Genome characteristics of a novel phage from Bacillus thuringiensis showing high similarity with phage from Bacillus cereus.
ABSTRACT Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three modules: the "late" region, the "lysogeny-lysis" region and the "early" region. BtCS33 exhibited high similarity with several phages, B. cereus phage Wβ and some variants of Wβ, in genome organization and the amino acid sequences of structural proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B. cereus phage Wβ and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wβ may have a common distant ancestor.
[show abstract] [hide abstract]
ABSTRACT: Bacillus cereus sensu lato, the species group comprising Bacillus anthracis, Bacillus thuringiensis and B. cereus (sensu stricto), has previously been scrutinized regarding interspecies genetic correlation and pathogenic characteristics. So far, little attention has been paid to analysing the biological and ecological properties of the three species in their natural environments. In this review, we describe the B. cereus sensu lato living in a world on its own; all B. cereus sensu lato can grow saprophytically under nutrient-rich conditions, which are only occasionally found in the environment, except where nutrients are actively collected. As such, members of the B. cereus group have recently been discovered as common inhabitants of the invertebrate gut. We speculate that all members disclose symbiotic relationships with appropriate invertebrate hosts and only occasionally enter a pathogenic life cycle in which the individual species infects suitable hosts and multiplies almost unrestrained.Environmental Microbiology 09/2003; 5(8):631-40. · 5.84 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: Three species of the Bacillus cereus group (Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis) have a marked impact on human activity. Bacillus cereus and B. anthracis are important pathogens of mammals, including humans, and B. thuringiensis is extensively used in the biological control of insects. The microbiological, biochemical, and genetic characteristics of these three species are reviewed, together with a discussion of several genomic studies conducted on strains of B. cereus group. Using bacterial systematic concepts, we speculate that to understand the taxonomic relationship within this group of bacteria, special attention should be devoted also to the ecology and the population genetics of these species.Canadian Journal of Microbiology 07/2007; 53(6):673-87. · 1.36 Impact Factor
Article: Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence.[show abstract] [hide abstract]
ABSTRACT: Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.Applied and Environmental Microbiology 07/2000; 66(6):2627-30. · 3.83 Impact Factor
Genome Characteristics of a Novel Phage from Bacillus
thuringiensis Showing High Similarity with Phage from
Yihui Yuan, Meiying Gao*, Dandan Wu, Pengming Liu, Yan Wu
Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People’s Republic of China
Bacillus thuringiensis is an important entomopathogenic bacterium belongs to the Bacillus cereus group, which also includes
B. anthracis and B. cereus. Several genomes of phages originating from this group had been sequenced, but no genome of
Siphoviridae phage from B. thuringiensis has been reported. We recently sequenced and analyzed the genome of a novel
phage, BtCS33, from a B. thuringiensis strain, subsp. kurstaki CS33, and compared the gneome of this phage to other phages
of the B. cereus group. BtCS33 was the first Siphoviridae phage among the sequenced B. thuringiensis phages. It produced
small, turbid plaques on bacterial plates and had a narrow host range. BtCS33 possessed a linear, double-stranded DNA
genome of 41,992 bp with 57 putative open reading frames (ORFs). It had a typical genome structure consisting of three
modules: the ‘‘late’’ region, the ‘‘lysogeny-lysis’’ region and the ‘‘early’’ region. BtCS33 exhibited high similarity with several
phages, B. cereus phage Wb and some variants of Wb, in genome organization and the amino acid sequences of structural
proteins. There were two ORFs, ORF22 and ORF35, in the genome of BtCS33 that were also found in the genomes of B.
