Bait

Limonene

Overview

We transferred plasmids into E. coli BL21 (DE3) to make it express a normally plant-expressed monoterpene: limonene. The engineered E. coli can thus produce limonene. There are researches showing that limonene can be an attractant to nematodes^[1][2], so limonene is used to attract plant parasitic nematodes in our project.

Structure

Terpenoids, which has more than forty thousand kinds of chemicals, is the largest family of natural products^[3]. Limonene is a kind of valuable terpenoids (isoprenoid) normally expressed in plants, especially in citrus and mentha plants. It has two enantiomers in natural source, d-limonene and l-limonene (Fig. 1), which have opposite optical activities (dextrogyrate for d-limonene and levogyrate for l-limonene). In our life, limonene has always been used as a flavoring or fragrance with aroma value. It is also used in the production of several commodity chemicals and medicinal compounds^[4]. In our research, limonene is expressed as a kind of bait to attract plant-parasitic nematodes as its special flavor could draw nematodes’attention.

Chemotaxis

There are researches showing that when plants are infected by herbivore insects, they will secrete many kinds of volatiles to induce nematodes that are harmful to these predators. For example, Tylenchulus semipenetrans are more attracted to Citrus spp. roots that infected by weevil larvae than uninfected plants^[1], which proves that the terpene secreted by the infected plants could attract some kinds of parasitic nematodes. This proves that many terpenes can attract plant-parasitic nematodes in natural circumstances, which probably serves as a self-defending mechanism.

Some researches showed that limonene is a kind of volatile that attracts nematodes such as Tylenchulus semipenetrans^[2]. As a result, we tried to use limonene to achieve our aim to attract some plant-parasitic nematodes.

Biosynthesis Pathways

Limonene is a kind of terpenoids (isoprenoids), and the precursor of limonene is geranyl pyrophosphate (GPP). GPP is synthesized by Isopentenyl diphosphate (IPP) and IPP’s isomer dimethylallyl diphosphate (DMAPP). IPP and DMAPP are the two essential building blocks to synthesize all terpenoids^[3]. The synthesis pathways of IPP and DMAPP in most eukaryotes and prokaryotes are slightly different. While MVA pathway occurs^[5] in most eukaryotes (Plants use both pathways), MEP pathway (Fig. 2) occurs in most bacteria including Escherichia coli.

Based on the synthesis of IPP and DMAPP in the pathways mentioned above, GPP synthase (GPPS) catalyzes the condensation between IPP and DMAPP to synthesize GPP, and then Limonene synthase (LS) catalyzes the intramolecular cyclization of GPP to synthesize limonene.

In E. coli, the levels of intracellular GPP expression are very limited, which will hinder the expression of limonene[4]. However, transferring the entire MEV pathway into E. coli may increase the burden for E. coli, so in our research, we transferred both GPPS gene and LS gene into the E. coli BL21 (DE3) to improve limonene expression.

Project

Design

In our project we decided to use E. coli, MEP pathway to express limonene. We cloned the GPPS and LS genes (for d-limonene and l-limonene respectively) into the pGEX-4T-1 plasmid. The GPPS gene was from Abies grandis (Genbank: AF513112), and d&l-limonene synthase genes were from Citrus unshiu (GenBank: AB110636.1) and Mentha spicata (GenBank: L13459) respectively. Then we transferred the plasmids into E. coli BL21 (DE3) to express the synthases. After that, we used the ultrasonication method to make E. coli homogenate and did SDS-PAGE analysis to identify the expression of the synthases.

After the successful expression of the synthases, we did GC-MS to identify the expression of limonene. As the limonene was expressed, we tried to verify the limonene’s attractivity to nematodes.

Verification

We conducted the following experiments^[6] to verify whether limonene can attract nematodes.

We divided the NGM medium dish into two even parts and drew a circle of 1cm diameter at the center of the plate. We put two small pieces of circular filter paper 2.5cm from the center of the circle (Fig. 3).

