Genome-wide screening reveals the genetic determinants of an antibiotic insecticide in Bacillus thuringiensis

Article abstract of DOI:10.1074/jbc.M110.148387

Thuringiensin is a thermostable secondary metabolite in Bacillus thuringiensis and has insecticidal activity against a wide range of insects. Until now, the regulatory mechanisms and genetic determinants involved in thuringiensin production have remained unclear. Here, we successfully used heterologous expression-guided screening in an Escherichia coli-Bacillus thuringiensis shuttle bacterial artificial chromosome library, to clone the intact thuringiensin synthesis (thu) cluster. Then the thu cluster was located on a 110-kb endogenous plasmid bearing insecticide crystal protein gene cry1Ba in strain CT-43. Furthermore, the plasmid, named pBMB0558, was indirectly cloned and sequenced. The gene functions on pBMB0558 were annotated by BLAST based on the GenBank and KEGG databases. The genes on pBMB0558 could be classified into three functional modules: a thuringiensin synthesis cluster, a type IV secretion system-like module, and mobile genetic elements. By HPLC coupling mass spectrometer, atmospheric pressure ionization with ion trap, and TOF technologies, biosynthetic intermediates of thuringiensin were detected. The thuE gene is proved to be responsible for the phosphorylation of thuringiensin at the last step by vivo and vitro activity assays. The thuringiensin biosynthesis pathway was deduced and clarified. We propose that thuringiensin is an adenine nucleoside oligosaccharide rather than an adenine nucleotide analog, as is traditionally believed, based on the predicted functions of the key enzymes, glycosyltransferase (ThuF) and exopolysaccharide polymerization protein (Thu1).

Full text of DOI:10.1074/jbc.M110.148387

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 50, pp. 39191–39200, December 10, 2010
2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
©
Genome-wide Screening Reveals the Genetic Determinants
□
S
*
of an Antibiotic Insecticide in Bacillus thuringiensis
Received for publication, May 26, 2010, and in revised form, September 22, 2010 Published, JBC Papers in Press, September 23, 2010, DOI 10.1074/jbc.M110.148387
1
1
Xiao-Yan Liu , Li-Fang Ruan , Zhen-Fei Hu, Dong-Hai Peng, Shi-Yun Cao, Zi-Niu Yu, Yao Liu, Jin-Shui Zheng,
2
and Ming Sun
From the State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural
University, Wuhan 430070, China
Thuringiensin is a thermostable secondary metabolite in vegetative insecticidal protein (4, 6), secret insecticidal protein
Bacillus thuringiensis and has insecticidal activity against a wide (7), thuringiensin (8), zwittermicin A (9), Mtx-like toxin (10),
range of insects. Until now, the regulatory mechanisms and Bin-like toxin (11), etc. The diversity of toxin is paralleled by a
genetic determinants involved in thuringiensin production have diversity in pesticidal activity. Some B. thuringiensis strains
remained unclear. Here, we successfully used heterologous found have novel biological activities other than insect toxicity,
expression-guided screening in an Escherichia coli–Bacillus like parasporin, which targets human cancer cells, and some
thuringiensis shuttle bacterial artificial chromosome library, to other strains are toxic to human-pathogenic protozoa (12).
clone the intact thuringiensin synthesis (thu) cluster. Then the Specific toxins, responsible for most of those activities, have not
thu cluster was located on a 110-kb endogenous plasmid bearing yet been identified or characterized (13). Most of the research-
insecticide crystal protein gene cry1Ba in strain CT-43. Further- ers in the B. thuringiensis field pay attention to the crystal pro-
more, the plasmid, named pBMB0558, was indirectly cloned tein toxins. So far, 460 kinds of crystal protein toxins, classified
and sequenced. The gene functions on pBMB0558 were anno- into 60 classes, and 27 kinds of cytotoxin, classified into two
TM
tated by BLAST based on the GenBank and KEGG databases. classes, have been reported (see the Bacillus thuringiensis
The genes on pBMB0558 could be classified into three func- Toxin Nomenclature site on the World Wide Web).
tional modules: a thuringiensin synthesis cluster, a type IV
Thuringiensin (␤-exotoxin) is a secondary metabolite of
secretion system-like module, and mobile genetic elements. By B. thuringiensis and has insecticidal activity against a wide
HPLC coupling mass spectrometer, atmospheric pressure ioni- range of insects (14–16). From the structural formula, thuring-
zation with ion trap, and TOF technologies, biosynthetic inter- iensin is composed of four precursors: adenosine, glucose, a
mediates of thuringiensin were detected. The thuE gene is phosphate group, and gluconic diacid (17). This kind of struc-
proved to be responsible for the phosphorylation of thuringien- ture is seldom reported in antibiotic compounds. Historically,
sin at the last step by vivo and vitro activity assays. The thuring- the consensus has been that it is an adenine nucleotide analog
iensin biosynthesis pathway was deduced and clarified. We (17), like ATP, and this similarity makes it an inhibitor of DNA-
propose that thuringiensin is an adenine nucleoside oligosac- dependent RNA polymerases (18). It is a nonspecific antibiotic
charide rather than an adenine nucleotide analog, as is tradi- insecticide and acaricide.
tionally believed, based on the predicted functions of the key
enzymes, glycosyltransferase (ThuF) and exopolysaccharide nants involved in thuringiensin production have been unclear.
polymerization protein (Thu1).
Using a B. thuringiensis strain mutant library, Espinasse et al.
