Transcript B. thuringiensis kurstaki

Microbial Insecticides
Ziad W Jaradat
Microbial Insecticides
Insects are the most abundant group of
organisms on Earth, and they
negatively affect humans in a variety of
ways:
 They cause massive crop damage,
 They act as vectors of both human
and animal diseases.
History of insecticides
During the 1940s, a number of chemical insecticides
were developed as a means of controlling the
proliferation of noxious insect populations.
 One of these was the chlorinated hydrocarbon
DDT (dichlorodiphenyltrichloroethane). DDT
proved to be exceptionally effective in killing
and controlling many species of pests by
attacking the nervous system and muscle tissue
of insects.
 Other chlorinated hydrocarbons such as
dieldrin, aldrin, chlordane, lindane, and
toxophene have since been synthesized and
applied on a massive scale.
Another class of chemical insecticides is called
organophosphates and includes malathion,
parathion, and diazinon.
The first generation of organophosphates were
developed as chemical warfare agents. Now they are
used to control insect populations by inhibiting the
enzyme acetylcholinesterase, which hydrolyzes the
nerve transmitter acetylcholine. These insecticides
disrupt the functioning of motor and brain neurons
of the insect.
By early 1960s, over 100 million acres of U.S. agricultural land
were being treated annually with chemical insecticides. However,
at a latter time, researchers realized that chlorinated hydrocarbon
insecticide and organophosphate insecticides had dramatic
effects on animals, ecosystems, and humans.
DDT, was found to persist in the environment for 15 to 20 years
and accumulates in increasing concentrations through food
chains.
This bioaccumulation in fatty tissues was having a significant
biological impact on many organisms.
For example, in North America, many species of birds including
peregrine falcons, sparrow hawks, bald eagles, brown pelicans,
and double-crested cormorants were severely depopulated.
Drawbacks of chemical insecticides;
Targeted insect pest populations be increasingly
resistant to treatment with many chemical insecticides
Chemical insecticides were found to lack specificity;
consequently, beneficial insects were being killed
along with those that were considered to be pests.
Some times the natural enemies of the insect pest
species were killed more efficiently than the target
organisms.
Given all the drawbacks associated with the use of
chemical inseciticides, alternative means of
controlling harmful insects have been sought over the
past 20 years.
Using insecticides that are produced
naturally by either microorganisms or plants
was an obvious choice, Why?
Highly specific for a target insect species
Biodegradable, Slow to select for resistance.
Researchers can manipulate genes that
encode insect pathogenic agents and
introduce them in target microbes that can
infect these insects.
Insecticidal Toxin of Bacillus thuringiensis
A microbial insecticide can be an organism that
either produces a toxic substance that kills an
insect species or has the capability of fatally
infecting a specific target insect.
The most studied, most effective, most often
utilized microbial insecticides are the toxins
synthesized by Bacillus thuringiensis.
This bacterium comprises a number of different
strains each of which produces a different toxin
that can kill certain specific insects, for example;
B. thuringiensis kurstaki is toxic to;
lepidopteran larvae including moths,
butterflies, skippers, cabbage worm, and
spruce budworm.
B. thuringiensis israelensis kills diptera such as
mosquitoes and black flies.
B. thuringiensis tenebrionis is effective against
coleoptera (beetles) such as the potato beetle
and the boll weevil.
Other B. thuringiensis strains with different
toxins that are specific toward certain insects.
Mode of action
To kill an insect pest, B. thuringiensis
must be ingested as the contact of the
bacterium or the toxin with the surface
of an insect has no effect on the target
organism.
B. thuringiensis is generally applied by
spraying, so it is usually formulated
with insect attractants to increase the
probability that the target insect will
ingest the toxin.
Advantage;
Because for the toxin to be effective it has to be
ingested, this limits the susceptibility of none target
insects and other animals to this insecticide.
Drawbacks;
Insects that attack plant roots are less likely to ingest a
B. thuringiensis toxin that has been sprayed on the
surface of a host plant.
B. thuringiensis toxin can only kill a susceptible insect
during a specific developmental stage.
It costs 1.5-3 times as much as chemical insecticides
Resistance of insects to the toxins produced by these
bacteria might occur.
Example of using Bacterial insecticides;
Bacillus thuringiensis sub specs. kurstaki was
used as major means of controlling spruce
budworm in Canada.
Its use was increased from 1% in 1979 to
around 74% in 1986 for treating spruce
budworm in Canada
In other countries, B. thuringiensis kurstaki has
been used against tent caterpillars, gypsy
moths, cabbage worms, cabbage loopers, and
tobacco hornworms.
How Does it Work
The insecticidal activity of B. thuringiensis
kurstaki and other strains is contained within a
very large structure called the parasporal crystal,
which is synthesized during bacterial
sporulation.
