hwajinpoensis #UST040801-008
77.0
(86.6)
B. litoralis #UST2006-BC006
81.3
(91.1)
B. sporothermodurans #UST200679.2
BC007
(89.3)
B. vietnamensis #UST040801-005
81.5
(91.5)
Bacillus sp. (asahii) #CU040510-015 81.2
(91.1)
Bacillus sp. (seohaenensis)
77.9
#UST2006-BC008
(91.5)
Bacillus sp. (lentus) #UST991130-010 78.5
(86.2)
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
94.7
92.2
99.8
93.2
95.5
92.2
93.9
95
94.5
93.8
95.1
92.3
93.0
94.6
95.5
94.8
93.7
93.8
96.6
99.1
94.8
97.6
91.5
92.1
93.8
91.7
91.1
93.5
95.4
92.7
93.7
94.0
95.5
93.1
95.4
92.1
93.8
94.9
94.4
93.7
95.0
92.3
93.9
91.8
93.1
95.8
94.9
96.5
95.2
93.9
92.3
94.7
94.1
94.6
94.7
94.3
91.5
90.3
94.0
93.7
92.8
93.3
93.5
93.9
93.4
93.3
92.5
92.3
96.2
96.2
96.6
97.1
94.4
97.6
91.1
95.5
93.9
78.5
(91.5)
79.2
(90.6)
78.9
(90.2)
77.6
(90.2)
76.0
(86.6)
78.6
(89.7)
79.5
(90.2)
92.3
(99.5)
78.6
(89.3)
81.5
(96.4)
77.5
(85.3)
76.1
(89.3)
78.8
(92.8)
77.2
(90.6)
74.1
(87.1)
75.7
(89.3)
78.2
(90.6)
80.1
(92.0)
78.5
(92.4)
80.1
(90.6)
75.4
(86.2)
80.7
(91.1)
82.5
(91.5)
77.0
(86.6)
81.5
(90.6)
79.4
(88.8)
80.6
(91.1)
80.9
(90.6)
77.9
(91.5)
78.6
(85.7)
80.3
(88.0)
77.6
(86.2)
77.5
(90.2)
79.1
(90.6)
80.4
(90.6)
87.8
(98.2)
81.2
(91.1)
77.5
(87.1)
73.9
(83.1)
77.9
(88.0)
77.0
(87.5)
79.5
(90.6)
77.8
(87.5)
76.9
(91.1)
75.1
(83.5)
73.8
(84.8)
75.8
(88.0)
75.4
(87.1)
75.8
(86.2)
77.9
(87.5)
74.4
(83.5)
78.8
(86.2)
78.9
(90.2)
78.4
(89.7)
76.4
(90.2)
76.3
(82.6)
79.4
(90.6)
78.6
(89.3)
78.4
(90.6)
94.8
(94.2)
79.8
(89.7)
82.2
(96.8)
77.6
(85.7)
78.6
(90.2)
77.0
(86.2)
92.7
76.1
(86.2)
The highest and lowest scores in each comparison are underlined in bold in the body of the table.
⁎Represents protein level.
found that their marine isolates were clustered separately with B. subtilis
B. amyloliquefaciens B. amyloliquefaciens and B. pumilus B. firmus, B. lentus,
B. megaterium B. megaterium B. fusiformis B. sphaericus. Recently, Yoon
and colleagues described several marine Bacillus species such as B.
aquaemaris (Yoon et al., 2003), B. hwajinpoensis (Yoon et al., 2004) and B.
litoralis (Yoon and Oh, 2005) by analyzing marine isolates from tidal flats
and sea water from the Yellow Sea in Korea. More recently, Miranda et al.
(2008) reported species of the genus Bacillus recovered from marine
sediments by conventional biochemical tests, sequencing analysis of 16S
rRNA genes and tDNA-intergenic spacer length polymorphism (tDNAPCR). Considering the number of recorded Bacillus (at least 222 species)
and the number of studies done on terrestrial isolates, marine Bacillus
have not yet been sufficiently investigated or classified. In the present
study we documented 13 Bacillus species from marine environments. Of
them, three Bacillus species, B. aquaemaris and B. hwajinpoensis and B.
litoralis, were previously described from marine environments, suggesting that they are marine forms. Of the other species identified in our
study, at least 10 species (e.g. B. badius, B. cereus group, B. firmus, B.
halmapalus, B. sporothermodurans, B. vietnamensis, Bacillus sp. [asahii],
Bacillus sp. [seohaenensis] and Bacillus sp. [lentus]) have been discovered
in marine habitats. In particular, we have detected certain pathogens (e.g.
B. anthracis, B. cereus/ anthracis) from marine Bacillus isolates that were
previously thought to be exclusive to terrestrial habitats.