19) reported that an ABC transporter, which might be related
to the secretion of thuringiensin, was essential for thuringiensin
Until now, the regulatory mechanisms and genetic determi-
(
Bacillus thuringiensis is a Gram-positive insect pathogen production.
that is extensively used in biological pest control (1). B. thuring-
The aim of this study was to elucidate the genetic determi-
iensis-based biopesticides are an environment-friendly prod- nant of thuringiensin in the strain CT-43. We adapted HPLC to
uct. B. thuringiensis could produce multi-insecticidal metabo- selectively detect the characteristic peak of thuringiensin in an
lites like insecticide crystal proteins (2–4), cytotoxin (3, 5), Escherichia coli–B. thuringiensis shuttle bacterial artificial
3
chromosome (BAC) library of the thuringiensin high produc-
tion strain, CT-43. The intact thu cluster was then isolated. The
*
This work was supported by National Basic Research Program (973 Pro-
gram) of China Grant 2009CB118902, National Natural Science Foundation thuringiensin biosynthesis pathway was deduced and clarified
of China Grants 30400003 and 30970037, National High Technology
by LCMS-IT-TOF detection and the identification of key gene
Research and Development Program (863 Program) of China Grants
thuE.
2
006AA02Z174 and 2006AA10A212, Genetically Modified Organisms
Breeding Major Projects of China Grant 2009ZX08009-032B, and Ministry
of Forestry Project (948 Program) of China Grant 2006-4-41.
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S7.
□S
3
The abbreviations used are: BAC, bacterial artificial chromosome; ACP, acyl
carrier protein; LCMS, HPLC coupling mass spectrometry; LCMS-IT-TOF,
HPLC coupling mass spectrometry, atmospheric pressure ionization with
ion trap, and time-of-flight technologies; ESI, electrospray ionization;
NRPS, non-ribosomal peptide synthetase; PKS, polyketide synthase; AT,
acyltransferase; contig, group of overlapping clones; T4SSs, type IV secre-
tion system.
Thenucleotidesequence(s)reportedinthispaperhasbeensubmittedtotheGen-
TM
Bank /EBI Data Bank with accession number(s) HM037272.
Both authors contributed equally to this work.
To whom correspondence should be addressed. Tel.: 86-27-87283455; Fax:
1
2
8
6-27-87280670; E-mail: m98sun@mail.hzau.edu.cn.
DECEMBER 10, 2010•VOLUME 285•NUMBER 50
JOURNAL OF BIOLOGICAL CHEMISTRY 39191

Genetic Determinants of an Insecticide
EXPERIMENTAL PROCEDURES
injected into a C18 end-capped column. A 5% methanol gradi-
ent in 50 mM potassium phosphate buffer (pH 3.0) was applied
for 15 min. The flow rate was 1.0 ml/min, and UV absorption
was monitored at 260 nm at 25 °C. Thuringiensin was eluted at
Materials—B. thuringiensis strain CT-43 (without a flagel-
lum) was isolated from Chinese soil by our group and showed
high production of thuringiensin. The details of bacterial
strains and plasmids used in this study are listed in Table 1. All
strains were grown at 28 °C in Luria-Bertani (LB) medium.
Antibiotics were added at the following concentrations: ampi-
cillin (100 ␮g/ml), chloromycetin (25 ␮g/ml), erythromycin (15
5
.5 min with a Hypersil C18 column (10 ␮m, 4.6 ϫ 150 mm;
Elite) and at 8.0 min with an Agilent TC-C18 column (5 ␮m,
4
.6 ϫ 250 mm; Agilent). The detection limit of this method for
thuringiensin was 2 ␮g/ml.
Extraction and Preparation of Thuringiensin—To extract
thuringiensin, B. thuringiensis strains were grown in LB at 200
rpm and 28 °C for 24 h. After centrifugation, the culture super-
natant was collected. Acetone was added to the supernatant to
␮
g/ml), and kanamycin (50 ␮g/ml).
Construction of the Shuttle BAC Library of B. thuringiensis
Strain CT-43—An E. coli to B. thuringiensis shuttle BAC vec-
tor, pEMB0557 (20), was used to construct the shuttle BAC
library. This vector incorporated the plasmid replication origin
9
0% final concentration, and the solution was then centrifuged
for 6 min at 12,000 rpm. The pellet was dissolved in 0.2 ml of
(
ori60) from the 100-kb plasmid of B. thuringiensis subsp.
ultrapure H O, acetonitrile was added to a final concentration
2
kurstaki strain YBT-1520, erythromycin resistance (B. thuring-
iensis), and chloromycetin resistance (E. coli) genes. The over-
night culture of strain CT-43 was inoculated (1% v/v) into 100
ml of sterile LB and incubated at 28 °C for about 3–4 h until the
of 40%, and the sample was centrifuged again for 6 min at
1
2,000 rpm. The pellet was discarded, and the acetonitrile con-
centration in the supernatant was increased to 90%. The pre-
cipitate was collected by centrifugation for 6 min at 12,000 rpm
at 4 °C, and the pellet was finally dissolved in 0.1 ml of HPLC
elution buffer (50 mM KH PO , 5% methanol, pH 3.0).
cell density reached an A
of 0.2–0.3. Cells were harvested
600 nm
by centrifugation, and agarose plugs were prepared as described
20). The genomic DNA was then extracted and separated. The
2
4
(
Intracellular Intermediates Extracted from B. thuringiensis
separated and recovered high molecular weight genomic DNA
was ligated into the cloning-ready BAC vector, pEMB0557,
digested with HindIII. The ligation mixture was then trans-
formed into B. thuringiensis host strain BMB171 by electropo-
ration with a GenePulser electroporator (Bio-Rad).
Ϫ
Strain CT-43 and thuE Mutant BMB0545—B. thuringiensis
Ϫ
strain CT-43 and the interrupted thuE mutant BMB0545 were
grown in LB medium. To analyze intracellular intermediates,
the cells from culture filtrates were washed twice in 20 ml of
sterile deionized water. The cells were collected and lysed by
mechanical disruption with liquid nitrogen. The cell lysates
were resuspended in 5 ml of sterile deionized water and centri-
fuged at 4,000 rpm for 10 min. The supernatants (5 ml) were
collected for LCMS-IT-TOF analysis or stored at Ϫ20 °C.