Our verification experiment was divided into two types of groups -- the experimental groups and the control groups. First we dilute the limonene by DMSO and the final concentration of the limonene is 5%. We then add 5μL 5% limonene (T) and DMSO (C) respectively at the two small circular filters in the experimental groups(Fig. 4-a). As for the control groups, both of the two small circular filters are added 5μL DMSO (Fig. 4-b) to eliminate the influence of the position of the nematodes. We also add 5 μL DMSO and M9 saline respectively (Fig. 4-c) at the filters in order to eliminate the influence of the attraction or the exclusion of DMSO. Later we add 30μL suspension of the nematodes at the center of the plate and cultivate them in the incubator under 20℃ for 1 hour. After the nematodes move dispersedly, we put the plate into the 4℃ refrigerator for 1 hour until the move of the nematodes slows down.

We observed the distribution of the nematodes and counted the number of the nematodes. After that, we did a statistic analysis to confirm the attractive function of limonene towards the nematodes. If in the verification experiment limonene attracts nematodes, the module we build can be used to attract nematodes successfully.

Killer

Bace 16

Backgrounds

• Bacillus nematocida

A novel bacterial strain named Bacillus nematocida has been isolated from soil in Yunnan, China by Huang Xiaowei in 2005. It’s an endospore-forming and Gram-positive bacterium. It can lure nematodes by emitting potent volatile organic compounds, and once the bacteria enter the intestine of nematodes, it can secrete proteases with broad substrate ranges but preferentially target essential intestinal proteins, leading to nematode death. Up till now, the research group has found that B. nematocida has remarkable nematotoxic activity against Panagrellus redivivus, which is a kind of free-living nematode, and Bursaphelenchus xylophilus, which are parasitic on the xylem of the pines.



• The processes for B. nematocida to kill nematodes

After Bacillus nematocida was isolated and testified for its nematotoxicity, the mechanism of the infection process of this strain has been explored, and its pathogenesis against nematodes is said to be a Trojan horse mechanism.

First, B. nematocida has a simple but effective strategy for attracting nematodes, it can use a mixture of VOCs as the lure in a kill-from-within nematocidal strategy.

Once inside the worm, the bacteria colonize the intestinal tract of the C. elegans and secretes extracellular proteases that kill the nematodes primarily through damage to the intestine of its host.







A serine protease bace16 was first reported as a pathogenic factor against nematodes, whose accession number is AY708655. It was identified by methods such as ammonium sulfate precipitation. ^[7] In vitro assay demonstrated that the recombinant protease Bace16 expressed in Escherichia coli presented a nematotoxic activity, and it has been verified by experiments that Bace16 has the ability to degrade a nematode cuticle, leading to the nematode’s death.^[9] To our knowledge, the nematode cuticle mainly consists of keratin, collagen, and fibers, which made it a rigid but flexible multilayered extracellular exoskeleton and a necessary barrier to prevent nematodes from damages.^[8] So Bace16 could be considered as a core component of this project to kill the nematode.




Bace 16

Structure

The molecular mass of a mature Bace16 protein is about 28kDa. And the protein has 275 residues, with a catalytic triad center containing His, Asp, and Ser residues and two calcium binding sites for stabilizing the three-dimensional structure. Characterization of the purified protease revealed the optimum activity of Bace16 is at pH10, 50℃. The deduced protein also contains a presequence signal peptide of 30 amino acids and a propeptide of 77 amino acids. The presequence signal peptide directs the secretion of subtilisin from the interior of cells, while the propeptide functions as a chaperon to facilitate the folding process of the active protease.^[10] By sequence alignment, researchers found that the whole amino acid residues of Bace16 showed only around 33% sequence identity between cuticle-degrading proteases produced by several fungi such as Beauveria bassiana, Cordyceps brongniartii, Metarhizium anisopliae, etc. And only several residues near the conserved catalytic triad that are probably essential for activity of cuticle degradation are in consensus in all the proteases. However, Bace16 and other subtilisins produced by several bacteria are found 62-98% homologous, much higher than relevant fungi. Besides, the deduced amino acids of Bace16 has 98% identity with subtilisin BPN’ from B. amyloliquefaciens. So the enzyme probably belongs to the subtilisin family of enzymes, subtilisin BPN’ (EC 3.4.21.14, also known as Novo, or Nagarse), based on the alignment of the amino acid sequence in NCBI.^[11]