cereus phage Wb and may be involved in regulating sporulation of the host cell. Based on these observations and analysis of
phylogenetic trees, we deduced that B. thuringiensis phage BtCS33 and B. cereus phage Wb may have a common distant
Citation: Yuan Y, Gao M, Wu D, Liu P, Wu Y (2012) Genome Characteristics of a Novel Phage from Bacillus thuringiensis Showing High Similarity with Phage from
Bacillus cereus. PLoS ONE 7(5): e37557. doi:10.1371/journal.pone.0037557
Editor: Adam Driks, Loyola University Medical Center, United States of America
Received December 25, 2011; Accepted April 25, 2012; Published May 23, 2012
Copyright: ? 2012 Yuan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the National Project (2009ZX08009-056B), the National Natural Science Foundation of China (No. 31170123) and the
projects of the Chinese Academy of Sciences (KSCX2-EW-G-16). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Bacillus thuringiensis (Bt) is a Gram-positive entomopathogenic
bacterium belonging to the B. cereus group. This group includes six
very closely related species: B. cereus, B. anthracis, B. thuringiensis, B.
mycoides, B. pseudomycoides, and B. weihenstephanensis [1,2]. Based on
multilocus enzyme electrophoresis (MEE) data  and DNA
sequence variations of the 16S–23S internal transcribed spacers
, B. thuringiensis, B. anthracis and B. cereus sensu stricto are
considered as members of a single species, B. cereus sensu lato.
As an important biological pesticide, B. thuringiensis (Bt) has been
widely used for biocontrol of insect pests for several decades.
During their sporulation, Bt strains produce insecticidal crystal
proteins (ICPs), which are highly toxic to larvae of numerous
Lepidoptera, Diptera and Coleoptera species, but are harmless to
human and vertebrates [5,6]. About 83% of Bt strains contain
lysogenic phage. During Bt fermentation, lysogenic phages can
caused failures in 15%–30% of the batches, resulting in severe
losses . Studies to resolve this problem found that chitosan
oligomer and derivatives can inactivate Bt phage 1–97A partices of
and inhibit its infection [8,9]. However, the exact mechanism
involved in this process is still unclear. To figure out the lysogeny
control mechanism and reduce losses during Bt fermentation,
more genetic information from Bt phages is needed.
At present, five genomes of phages originating from B.
thuringiensis, have been completely sequenced, namely Bam35c
, GIL01 , GIL16c , 0305phi8-36  and MZTP-02
. Phage Bam35c, GIL01 and GIL16c are all Tectiviral phages
with high sequence identity and genomes about 15 kb in size.
Bam35c is different from GIL01 by only 11 bp, while GIL16c has
83.6% of sequence identity with GIL01 [12,14]. Phage MZTP02,
isolated from a Bt subsp. kurstaki strain, is a tailed phage with
15,717 bp genome with 40 bp inverted terminal repeats .
Phage 0305phi8-36, which has 218,948 bp genome with low
homology to other sequenced phages, is a atypical Myovirus phage
[13,15]. Based on genome analysis, Hardies et al classified
0305phi8-36 as a novel ancient phage lineage .
Besides the five Bt phages, one B. anthracis (Ba) phages and five
B. cereus (Bc) phages have been sequence. Thus far, comparisons of
phages have focused primarily on isolates that share the same host
species. For example, five Bc phages were compared with each
other, and four of them (Wb, Gamma, Cherry and Fah) were
closely related [16,17]. Comparison of phages from different but
closely related hosts can provide more information. Ba phage
AP50 was found to be closely related to Bt phages GIL16c and
Bam35c . These discoveries provide insight into the evolution
of the phages as well as the hosts.
In the present study, a novel lysogenic phage named BtCS33,
was isolated from Bt strain CS33, which has high toxicity to
PLoS ONE | www.plosone.org1 May 2012 | Volume 7 | Issue 5 | e37557
Lepidopteran and Diptera larvae. This is the first report of
a Siphoviridae family isolate among the sequenced B. thuringiensis
phages. The complete genome of phage BtCS33 was sequenced,
characterized and compared with other phages that infect the
same or closely related species hosts. This is also the first report of
a phage isolated from B. thuringiensis exhibiting high sequence
similarity to B. cereus phage Wb and some of its variants. But, the
host range is quite different, for phage Wb and it’s variants can
infect Ba and BtCS33 can’t infect Ba. This study may provide
more information about evolutionary relationship among these
Isolation and morphology of the bacteriophage
A bacteriophage designated BtCS33 was isolated from B.
thuringiensis subsp. kurstaki strain CS33, which has high toxicity to
Lepidoptera larvae. Transmission electron microscopy showed
that BtCS33 had an icosahedral head (61 nm667 nm) and a long
tail (204 nm65.7 nm) (Figure 1). The phage BtCS33 was similar
in shape to B. cereus phage Wb  and its variant, Gamma ,
and was considered to be Siphoviridae.