As a DNA-sequence-based-identification scheme for Bacillus (Goto
et al., 2000; Blackwood et al., 2004), we consider 16S rRNA sequences
appropriate for the identification of marine Bacillus members,
excluding the B. cereus group. In the present study, 20 isolates were
distributed among the five clades (Fig. 1) depending on their
taxonomic positions. In addition, similarity scores were recorded as
high between matched species but low between neighbor or sister
species (Table 2). The results of this identification scheme based on
16S rRNA sequences were well in accordance with those of Goto
et al. (2000) and Blackwood et al. (2004). However, certain clades,
such as the B. cereus group, were not clearly separated into individual
species. Also, the identities of two of the species we detected
(B. anthracis and B. cereus) could not be determined by comparisons
of their 16S rRNA sequences alone. As noted previously, in this study
we could not discriminate between B. cereus and B. anthracis clearly
according to a phylogeny of rpoB sequences. However, apart from
those two species, all of the clades were separated clearly with high
phylogenetic resolution (Figs. 1–3). Although the rpoB gene could be
used to identify some members of the B. cereus group, for example
B. anthracis (Blackwood et al., 2004), our data demonstrate that
certain relationships still remain unclear, particularly the relationship
between B. thuringiensis and B. cereus.
Sequencing rpoB genes has been found to be useful in identifying
species of the genus Bacillus (Qi et al., 2001; Ko et al., 2003;
Blackwood et al., 2004; Palmisano et al., 2001; present study) with
some exceptions for certain clades such as the B. cereus group (Fig. 2).
Fig. 4. Similarity of each 16S rRNA, rpoB gene sequences of the 13 selected Bacillus
species (see Table 3). AA, amino acid.
56
J.-S. Ki et al. / Journal of Microbiological Methods 77 (2009) 48–57
Table 4
Sequence characteristics of 13 marine Bacillus 16S rRNA and corresponding rpoB gene
sequences
Characteristics
Nt
Nc
Nv
PI (%)
p-distance
16S
rpoB
872
872
761
491
111
381
70 (8.0)
309 (35.4)
0.043
0.248
Note: Nt, total number of sites compared; Nc, total number of conserved sites; Nv, total
number of variable sites; PI, parsimony informative site.
DNA sequences were identical to those in Table 3.
The results of the phylogenetic analysis and similarity analysis we
conducted undoubtedly reflect a relatively high level of sequence
diversity within the targeted DNA regions of the Bacillus isolates we
examined, supporting the suggestion that rpoB genes are useful,
precise markers that can supplement other analyses and make up for
the limited utility of 16S rRNA sequence typing within a given bacterial
species (Blackwood et al., 2004; Mollet et al., 1997; Dahllöf et al.,
2000; Khamis et al., 2003; Walsh et al., 2004; Marianelli et al., 2006).
In addition, our data, based on rpoB sequences of these bacteria,
suggest that this gene is probably polymorphic enough to replace the
16S rRNA gene for definitive identifications of Bacillus (Table 3, Fig. 4).
Currently, many rpoB gene sequences, including bacterial genome
sequencing projects, are available in databases; however, data are
present for only a very few Bacillus species (primarily pathogens such
as B. cereus, B. thuringiensis, B. anthracis) out of the 222 recorded
species. Apart from those of pathogens, rpoB sequences from seven
species (e.g. B. clausii, B. halodurans, B. licheniformis, B. pumilus, B.
subtilis, B. weihenstephanensis, B. thuringiensis) are currently available
in the NCBI database (see Table 1). Based on available data and our
new rpoB sequences, the relatively long rpoB gene (approximately
3,540 bp) shows a higher degree of variability and has more
informative characters than 16S rRNA (Tables 3 and 4). In this study,
parsimony analysis showed that the number of parsimony informative
(PI) sites is considerably higher for the rpoB gene (35.4%) than for the
16S rRNA gene (8.0%). That is, the rpoB gene of marine vibrios may
evolve 4.43 times more rapidly than the 16S rRNA gene, as judged
from the % PI values. These differences agree with the results of our
phylogenetic comparison of rpoB and 16S rRNA genes (Figs. 1 and 3),
in which the rpoB gene differentiated the Bacillus phylogeny with high
resolution.
In addition, the rpoB gene is present as a single copy in all bacterial
genomes, while microbial genomes have multiple copies of the 16S
rRNA gene, and the numbers and nucleotide sequences of these 16S
rRNA gene copies are not identical (Dahllöf et al., 2000). In the present
study, we screened the copy numbers of the 16S rRNA gene in the
Bacillus genomes (Table 1). Overall, Bacillus had four or more copies
of the 16S rRNA gene in all the known genomes: 14 copies in B.
weihenstephanensis, 13 in B. cereus # ATCC 14578, 10 in B. anthracis, 8
in B. clausii, and 7 in B. pumilus and 4 in B. amyloliquefaciens listed in
Table 1. As noted by Dahllöf et al. (2000), the heterogeneity of the 16S
rRNA gene hampers the quantification of bacterial species by PCRbased assays. In contrast, the rpoB gene is common to all bacteria and
occurs as a single copy in the genome (Mollet et al., 1997). This gene
allows us to quantify bacterial cell numbers in rpoB gene-targeting
quantification studies (Dahllöf et al., 2000). Our results with Bacillus
rpoB gene sequencing support the usefulness of this gene and also
demonstrate that the polymorphism of the rpoB gene can be used to
identify Bacillus species, including those found in marine isolates. This
represents an improvement over conventional typing methods and a
better identification scheme for Bacillus species.
Acknowledgements
We are grateful to Dr T.Y. Ki and Miss M.Y. Tsoi for technical help
and bacterial cell cultures. This work was supported by a HKSAR
governmental grant AoE04/04-02 awarded to P-Y Qian.
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