Five Distinctive Phages from an Egyptian Industrial Strain of Bacillus
thuringiensis subsp. Aegypti
A.A. El-Morsi ,
The production of the biocontrol agent, Bacillus thuringiensis (Bt)
subsp. aegypti C18 (BtaC18), suffers huge economic losses due to phage infection.
To improve it, an isolation and characterization of the causative agent are
necessary, using gradient centrifugation, electron microscopy and protein analysis.
Five morphologically distinctive phages were isolated from this strain belonged
to the order Caudovirales. Moreover, they been classified: Tailed group (TP3-01,
TP3-02, TP3-03 and TP3-05) and pseudotailed group (TP3-04) and fell into three
families: Siphoviridae, Tectiviridae and Podoviridae. TP3-01 phage showed hexagonal
head and slight long, non-contractile, relatively straight tail. TP3-02 has
an isometric head with a long non-contractile, flexible tail while the morphologically
similar TP3-03 has a slightly longer head and shorter tail. TP3-04 virus profile
showed a rigid polyhedral protein capsid surrounded with a thick, flexible lipoprotein
vesicle which sometimes became tail like tube, while TP3-05 has a rounded head
and short non-contractile tail. These results suggest that TP3-01, TP302 and
TP3-03 are members of the Siphoviridae family, TP3-04 belongs to Tectiviridae
and TP3-05 is a member of Podoviridae. Protein banding analysis of the five
phages revealed four major bands: 112.20, 70.79, 61.66, 50.12 and 39.81 kDa.
The 112.20 kDa protein band does exist in all phages and it may represent the
capsid protein. The other proteins are not universal and may be present in one
phage and absent from another. In conclusion, BtaC18 was the host of at least
five morphologically diversified phages which needs a lot of work to make a
phage resistant bacterium.
Received: June 12, 2013;
Accepted: August 27, 2013;
Published: March 10, 2014
The Gram positive entomopathogenic bacterium Bacillus thuringiensis
(Bt), is a common soil microorganism that has been widely used for biocontrol
of insect pests belong to the orders: Lepidoptera, Diptera and Coleoptera (Pang
et al., 1998; Agaisse and Lereclus, 1995;
Schnepf et al., 1998). Most of wild type strains
of Bt are sensitive to phages and/or carry prophages either integrated into
the bacterial genome or as a repressed plasmid. The prophages may be converted
into a lytic phage causing host cell lyses under certain cultural conditions
(Hendrix et al., 1999). It is reported that
about 83% of Bt strains contain lysogenic phage (Ackermann
et al., 1994) and during their fermentation, lysogenic phages might
cause failure in 15-30% of the batches, resulting in severe economic losses
(Liao et al., 2008).
Characterization of phages depended mainly on their morphology but the introduction
of the negative staining in (1959) had facilitated their studies; especially
when examined microscopically. According to Ackermann (2007),
96% of the virus particles appeared to be tailed and they constitute the order
Caudovirales and about 3.7-4.0% are recorded as polyhedral, filamentous, or
The tailed viruses are extremely large group and characterized by having diverse
genomes, virion and replication patterns (Fauquet et al.,
2005). Caudovirales, one of the largest orders of the tailed viruses, consisted
of three families (Myoviridae, Podoviridae and Siphoviridae) where their structural
tails are unique and have large implication on taxonomic properties of these
bacteriophages. This order harbors more than 350 species of bacteriophages with
homologous genes which perform similar function. However, their nucleotide sequences
can vary significantly but still believe they have the same origin. Myoviruses
is the largest among the tailed viruses (~25%) which include the largest and
most complex tailed bacteriophages. For example, the Myoviridae or contractile
tailed bacteriophage; is one of the families where the virus consisted of a
head and a tail separated by a neck. The tail has a contractile sheath, a central
tube and an adsorption apparatus. The Siphoviridae has long simpler, noncontractile
tails with either a rigid or flexible tubes. However, Siphoviruses are the most
abundant tailed phages (61% of tailed viruses). The Podoviridae, the third family
of the caudivirales, characterized by short noncontractile tails and consisted
about 14% of the members of this order (Fauquet et al.,
2005; Ackermann, 2009).
The importance of viruses has been recognized by the industrial and scientific
community. Viruses have been used academically for bacterial typing or identification
and recently, they been recognized to control pathogenic bacteria in food chain.