Host range of phage BtCS33
To investigate the sensitivity of Bacillus strains to BtCS33, 78 B.
thuringiensis strains, 4 B. sphaericus strains, 2 B. anthracis strains, and 1
B.cereus strain were tested. The tests showed that only two B.
thuringiensis strains, CS33 (H-serotype 3) and C-3 (H-serotype 3),
were sensitive to phage BtCS33, indicating that BtCS33 had
a narrow host range.
Overview of phage BtCS33 genome
The complete genome of phage BtCS33 was 41,992 bp in
length with an overall G+C content of 35.22%, about the same as
the 35.29% G+C% content of the geome of B. thuringiensis subsp.
NC_014171). Exonuclease III treatment (data not show) indicated
the physical structure of the genome DNA was linear. Sequence
analysis revealed 57 putative ORFs (Table S1). The combined
length of all ORFs covered 35,432 bp, about 84.4% of the whole
genome. The average length of each ORF was 737 bp with ATG
as the main start codon, except for ORF1 which had a GTG start
codon. Among the 57 ORFs, 51 were transcribed forward,
whereas 6 were transcribed in the opposite direction (Figure 2).
The genes transcribed rearward were ORF19, ORF20, ORF24,
ORF25, ORF28 and ORF41. Furthermore, the start codons of 13
ORFs (22.8% of the total) overlapped with the stop codon of the
previous gene. Several promoters of the s70family were identified
by using Bprom (data not show). No ORFs encoding tRNA were
found by analysis with tRNAscan-SE 1.21.
On the basis of homology comparisons, 26 ORFs were assigned
putative functions. As in other sequenced phages, the major
functions were organized in gene clusters (Figure 2). The BtCS33
genome contained three main clusters: the ‘‘late’’ region (encoding
structural, assembly, DNA packaging and lysis proteins), the
‘‘lysogeny-lysis control’’ region (encoding proteins for controlling
the lysogeny-lysis process) and the ‘‘early’’ region (encoding
proteins for phage DNA replication, recombination and modifi-
The structural and lysis module
This module corresponded to the ‘‘late’’ region, encoding
proteins for phage structure, assembly and DNA packaging. The
module could be divided into five submodules, representing DNA
packaging, head morphogenesis, head-tail joining and tail
morphogenesis functions (Figure 2). The module included proteins
GP1 and GP2 encoded by ORF1 and ORF2 that homologous
with (or that had domains typical of) the small and large subunits
of terminase. The module also included ORFs encoding portal
protein (GP3, gene product of ORF3), major capsid protein (GP5),
major tail protein (GP9), tail tape measure protein (GP12) and tail
fiber protein (GP13). Comparing the amino acid sequence of
GP18 with the CDD database revealed similarity to the GH25-
PlyB-like protein, which is a bacteriophage endolysin with
potential lytic activity toward B.anthracis . Endolysins are
produced by phages at the end of their life cycle and participate in
lysing the bacterial cell wall to release the newly formed virions
The lysogeny control module
This module controls the lysogeny-lysis process of the phage.
