Insect Control: Biological and Synthetic Agents - Index of

314 9: Insecticidal Toxins from Photorhabdus and Xenorhabdus
W14, P. luminescens subsp. laumondii strain TT01,
and P. temperata strains K122 and NC1. For brevity
these strains will be referred to as Photorhabdus
strains W14, TT01, K122, or NC1, or simply W14,
TT01, K122, and NC1, except where reference to
their different specific designation is relevant to the
discussion.
9.1.2. The Need for Alternatives to Bt
To date, the majority of transgenes deployed in
insect resistant crops are Cry genes from Bacillus
thuringiensis or ‘‘Bt.’’ Despite the diversity of Cry
genes cloned from B. thuringiensis, only a few Cry
proteins are toxic towards specific pests. This observation
has led to concerns about the development
of resistance to specific Cry toxins and over the
subsequent management of cross-resistance to other
toxins occupying the same or similar binding sites
(Ives, 1996; McGaughey et al., 1998). These concerns
are increasing as specific cases of laboratorydeveloped
Bt resistance are documented in pest
insects (McGaughey, 1985; Gahan et al., 2001).
Bacillus thuringiensis has also proved to be a useful
source of other non-Cry genes such as the vegetative
insecticidal proteins or ‘‘Vips’’ (Estruch et al., 1996;
Yu et al., 1997); however, this organism clearly has
a limited capacity to produce further toxins. The
work described here on the isolation and characterization
of insecticidal toxins from Photorhabdus
and Xenorhabdus has been performed partly in the
search for alternative novel insecticidal proteins for
insect control.
9.2. The Toxin Complexes
9.2.1. Discovery of the Toxin Complexes
9.2.1.1. Purification and cloning of Photorhabdus
toxins Despite the fact that Photorhabdus is released
directly into the insect hemocoel by its
nematode host, the culture supernatant of Photorhabdus
strain W14 shows unexpected oral toxicity
to the lepidopteran model Manduca sexta (Bowen
and Ensign, 1998). A toxic high molecular weightprotein
fraction was purified from the W14
supernatant by sequential ultrafiltration, dimethyl
aminoethyl (DEAE) anion-exchange chromatography,
and gel filtration (Bowen and Ensign, 1998). As
a final purification step, high-performance liquid
chromatography (HPLC) anion-exchange chromatography
was used to separate four peaks or ‘‘Toxin
complexes’’ A, B, C, and D (Bowen et al., 1998).
Purified Toxin complex A (Tca) has a median lethal
dose of 875 ng cm 2 of diet against M. sexta, and is
therefore as active as some Bt Cry proteins (Bowen
et al., 1998). The histopathology of orally ingested
Tca shows the primary site of action to be the
midgut, where cells of the midgut epithelium produce
blebs as the epithelium itself disintegrates
(Blackburn et al., 1998). Interestingly, injection of
purified Tca also results in destruction of the midgut
with a similar histopathology, suggesting that
Tca can act on the gut from either the lumen or
the hemocoel (Blackburn et al., 1998).
Each of the HPLC purified complexes migrates as
a single or double species on a native gel but resolves
into numerous different polypeptides on a denaturing
sodium dodecylsulfate (SDS) gel (Bowen et al.,
1998). The toxin complex (tc) encoding genes were
cloned by raising both monoclonal and polyclonal
antisera against the purified complexes and using
these to screen an expression library. Each of the
four complexes Tca, Tcb, Tcc, and Tcd is encoded
by four independent loci tca, tcb, tcc, and tcd
(Bowen et al., 1998). Each of these loci consists
of an operon with successive open reading frames,
for example, tcaA, tcaB, tcaC, and tcaZ (Figure 1).
These open reading frames encode the different
polypeptides that can be resolved from the native
complexes as confirmed by N-terminal sequencing
of the individual polypeptides resolved on an SDS
gel (Bowen et al., 1998). Genetic knockout of each
of the tca, tcb, tcc, and tcd loci in turn showed that
both tca and tcd contribute to oral toxicity to
M. sexta and that removal of both these loci in the
same strain renders the double mutant nontoxic
(Bowen et al., 1998).
Similar independent purification approaches to
the supernatant of strain W14 identified two high
molecular weight complexes, confusingly termed
‘‘toxin A’’ and ‘‘toxin B,’’ with oral activity to the
coleopteran Diabrotica undecimpunctata howardi
(Guo et al., 1999). These two toxins correspond to
the species TcdA and TcbA, respectively, therefore
confirming that both Tcd and Tcb also have activity
against Coleoptera. The native molecular weights
of TcdA and TcbA were estimated at 860 kDa, leading
to the suggestion that they are tetramers of
the 208 kDa species observed on an SDS gel (Guo
et al., 1999). These species can also be proteolytically
cleaved by proteases found in the culture supernatant
and an increase in insecticidal activity
associated with the cleaved form of the toxin was
reported (Guo et al., 1999). However, the nature
of the protease responsible and the relevance of
the cleavage in the biological activity of the toxins
remain obscure.
9.2.1.2. Cloning of Xenorhabdus toxins Supernatants
of some Xenorhabdus strains also show oral