Accordingly, the isolation, characterization and identification of viruses infecting
industrial strains of bacteria is central to any industrial process (Ackermann
et al., 1995; Rosovitz and Leppla, 2000).
The phages infecting Bt were not always from one morphogroup and this constitutes
a challenge to the industrial people. Therefore, isolation, purification and
identification of the phages represent a step forward in controlling the economic
damages to the biopesticide industry. The objective of this study was designed
to characterize the five different phages attacking the industrial Egyptian
isolate of Bt; the BtaC18; under laboratories condition.
MATERIALS AND METHODS
Bacterial strains: There were two types of Bt wild type strains used
in this study, 1-phage sensitive strain (BtaC18) which was used for phage isolation
and propagation and 2-phage-sensitive strain (tolworthi) which has been used
as hosts for phage senstivity assays (Osman and Madkour,
Bacterial propagation: The selected Bacillus thuringiensis (Bt)
strains used in this research were inoculated in Luria Bertani (LB) media, containing:
Peptone 10 gm L-1, yeast extract 5 g L-1 and sodium chloride
10 g L-1. The inoculated media were shacked at 250 rpm and 30°C
for 24 h. For sporulation, the Bt strains were grown in T3 medium
containing: Peptone (5 g L-1), yeast extract (1.5 g L-1),
MnCl2 (0.5 m L-1), sodium phosphate buffer (50 mM) and
was adjusted to pH 6.8). The cultures were shacked for 72-96 h at 250 rpm and
30°C as described by Osman and Madkour (1999).
Preparation of phage-free-Bt strain: Forty five strains of the wild
types Bt were cultured on LB broth and detected for viral infection; lysis.
Each lytic resistant strain was cultured again in T3 broth media,
incubated at 30°C on a rotatory shaker for 72 h until complete sporulation.
To get rid of all potential prophages, sporulated cells were boiled for 30 min
and recultured on LB broth. The sporulation was detected by endospore stain
and examined microscopically.
Phage isolation and propagation: Isolation of bacteriophages were carried
out from wild-type Bt aC 18 (phage-sensitive) according to the methods of Lu
et al. (2003) and Carey-Smith et al. (2006)
with slight modification. The bacterium was grown in 5 mL LB broth at 30°C
for 24 h on a rotatory shaker (250 rpm). A pure phage stock was obtained by
adding 0.25 mL of chloroform to each 5 mL of bacterial lysate and the supernatant
containing the virus was collected by centrifugation at 5,000 rpm for 13 min.
The viral solution was further purified from any bacterial remains by filtrations
(0.45 μm), then the viral stock was kept at 4°C for subsequent experiments.
Phage detection: Bacterial sensitivity to the phage infection was carried
out by observing bacterial lyses at the spot where the phage was deposited on
the bacterial lawn as reported by Chang et al. (2005).
Phages concentration: The phages containing supernatant were concentrated
according to the method of Gill and Abedon (2003). The
filtrate was separated and centrifuged at high speed of 16000 rpm for 90 min
at 5°C, the pellets were resuspended in SM buffer using sterile Eppendorf
Precipitation of phages using polyethyleneglycol (PEG8000): The concentrated
phages were precipitated using the method of Yamamoto et
al. (1970) and Sambrook et al. (1989) while
some modifications were done. A 7.5 mL of phage was added to 1.8 mL of PEG 8000
in a sterile test tube, incubated in ice for 2 h, then centrifuged at 16000
rpm for 30 min at 4°C, respin 2-3x to remove all of PEG solution (using
a micro-pipet tip facilitates removal of all solution). Resuspend phages in
SM buffer (250 μL) mixed well then transfer to eppendorf tubes and centrifuged
at (13000 rpm for 15 min), the supernatants were transferred to new eppendorf
tubes and kept for further studies.
Separation of precipitated phages: A half gram of solid CsCl was added
to 1 mL of purified phage suspension, inverted gently until they completely
dissolved, then stored in ice. A series of CsCl gradients were used to separate
the phages; these were 1.15, 1.35, 1.55 and 1.70 g mL-1 of SM buffer.
