Download Physiological and Molecular Characterization of a Newly Identified

J. Microbiol. Biotechnol. (2012), 22(12), 1605–1612
http://dx.doi.org/10.4014/jmb.1203.03068
First published online October 4, 2012
pISSN 1017-7825 eISSN 1738-8872
Physiological and Molecular Characterization of a Newly Identified
Entomopathogenic Bacteria, Photorhabdus temperata M1021
Jang, Eun-Kyung1,2, Ihsan Ullah1, Jong-Hui Lim1, In-Jung Lee1, Jong-Guk Kim3, and Jae-Ho Shin1*
1
School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Korea
Food and Biological Resources Examination Division, Korean Intellectual Property Office, Daejeon 302-701, Korea
3
Department of Life Sciences and Biotechnology, College of National Sciences, Kyungpook National University, Daegu 702-701,
Korea
2
Received: March 29, 2012 / Revised: July 4, 2012 / Accepted: July 26, 2012
The present study concerned the identification and
characterization of a novel bacterial strain isolated from
entomopathogenic nematodes collected from different
regions in Korea. The bacterial isolate M1021 was Gramnegative, bioluminescent, and produced red colonies on
MacConkey agar medium. A rod-shaped structure was
confirmed by the electron micrograph. Fatty acid
composition was analyzed by using the Sherlock MIDI
system. The identification was further supported by 16S
rDNA sequence analysis, which revealed 96-99% sequence
homology with strains of Photorhabdus temperata. The
location of the isolated strain of P. temperata in the
phylogenetic tree was confirmed and it was named P.
temperata M1021. P. temperata M1021 exhibited catalase,
protease, and lipase activities when grown on appropriate
media supplemented with respective substrates. The culture
of P. temperata M1021 exhibited insecticidal activity
against the larvae of Galleria mellonella and the activity
was the highest after 3-4 days of cultivation with agitating
at 28oC under 220 rpm. Antibacterial activity was also
observed against Salmonella Typhimurium KCTC 1926
and Micrococcus luteus KACC 10488.
Keywords: Antibacterial activity, entomopathogenic, Galleria
mellonella, insecticidal toxicity, Photorhabdus temperata
Photorhabdus spp. are symbiotically associated with
entomopathogenic nematodes (EPNs) of the family
Heterorhabditidae [7]. The Photorhabdus-Heterorhabditidae
symbiotic complex passes through three stages during a
complete life cycle. In the first symbiotic life cycle stage,
*Corresponding author
Phone: +82 53 9505716; Fax: +82 53 9537233;
E-mail: [email protected]
the infective form of the EPNs, called infective juveniles
(IJs) [17], take Photorhabdus bacteria into their guts and
then actively hunt for insect hosts. In the second stage
(pathogenic stage), the nematodes enter into the hemocoel
of an insect host through its respiratory spiracles or
digestive tract [33], and the combined action of the
nematodes and bacteria kills susceptible insect hosts within
48 h of infection [26, 33]. In the third stage (replicative
stage), the bacterial cells multiply and convert the tissues
of the insect cadaver into bacterial biomass using
hydrolytic enzymes [8]. The insect dies of septicemia and
the IJs feed on the multiplying symbiotic bacteria, and in
this way complete 1-3 generations in the insect host
cadaver [19]. Upon depletion of food resources, a large
number of IJs have been produced, which contain colonies
of the symbiotic bacteria in their guts, and this symbiotic
association is then dispersed in search of new hosts [22].
During infection, the symbiotic bacteria show pathogenicity
against the insects [8, 21]. They inhibit the insect immune
response by suppressing phospholipase A2, which catalyzes
phospholipids at the sn-2 position to release arachidonic
acid [20]. The Photorhabdus bacteria also inhibit humoral
immunity by down-regulating gene expression of antibacterial
peptides including cecropin. They can directly induce
apoptosis of insect immunocytes and hemocytes, resulting
in complete immune suppression and septicemia [32].