Identification of the Intracellular Intermediates by LCMS
Analysis—LCMS was performed by using an Agilent 1100
series LC/MSD trap, and the analytical column was ZORB-
AXSB-C18 (5 ␮m, 2.1 ϫ 150 mm; Agilent). The MS operating
conditions were optimized as follows: electrospray ionization
The Screening and Identification of the BAC Library Clones—
The extracted plasmid DNA of the BAC clones was digested
with NotI and HindIII and separated by PFGE with a Bio-Rad
CHEF III instrument that could estimate the inserted fragment
sizes.
Location of the thu Cluster—To locate the thu cluster, a
Southern blot assay was carried out. Plasmids were extracted
from strain CT-43 and HD-2 and then transferred to an Immo-
bilon-Nyϩ nylon membrane (Millipore). Southern blotting was
performed according to a standard protocol (21). Probe cry1Ba
on plasmid pBMB0558 was amplified from strain CT-43. The
DIG High Primer DNA Labeling and Detection Starter Kit I
(
ESI) source set at the negative mode; m/z 100–1000; drying
gas, 8.0 liters/min; drying gas temperature, 350 °C; and spray
gas detector voltage, 30 ␺.
(
Roche Applied Science) was used for Southern blotting.
Identification of the Intracellular Intermediates by LCMS-IT-
TOF Analysis—LCMS-IT-TOF had an ITTOFMS system
The Indirect Cloning of Endogenous Plasmid, pBMB0558—
According to the physical map, two BAC clones that covered
the whole plasmid were selected to construct a subcloning
library. The BAC clones were partially digested with EcoRI and
HindIII separately and then ligated with plasmid pHT304 and
transformed into E. coli DH5␣. Clones were sequenced using
Megabace 1000 automated sequencers (GMI). The Phred/
(
Kyoto) coupled with a high performance liquid chromatogra-
phy (HPLC) system. The LC system (Shimadzu) was equipped
with a solvent delivery pump (LC-20AD), an autosampler (SIL-
2
0AC), a DGU-20A3 degasser, a photodiode array detector
(
SPD-M20A), a communication base module (CBM-20A), and
a column oven (CTO-20AC). The separation was performed on
Phrap/Consed (22) software package and DNASTAR 7.10 soft- a VP-ODS column (5 ␮m, 2.0 ϫ 150 mm) using a gradient
ware package were adopted for quality assessment and elution consisting of mobile phase B (acetonitrile/water/formic
sequence assembly. During the process of sequence assembly, acid (80:20:0.1)). The gradient was as follows: 0–5 min, a linear
the pBMB0558 nucleotide sequence with an average coverage gradient from 2% B to 5% B; 5–10 min, a linear gradient to 20%
of 5-fold was obtained. To confirm the position of these contigs B; 10–15 min, a linear gradient to 20% B; 15–20 min, 2% B. The
in pBMB0558, a BAC-end sequencing technique was per- injection volume was 50 ml, the flow rate was 0.2 ml/min, and
formed. The leaks between contigs were filled by PCR.
PDA detection was performed from 260 nm. The sample cham-
HPLC Analysis for Thuringiensin Detection—HPLC analysis ber in the autosampler was maintained at 4 °C, whereas the
was carried out on a system consisting of a UV-visible detector column was set at 40 °C. The whole analysis lasted 20 min. Mass
(
CapLC 2487, Waters), Rheodyne manual sample injector valve spectral data for the metabolites were obtained using a Shi-
7
725i, and a Waters 515 HPLC pump. 20 ␮l of the sample was madzu ITTOF mass spectrometer. It was equipped with an ESI
3
9192 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 50•DECEMBER 10, 2010

Genetic Determinants of an Insecticide
TABLE 1
Bacterial strains and plasmids used in this study
Strains and plasmids
Features
Source/Reference
DH10B
E. coli(⌬mrr_hsdRMS_mcrBC) mcrA recA1
New England Biolabs
BMB171
A plasmid-cured derivative of B. thuringiensis YBT-1463, which lacks the endogenous
plasmid and the thu cluster
Ref. 43
CT-43
B. thuringiensis subsp. chinensis strain, no flagellum
Ref. 20
Ref. 8
HD-2
B. thuringiensis subsp. thuringiensis strain
EMB1228
EMB1234
BMB0542
BMB0543
BMB0545
BMB0546
BMB0547
pMD19-T
pHT304
E. coli recombinant strain with BAC plasmid harboring cry1Ba gene
E. coli recombinant strain with BAC plasmid harboring cry1Ba gene
B. thuringiensis recombinant strain producing thuringiensin
B. thuringiensis recombinant strain producing thuringiensin
Mutant of strain CT-43 in which the thuE gene was knocked out
Recombinant of BMB0545 in which the knocked out thuE gene was retrocomplemented
Recombinant of BMB0545 in which there was a pHT304 plasmid
PCR product cloning vector
This work
This work
This work
This work
This work
This work
This work
TaKaRa
E. coli–B. thuringiensis shuttle BAC vector
Ref. 26
pHT304-ts
pEMB0572
pEMB0557
pBMB0558
pHT304 derivative with a thermosensitive replicon of B. thuringiensis
Plasmid-interrupted thuE gene by kanamycin gene inserted in pHT304-ts
An E. coli–B. thuringiensis shuttle BAC vector
Ref. 24
This work
Ref. 20
Plasmid harboring cry1Ba gene of strain CT-43
This work
source operated in the negative ionization mode. Liquid nitro- GATATATATTGA-3Ј; thuE-2, 5Ј-CGCTCGAGTCATAG-
gen was used as nebulizing gas at a flow rate of 1.5 liters/min. TACTTCTTCCTTAAA-3Ј. The amplified fragments were
The interface and detector voltages were set at 4.5 and 1.6 kV, purified and digested with BamHI and XhoI and then sub-
respectively. The CDL and heat block temperatures were both cloned into expression vector pGEX-6P-1 (Amersham Bio-
2
00 °C. The MS/MS spectra were produced by collision-in- sciences) digested with BamHI and XhoI, resulting in
duced dissociation of the selected precursor ions with argon as pEMB1101. The coding sequence was identified by sequencing.
