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Cite this: DOI: 10.1039/c5sc00101c
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Two more pieces of the colibactin genotoxin
puzzle from Escherichia coli show incorporation of
an unusual 1-aminocyclopropanecarboxylic acid
moiety†
Xiaoying Bian,ab Alberto Plaza,a Youming Zhang*b and Rolf Müller*a
Colibactin represents a structurally undefined class of bacterial genotoxin inducing DNA damage and
genomic instability in mammalian cells, thus promoting tumour development and exacerbating
lymphopenia in animal models. The colibactin biosynthetic gene cluster (clb) has been known for ten
years and it encodes a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS)
assembly line. Nevertheless, the final chemical product(s) remain unknown. Previously, we and others
reported several colibactin pathway-related metabolites including N-myristoyl-D-asparagine (1) as part of
a prodrug precursor that is cleaved from the putative precolibactin to form active colibactin by the
peptidase ClbP. Herein, we report two new colibactin pathway-related metabolites (2 and 3) isolated
from a clbP mutant of the probiotic E. coli Nissle 1917 strain. Their structures were established by HRMS
and NMR. Compound 2 shows an additional 4-aminopenatanoic acid moiety with respect to 1, while 3 is
characterized by the presence of an unusual 7-methyl-4-azaspiro[2.4]hept-6-en-5-one residue.
Moreover, we propose the biosynthetic pathway towards both intermediates on the basis of extensive
Received 11th January 2015
Accepted 23rd March 2015
gene inactivation and feeding experiments. The identification of 2 and 3 provides further insight into
DOI: 10.1039/c5sc00101c
colibactin
biosynthesis
including
the
involvement
and
formation
of
a
rare
1aminocyclopropanecarboxylic acid unit. Thus, our work establishes additional steps of the pathway
www.rsc.org/chemicalscience
forming the bacterial genotoxin colibactin.
Introduction
Human gut microbiota play an important role in health and
disease.1,2 As the predominant aero-anaerobic Gram-negative
bacteria in the normal intestinal microbiota, most of the
Escherichia coli are harmless or of benet to the mammalian
host and rarely cause disease. However, some virulent E. coli
have acquired pathogenicity islands and can induce disease.
Recently, it has been shown that many commensal and extraintestinal pathogenic E. coli strains of the B2 phylogenetic
group and closely related Enterobacteriaceae harbour a pks
genomic island that is responsible for the production of a
a
Department of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical
Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI),
Department of Pharmaceutical Biotechnology, Saarland University, Campus C2 3,
66123 Saarbrücken, Germany. E-mail: rolf.mueller@helmholtz-hzi.de; Tel:
+49-681-30270201
b
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of
Microbial Technology, School of Life Science, Shandong University, Zhuzhou Road
168, 266101 Qingdao, P. R. China. E-mail: zhangyouming@sdu.edu.cn; Tel: +86531-88363082
† Electronic supplementary information (ESI) available: Experimental methods,
supplementary tables and gures including information on mutants and NMR
spectra of compounds. See DOI: 10.1039/c5sc00101c
This journal is © The Royal Society of Chemistry 2015
bacterial genotoxin termed colibactin.3,4 Colibactin-producing
E. coli (clb+) induce DNA damage, cell cycle arrest, and genomic
instability of mammalian cells in vitro and in vivo, and thus
promote development of tumours, e.g. colorectal cancer, and
exacerbate lymphopenia in animal models.3,5–11 To reveal the
molecular mechanism underlying the genotoxic activity of colibactin and to design drugs that inhibit its activity, information
regarding colibactin's chemical structure, biosynthetic
assembly and transfer to the host cell is required.12
The colibactin biosynthetic gene cluster (clb) was described
about a decade ago and encodes a hybrid nonribosomal peptide
synthetase (NRPS)/polyketide synthase (PKS) assembly line as
reported for many secondary metabolite biosynthetic pathways.3,13 However, the product(s) of this pathway remain a
mystery which is at least in part due to the fact that the cytopathic activity is contact-dependent and therefore neither the
clb+ bacterial culture supernatant nor the bacterial lysate are
cytopathic.