The crystal is an aggregate of one kind of protein
that can be dissociated by mild alkali treatment
to yield two subunits of 130 kDa each (Fig. 12.1).
The parasporal crystal is not the active form of
the insecticide; rather, it is a protoxin, a precursor
of the active toxin.
When the parasporal crystal is ingested by a target
insect, the protoxin is activated within its gut by the
combination of alkaline pH (7.5 to 8.0) and specific
digestive proteases, which converts the protoxin into
an active form with 68 kDa (Fig. 12.1).
When the toxin changes to its active form, it inserts
itself into the membrane of the gut epithelial cells of
the insect and creates an ion channel through which
excessive loss of cellular ATP occurs (Fig. 12.2).
About 15 min after the channels forms cellular
metabolism ceases, the insect stops feeding,
becomes dehydrated, and eventually dies.
Two things make this process very specific;
1. The need for an alkaline media
2. The need for specific proteases
B. thurigienis kurstaki is applied by spraying
approximately
1 .3 to 2.6 X 108 spores per square foot of the target
area at the peak of the larval population of the
target organism.
The crystal is short lived as it breaks down after
exposure to sunlight so it is appropriate to spray it
in cloudy days.
Toxin Gene Isolation
Isolate and characterize the protoxin gene(s).
determine whether the toxin genes are located
on a plasmid or on the chromosomal DNA
Test for plasmid-borne toxin genes; a toxin
producing strain can be conjugated to a strain
that lack the insecticidal activity. If the latter
strain acquires the ability to synthesize the
insecticidal toxin, then the toxin gene(s) is most
likely present on a plasmid because the transfer
of chromosomal DNA during conjugation is a
rare event.
Isolation of the protoxin encoding DNA sequence
Total cellular DNA is isolated and separated into
plasmid and chromosomal DNA using cesium
chloride gradient centrifugation.
A clone bank is constructed from the chromosomal
DNA
When toxin gene is plasmid encoded, the plasmid
can be further fractionated by sucrose gradient to
separate the plasmids to different sizes. Figure 12.3
Bacillus thuringiensis kurstaki contains an
insecticidal protoxin gene on one of 7 different
plasmids (2, 7.4, 8.2, 14.4, 45 and 71 kb in length.
To determine which B. thuringiensis kurstaki
plasmid carries the protoxin gene, following sucrose
gradient centrifugation, the plasmid DNA sample is
divided into three fractions
Small (2.0 kb)
Medium (7.4, 7.8, 8.2, and 14.4 kb)
Large (45 and 71 kb)
The fraction with the small plasmid (2.0 kb) is
discarded, because this plasmid is too small to
encode a protein equivalent to the 130-kDa protoxin.
The medium and large plasmid fractions are each
partially digested with the restriction enzyme
Sau3AI and then ligated into the BamHI site of
plasmid pBR322.
These clone banks were transformed into E. coli and
then screened immunologically using the following
procedure:
Colonies are transferred from agar plates to a
nitrocellulose membrane.
•
The transferred colonies are partially lysed with
organic solvents.
•
All available sites on the membrane to which primary and
secondary antibodies could bind are blocked by treating the
membrane with bovine serum albumin.
•
The bovine serum albumin-treated membranes are treated
with rabbit antiserum that contains antibodies against the
insecticidal toxin.
•
The membranes are washed to remove unbound antibodies
and then treated with 251-labeled S. aureus protein A, which
binds to the Fc portion of the bound antibodies.
•
Spots on the membrane corresponding to colonies that
actively synthesize the insecticidal toxin are visualized by
autoradiography.
•
Using the protoxin gene, the 71-kb plasmid of
B. thuringiensis. kurstaki was found by DNA
hybridization to encode the toxin gene.
Genetic Engineering of B. thuringiensis
Strains
First step is to isolate and sequence the toxin gene,
then to obtain the amino acid sequence of toxin.
When the amino acid sequence was compared for
other toxins they all showed a common toxin
domain.
It was found that the whole gene is not necessary for
the toxin to have its insecticidal activity, rather a
portion, a chemically synthesized coding sequence
or of course the whole gene can be used for further
genetic manipulation
The B. thuringiensis protoxin protein is only
synthesized during the sporulation phase of
growth therefore,
It might therefore be advantageous to have the
toxin gene transcribed and translated during
vegetative growth.
This would permit the toxin to be synthesized
by a continues fermentation process which
decreases the cost of toxin production
However, when a continuously active
(constitutive) promoter form tetracycline
resistant gene was introduced into B.
thuringiensis the active toxin protein was
produced continuously through out the
whole growth cycle ( spore and
vegetative phases)
Figure 12.4, and even the toxin synthesis
occurred in B. thuringiensis defective in
sporulation gene.