Phage BtCS33 is a lysogenic phage that begins the lysis cycle
spontaneously. At least seven putative ORFs were involved in the
lysogeny control module. Besides ORF49, six other ORFs were
physically related to each other but were transcribed in different
directions (Figure 2). ORF49 was predicted to encode integrase,
a DNA breaking-joining enzyme that catalyzes site-specific
integration of the DNA [23,24]. Other genes of this module
associated with the lysis/lysogeny switch function are are
commonly present in temperate Siphoviridae phages. GP28 dis-
played homology to the Cro/CI family proteins, which contain
a classical helix-turn-helix domain and can be assigned to the
XRE family of transcriptional regulators. GP30 showed identity to
many DNA binding proteins and might represent a Cro analogue.
The closest BLASTP match for GP32 was an antirepressor that
can inactivate the CI repressor [25,26].
The DNA replication and recombinant module
This module corresponded to the ‘‘early’’ region, encoding
proteins for phage DNA replication, recombination and modifi-
cation processes. At least three gene products (GP36, GP37, and
GP57) of phage BtCS33 were involved in the replication and
recombinant process. The amino acid sequence of GP36 was
exactly matched with the replication protein O from Bacillus phage
lambda Ba01. ORF37 was predicted to encode a DNA replication
protein (GP37) with an ATP/GTP binding P-loop motif. GP57
Figure 1. Morphology of phage BtCS33 particles under TEM.
The virion was negatively stained with 2% potassium phosphotung-
state. The white arrows indicate the putative tail fiber structure.
Genome of a Novel Bacillus thuringiensis Phage
PLoS ONE | www.plosone.org2 May 2012 | Volume 7 | Issue 5 | e37557
demonstrated similarity with various HNH endonucleases from
phage and bacteria. HNH endonuclesases belongs to the homing
endonuclease family and confers the mobility or duplication of
their coding and flanking sequences by a recombination-based
process [27,28]. Genes encoding for HNH endonucleases in phage
genomes are considered to be analogous to insertion or transposon
elements in bacterial genomes .
The genes associated with host cell sporulation
The genome of BtCS33 contained two genes of particular
interest, ORF35 and ORF22. The genes encode proteins with
83% and 100% amino acid sequence similarity, respectively, to
proteins from Bacillus thuringiensis subsp. kurstaki strain T03a001.
ORF35 was predicted to encode an RNA polymerase sigma factor
(s70) with about 23% similarity to sigmaF from Bacillus thuringiensis
97-27. SigmaF can direct expression of sporulation genes in
Bacillus strains. Many phage RNA polymerase s factors have been
analyzed that can drive phenotypic alteration of the host bacteria
. Therefore, we inferred that ORF35 has the ability to regulate
the host gene transcription during the sporulation phase
[16,18,31]. ORF22 encoded a FtsK/SpoIIIE ATPase, an enzyme
that plays important roles in intercellular chromosomal DNA
translocation and asymmetric division during sporulation [32,33].
In B. subtilis, FtsK/SpoIIIE ATPase was also involved in
transferring missegregated DNA during vegetative growth .
The cross talk between the phage BtCS33 and the host cell may
influence the division of the host cell as well as the lytic life cycle of
the phage. Confirming the exact functions of the ORF35 and
ORF22 proteins in the phage requires further investigation.
Comparison of phage BtCS33 and B. cereus phage Wb
BLASTN analysis of the genome ofphage BtCS33 revealed the
closest matches to be the genomes of B. cereus phage Wb
(NC_007734), and some variants of phage Wb, such as phage
(NC_007814). A dot plot of the BtCS33 and Wb genomes
(Figure 3) showed high co-linearity mainly in the ‘‘late’’ regions of
each genome encoding the head-tail joining proteins and tail
morphogenesis proteins. The five most similar fragments in their
‘‘late’’ regions were shown in Table 1. Raymond Schuch et al.
reported high sequence similarity between phage Gamma and Wb
[17,18]. BtCS33 and Gamma also have co-linearity in the ‘‘late’’
region of both genomes (data not shown). Pairwise alignment of
the proteomes of phage BtCS33 and phage Wb also revealed
similar genome organization and high homology of ORFs in the
‘‘late’’ region (Figure 4). The similarity between proteins encoded
by ORFs 6 to 12 in BtCS33 and ORFs 7to 13 in Wb was more
than 80%, while proteins encoded by ORFs 13 and 14 in BtCS33
and ORFs 14 and 15 in Wb was more than 50% similar.