Although the probiotic commensal strain E. coli Nissle 1917
is widely used for the treatment of intestinal disorders, it
surprisingly also contains a functional clb locus.14 Unexpectedly, the product(s) of the colibactin pathogenicity island here
possess anti-inammatory activity and are required for the
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probiotic activity of E. coli Nissle 1917.15 A possible explanation
for these entirely different activities might be that colibactin
may not be a single compound but rather a class of metabolites
differentially expressed and formed under various culture
conditions. Thereby the clb biosynthetic gene cluster may
encode for the formation of additional compounds that could
mediate the probiotic activity observed in the gut.15 These
intriguing but ambiguous results motivated our efforts to learn
more about the chemical structure and the biosynthesis of
colibactin(s).
Our previous genetic and comparative metabolomic efforts
led to the identication of several colibactin pathway-dependent small molecules including N-myristoyl-D-asparagine (1,
Fig. 1).16 Compound 1 is believed to represent a fragment of a
prodrug precursor cleaved from the putative precolibactin to
form active colibactin by a peptidase ClbP (see Fig. 4Ba).16–19
Such a prodrug activation mechanism was described recently in
several natural product biosynthetic pathways, e.g. xenocoumacins,20 zwittermicins,21,22 and didemnins.23 The in vitro
biochemical characterization of ClbP and in vivo identication
of 1 provided evidence for a prodrug mechanism during colibactin maturation.16,17 More recently, a handful of colibactin
pathway intermediates including 1 and 2 (Fig. 1) were identied
from the wild type and/or clbP mutants of E. coli Nissle 1917 and
E. coli IHE3034 by comparative metabolomics and targeted
structural network analyses.24 However, the structure of 2 was
only proposed based on MS2 fragmentation analysis.
Herein, we identify two new colibactin pathway-related
products (2 and 3) isolated from the clbP mutant of E. coli Nissle
1917. Their structures were established by HRMS and NMR and
their biosynthetic pathway(s) was proposed by a combination of
gene inactivation and feeding experiments revealing further
insight into colibactin formation.
Results and discussion
We applied Red/ET recombineering to construct multiple single
or double gene mutations/deletions in the native colibactin
Fig. 1
Structures of colibactin pathway-related metabolites 1–3.
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producer E. coli Nissle 1917.25,26 Each inactivation of the
respective clb biosynthetic gene by replacement with an antibiotic resistance gene was performed as previously reported (see
Experimental section, Tables S1 and S2, Fig. S1†).16 The gene
clbL encodes a putative amidase that might catalyse the
hydrolysis of an amide in the putative precolibactin,3,24 whereas
clbP encodes a peptidase which cleaves 1 off from precolibactin.16–19 Thus, clbL or clbP single deletion mutants were
constructed to investigate the presence of colibactin-related
products. Mutants and wild type strains were cultured, extracted, and analysed by LC-MS. We observed that the yield of the
precursor fragment N-myristoyl-D-asparagine (1) is greatly
decreased in the clbP mutant but slightly increased in the clbL
single mutant (Fig. S2†). These results were consistent with our
previous reports.16
Intriguingly, the LC-MS chromatograms of the clbP mutant
showed additional metabolites when compared to the wild type
and the clbL mutant (Fig. S2†). To shed light on their molecular
structures, EtOAc extracts prepared from 20 L cultures of E. coli
Nissle 1917 DclbP were chromatographed by sephadex LH-20
followed by reversed-phase HPLC to yield compounds 2 (4.9 mg)
and 3 (0.7 mg).
The molecular formula of compound 2 was determined to be
C23H43N3O5 by HR-ESI-MS (m/z 442.3273 [M + H]+). Its structure
was characterized by 2D NMR and HRMS experiments and it
exhibits an additional 4-aminopentanoic acid (App) residue in
comparison to 1 (Fig. 1, NMR data see Table S3, Fig. S3–S6†).