Function

According to relevant research, Bace16 is the key reason for the high infection toxicity of Bacillus nematocida to Panagrellus redivivus. Bioassay with purified Bace16 has showed that 90% of the nematodes could be killed within 24 h at the concentration of 1.79 μg/ml; after 48 h, all of the tested nematodes were almost killed and degraded.^[11] Researchers found that recombinant protease rm-Bace16(whose molecular weight is 34kDa) expressed in Escherichia coli also presented a nematotoxic activity. And both Bace16 and rm-Bace16 could degrade a broad range of substrates including casein, denatured collagen, and nematode cuticle. In addition, the corresponding extract of the B. nematocida strain with a bace16 knockout mutant decreased significantly proteolytic activity and nematotoxic activity compared with both rm-Bace16 and the wild-type strain under various physiological conditions. ^[9] And the table below compares the proteolytic activities between wild strain, recombinant strain and bace16 mutant.



Due to the complicated renaturation process of recombinant protein, the conformation of rm-Bace16 has some differences, so the enzyme activities are always lower than Bace16, but it still has significant nematotoxity comparing to Bace16 extract. And the research has set a precedent of expressing Bace16 in engineering bacteria for us to refer.

Why we choose E. coli expressing bace16 to kill nematodes ?

1. The needs for biocontrol agents

Plant-parasitic nematodes cause serious losses to a variety of agricultural crops worldwide. Since the traditional methods based on the use of nematocides and antihelminthic drugs are associated with major environmental and health concerns, the development of biocontrol agents of control nematodes is of major importance ^[12].

2. Bacteria are easy to culture

Bacteria are suitable for their rapid culturing and production compared with fungi, which has been used extensively as bioinsecticides against nematodes in soil. And E. coli is especially easy to culture and conduct gene manipulation in the lab stage.

3. Protease is widely used for killing nematodes

A common group of virulence factors shared among bacterial pathogens are the proteases, and the primary function of proteases in the bacterial kingdom is to provide a source of free amino acids for bacterial survival and growth, but there is accumulating evidence that proteases also play a role in bacterial pathogenesis during the invasion and destruction of host tissues. The prevalent view regarding the mode of action of the extracellular proteases during nematode infection is that these proteases participate in cuticle penetration. So we choose a serine protease Bace16 in our project to kill nematodes, meanwhile, protease is easy to express in bacteria.

Design

The DNA sequence of bace16 precursor is from NCBI(GenBank: AY708655.1), taking the fold and secretion of Bace16 into consideration, the presequence signal peptide of 30 amino acids and propeptide of 77 amino acids are retained before the mature peptide. And in order to produce this toxin, a pBAD promoter(BBa_K206000), which is said to be suitable for toxic protein expression is add upstream the functional gene. bace16-pSB1C3 plasmid is transformed into E. coli strain BW25113 to express and purify Bace16 protein. Then nematocidal activity test is conducted. (protocol).

rMpL

Introduction of rMpL

MPL is a novel β-trefoil lectin isolated from parasol mushroom（Macrolepiota procera）, the general function of which is to protect the plants themselves from the predators, such as stopping or even killing the predators. It is also thought to be a survival strategy that the plants have evolved to protect themselves. A specific feature of these defensive fruiting body lectins is their cytoplasmic localization. Lectins appear to be capable of distinguishing between self and nonself on the basis of the interspecies variation of glycosylation patterns. The mechanism of the lectin is that it can specifically combine with the glycosyl of the predators’ intestine in order to destroy the digestive system^[13]. β-trefoil lectins CNL, CCL2, MOA and SSA are nematotoxic, and the nematotoxicity has been shown to be dependent on specific binding of glycoconjugates displayed in the organism’s intestines. MpL also has the same effect. According to Jerica’s research, MPL can specifically bind with glycan of the nematodes^[13], which therefore is able to stop the growth of the nematodes from L1 phase to adults.