Phylogenetic tree analysis of phage BtCS33
To analyze the evolutionary relationship between phage
BtCS33 and other phages originating from Bacillus species,
a phylogenetic tree based on the complete genome sequences
from 13 Bacillus Siphoviridae phages was constructed (Figure 5A).
BtCS33 together with B. cereus phages Wb, Fah, Cherry, Gamma
clustered together, while some other phages from Bacillus were
clustered into different subgroups. Another phylogenetic tree was
constructed based on amino acid sequences of the major capsid
proteins, which are relatively conserved in phage genome of the
Siphoviridae family (Figure 5B). Notably, these two phylogenetic
trees were in perfect accord showing the same cluster of phage Wb
and BtCS33. The collective results above indicated that, among
the sequenced phages from B. thuringiensis, BtCS33 was the first
reported member of the Siphoviridae family and was closely releat to
the sequenced phages from Bc.
The tail fiber proteins of phages were reported to be essential for
cell wall receptor recognition and binding, which can determine
the host specificity of the phages . Because of the extremely
narrow host range of phage BtCS33, a phylogenetic tree based on
amino acid sequences of the tail fiber proteins was constructed. In
total, sequences from 5 Bacillus Siphoviridae phages and 13 B.
thuringiensis prophages were used. The tree showed that the tail
fiber protein of BtCS33 was closely related to the tail fiber proteins
from many B. thuringiensis prophages, but was distantly releated to
the tail fiber protein of Wb (Figure 5C).
In this study, a novel phage BtCS33 isolated from B. thuringiensis
kurstaki strain CS33 was sequenced and characterized. The
complete genome of BtCS33 exhibited some interesting features.
First, phage BtCS33 is different from other sequenced B.
thuringiensis phages. Until now, five phages originating from B.
thuringiensis had been sequenced. Three of them (GIL16c, GIL01
and Bam35c) belonged to the Tectiviral family and were clustered
in the same lineage; Phage 0305-phi8-36, an atypical Myovirus,
possibly represented a novel, ancient phage lineage, and MZTP02,
a Podoviridae phage belonging to the phi29 family, was clustered
with other lineages. So BtCS33 was the first reported member of
the Siphoviridae family from B. thuringiensis. It clustered with phages
from Bc and should be considered a Wb-group phage (Figure 5).
Second, it was reported that the recognition between phage and
host is mainly determined by the phage tail fiber; mutations in the
tail fiber gene change the infective activity [18,36]. Although
BtCS33 and Wb had similar genome organizations and high
sequence identify in the structural proteins, they only shared 65%
amino acid sequence identity in the tail fiber proteins and the host
ranges were different. The low identity of the tail fiber proteins
between BtCS33 and Wb might be the reason for their different
Figure 2. Genome organization of phage BtCS33. The schematic represents the whole genome with the ORFs numbered from left to right.
Different colors indicate three regions: the ‘‘late’’ region (red color), the ‘‘lysogeny-lysis control’’ region (green color) and the ‘‘early’’ region (blue
color). Gray indicates the genes involved in host cell sporulation. Genes with unknown functions are indicated by white. The orientations of the
arrows indicate the direction of transcription.
Genome of a Novel Bacillus thuringiensis Phage
PLoS ONE | www.plosone.org3 May 2012 | Volume 7 | Issue 5 | e37557
host ranges. The tail fiber protein of BtCS33 had high homology
with proteins from several B. thuringiensis prophages (Figure 5C),
but BtCS33 had an extremely narrow host range and did not
infect 77 tested B. thuringiensis strains except of its host (CS33) and
C-3. Therefore, the cause of the narrow host specificity of BtCS33
among Bt strains is still unclear. The tail fiber proteins may not be
the key or single factor determining the host specificity of BtCS33.
Efforts to understand the cause of the narrow host range of
BtCS33 are ongoing.