The structure of 2 was rst proposed by Vizcaino et al. on the
basis of MS fragmentation analysis but the compound was not
isolated or characterized by NMR.24 Our structural elucidation
of 2 supports the hypothesis that the NRPS module (C–A–T,
Fig. 4Ba) found on ClbB activates alanine and incorporates this
residue into an N-myristoyl-D-asparagine precursor (1) as
formed by ClbN (Fig. 4Ba). This process is followed by extension
of the intermediate by the PKS module (KS–AT–KR–DH–ER–T)
on ClbB by one malonyl-CoA unit including b-carbon processing (Fig. 4Ba), and subsequent hydrolytic release of 2 by a
currently unknown mechanism. This hydrolysis pattern is also
described in other colibactin pathway intermediates.24
HR-ESI-MS of 3 displayed an [M + H]+ ion peak at m/z
547.3853 consistent with the molecular formula C30H50N4O5. A
mass consistent with 3 was detected by MS before,24 but it was
never isolated nor structurally characterized. Its 1H NMR spectrum in comparison to that of 2 showed a few additional signals
including an exchangeable NH proton at d 8.53 (1H, s) and a
methyl vinyl at d 1.97 (3H, s) (see Table S4†). Comparison of the
HSQC spectrum of 3 to that of 2 indicated that the signals
corresponding to the methylene C-24-Apn (dH 2.14, 2.17, dC 30.4)
in 2 were shied downeld in 3 (dH 2.92, 2.83; dC 38.5). Additionally, analysis of 13C NMR, HSQC, and DET 135 spectra of 3
indicated the presence of two equivalent methylene groups (dH
1.49, dC 13.6, and dH 1.41, dC 13.7) that were absent in 2. A
detailed analysis of the 2D NMR data (HSQC, HMBC, DQFCOSY, ROESY) of 3 showed that the C-terminus of the fragment
N-myristoyl-asparagine was connected to a 4-aminopentanoyl
(4-Apn) residue. The structure of the remaining C7H8NO fragment was assembled as follows: HMBC correlations from the
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methyl signal at d 1.97 (Me-7Azh) to the quaternary carbon
resonance at d 45.3 (C-3Azh) and to the olenic carbon resonances at d 169.5 (C-7Azh) and 128.9 (C-6Azh), together with
HMBC correlations from the amide proton at d 8.53 (NH-4Azh) to
C-3Azh, C-6Azh, and to the carbonyl resonance at d 169.2 (C-5Azh),
determined the presence of a 4-methyl-1,5-dihydro-2H-pyrrol-2one ring (see Fig. 2). Furthermore, long-range correlations from
the methylene protons at d 1.49 (H-1Azh) and 1.41 (H-2Azh) to C3Azh and C-7Azh, COSY correlations between H-1Azh and H-2Azh,
together with ROESY correlations from H-1Azh to Me-7Azh and
from H-2Azh to H-4Azh indicated that a cyclopropane was spiro
fused to the dihydropyrrolone ring forming a 7-methyl-4-azaspiro[2.4]hept-6-en-5-one residue (Azh). Finally, connectivity
between Azh and 4-Apn residues was deduced from four-bond
HMBC correlations from Me-7Azh and NH-4Azh to the ketone
carbon at d 197.7 (C-14-Apn) thereby completing the structure of 3
as depicted in Fig. 1 (NMR spectra see Fig. S7–S15†). Tandem
mass spectrometry provided further evidence to support the
structure of 3 (Fig. S16†). MS2 fragmentation of the ion peak at
m/z 547 displayed ions at m/z 530 [M + H–NH3]+, 320 [M + H–
NH2–Myr]+, and 205 [M + H–Myr–Asn–NH2]+. Thus the MS2
fragmentation patterns in complete agreement with the structure of 3 determined by NMR.
The absolute conguration of D-Asn was determined by MS
detected chromatographic comparison with appropriate amino
acid standards of the acid hydrolysate of 2 and 3 aer derivatizing with L- and D-FDLA (L-uoro-2,4-dinitro-phenyl-5-L/D-leucinamide).27,28 Similarly, the absolute conguration of C-4 of
App in 2 was established as S. The S conguration of C-4Apn in 3
was assumed as S on the basis the structural and NMR data
similarities to those of 2.
To investigate the question which gene(s) from the clb
pathway are responsible for biosynthesis of the 7-methyl-4azaspiro[2.4]hept-6-en-5-one moiety in 3, eleven mutations were
performed on the clbP mutant of E. coli Nissle 1917 by one more
step of Red/ET recombination. Each target gene was replaced by
a kanamycin resistance gene (KmR) for clbC to clbK and clbO,
whereas inactivation of clbL was performed by replacement with
a spectinomycin resistance gene (SpectR) (Table S1†). All
mutants were veried by colony PCR (data now shown). LC-MS
analyses of the clbP mutant and the double mutants showed
that inactivation of clbC, clbH or clbI resulted in abolishment of
the production of 3 indicating that these three genes were
involved in the biosynthesis of 3 (Fig. 3). On the other hand,
gene inactivation of each of clbD, clbE, clbF, clbG, clbJ, clbK or
clbO had no signicant effect on the production of 3 (Fig. 3). The
Fig. 2
NMR-based connectivity of the fragments of 3.