Various antibiotics are synthesized from cultures of symbiotic
bacteria whereby xenorhabdins and xenocoumacins are
commonly produced by Xenorhabdus spp., whereas
hydroxystilbenes and anthraquinones are produced by
Photorhabdus spp. [29]. Currently, three species of
Photorhabdus have been described to be associated with
Heterorhabditis nematodes: P. asymbiotica, P. luminescens,
and P. temperata. Based on their 16S rDNA sequences and
metabolic properties, P. luminescens has been divided into
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Jang et al.
five subspecies [10]. Four out of five subspecies including
P. luminescens subsp. luminescens, P. luminescens subsp.
laumondii, P. luminescens subsp. kayaii, and P. luminescens
subsp. thracensis have been isolated from H. bacteriophora,
and one P. luminescens subsp. akhurstii has been isolated
from H. indica. Nematode association with P. temperata
appears to be more diverse. It has been found to be
associated with H. megidis, H. zealandica, H. marelatus,
and H. downesi [12]. P. temperata has also been observed
to be harbored by H. bacteriophora [4]. According to
Boemare et al. [7], these reports do not modify the wellestablished concept of a one-to-one association between
EPNs and their symbiotic bacteria, but it does represent a
problem for bacterial classification. In fact, DNA-DNA
hybridization and 16S rDNA sequence information
suggest that Photorhabdus is a heterogeneous genus [3],
and thus, subspecies definition is necessary [12]. Thus far,
two P. temperata subspecies have been described: P.
temperata subsp. temperata associated with H. megidis
[12] and P. temperata subsp. cinerea associated with H.
downesi [30]. Indigenous EPNs are perhaps more suitable
for inundatory release against local insect pests because
they have already adapted to the local climate and other
population regulators [24] [5]. In addition, many countries
are concerned about the introduction of exotic EPNs
because they may have a negative impact on non-target
organisms [5].
The main objective of the present study was to identify a
novel bacterial strain associated with an endogenous EPN
of Korea and investigate its pathogenicity against harmful
insects. In addition, we aim to introduce our newly
discovered strain into an agriculture field for use as a
biocontrol agent.
MATERIALS AND METHODS
Collection of the Nematodes
Approximately 200 soil samples were collected from different
locations in South Korea. Each sample location consisted of a 50 m2
area. Approximately 250 g of soil from each sample location was
placed in a pre-sterilized plastic bag and transported to the
laboratory. EPNs were isolated from the soil samples through the
insect baiting method [7]. The insect bait (G. mellonella larvae) was
placed in 250 ml plastic containers along with a moist soil sample.
The plastic containers containing the bait and soil sample were
covered with a lid and maintained at room temperature (20 ± 2oC).
Samples were moistened with water if they appeared to be dry at
any point during baiting. Larvae bait was checked daily and any
dead larvae were removed from the container. After 7-9 days of
baiting, the dead larvae (brick red color) were carefully washed with
distilled water and put in White traps until emergence of third-stage
IJs [16]. Emerging nematodes were collected and then used to infect
fresh G. mellonella larvae in order to produce nematodes for
identification and establishment of bacterial cultures.
Isolation and Identification of Symbiotic Bacteria
Symbiotic bacteria were secluded by streaking insect larvae
hemolymph on MacConkey agar medium. The culture plates were
incubated at 28 ± 2oC for 48 h and then observed for colony growth.
Bright-pink colonies grown on MacConkey medium (Becton,
Dickinson and Company, USA) were transferred to NBTA plates
(nutrient agar with 0.004% triphenyltetrazolium chloride and
0.025% bromothymol blue) and presumed Photorhabdus spp. were
identified by their characteristic adsorption of blue dye [2]. The
bacterial culture in Luria-Bertani (LB) medium was incubated at a
temperature range of 28-36oC for 48 h in a shaking incubator at
220 rpm. Bacterial growth was assessed using a spectrophotometer
to measure absorbance at OD600. The optimal incubation time was
determined by incubating the culture broth at 28 ± 2oC for 1 to
8 days in a shaking incubator at 220 rpm. Gram staining was
performed by application of the standard Gram stain procedure. The
culture broth was deposited on formvar-coated grids for 15 s and
followed by staining with 2% uranyl acetate for 25 min. The stained
grids were evaluated using a transmission electron microscope.