Third, new evidence for the evolution of Bacillus phage was
found. Two genes, named ORF22 and ORF35 encoding FtsK/
SpoIIIE ATPase and RNA polymerase factor, respectively, were
found in the genome of BtCS33, and had high similarty to the
correspondence genes in the geneome of B. thuringiensis subsp.
kurstaki strain T03a001, which belongs to the same subspecies as
the host of BtCS33. Furthermore, genes with similar functions
were also found in B. cereus phage Wb, phage Gamma and some
other Gamma isolates [16–18]. The phage-encoded RNA poly-
merase sigma factor regulates vegetative growth as well as
sporulation of the host bacteria. This represents a kind of cross-
talk between the phage and host, and has been reported previously
. Obtaining genes involved in sporulation may be a common
phenomenon for phages from sporulating bacteria. The exact
function of the putative sporulation genes in the genome of
BtCS33 will be further studied. From the combined evidences of
host-related genes and the phylogenetic trees, it can be inferred
that these phages may have a common distant ancestor and
BtCS33 should be considered as a Wb-group phage.
Fouth, from a different perspective, the similarity of these
phages was evidence of the evolution of the host bacterial species.
Classifying of B. anthracis, B. thuringiensis and B. cereus as a single
species, Bacillus cereus sensu lato, remains a matter of debate [37,38].
The genome similarity of these phages provides more evidence for
classifying the three Bacillus species as a single species.
Figure 3. Dot plot alignment of genomes from B. thuringiensis phage BtCS33 and B. cereus phage Wbeta (Wb). Arrows indicate the start
and the end positions of the most similar fragments on both genomes, corresponding to the dot plot. Both genomes were ranked in the orders of
‘‘late’’ region, the ‘‘lysogeney control’’ region and the ‘‘early’’ region.
Figure 4. Alignment of the proteomes of B. cereus phage Wbeta (Wb) and B. thuringiensis phage BtCS33. The putative proteins are
numbered and different color arrows show the levels of amino acid identity: green indicates 20%–50%; blue, 50%–80%; and red, 80%–100%.
Genome of a Novel Bacillus thuringiensis Phage
PLoS ONE | www.plosone.org4 May 2012 | Volume 7 | Issue 5 | e37557
Materials and Methods
Culture media and bacterial strains
Bacillus thuringiensis subsp. kurstaki CS33 was isolated in our lab
and is highly toxic to insects larvae of Lepidopteran and Dipteran
Isolation and propagation of the phage
Bacteriophage was isolated from B. thuringiensis strain CS33
according to the modified method described by Carey-Smith et al
. Bt strain CS33 was incubated on a nutrient plate to form
plaques. One plaque was picked and suspended in 1 ml SM buffer
[0.58% (w/v) NaCl, 0.2% (w/v) MgSO4, 50 mM Tris-HCl,
pH 7.5] as a bacteriophage suspension. To get pure phage, 100 ml
of phage suspension was mixed with 300 ml of CS33 culture
during exponential phase growth (OD600 about 1.0), and the
mixture was added into 5 ml of molten semisolid medium (at
about 45uC). After thoroughly mixing, the semisolid medium
containing bacteria and phages was poured onto a solid medium
plate as an overlay and incubated at 30uC overnight (12–
16 hours). After plaques formed on the upper medium, one
plaque was picked, and the process above was repeated at least five
times until homogeneous plaques formed. Finally, the pure
bacteriophage was harvested and designated BtCS33.
Propagation of the bacteriophage was performed by using the
method as described above, except a bacteriophage suspension at
a titer of about 106PFU/ml was used. After incubating the
suspension at 30uC overnight, 5 ml of SM buffer was added onto
each plate and left at 4uC at least 4 h with morderate rotation.
Then the suspension was recovered and centrifuged at 8,000 g at
4uC for 10 minutes. After the supernatant was filtrated through
a 0.22 mm sterile filter, the concentrated phage preparation was
stored at 4uC for use.