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clbL/clbP double mutant showed a great decrease of 3 implying
that the putative peptide amidase ClbL might be involved in
hydrolysis of 3 from a larger precolibactin molecule. Unfortunately, we were not able to detect any larger colibactin-pathway
dependent metabolites in these mutants.
According to the gene inactivation experiments and bioinformatics analysis of the modular organization of the
pathway, a proposal for the biosynthesis of 3 is shown in Fig. 4B.
Initially the presence of the 7-methyl-4-azaspiro[2.4]hept-6-en-5one residue in 3 seemed difficult to rationalize biosynthetically.
However, when taking into account the possibility of incorporation of a nonproteinogenic 1-aminocyclopropane-carboxylic
Fig. 3 UPLC-HRMS analysis of the E. coli Nissle double mutants.
Shown is a base peak chromatogram (BPC) m/z for 442.3 [M + H]+ and
547.4 [M + H]+ of compounds 2 and 3, respectively.
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Fig. 4 (A) HR-ESI-MS spectra of 3 obtained from feeding experiments. (a) Control; (b) after feeding of L-[U-13C, 15N]methionine (only four
carbons and one nitrogen are found in the final product, Fig. S17†); (c) after feeding of L-[methyl-2H3]methionine (no D incorporated) showing
that the aminobutyryl moiety of methionine (without S-methyl) is incorporated into the final product 3. (B) (a) Proposed biosynthetic pathway of
colibactin up to intermediate 4 involving 1 and 2 tethered to the assembly line. After release of 4 and decarboxylation of the b-keto-acid, 3 is
formed by intramolecular cyclization analogous to Dieckmann condensation. (b) A hypothesis for ACC biosynthesis and then loading on ClbH; (c)
alternative hypothesis for loading of methionine onto ClbH and then conversion to ACC on the T domain catalyzed by A2 domain. (C)
Biosynthesis of ACC moiety in other organisms. (a) Biosynthesis of ACC in the ethylene pathway in higher plants;30,31 (b) incorporation of L-[U-13C]
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acid (ACC) residue in conjunction with hydrolysis and possible
shunt product formation the following scenario was developed:
compound 2 undergoes extension by one PKS module (encoded
by ClbC or ClbI; KS–AT–T) to incorporate one malonyl-CoA unit,
one NRPS module (ClbH, A1–C–A2–T) is needed for incorporation of the unusual ACC, and one more PKS module (encoded by
ClbI or ClbC; KS–AT–T) is needed to incorporate an additional
malonyl-CoA unit eventually giving rise to 4 attached to the
biosynthetic assembly line (Fig. 4Ba). We hypothesize that 4
might be hydrolysed enzymatically (e.g. TE catalysed, like 2) to
form the corresponding free acid undergoing decarboxylation
to build 5, which in turn can engage in an intramolecular
cyclization analogous to a Dieckmann condensation forming
the dihydropyrrolone ring found in 3 (Fig. 4Ba). A similar Dieckmann cyclization catalyzed by a Dieckmann cyclization
domain (DKC) was proposed to form the pyrrolone ring in
pyranonigrin E,29 but no homologous DKC domain was found
in the clb gene cluster leading to the speculation that the
condensation in 3 may occur spontaneously.