Genomic Analysis of the Isolated Bacteria
Chromosomal DNA was extracted from the symbiotic bacteria using
a QIAprep Spin Miniprep kit (Qiagen, Valencia, CA, USA). The
extracted DNA segment containing the 16S rDNA gene (approximately
1.5 kb) was amplified by polymerase chain reaction (PCR) using
primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and
1492R (5'-GGT TAC CTT GTT ACG ACT T-3'). The PCR mixture
(50 µl) consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM
MgCl2, 0.2 mM dNTPs, 0.2 pmol of each primer, and Taq DNA
polymerase (Takara, Japan). Amplification was performed using a
protocol consisting of 30 cycles that included 1 min of denaturation
at 95oC, 30 s of annealing at 55oC, 1 min of elongation at 72oC, and
a final extension of 5 min at 72oC. The PCR product was then
purified (PCR purification kit; Solgent, Daejeon, Korea) and
sequencing was conducted by a commercial laboratory (Solgent,
Daejeon, Korea). The 16S rDNA gene sequences obtained from the
isolated bacterial strain was edited using a multiple sequence editor
(DNASIS, Hitachi, America, Ltd) and nucleotide comparison was
investigated using the BLASTN program from the National Center
for Biotechnology Information (NCBI). The nucleotide sequences
were aligned using ClustalW2 from EBI (http://www.ebi.ac.uk/
Tools/clustalw2). Pair-wise evolutionary distances were computed
using Poisson correction for multiple substitutions. The phylogenetic
tree of the isolated strain was constructed using the maximun
parsimony method [28] in the Mega 4.0 program.
Lipase and Protease Activities
The lipase activity produced by the bacterial isolate was determined
using LB agar plates. Two sets of LB media were prepared such
that one included Tween 20 and the other included Tween 80. Using
autoclaved toothpicks, colonies of bacterial isolate were inoculated
on the media. Escherichia coli DH5α (negative control) was
inoculated on the same media and incubated at 28 ± 2oC for 48 h.
LB agar supplemented with 10% (w/v) skim milk was used to
determine the protease activity of the bacterial isolate. Using
autoclaved toothpicks, bacterial colonies (test sample) were inoculated
on culture plates and incubated at 28oC for 48 h. Bacillus subtilis
2232 was used as a positive control and E. coli DH5α was used as
a negative control.
ENTOMOPATHOGENIC BACTERIA, PHOTORHABDUS TEMPERATA M1021
Antibiotic and Catalase Activities
An experiment was conducted to investigate the antibiotic activity
of the bacterial isolate. Thirty microliter cultures of S. typhimurium
KCTC 1926 (Korean Collection for Type Cultures) and M. luteus
KACC 10488 (Korean Agricultural Culture Collection) were spread
over the surface of two separate plates containing Tryptone soy agar
(TSA) media [tryptone (Bacon) 17.0 g/l, soytone (Bacon) 3.0 g/l,
glucose 2.5 g/l, sodium chloride 5.0 g/l, dipotassium hydrogen
phosphate 2.5 g/l, and agar 15 g/l]. The bacterial isolate (test
sample) and E. coli DH5α (negative control) were inoculated on the
S. typhimurium and M. luteus culture plates. The culture plates were
then incubated at 28oC for 48 h. The catalase activity was
determined by growing the bacterial isolate on tryptone soy agar
media. The culture plates were incubated at 28 ± 2oC for 48 h, and
then 30 µl of 3% (v/v) H2O2 was added to the fully grown colonies
of the bacterial isolate. E. coli DH5α was used as the positive
control and Streptococcus agalactiae ATCC13813 (American Type
Culture Collection) was used as the catalase negative control.