the collision gas. The ion accumulation time and relative colli- The identified plasmid pEMB1101 was transformed into E. coli
sion energy were set at 50 ms and 50%, respectively. Data acqui- strain BL21 (DE3) (Amersham Biosciences), and the positive
sition and processing were carried out using the LCMS solution transformants were selected on a Luria-Bertani plate contain-
version 3.41 software supplied with the instrument (23).
ing 100 ␮g/ml ampicillin. subsequently, ThuE was overex-
Interrupting the thuE Gene in the thu Cluster—The pressed and purified using a GSTrap FF column (Amersham
B. thuringiensis mutant strain BMB0545 was constructed via Biosciences). Purified ThuE was incubated with precursor C in
allelic exchange. The mutant allele was constructed via PCR reaction buffer (10 mmol/liter Tris-HCl, pH 8.5, 10 mmol/liter
using LA-taq polymerase. The primer sets were as follows: thuE MgCl , 1 mmol/liter dithiothreitol, 2.5 mM ATP). The reaction
2
upstream 1, 5Ј-GGATCCACCCTGATCATCTTGAAATG- product was identified by following the HPLC and LCMS-IT-
GTG-3Ј; thuE upstream 2, 5Ј-GGTACCCTTTCGATTCT- TOF assays.
GATAATCGCTGC-3Ј; thuE downstream 1, 5Ј-AAGCTTT-
Thuringiensin Bioassay—Thuringiensin was purified from
TCCCGAAACTAGGGTTATGTTC-3Ј; and thuE down- the supernatant of strain CT-43 and recombinant strain
stream 2, 5Ј-CTCGAGCCAAGCATAAATCGTGATAA- BMB0542 (Table 1) separately. Bioassays were carried out by
GGC-3Ј. The PCR product was cloned into pMD19-T vector the diet incorporation method with the larvae of Helicoverpa
(
TaKaRa), and then a kanamycin coding gene was inserted armigera, Plutella xylostella, Musca domestica, and Meloido-
between two arms of the allelic gene. Subsequently, the allelic gyne incognita. Thuringiensin was mixed with the diet at differ-
gene interrupted by the kanamycin coding gene was cloned into ent concentrations. Three replicates were conducted with each
a thermosensitive shuttle vector pHT304-ts (24), which had a dilution. After 6 days of incubation at 25 °C, mortalities for each
thermosensitive replicon and was designated pEMB0572. The treatment were recorded. Data were analyzed by the SPSS 13.0
constructed plasmid contained 630 bp upstream and 800 bp software. All of the bioassays were conducted three times for
downstream of the target gene with the DNA sequences each treatment.
GGATCC and CTCGAG (the recognition sequences for the
RESULTS
restriction endonuclease BamHI and XhoI) introduced
between these two flanking regions. For the ⌬thuE mutation,
Genome-wide Mining for the Gene (Cluster) Responsible for
the first 144 nucleotides were retained as well as the last 543 Thuringiensin Synthesis in B. thuringiensis Strain CT-43—The
(
this number includes the predicted stop codon) in the coding B. thuringiensis strain CT-43 was isolated from Chinese soil by
sequence. This resulted in a deletion of 3% (18 of 687 nucleo- our group. The thuringiensin produced by B. thuringiensis
tides) of thuE. The plasmid pEMB0572 was then introduced strain CT-43 was purified. The bioassay showed that the
into B. thuringiensis strain CT-43 by electroporation and cul- thuringiensin was toxic to Caenorhabditis elegans, H. armigera,
tured at 43 °C and 200 rpm for 5 days. The strain with allelic and P. xylostella (data not shown). To isolate the gene (cluster)
double exchange was selected by PCR and named BMB0545.
responsible for thuringiensin synthesis in B. thuringiensis strain
Extracellular Activity Assays for Purified ThuE—The ThuE- CT-43, a shuttle BAC library of this strain was established.
coding gene was amplified from the genomic DNA of B. thuringiensis strain BMB171, which does not produce
B. thuringiensis CT-43 by the PCR technique. The primer sets thuringiensin (Fig. 1D), was used as the host for the library
were as follows: thuE-1, 5Ј-GCGGATCCATGGAAAA- construction. An E. coli–B. thuringiensis shuttle BAC vector,
DECEMBER 10, 2010•VOLUME 285•NUMBER 50
JOURNAL OF BIOLOGICAL CHEMISTRY 39193

Genetic Determinants of an Insecticide
FIGURE 1. HPLC analysis of heterogeneous expression of thuringiensin in recombinant BMB0542 (25-kb insert). A, trace of the supernatant from strain
CT-43 (positive control). B, trace of the supernatant from recombinant BMB0542 (25-kb insert). C, trace of the supernatant mixture from recombinant BMB0542
(25-kb insert) and strain CT-43. D, trace of supernatant from host strain BMB171 (negative control). The characteristic peak of thuringiensin is indicated by an
arrow. The red lines were produced by the MS system to fix on the range of a peak and facilitate the peak area calculation by the (MS soft, Varian workstation).