One milliliter of each phage stock was pipetted onto the top layers of the CsCl
layers and centrifuged at 45,000 rpm at 4°C for overnight (XL-90 Ultracentrifuge,
Beckman). When the centrifugation was completed, the phage particles (bands)
were collected using a hypodermic needle (18 gauge). The CsCl was removed by
dialysis against phosphate buffer saline (pH 7.0) and the purfied phages were
stored at 4°C (Sambrook et al., 1989).
Phage titration: The concentration of phage was measured using the formula
developed by Wiseman and Day (1977) at 269 and 320
Morphological characteristics of the isolated phages: Morphological
assessments of the isolated phages were examined under a Transmission Electron
Microscope (TEM). A drop of purified phage particles (about 1012)
were spotted on carbon film picked up on copper grids and then the phage particles
were negatively stained with 2% (w/v) uranyl-acetate. After 1 min the excess
liquid was removed with a filter paper. The grids were air dried for about 5
min. Phage morphology was examined and photographed with TEM (JEOL JEM 1010
at Regional Centre of Mycology and Biotechnology, Al-Azhar University, Cairo,
Analysis of phages proteins: Purified phage suspensions were precipitated
in 4 volumes of ice- cold acetone. After a centrifugation at 10000 rpm, 10 min,
4°C, the pellet was air-dried and resuspended in PBS buffer. Phage structural
proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (Laemmli, 1970).
Screening of several strains of a local collection of wild type Bt revealed
that the cultural techniques induced complete cell lyses of some strains after
24 h these strains especially BtaC18 was used for phage propagation. An authentic
strain was used to test the phage sensitivity assay; Bt subsp. tolworthi. The
filtrates of BtaC18 were found to produce prominent clear spot by the drop test
and cause numerously irregular plaques differ in their transparency on the bacterial
lawn by the double layer agar plaque method (Fig. 1).
The culture filtrates of the BtaC18 revealed the presence of five different
morphotypes of phages as judged from transmission electron micrographs which
named: TP3-01, TP302, TP3-03, TP3-04 and TP3-05 (Fig. 2a-e).
TP3-01 was fairly small phage with hexagonal head of 103.33 nm diameters and
slight long, non-contractile, relatively straight tail of 183.33 and 23.33 nm
diameters. TP3-02 showed that isometric head of 111.11 nm with a long non-contractile,
flexible tail of 388.88 nm length and 17.7 nm wide. TP3-03 appear to be similar
TP3-02 morphologically with dimensions 113.04 nm for head and 188.5x8.3 nm for
||Wild-type Bt strain C18 (BtaC18) showing numerously irregular
||Transmission electron micrographs of the five different morphotypes
of the viruses isolated from Bt subsp. aegypti C18 (TP3-virions), panels
from A-E. The bar represents 100 nm. TP3-01phage had hexagonal head and
non-contractile. TP3-02 had isometric head with a long non-contractile,
flexible tail. TP3-03 had TP3-02 morphologically with dimensions 113.04
nm for head and 188.5x8.3 nm for tail. TP3-04 had a rigid polyhedral protein
|| Morphological features and taxonomical position of the five
isolated phage from the local BtaC18 strain of B. thuringiensis
The TP3-04 showed a rigid polyhedral protein capsid (93.3 nm) surrounded with
a thick, flexible lipoprotein vesicle which sometimes became tail like tube
while TP3-05 was observed as rounded head of 80.0 nm and short non-contractile
tail of 53.3x20.0 nm (Fig. 2). The general morphology of the
five TP3-phages is summarized in Table 1.
||Protein banding pattern of the five phages lanes (1, 2, 3,
4 and 5) isolated from BtaC18. Lane M: Protein molecular weight marker (from
top to bottom 250, 130, 100, 70, 55, 35 and 27 KDa)
|| Proteins found in each of the five isolated phage as seen
in the SDS-PAGE
|+: Present, -: Absent
Total protein analysis of the purified phage particles was performed using
denatured polyacylamide gel, SDS-PAGE electrophoresis (Fig. 3).
A total of five protein bands were observed in the gel, with the molecular weights
of 112.20, 70.79, 61.66, 50.12 and 39.81 KDa (Table 2).
The major proteins of the five phages showed different degrees of similarities
but not identical. A 112.2 KDa protein appeared to be highly conserved between
all phages, a sub-major protein was 50.12 KDa, present in three of the five
phages: TP301, TP3-02 and TP3-04. The highest degree of similarity was observed
between phages TP301 and TP3-02. The phage protein profiles also showed two
sub-minor proteins with no particular similarity between the phages.