Analysis of Fatty Acid Composition
The fatty acids were analyzed by gas chromatography (Model 6890;
Hewlett Packard, Canada) using the Microbial Identification
software package [25]. Four different types of reagents were used
for fatty acid analysis: Reagent I (45 g sodium hydroxide, 150 ml
methanol, and 150 ml distilled water), Reagent II (325 ml certified
6.0 N hydrochloric acid and 275 ml methyl alcohol), Reagent III
(200 ml hexane and 200 ml methyl tert-butyl ether), and Reagent IV
(10.8 g sodium hydroxide dissolved in 900 ml distilled water). The
sample was prepared according to the requirements of the Sherlock
microbial identification system (MIDI). (1) Harvesting: A 4 mm
loop was used to harvest about 40 mg of bacterial cells from the
third quadrant (second or first quadrant if slow growing) of the
quadrant streaked plate. The cells were placed in a clean 13×100
culture tube. (2) Saponification: 1.0 ml of Reagent I was added to
each tube containing harvested cells. The tubes were securely sealed
with Teflon-lined caps, vortexed briefly, and then heated in a boiling
water bath for 5 min. The tubes were vigorously vortexed for 510 s and then returned to the water bath in order to complete the
30 min heating. (3) Methylation: The cooled tubes were uncapped
and 2 ml of Reagent II was added. The tubes were then capped and
briefly vortexed. After vortexing, the tubes were heated for 10 ±
1 min at 80 ± 1oC. Note that this step was critical in terms of time
and temperature. (4) Extraction: Addition of 1.25 ml of Reagent III
to the cooled tubes was followed by recapping and gentle tumbling
on a clinical rotator for approximately 10 min. The tubes were then
uncapped and the aqueous (lower) phase was pipetted out and
discarded. (5) Base Wash: Approximately 3 ml of Reagent IV was
added to the organic phase remaining in the tubes. The tubes were
recapped and tumbled for 5 min. Following uncapping, approximately
2/3 of the organic phase was pipetted into a GC vial, which was
then capped and ready for analysis. Fatty acids produced by the
bacterial isolate were measured by GC (Model 6890; Hewlett
Packard, Canada).
Insect Breeding
G. mellonella larvae were bred from eggs of wax moth (collected
from Daegu, Korea) using artificial media. The media ingredients
consisted of wheat bran (600 g), rice bran (600 g), yeast extract
(4.5 mg), CaCO3 (2 mg), glycerol (250 ml), water (175 ml), honey
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(600 ml), and vitamin B-complex (600 mg). All the ingredients,
except the honey and vitamins, were mixed thoroughly. Then the
mixture and honey were autoclaved in separate containers. After
sterilization, the honey, vitamins, and other ingredients were all
mixed together. Eggs laid by wax moth on butter paper were added
into 150 g of the media and then incubated at 27 ± 1oC and relative
humidity 50 ± 5%, whereby the eggs hatched. The small larvae
were then transferred to a larger container containing a large amount
of medium.
Toxicity Test of the Bacterial Culture Supernatants
An experiment was designed to examine the pathogenicity of the
isolated bacteria. The bacteria were cultured in LB medium for 48 h
at 28 ± 2oC, and then the culture broth was centrifuged at 12,000 rpm
in order to harvest the supernatant. Three microliters of the culture
supernatant was injected into the hemocoel of individual G.
mellonella larvae using a 10 µl Hamilton syringe. Each injected
larva was transferred to a 90 mm Petri dish and then incubated at 25
± 2oC and 50% relative humidity. The larva mortality rate was
evaluated after five days. Ten larvae were tested using each bacterial
sample and the experiment was repeated three times.
RESULTS
Morphological Characteristics of the Isolate
The bacterial strain isolated from the EPNs was grown on
NBTA at 28 ± 2oC, producing colonies of up to 1.0 mm in
diameter after 48 h. The bacterial isolate grew well on all
Fig. 1. Biochemical analysis of P. temperata M1021.