AU, absorbance unit.
pEMB0557, with a large loading capacity was constructed for
this purpose (21). The average insertion size of the library was
ϳ80 kb, and assuming that the size of the strain CT-43 genome
is 5 Mb, the library would have an 80-fold coverage of the
genome. 5,000 clones were obtained in total and were divided
FIGURE 2. The thu cluster. thuA, glucose-6-phosphate 1-dehydrogenase
into 250 groups. Twenty clones from each group were co-cul- gene; thuB, racemase gene; thuC, PEP-protein phosphotransferase gene;
thuD, UDP-glucose-6-dehydrogenase gene; thuE, shikimate kinase gene;
tured and analyzed by HPLC. By this screening strategy, a char-
thuF, glycosyltransferase gene; thuG, N-acyl-D-glucosamine 2-epimerase
acteristic peak of thuringiensin was detected in the purified gene; thu1, putative exopolysaccharide polymerization protein gene; thu2,
non-ribosomal peptide synthetase gene; thu3, bacterial gene of unknown
function; thu4, SAM-dependent methyltransferase.
supernatant (Fig. 1B) of one clone, BMB0542 (25-kb insert), and
compared with that of strain CT-43 (Fig. 1A). The identity of
the product was confirmed by co-injection of a sample contain-
TABLE 2
ing equal volumes of strain CT-43 and BMB0542 (25-kb insert) Predicted proteins involved in thuringiensin production
supernatant mixture. The HPLC analysis showed that the sus- The ORF sequences were analyzed by the protein-protein BLAST of NCBI database.
The protein functions were predicted based on the identities of amino acids.
pected peak from BMB0542 (25-kb insert) overlapped the char-
Protein Amino acids
Predicted protein function
acteristic peak of thuringiensin from strain CT-43 (Fig. 1C).
The single peak was collected and further identified by MS
detection (supplemental Fig. S1), which showed that this com-
pound shared the same molecular mass (701 daltons) with
thuringiensin. Therefore, we deduced that the intact cluster
responsible for thuringiensin synthesis was included in the DNA
insert of BMB0542 (25-kb insert). Subsequently, the 25-kb
inserted fragment in BMB0542 (25-kb insert) was sequenced and
ThuA
ThuB
ThuC
ThuD
ThuE
ThuF
ThuG
Thu1
Thu2
Thu3
Thu4
120
105
575
205
228
375
226
101
561
311
143
Glucose-6-phosphate 1-dehydrogenase
Racemase
PEP-protein phosphotransferase
UDP-glucose-6-dehydrogenase
Shikimate kinase
Glycosyltransferase
N-Acyl-D-glucosamine 2-epimerase
Exopolysaccharide polymerization protein
Non-ribosomal peptide synthetase
ABC transporter membrane-spanning permease
SAM-dependent methyltransferase
TM
annotated based on data from the GenBank database.
The Minimized Assay of the Cloned 25-kb Insertion Fragment
and Bioinformatics Analysis—Bioinformatics revealed that a synthesis on host strain BMB171. This cluster was named the
1
2-kb acyl carrier protein-dependent cluster and an insecticidal thu cluster.
crystal protein cry1Ba gene were present on the 25-kb insert. The thu cluster comprised 11 ORFs (Fig. 2). Their gene func-
TM
The 12-kb acyl carrier protein-dependent cluster was sus- tions were predicted based on matches in the GenBank data
pected to be related to thuringiensin synthesis. To isolate a base. Some genes that showed low homology to the known
fragment comprising the 12-kb cluster, the 25-kb insert of genes in the databases were analyzed for their functional
BMB0542 (25-kb insert) was digested with BamHI because a domains and realigned; thus, their deduced functions were
BamHI site was observed to be present at either end of the based only on their functional domains. The detailed results are
1
2-kb cluster. The 12-kb acyl carrier protein-dependent cluster shown in Tables 2 and 3. Based on gene function, we deduced
was thus isolated, cloned into BAC vector, pEMB0557, and that thuA, thuC, and thuD might encode proteins responsible
electroporated into B. thuringiensis host BMB171, for heterol- for the synthesis of the key precursor, gluconic diacid (precur-
ogous expression, resulting in a recombinant termed BMB0543 sor A), from glucose 6-phosphate, whereas thuF and thu1 might
(
12-kb insert). HPLC analysis confirmed that the 12-kb acyl encode proteins responsible for the assembly of thuringiensin.
carrier protein-dependent cluster could confer thuringiensin The predicted Thu2 protein, which comprises an adenylation
3
9194 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 50•DECEMBER 10, 2010

Genetic Determinants of an Insecticide
TABLE 3
NRPS/PKS-like components encoded by thu2
The ORF sequences were analyzed by the protein-protein BLAST of NCBI database.
The domain functions were predicted based on the identities of amino acid.
Proposed domain
Amino acids
Domain function
A
ACP
C
101
60
400
Recognition and activation
Acyl carrier protein
Condensation
(
A) domain, a condensation (C) domain, and an acyl carrier
protein (ACP) domain, might serve as the location for the
assembly of thuringiensin. The thuE gene might encode the
enzyme in charge of the final modification (phosphorylation),
and thu3 might encode a protein that takes part in releasing
mature thuringiensin.
The thu Cluster Is Located on an Endogenous Plasmid of
Strain CT-43—A genome walking strategy was adopted to
clone the flanking sequence of the thu cluster. The known
cry1Ba gene sequence at the end of the 25-kb insert was used as
a probe to isolate all the target clones harboring this gene from
the whole genome library. Thus, six clones harboring the
cry1Ba gene, with insertion sizes ranging from 40 to 120 kb,
were selected. Interestingly, sequence analysis showed that the
cluster was not located on the bacterial chromosome but on an
endogenous plasmid named pBMB0558. The cry1Ba gene was
FIGURE 3. Location of the thu cluster. A, the electrophoretic profile of
endogenous plasmids from B. thuringiensis strain HD-2 and CT-43. Lane 1,
HD-2 (the molecular weights of endogenous plasmids are indicated); lane 2,
used as a Southern blotting probe to confirm this result, and a CT-43. B, Southern blot of B. thuringiensis strain HD-2 and CT-43 using cry1Ba
asprobe. Lane1, HD-2;lane2, CT-43. Theplasmidof75MDainstrainHD-2and
positive signal was detected for one of the six endogenous plas-
CT-43 showed a positive signal with the cry1Ba probe.
mids. The standard B. thuringiensis strain HD-2, the molecular
size of whose endogenous plasmids has been established, was
used as the molecular weight marker (25). The positive signal glucose dehydrogenase (ThuD), which can directly oxidize a
was also detected for a 75-MDa endogenous plasmid of stan- hydroxyl group to a carboxyl(ic) group. In step 2, gluconic
dard B. thuringiensis strain HD-2 (Fig. 3).
diacid, glucose, and adenosine are assembled by thuF and thu1.