Therefore, under these conditions the toxin will be
produced in high quantities.
Many crops might be attacked by different insect
species, therefore, it would be advantageous if we
could create microbial insecticides with broad
spectrum of target insects. This could be done by;
Transferring a gene against one insect into a B.
thuringiensis that already produced another toxin
against another insect
Fusing portions of two different species-specific
toxin genes to one another so that a unique hybrid
toxin is produced and is effective against these to
different insects.
Testing whether the insect target can be
widened
Toxin genes were taken from B. thuringiensis
subspecies aizawai and tenebrionis and used to
make one construct (vector)
The vector was introduced onto B. thuringiensis
subspecies aizawai, tenebrioinis, kurstaki,
israelensis
All the transformed strains were tested for their
toxicity toward three different larvae.
Table 1 shows the results of this experiment.
Some times B. thuringiensis might not be the best
bacteria to be sprayed to combat an insect, so the
toxin genes must then be introduced into anther
suitable vector such as Caulobacter crescentus or
cyanobacteria.
Another example is that some insects attack the roots
of the plants which makes them inaccessible to the
B. thuringiensis –based insecticidal therefore, it is
possible to introduce the toxin gene from the
Bacillus to a bacteria that colonizes close to the
roots.
The engineered bacteria could be introduced into
the soil so they;
release the toxin close to the roots
Since they colonize the soil, they keep
producing the toxin continuously thus
obviating the need for continuous spraying.
Example; the B. thuringiensis kurstaki
insecticidal toxin was introduced into the
chromosomal DNA of Pseudomonas
fluorescens that colonize corn roots.
1.
A transposon Tn5 element that had been
cloned into a plasmid was genetically modified by
altering portions of its left and right borders and
deleting its transposase. Such an altered Tn5
element cannot be excised from the plasmid, even
by exogenous transposase.
Transposes is an enzyme that is encoded by
transposon gene and facilitates the insertion or
excision of the transposon form a chromosomal site.
2.
An isolated B. thuringiensis subsp. kurstaki
insecticidal toxin gene was spliced into the middle
of the altered Tn5 element on the plasmid and
placed under the control of a constitutive promoter.
3.
A wild-type Tn5 element was transposed into
the chromosome of the root-colonizing strain of P.
fluorescens.
4.
The plasmid carrying the altered Tn5 element
with the insert toxin gene was introduced into the
host bacterium that had the integrated wild-type
Tn5 element.
5.
Homologous recombination by means of a
double crossover between the nontransposable Tn5
element on the plasmid that carries the toxin gene
and the chromosomally integrated wild-type Tn5
led the integration of the altered Tn5 with the toxin
gene into the chromosomal DNA, with the
concomitant loss of the wild-type Tn5 element.
This form of engineering controls two things;
toxin gene is unlikely to be lost either during the
large-scale laboratory growth or after release of
the engineered microorganism to the
environment.
Probability of transfer of the toxin gene to other
microorganisms in the environment is very low.
Baculoviruses as Biocontrol Agents
Baculoviruses are rod-shaped, double-stranded
DNA viruses can infect and kill a large number of
different invertebrate organisms. Sub groups of this
viral family are pathogenic to several classes of
insects including Lepidoptera, Hymenoptera,
Diptera, Neuroptera, Trichoptera Coleoptera, and
Homoptera.
Therefore, some of the baculoviruses are important
for the control of certain pests and thus are
registered as pesticides.
Problems!!!
These viruses kill insects slowly within days or
weeks.
Solution !!!
Enhance the virulence of the virus by introducing
foreign genes that will severely impair or kill the
target insect such using the gene that disrupts the
cell cycle of the insect.
During the insect development, larva juvenile
hormone is needed for the metamorphosis to
happen, now this hormone is degraded by the action
of the juvenile hormone estrase which is an enzyme
that inactivate the juvenile hormone. Therefore, an
increase in the production of this hormone will
interrupt the insect life cycle and leads to its death.
The gene for the juvenile estrase was
purified from the insect Heliothis virescens (
tobacco budworm) and the coding sequence
was isolated from the cDNA library and
inserted into the genome of a baculovirus
under the control of the baculovirus
transcription signals.
When this virus was fed to target insects, the
larva feeding and growth was severely
limited relative to the control.
Problems of this method;
This method is specific for the control of the insects at
this larval stage only so it has a limited effect.
Therefore, the incorporation of a toxin gene in the
genome of the virus that will be transcribed and
translated during the viral normal cycle in the insect
would help in killing the insect any time regardless of
its developing stage.
Example, the gene that encodes the insect specific
neurotoxin produced by the North American scorpion
was cloned into a baculovirus and was tested against
target insects and found to decrease the damage to crops
by 50% . The cost of propagating this virus is high