Observation of bacteriophages by electron microscopy
A 5 ml aliquot of SM buffer containing 0.01% of gelatin was
added onto the bacterium-phage plate, the plate was morderately
rotated at 4uC for 2 h, and then the suspension was collected.
After centrifugation for 10 min at 8,000 g at 4uC, the supernatant
was immediately deposited on cuprum grids with carbon-coated
Formvar films, and stained with 2% potassium phosphotungstate
(PT, pH 7.2) [7,40]. After the film was dried in air, it was observed
by TEM (HITACHI H-7000FA transmission electron microscope)
at an acceleration voltage of 100 kV.
Investigation of host range
The host range of BtCS33 was investigated using the method
described above for the isolation of the phage. A suspension of
bacteriophage at about 106PFU/mL was used to infect the tested
Bacillus spp. strains. After incubation overnight, plaques on the
bacterial plates were observed. These experiments were repeated
for three times. In total, 79 B. thuringiensis strains including 21 Bt
reference strains and 58 isolates belonging to 17 of H-serotypes, 4
B. sphaericus (Bs) strains (2 reference strains and 2 isolates), 1 B.
cereus strain and 2 B. anthracis (Ba) strains were tested. B. anthracis
strains were provided by Dr. Yuan in Wuhan Institute of Virology,
Chinese Academy of Sciences, and pXO1 and pXO2 were
eliminated in the two B. anthracis strains, respectively. Bt and Bs
strains tested were kept by our lab.
Extraction of phage DNA
DNA extraction from phage particles was performed according
to the method described by Santos with modifications . Each
milliliter of phage suspension was treated with 6 units/ml of
DNase and 20 mg/ml of RNase at 37uC for 30 min, 20 ml of a 2 M
solution of ZnCl2 was added and the phage suspension was
incubated at 37uC for 5 min. After centrifugation for 1 min at
10,000 g, the supernatant was removed and the pellet was
suspended in 500 ml TES buffer (0.1 M Tris/HCl, pH 8.0;
0.1 M EDTA; 0.3% SDS) and incubated at 60uC for 15 min.
After incubation with 20 ml proteinase K (20 mg/ml) at 37uC for
90 min, 60 ml of 3 M potassium acetate solution (pH 5.2) was
added to the suspension and completely mixed; then, the mixture
was kept on ice for 15 min. The mixture treated with phenol/
chloroform/isoamyl alcohol (25:24:1, v/v) twice, and then with
chloroform/isoamyl alcohol (24:1, v/v) once. Phage DNA was
precipitated with an equal volume of isopropanol washed with
70% ethanol twice, and dissolved in 10 ml distilled water. The
DNA was checked by 0.6% agarose-gel electrophoresis.
Proteinase K and exonuclease III treatment of the BtCS33
DNA preparations of BtCS33, which were prepared proteinase
K pretreatment, were incubated with variable amounts of
proteinase K (0.01 and 0.1 mg/ml) for 4 h at 37uC . BtCS33
DNA preparations were also treatment with exonuclease III
according to the manufacturers’ instructions. The effect of the
treatment was analyzed by running the BtCS33 DNA preparation
on a 0.6% agarose gel
Genomic DNA sequence and bioinformatics analysis
Genomic DNA sequencing was performed by BGI Co. (Beijing,
China) with a shotgun sequencing method and the genome was
assembled with phrap version 1.080812. Each base had at least
five-fold coverage. Open reading frames (ORFs) were predicted
with FGENE SV software (http://linux1.softberry.com/berry.
Table 1. The five most similar nucleotide sequence fragments between genomes of phage BtCS33 and phage Gamma.
Length(bp) (BtCS33/Gamma)Similarity (%)
Position (From..to) Position (From..to)
10566..12713 10266..124132148/2148 82
20013..2005018487..18524 38/38 97
Genome of a Novel Bacillus thuringiensis Phage
PLoS ONE | www.plosone.org5May 2012 | Volume 7 | Issue 5 | e37557