The sequencing results revealed that pBMB0558 was a circu- First, gluconic diacid binds to the ACP region of the non-ribo-
lar plasmid with a molecular weight of 109,464 base pairs and somal peptide synthetase (Thu2); a hydroxyl group on the C-1
TM
1
02 putative ORFs (GenBank accession number HM037272). of gluconic diacid forms a thioester bond with a sulfhydryl
The functions of the gene were predicted by BLAST based group of the ACP protein. Second, a UDP-glucose moiety is
TM
on the GenBank
and KEGG databases. The genes on added onto the gluconic diacid-ACP complex by a glycosyl-
pBMB0558 could be classified into three functional modules: 1) transferase (ThuF), and a 1,5-glycosidic bond is formed
a 12-kb thuringiensin synthesis cluster (between 30 and 42 kb), between glucose and gluconic diacid. The resulting product
2
) a 30-kb type IV secretion system-like (0–30 kb), and 3) (precursor B) is polymerized with the ribose of adenosine from
mobile genetic elements of about 67 kb, which included a ATP/ADP/AMP by an exopolysaccharide polymerization pro-
prophage and the transposase (42–109 kb) (supplemental tein (Thu1). A 4,5-glycosidic bond is then formed between
Fig. S2).
ribose and glucose, producing precursor C. Finally, precursor C
The Deduced Thuringiensin Biosynthesis Pathway and is released from ACP. In step 3, precursor C is phosphorylated
Assembly Process—The thuringiensin biosynthesis pathway by ThuE, and the mature thuringiensin is released. The mature
was deduced according to the predicted genes’ functions (Fig. thuringiensin molecule can then be secreted by the cell.
4
). From the structural formula, it is assumed that thuringiensin
Identification of the Biosynthesis Pathway by LCMS-IT-
is composed of four precursors: adenosine, glucose, a phos- TOF—The LCMS-IT-TOF was conducted to clarify the
phate group, and gluconic diacid. The pathway could be divided deduced pathway of thuringiensin. The extracellular and intra-
into three steps. In step 1, the key precursor gluconic diacid cellular contents of a culture of strain CT-43 were detected by
(
precursor A) is synthesized by the products of thuA, thuC, LCMS-IT-TOF. In the intracellular fraction, all of the proposed
and thuD. The initial substrate glucose 6-phosphate is oxidized intermediates could be detected (molecular weights in paren-
to 6-phosphoglucono-␦-lactone by a glucose-6-phosphate theses), i.e. to glucose 6-phosphate (m/z 259.0517), 6-phospho-
1
6
-dehydrogenase (ThuA) and subsequently hydrolyzed to gluconic acid (m/z 275.1091), gluconic acid (m/z 195.1083),
-phosphogluconic acid. The dephosphorylation of 6-phos- gluconic diacid (precursor A) (m/z 209.1249), precursor B with-
phogluconic acid is catalyzed by a phospholipid exchange out a hydroxy group (m/z 354.0271), and precursor C without a
protein-protein phosphotransferase (ThuC). The resulting glu- hydroxy group (m/z 603.2548) (Fig. 5, A–F). The correct molec-
conic acid is subsequently oxidized to gluconic diacid by UDP- ular weight should be increased by 1 based on the m/z value
DECEMBER 10, 2010•VOLUME 285•NUMBER 50
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Genetic Determinants of an Insecticide
FIGURE 4. Schematic of the thuringiensin biosynthesis pathways. Step 1, synthesis of key precursor gluconic diacid (precursor A) by thuA, thuC, and thuD;
step 2, assembly of gluconic diacid, glucose, and adenosine by thuF and thu1; step 3, phosphorylation of precursor C by ThuE. Numbers have been added to
designate the molecular weight of each intermediate.
Ϫ
under the [M Ϫ H] pattern according the principle of LCMS- extracellular supernatant (Fig. 7D; compare with negative con-
IT-TOF. Only the mature thuringiensin could be detected in trol (Fig. 7, B and C) and positive control (Fig. 7A)). Thus, the
the extracellular supernatant of strain CT-43, by both HPLC insertion of the wild-type thuE gene was able to complement
Ϫ
(
supplemental Fig. S3) and LCMS analysis (supplemental Fig. the thuE mutant BMB0545. Taken together, these results
S4). All of the proposed intermediates could not be found in the indicate that the thuE gene is responsible for the phosphoryla-
non-expressing strain BMB171, whereas the origin of struc- tion of thuringiensin at the last step.
tural assignments and predictions, such as glucose fragment,
glucose 6-phosphate, and adenosine could be found based on
the LCMS-IT-TOF data (supplemental Figs. S5 and S6).
The Biological Function of the Key Gene thuE—The function
of the key gene thuE was confirmed by a knock-out experiment.