(A) Colony morphology of P. temperata M1021. (B) Catalase activity of P.
temperata M1021, forming bubbles in response to the addition of H2O2.
(C) Antibiotic activity of P. temperata M1021 against S. typhimurium. (D)
Antibiotic activity of P. temperata M1021 against M. luteus. (E) Lipase
activity of P. temperata M1021 in medium supplemented with Tween. (F)
Protease activity of P. temperata M1021 in medium supplemented with
skim milk. Clear zones around the colonies of P. temperata M1021 in
Figures C, D, E, and F confirmed the positive results.
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Jang et al.
Fig. 2. Morphological characterization of P. temperata M1021.
(A) Rod-shaped P. temperata strain M1021 as viewed on an electron
micrograph. (B) Gram-negative nature of the P. temperata strain M1021
isolated from EPNs.
the media tested in this study. The colony morphology was
granulated, convex, and opaque (Fig. 1A). The bacterial
isolate was streaked on NBTA plates and the potential
Photorhabdus sp. was identified based on phenotypic criteria
associated with bioluminescence and colony morphology.
The primary colonies appeared as mucoid, convex, and
greenish or dark blue with a clear zone due to dye
absorption and TTC reduction. Electron micrograph (Fig. 2A)
results revealed the rod shape of the bacterial isolate and
Fig. 2B illustrates their Gram-negative characteristic.
Genotypic Characteristics of the Isolate
The PCR product was purified and sequenced, revealing a
length of 1,498 bp. The 16S rDNA sequence from the
present study was aligned against a database available at
the NCBI. The identification of bacterial isolate was
carried out by the maximum parsimony (MP) method. Nine
sequences (8 references and 1 clone) were selected for the
construction of a phylogenetic tree with 1,000 bootstrap
replications. These strains were selected from BLAST
search showing maximum sequence homology and query
coverage, as well as lowest E values. Xenorhabdus was
used as the out-group. BLAST search showed that bacterial
isolate M1021 has 99.8% sequence homology against
Fig. 3. Phylogenetic tree of the selected Photorhabdus was
constructed by maximum parsimony with bootstrap analysis of
1,000 replicates.
The tree was constructed using 9 taxa (8 references and 1 clone). Numbers
at branch points indicate different clones having the same, or nearly same,
sequence. M1021 formed a subclade with P. temperata with a bootstrap
support of 100%; therefore, M1021 was identified as a new strain of P.
temperata.
P. temperata strain XINachT, 98% against P. luminescens
subsp. luminescens strain HbT, 97% against P. asymbiotica
strain ATCC 43950T, and 96% against P. luminescens
subsp. luminescens strain ATCC 29999T. In the dendrogram,
bacterial isolate M1021 formed a clade (99% bootstrap
support) with P. temperata (Fig. 3). On the basis of
sequence homology and phylogenetic analysis, isolate
M1021 was thus identified as a new strain of P. temperata.
It was named Photorhabdus temperata M1021. The 16S
rDNA sequence was deposited into GenBank under the
accession number HQ647119.
Catalase Activity
The bacterial catalase activity was determined by growing
the P. temperata M1021 on a TSA plate at 28 ± 2oC for
48 h, and then 30 µl of 3% H2O2 (v/v) was poured over the
fully grown colonies (Fig. 1B). Bubble formations over the
surface of colonies indicated the presence of catalase. S.
agalactiae was used as a catalase negative control and E.
coli DH5α was used as a catalase positive control.
Antibiotic Synthesis
Antibiotic synthesis by P. temperata M1021 was confirmed
using a simple experiment. Thirty microliter cultures of
S. typhimurium and M. luteus were spread over the surfaces
of two different media plates containing tryptone soy agar,
and the P. temperata M1021 and E. coli DH5α colonies
were streaked over the plates. The results (Fig. 1C and 1D)
indicated that antibiotics were produced by P. temperata
M1021, which killed both S. Typhimurium and M. luteus in
their respective culture plates. The secretion of antibiotics
functioned to form clear zones around the P. temperata
M1021 colonies. No clear zones formed around the E. coli
DH5α colonies, which were included as a negative control.