Particles with a tail-like: TP3-05 had rounded head of 80.0 nm and short
non-contractile tail of 53.3x20.0 nm, Podoviridae family (X-70000).
The number of distinctive phages isolated from BtaC18, in this study, emphasized
the urgency for a smart development program of the industrial strain to continue
its effectiveness as a bioinsectcide. Previous studies have shown that the bacteriophages
associated with this particular bacterium could be the main factor for the troubles
associated with the manufacturing process (Al-Heeti and
Osman, 2008; Osman et al., 2013). Electron
microscopy photos demonstrated not only the different phages parasitize on the
BtaC18 but also their morphological diversities. Dependence on the morphology
of phages, a widely used system to classify phages (Bradley,
1967; Ackermann, 2007), is probably one of the most
prominent criteria in this field (Yoon et al., 2007).
Accordingly, the five phages isolated during this study were found to belong
to the order Caudovirales. However, four of them (TP3-01, TP3-02, TP-03 and
TP-05) are tailed and one (TP-04) has pseudo-tail. Therefore, they were further
classified as members of families: Siphoviridae, Tectiviridae and Podoviridiae;
based on the system of Bradley (1967) and Ackermann
The phages TP3-01, TP3-02 and TP3-03 of hexagonal head and long flexible, non-contractile
tails are considered as members of the family Siphoviridae or Bradleys
group B1. These phages were similar to phages TP-16c, TP-17c and TP-19c of Reynolds
et al. (1988); MZTP02 of Liao et al.
(2008) and BtCS33 of Yuan et al. (2012). While,
TP3-04 has a rigid polyhedral capsid with a thick, flexible lipoprotein vesicle
and sometimes became tail like tube, so far the authors considered it in the
family Tectiviridae (Bradleys group D4). These above results are in agreement
with the finding of Ackermann et al. (1978)
and Verheust et al. (2005); for phage Bam35
and GIL16, respectively. On the other hand, the phage TP3-05 which has a rounded
head and a short non-contractile tail is typically similar to the members of
family Podovoridae or Bradleys group C1. These results are in agreement
with Ito (1978) for phi 29 phage.
Overall, about 96% of all phages reported in the literature were tailed and
belonged to the three major families: The Myoviridae (24.5%), Siphoviridae (61%)
and Podoviridae (14%) (Ackermann, 2007). Therefore,
It was not surprising to find that four of the isolated phages (80%) belonged
to these morphological families.
The phage protein patterns supported the notion of diversity among the phages
isolated from the local strain of Bt; BtaC18. The numerous viruses feast on
the same bacterial reflects the dynamics between the host and the parasite.
Meaning the bacterial tries to escape the viral infection by changing its receptors,
a new modified viruses interacts with the newly evolved surface protein. Dependence
on the electrophoretic profiles of the viral structural proteins for phages
differentiation and characterization is an established procedure (Stewart
et al., 1985; Coveney et al., 1987).
In this study, the protein banding pattern showed two main clusters: Cluster
one contains TP3-01, TP3-02 and TP3-05; while cluster two includes TP3-03 and
TP3-04. This grouping is slightly different from the straightforward morphological
data, where, TP3-5 is placed in a separate group, pseudotailed one. Moreover,
TP3-01, TP3-02 and TP3-03 were placed in the family Siphoviridae, TP3-05 belonged
to the family Podoviridae and TP3-04 is included in families Tectiviridae, respectively.
This apparent discrepancy between the morphological and protein data is understandable
because of the rapid mutation trends seen in viruses (Osman
et al., 2013). Moreover, possessing protein with the same the molecular
weights is not necessarily for the same protein but the function and the amino
acid sequences of the same molecular weight protein band could be totally different.
Therefore, further consideration should be taken and more characters needed
for accurate and definitive differentiations between the closely related phages
as suggested by Osman et al. (2013), Grimont
et al. (1978) and Loessner et al. (1993).
In conclusion, BtaC18 was the host of at least five morphologically diversified
phages which needs a lot of work to make a phage resistant bacterium. Especially,
if this bacterium is already industrial strain such as BtaC18.
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