Extracellular Activity of Purified ThuE—The thuE gene was
heterologously expressed in E. coli strain BL21 (DE3) by expres-
sion vector pGEX-6P-1. Then the ThuE was purified using the
GSTrap FF column. The predicted precursor C was obtained by
digesting purified thuringiensin with alkaline phosphatase and
then identified by HPLC (Fig. 8B). The HPLC revealed a new
peak with a retention of about 5.3 min. The LCMS-IT-TOF was
performed to determine the presence of precursor C, whose
Ϫ
HPLC and LCMS-IT-TOF results for the thuE mutant
BMB0545, carrying an interrupted thuE gene, demonstrated
that this mutant could not produce thuringiensin (supplemen-
tal Fig. S3 and S4) in the extracellular supernatant, although it
could produce all of the predicted intermediates in the cells
Ϫ
molecular weight is 620 under the [M Ϫ H] pattern (supple-
mental Fig. S7). The purified ThuE was incubated with precur-
sor C in reaction buffer and analyzed by HPLC. The HPLC
result showed the conversion of precursor C to thuringiensin
after a 3-h incubation (Fig. 8C).
(
Fig. 5, G–L). Disruption in the thuE gene led to the complete
loss of phosphorylation activity and resulted in the build-up of
precursor C (m/z 603.2548) in the intracellular fraction (Fig. 5,
F and L). MS/MS was further conducted to confirm the struc-
tural formula of the enriched precursor C (m/z 603.2548) in this
mutant. The following components (with expected molecular
weights) were detected: adenine without a hydrogen group
Insecticidal Activities of Thuringiensin—The bioactivity of
thuringiensin extracted from strain CT-43 and BMB0542 was
evaluated against the larvae of H. armigera, P. xylostella, M. do-
mestica, and M. incognita separately. The results (Table 4)
showed that thuringiensin was toxic to them. The LC values
(
m/z 133.0145), ribose without a C5Ј-hydroxy group (m/z
5
0
1
15.0057), glucose without a C1Ј-hydroxy group and C4Ј-hy-
of thuringiensin from strain CT-43 against the larvae of H. ar-
drogen group (m/z 161.0478), and gluconic diacid without a
C1Ј-hydroxy group and C5Ј-hydrogen group (m/z 191.0185)
migera, P. xylostella, M. domestica, and M. incognita were 19.3,
0
.9, 47.7, and 25.5 ␮g/ml, whereas the LC values of thuring-
5
0
(
Fig. 6). The complementation experiment of thuE was also
iensin from BMB0542 were 23.2, 1.2, 49.2, and 23.7 ␮g/ml. The
data showed that the expressed product from strain BMB0542
exhibited thuringiensin-like specific activity.
performed. The primer sets thuE1 and thuE2, were used to
amplify the full-length thuE gene contained in a 1.5 kb DNA
fragment. The double digestion product (BamHI and HindIII)
of the PCR fragment was subcloned into the shuttle vector
pHT304 (26). The obtained recombinant plasmid was electro-
DISCUSSION
Ϫ
porated into the thuE mutant BMB0545, which lead to recom-
In this study, genome screening revealed the genetic deter-
ϩ
binant BMB0546 (thuE ). HPLC analysis of the extracellular minants of a nonspecific antibiotic insecticide thuringiensin in
supernatant of this recombinant revealed thuringiensin in the B. thuringiensis strain CT-43.
3
9196 JOURNAL OF BIOLOGICAL CHEMISTRY
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Genetic Determinants of an Insecticide
؊
FIGURE 5. Intracellular metabolites produced in strain CT-43 and thuE mutant BMB0545 detected by LCMS-IT-TOF. A–F, the LCMS-IT-TOF profile of strain
Ϫ
CT-43. G–L, the LCMS-IT-TOF profile of thuE mutant BMB0545. Each intracellular metabolite is indicated (m/z values are shown). Inten., ion signal strength detected
by the detector.
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Genetic Determinants of an Insecticide
Ϫ
FIGURE 6. Identifying the structure of precursor C. A, MS/MS result of fragments of precursor C (m/z 603.2548, [M Ϫ H] ). Each component (with its
corresponding molecular weight) is highlighted by a circle. B, the possible fractions of precursor C formed in MS/MS are separated by a line. Black numbers are
the molecular weights of each component.
An alternative viewpoint con-
cerning the structure of thuringien-
sin was proposed in this study.
Thuringiensin has historically been
believed to be a thermostable ade-
nine nucleotide analog, like ATP. If
this were true, it would be an inhib-
itor of DNA-dependent RNA poly-
merases (18). The biosafety of
thuringiensin to mammals is still
debated due this potential toxicity
(
27, 28). Our results show that
thuringiensin is synthesized by the
polymerization of three kinds of
monosaccharides: gluconic diacid,
glucose, and ribose. An antibiotic
substance with this structure has
been seldom reported before. This
new finding concerning the struc-
ture of thuringiensin will lead to a
definitive establishment of its toxic-
؊
FIGURE 7. HPLC analysis of the supernatant from strain CT-43, thuE mutant BMB0545, complementary
؉
strain BMB0546 (thuE ), and BMB0547 (vector only). A, trace of the supernatant from strain CT-43. The ity mechanism. The three-dimen-
Ϫ
position of the arrowhead is the characteristic peak of thuringiensin. B, trace of the supernatant from thuE
sional structure of thuringiensin is
mutant BMB0545. C, trace of the supernatant from BMB0547 (vector only). D, trace of the supernatant from
ϩ
cryptic, and we supposed that
thuringiensin possesses a unique
complementary strain BMB0546 (thuE ). The characteristic peak of thuringiensin is observed when the lack of
expression of the ThuE enzyme is compensated for.
3
9198 JOURNAL OF BIOLOGICAL CHEMISTRY
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Genetic Determinants of an Insecticide
combination of special functional elements might represent a
novel mode of antibiotic synthesis.
During the process of pathway prediction, we considered
several different ideas. Finally, the pathway was deduced based
on the LCMS-IT-TOF data, function of genes, and structure of
thuringiensin. The origin of structural assignments and predic-
tions could be initially clarified based on the LCMS-IT-TOF
data (supplemental Fig. S6). The origin of structural assign-
ments should be the basal metabolism, such as Glc-6-PO4,
UDP-glucose, and adenosine, in order to economize the energy.