Lipase and Protease Activities
Lipase and protease activities were found in the P.
temperata strain M1021. LB media containing either
Tween 20 or Tween 80 were used to determine the lipase
activity. Results (Fig. 1E) indicated that P. temperata
M1021 was capable of producing the lipase, which
degraded both the Tween 20 and Tween 80 present in the
LB media, forming digestion zones around the colonies.
The protease activity was determined by incubating the P.
temperata M1021 on LB agar supplemented with 10% (w/v)
skim milk. The formation of clear zones around the P.
temperata M1021 colonies confirmed the secretion of
proteases (Fig. 1F).
Fatty Acid Composition
The fatty acid composition of P. temperata M1021was
analyzed using a Sherlock MIDI system. The results (Fig. 4)
revealed 14 different fatty acids (even and odd numbered,
normal, iso, and anteiso; fatty acids are represented by a
ENTOMOPATHOGENIC BACTERIA, PHOTORHABDUS TEMPERATA M1021
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Fig. 4. Fatty acid composition of P. temperata strain M1021.
The fatty acids were analyzed with a Sherlock MIDI system following the
manufacturer instructions.
binumeric system in which the first number refers to the
chain length and the second number refers to the number
of double bonds) emerging between 12:0 and 18:0.
Growth Conditions
Based on our results, the optimum growth conditions for P.
temperata M1021 (Fig. 5A) indicated that P. temperata
M1021 showed best growth activity at 28 ± 2oC and 220 rpm.
The effect of the incubation time was also evaluated, with
the highest growth rate recorded at the fourth and fifth
days of incubation (Fig. 5B). After a long exponential
phase, the growth curve for P. temperata M1021 reached a
stationary phase on the fifth day of incubation. A declination
in growth was observed beyond 5 days of incubation.
Pathogenicity of P. temperata M1021
Approximately 3 µl of the filter extract was injected into
fifth instar larvae of G. mellonella. The results (Fig. 5C)
indicated that P. temperata M1021 was highly virulent and
caused 100% mortality within 48 h. The LB medium used
as a negative control showed no evidence of pathogenicity
at any stage of larval incubation. The majority of the larvae
injected with the filtered extract died 24-36 h after
injection and turned a red-brown color typical of insects
infected with P. temperata. After a long exponential phase,
the growth curve of the P. temperata M1021 culture
reached a stationary phase on the sixth day of incubation
(Fig. 5C). The insecticidal activity contained in the
supernatant gradually increased as the incubation time
increased, and maximum pathogenicity was noticed in the
mid and late stages of the exponential phase (Fig. 5C).
Fig. 5. Optimal conditions for the growth of P. temperata
M1021.
(A) P. temperata M1021 was incubated in LB medium at different
temperatures (25-36oC) for 48 h. (B) The growth curve indicates that P.
temperata M1021 reached an exponential phase on the fourth day of its
life cycle. The culture was incubated at 28oC in a shaking incubator at
220 rpm. (C) Pathogenicity patterns of the P. temperata M1021 against the
fifth instar larvae of G. mellonella. Three microliters of bacteria was
injected into the hemocoel. Ten larvae were injected and their mortality
was assessed after 24 h. The histogram shows the mortality rate of G.
mellonella, and closed circles ( ● ) show the growth curve at 600 nm.