The ACC moiety is quite an unusual moiety in bacterial
secondary metabolites, and to the best of our knowledge this is
the rst occurrence in an E. coli natural product. However, it is
well known from plant metabolites such as in the biosynthesis
of the higher plant hormone ethylene where ACC serves as key
intermediate,30 ACC is synthesized from S-adenosylmethionine
(SAM) via the pyridoxal phosphate (PLP)-dependent ACC synthase catalysing a g-elimination reaction (Fig. 4Ca).30,31 The
ansa-bridged cytotrienin from the soil bacterium Streptomyces
sp. RK95-74 also contains an ACC moiety32 and stable isotope
feeding experiments showed that the ACC moiety in this
producer is also derived from L-methionine (Fig. 4Cb),33 but the
cytotrienin biosynthetic gene cluster has not been published
yet. An alternative strategy was described for the enzymatic
formation of the ethyl-substituted ACC moiety coronamic acid
(CMA) in the phytotoxin coronatine produced by Pseudomonas
syringae strains. Here, biosynthesis follows a cryptic chlorination pathway relying on an aminoacyl thioester intermediate
involving ve dedicated proteins CmaABCDE (Fig. 4Cc).34 CmaB
and CmaC execute g-halogenation of L-allo-isoleucine followed
by intramolecular g-elimination to form the cyclopropyl
ring.34,35 As no homologues of ACC synthase genes or coronamic
acid biosynthesis genes were found in the colibactin gene
cluster, we performed feeding experiments to investigate the
hypothetical biosynthesis of ACC in 3. Methionine labelled by
stable isotopes was added to the growth medium resulting in
the following incorporation patterns in 3: feeding of L-[U-13C,
15
N]methionine gave m/z 552.3959 [M + H]+ (5 amu more than
547.3853) corresponding to the chemical formula of 3-13C4, 15N
(C2613C4H50N315NO5, m/z 552.3959 [M + H]+) while feeding of L[methyl-2H3]methionine did not result in deuteration of 3
(Fig. 4A, Fig. S17A†). Taken together, these data indicated that
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the aminobutyryl moiety (accounting for four carbons and one
nitrogen without the S-methyl carbon) of L-methionine was
introduced into the ACC unit of 3 which suggests that ACC
originates from L-methionine in E. coli (Fig. 4AB, Fig. S17B†).
Nonetheless, the L-methionine-ACC pathway including ACC
synthase has to the best of our knowledge not been detected in
prokaryotic microorganisms.33,36 As stated above we did not nd
a homolog of the ACC synthase gene in the E. coli Nissle 1917 clb
gene cluster. An additional search for homologs in the genome
of E. coli Nissle 1917 (GenBank: CP007799.1) using ACC synthases from apple (Malus domestica) (GenBank: AAB68617.1,
PDB: 1B8G)37 and from Penicillium citrinum (GenBank:
AB038512)38 as queries was performed using BlastP.39 This
resulted in the identication of some homologs belonging to
the family of PLP-dependent aminotransferases/transaminases/
lyases exhibiting low identities on the amino acid level (<30%,
Table S5†). As either one of these proteins or any other ORF of
unknown function encoded in the E. coli genome may be
responsible for the conversion of SAM to ACC, clearly further in
vivo gene inactivation and in vitro biochemical experiments are
required to study the formation of ACC in E. coli.
Intriguingly, the NRPS module encoded on ClbH hypothesized to be in charge of ACC incorporation contains two A
domains; the rst A domain (A1) is predicted to activate a serine
residue according to the specicity-conferring code of adenylation domains in NRPSs (Table S6†).40,41 A subsequent feeding
experiment employing L-[2,3,3-2H3]serine led to an isotopic
pattern compatible with incomplete incorporation of serine
into 2 and 3 (Fig. S18A†). This nding may be explained by
serine degradation via pyruvate to acetyl-CoA and malonyl-CoA
for incorporation into 2 and 3 by the PKS or fatty acid synthetase
(FAS) which may be responsible for the fatty acid side chain
(Fig. S18B†). Thus, we assume that the A1 domain is not
required for serine activation but may play a role in ACC
formation as discussed below. The second A domain (A2) of
ClbH is predicted to be specic for a hydrophobic/aromatic
residue (phenyalanine/tryptophan/tyrosine/valine/leucine) or
may represent an A domain with novel specicity (Table S6†).