The similar enzymatic reactions were proved in other organ-
isms. For example, from 6-phosphate-glucose to 6-phospho-
glucono-lactone there needs to be a glucose-6-phosphate 1-de-
hydrogenase action (31–33). In the process of ATP ϩ
D-gluconic acid [dharrow] ADP ϩ 6-phospho-D-gluconate,
phosphorylation and dephosphorylation were produced (34,
ϩ
3
5). In UDP-glucose ϩ H O ϩ 2 NAD [dharrow] UDP-glucu-
2
ϩ
ronate ϩ 2 NADH ϩ 2 H , an oxidation reaction is produced
with a UDP-glucose-6-dehydrogenase (36, 37). Glycosyl trans-
ferase family 2 could transfer sugar from UDP-glucose, UDP-
N-acetyl-galactosamine, GDP-mannose, or CDP-abequose, to
a range of substrates, including cellulose, dolichol phosphate,
and teichoic acids (38–40). Shikimate kinase catalyzes the
phosphorylation of the 3Ј C terminus during the shikimate acid
FIGURE 8. HPLC analysis for extracellular activity of purified ThuE. A, trace
of the supernatant from strain CT-43; B, trace of thuringiensin digested by pathway (41, 42). Non-ribosomal peptide synthetase mainly
alkalinephosphatase;C, traceofthereactionproductaftera3-hincubationof
purified ThuE and precursor C.
catalyzes the antibiotic synthesis, and the key functional ele-
ments responsible for assembly in NRPS are the AC domain
and peptidyl carrier protein, whereas the key elements in PKS
TABLE 4
Activities of thuringiensin from strain CT-43 and BMB0542 against
H. armigera, P. xylostella, M. domestica, and M. incognita
are the acyltransferase domain and ACP (29, 44). The current
genome-wide screening work confirms the structure predicted
and confirmed by chemical synthesis in the work of Kalvoda et
al. (see Ref. 17 and references cited therein).
The second stage juveniles of H. armigera, P. xylostella, M. domestica, and M. incog-
nita were tested, and LC values were determined on day 6.
5
0
Origin of
LC50a
Insect name
Regression equation
S.E.
thuringiensin
An immune mechanism related to thuringiensin was pro-
posed in this work. A typical type IV secretion system (T4SSs),
which is seldom reported in Gram-positive bacterium, was
revealed adjacent to the thu cluster. Type IV secretion systems
in many Gram-negative pathogens are involved in the delivery
of protein and/or DNA substrates (45). The vir-encoded T4SSs
typically include 12 proteins: VirB1–VirB11 and VirD4 (46).
The VirD4 protein, which is responsible for the recognition and
binding of the substrate, is a key gene in a type IV secretion
␮
g/ml
Strain CT-43
Strain CT-43
Strain CT-43
Strain CT-43
H. armigera
P. xylostella
y ϭ Ϫ0.85314 ϩ 0.66321x 19.3 0.15063
y ϭ 0.06337 ϩ 1.09866x
0.9 0.31076
M. domestica y ϭ Ϫ5.11486 ϩ 3.04741x 47.7 1.46069
M. incognita y ϭ Ϫ3.74985 ϩ 2.66451x 25.5 1.67528
Strain BMB0542 H. armigera
Strain BMB0542 P. xylostella
y ϭ Ϫ1.17306 ϩ 0.85965x 23.2 0.15729
y ϭ Ϫ0.09482 ϩ 1.30222x
1.2 0.23255
Strain BMB0542 M. domestica y ϭ Ϫ2.19691 ϩ 1.29870x 49.2 0.96294
Strain BMB0542 M. incognita y ϭ Ϫ0.62790 ϩ 0.45646x 23.7 0.75605
a
Water was used as the negative control.
structure. Thuringiensin, as a polymer of monosaccharides, system. Our preliminary data showed that the interruption of
possesses asymmetric carbon atoms. Two putative enzymes VirD4 in strain CT-43 resulted in absent production of thuring-
4
encoded by thuB and thuG in the thu cluster, a racemase and an iensin. Moreover, Thu3, which is homologous to an ABC
epimerase, might be involved in the stereochemical inversion of transporter membrane-spanning permease, is possibly
thuringiensin. A unique ACP was also revealed in the thu clus- involved in the secretion of thuringiensin. In this work, the key
ter. There are two classic antibiotic synthesis systems: the non- gene, thuE, was proved to be responsible for thuringiensin
ribosomal peptide synthetase (NRPS) and polyketide synthase phosphorylation. This would happen during the last step of the
(
PKS) systems. The key functional elements responsible for membrane translocation process; the mature thuringiensin
assembly in NRPS are an A domain, a C domain, and a peptidyl would then be excluded by a combination of Thu3 and T4SSs.
carrier protein. In the PKS system, an acyltransferase domain, This mechanism protects the cell from possible damage by
ketoacyl synthase domain, and ACP are required (29, 30). thuringiensin.
Bioinformatic analysis showed that the Thu2 protein encoded
Insecticidal factor (thuringiensin) is encoded on an endoge-
by the thu cluster contains three domains: an A domain (101 nous plasmid related to the evolution of B. thuringiensis. Some
amino acids), a C domain (400 amino acids), and an ACP investigators consider that B. thuringiensis has acquired insec-
domain (60 amino acids) (Table 3), showing similarities to both
the NRPS and PKS systems. This kind of hybrid protein for
antibiotic synthesis has not been previously reported. This
4
X.-Y. Liu, L.-F. Ruan, Z.-F. Hu, D.-H. Peng, S. -Y. Cao, Z.-N. Yu, Y. Liu, J.-S. Zheng,
and M. Sun, unpublished data.
DECEMBER 10, 2010•VOLUME 285•NUMBER 50
JOURNAL OF BIOLOGICAL CHEMISTRY 39199

Genetic Determinants of an Insecticide
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