DISCUSSION
Photorhabdus spp. are Gram-negative, bioluminescent
bacteria that live in symbiotic association with soil
nematodes (Heterorhabditidae). In nature, the complex
formed by the bacterium-nematode has evolved to be an
obligate insect pathogen. EPNs require their own symbiotic
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Jang et al.
bacteria for successful parasitism against their insect hosts
[1]. Two genera of symbiotic bacteria, Photorhabdus and
Xenorhabdus, are found in EPNs [7]. This study was
designed to identify and characterize the bacteria isolated
from soil nematodes (Heterorhabditis) collected from different
locations across Korea. Our morphological identification
results coincided with positive key characteristics associated
with P. temperata, including motility, Gram negativity, rod
shape, highly bioluminescent, and adsorbing dye on NBTA
and MacConkey plates [11, 18, 27]. Optimal bacterial
growth for Photorhabdus spp. was observed at 28 ± 2oC.
This temperature had been previously optimized by Akhurst
and Boemare [2]. Morphological characteristics of the
bacteria provide some information regarding the
identification, but nowadays molecular and phylogenetic
approaches have absolutely changed the dynamics of the
identification techniques. Genomic DNA sequencing is an
objective, reproducible, and rapid technique for
identification of bacterial strains. Sequencing of 16S
rDNA has gained an immense importance in bacterial
identification in recent times, due to the presence of highly
conserved sequence of nucleotides in this part of the DNA.
Genotypic characteristics strongly supported the
hypothesis that the bacterial strain isolated from the EPN
belonged to P. temperata. Phylogenetic analysis of our
discovery confirmed its location in the phylogenetic tree
and it was named P. temperata M1021. The identification
was further confirmed by the fatty acid composition as
analyzed by the Sherlock microbial identification system.
The fatty acid profile of P. temperata M1021 was
comparable with the previous analysis of fatty acid
composition of P. temperata subsp. temperata [20, 31]. P.
temperata releases secondary metabolites such as
antibiotics, bacteriocins, lipases, and proteinases, and it is
tempting to speculate that the more rigid membranous
structures associated with P. temperata may facilitate the
release of these secondary metabolites [34] as compared
with other bacteria. This hypothesis may also explain the
increase, instead of the expected decrease, of the structural
order of the fatty acids released from P. temperata cultured
at reduced temperatures. The surface area that a given
quantity of phospholipids occupy in their respective
membranes decreases at lower temperatures because of the
reduced thermal motion in their component acyl chains
[23]. This reduction in surface area may induce the
opening of additional channels on the cell surface through
which different secondary metabolic products may pass. In
this manner, the bacteria may maintain proper levels of
these compounds in the insect host, thus protecting the
cadaver and the bacteria from colonization by other
bacterial species as well as providing digestible food for
the nematode host at reduced temperatures [13]. The P.
temperata M1021 discovered during the present study
secreted insecticidal toxin into its growth medium. The
toxin was extremely lethal when injected into the fifth
instar larvae of G. mellonella. The hemocoelic injection of
the bacterial extract resulted in complete mortality of the
insect larvae within 48 h. In typical insect infections, upon
entry into the host hemocoel, the nematodes release the
symbiotic bacteria in their guts [15, 29]. The presence of
the bacterial symbiont is required in order to kill the insect
host and to digest the host’s tissues, thereby providing
nutrients for nematode growth and development [9]. It was
observed during the incubation of P. temperata M1021 that
after a long exponential phase, the bacteria entered into a
stationary phase at the fifth day of the incubation period.
The insecticidal activity of P. temperata M1021 gradually
increased with culture time and reached a peak at the mid
and late stages of the exponential phase. The insecticidal
activity of entomopathogenic bacteria could be due to the
secondary metabolites they produce, such as toxins and
enzymes [6, 33]. It is notable that there was a conflict
between the results of the present study and previous
reports, which observed that culture extracts after 24 h of
incubation exhibited the highest insecticidal activity [14].
Considering the combined results, we inferred that the
toxins were extracellular in nature and secreted in the mid
exponential phase by the strain used in the present study,
and secreted in the early exponential phase in the strains
used previously [6, 14]. The proposed strain of the present
study was named as P. temperata M1021.
Acknowledgment
This research was supported by Basic Science Research
Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science
and Technology (2010-0009969) and Korea Ministry of
Environment as the Eco-Innovation project.
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