Feeding of L-[U-2H]valine, L-[U-2H]leucine, L-[U-13C, 15N]phenyalanine and L-[ring-2H4]tyrosine did not show any incorporation into 2 or 3 (data not shown). The nonribosomal code of
the A2 domain of ClbH showed that the strictly conserved
aspartic acid (D235) interacting with the a-amino group of
amino acids in peptide synthetases is mutated to alanine (Table
S6†), indicating that the substrate of the A2 domain may not
have an a-amino group.42 Thus, according to the chemical
structure of 3, we speculate that the A1 domain may load an ACC
unit (Fig. 4Bb) or methionine (Fig. 4Bc) onto ClbH. The former
hypothesis regarding loading of ACC by the A1 domain would
lead to the requirement of ACC biosynthesis in E. coli and make
the A2 domain ‘superuous’ (Fig. 4Bb). The latter hypothesis
methionine into cytotrienin A showed that the ACC unit originates from methionine in Streptomyces sp. RK95-74;33 (c) biosynthetic pathway of
the coronamic acid (CMA) moiety of coronatine in P. syringae strains.34,35 C, condensation domain; A, adenylation domain; T, thiolation domain;
E, epimerization domain; KS, ketosynthase domain; AT, acyltransferase domain; DH, dehydratase domain; KR, ketoreductase domain; ER, enoyl
reductase domain; SAM, S-adenosylmethionine; ACC, 1-aminocyclopropanecarboxylic acid.
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postulates that ClbH might function as a SAM synthetase aer
methionine loading by the A1 domain to the T domain of ClbH.
Subsequently, ClbH may also function as an ACC synthase
(Fig. 4Bc). According to this hypothesis the SAM synthetase and
ACC synthase activity may be encoded within the NRPS ClbH. In
light of this hypothesis the biosynthetic function of each
domain found on ClbH (A1–C–A2–T) during loading, modication and ACC incorporation needs to be characterized in
upcoming experiments in detail. To achieve this goal the whole
protein and/or the respective domains need to be expressed in
their active forms, but this is a goal for future experiments.
We measured the in vitro bacterial growth inhibitory activity of
2 and 3, as 1 has been reported to exhibit mild growth inhibitory
activity against Bacillus subtilis NCIB 3610.24 Both, 2 and 3, also
showed weak growth inhibitory activities (EC50 30–70 mg mL 1)
against E. coli (TolC-decient) and B. subtilis DSM-10 (Fig. S19†).
According to our gene inactivation experiments, further
assembly of precolibactin and colibactin most likely requires
additional proteins encoded on the clb pathogenicity island.
Whereas this study provides evidence on the function of ClbC,
ClbH and ClbI in vivo on route to intermediate 4, it does not
determine any activity for ClbDEFG, ClbJ, ClbK and ClbO which
are clearly not required for the formation of 3 according to the
mutagenesis experiments reported here. Consequently, further
experiments in vitro and/or in vivo are required not only to
determine the structure of colibactin and precolibactin but also
to decipher each biosynthetic step in greater detail.
Conclusions
Colibactin represents a chemically unknown class of bacterial
genotoxin present in most species of the B2 group of E. coli. Its
presence in the probiotic E. coli Nissle 1917 not only contributes
to probiotic activity but also induces chromosomal instability of
mammalian cells. With the aim to eventually determine the
structure of colibactin, two colibactin pathway-related metabolites were identied by HRMS and NMR analyses from E. coli
Nissle 1917 aer extensive mutagenesis studies. Compound 3 is
a linear lipopeptide containing a novel 7-methyl-4-azaspiro[2.4]
hept-6-en-5-one (Azh) residue and its biosynthetic pathway was
proposed based on gene inactivation, in silico analysis of
biosynthetic pathway proteins and feeding experiments.
Surprisingly, the latter indicated that the uncommon nonproteinogenic amino acid ACC, which is involved in the
formation of Azh, might be derived from L-methionine in E. coli
and possible scenarios for its biosynthesis during colibactin
assembly are discussed. The structural elucidation and the
biosynthetic proposal regarding these two colibactin pathwaydependent biosynthetic intermediates (2 and 3) provides additional knowledge required on route to eventually establish the
structure of colibactin and thus deepens our understanding of
this intriguing probiotic and genotoxic compound class.
Acknowledgements
The authors thank Dr Thomas Hoffmann, Suvd Nadmid and Fu
Yan from R.M.'s laboratory for HRMS measurements and
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experimental assistance. Research in the laboratory of R.M. was
funded by the Deutsche Forschungsgemeinscha (DFG) and
the Bundesministerium für Bildung und Forschung (BMBF).
Y.Z. is supported by Program for the Recruitment Program of
Global Experts in Shandong University. The authors acknowledge Dr Kevin Sours's help in proofreading this manuscript.
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