The three-dimensional structure and carbohydrate-binding properties

rMpL has a typical β-trefoil fold, consisting of α-,β- and γ- repeats. （图6B）The β-trefoil fold seems like a tree, which has a short trunk(in red) and an expanded crown(in blue).(fig 6A) The trunk is a six stranded β-barrel composed of β-strands(β1,β4,β5,β8,β9,β12).And the crown is constituted by the other three pairs of β-strands(β2,β3,β6,β7,β10andβ11) and its connective loops. rMpL has a typical β-trefoil fold, consisting of α-,β- and γ- repeats. （图6B）The β-trefoil fold seems like a tree, which has a short trunk(in red) and an expanded crown(in blue).(fig 6A) The trunk is a six stranded β-barrel composed of β-strands(β1,β4,β5,β8,β9,β12).And the crown is constituted by the other three pairs of β-strands(β2,β3,β6,β7,β10andβ11) and its connective loops.

Nematotoxicity of rMpL

According to the related literature，rMpL is toxic to C.elegans alrvae. Only 20% of rMpL-expressing E. coli is sufficient to inhibit the development of most N2 larvae into adulthood.（图7A） The potential glycan targets in the nematode is ‘GalFuc’, for 30% of the worms developed to adulthood when nematodes lacks additional modifications in the antennae of N-glycans, and 20% of worms reach adulthood when nematodes lack the ‘GalFuc’ epitope in the N-glycan core, compared with almost all the nematodes which cannot reach to L4-adults with normal N-Glycans.

Design

In Macrolepiota procera, the mpl gene is 791 bp long (including start and stop codons) which is composed of four exons and three introns. By knocking out the introns, we will optimize this gene which comes from eukaryotic cells so that it can express efficiently in E. coli. Furthermore, we will add the pBAD promotor (K206000) induced by L- arabinose as well as the RBS (B0034) in the upstream of rmpl gene sequence, for pBAD promoter is suitable for the expression of the toxin.(参考文献) At the same time, the Xho I restrict enzyme site will also be added between the RBS and the initiation codon, which will give us a lot of convenience to replace different promoter with different intensity.

After acquiring the recombinant vector successfully, we will firstly transfer the vector into the E.coli DH5α to clone and preserve the plasmid, and then we will transfer the recombinant vector into the E. coli BW25113, which are the competent cells in order to express the rMpL protein. Then we will design a series of concentrations of the arabinose to induce the expression of the rMpL. Next we will centrifuge the E. coli BW25113 and use ultrasonication to break the cells. Finally we will analyze the protein both in homogenate and supernate by SDS/PAGE.

When the expression of the rMpL is detected by SDS-PAGE, we will do the replication experiment from the literature, which is to verify whether it had the effect of stopping the growth of the nematodes. Firstly, mix the recombinant strains and the OP50 according to a certain proportion; then put the bacterial suspension and the synchronized nematodes into the 96-well plate at a special proportion and cultivate for a time. However, this method has two disadvantages. One is that the nematodes will preferentially select the OP50 as their food. In this case, most of rMpL will not be eaten by the nematodes, so the nematodes can become L4- adult successfully. As for the other advantage, when compared to the solid medium, using the liquid medium to cultivate the nematodes will cost a lot more time, and it it also difficult for us to operate. Therefore, the experiment will be improved. We will plate 100μL the recombinant E. coli which can express the rMpL with chloramphenicol(35μg(/mL) on the NGM medium to cultivate under 37℃ for 12 hours. Then we will inoculate the eggs at the center of the plate. The growth condition of the nematodes will be observed after 24 hours.

In conclusion, we plan to link the rmpl gene to our photo induced bidirectional transcription system in the future, which will achieve the bidirection expression of the toxic protein and the baits.

Suicide

Background

In nature, some bacterial cells sense the density of their population through a sophisticated cell–cell communication system. When both the cell density reaches a specific threshold and the signal molecules accumulate to a certain concentration, they will do a series of measures to regulate their group behavior. For example, they can trigger the expression of certain genes to induce apoptosis so the population density can be controlled. Thus the amount of population will keep dynamic balance. The phenomenon above is called quorum sensing.

Referring the research did by Lingchong You^[13] and using the quorum sensing principle, we construct a genetic circuit which can control the population density of engineering bacteria artificially.




AHL luxI luxR MazF

N-acyl-homoserine lactone(AHL) is a signalling molecule found in Vibrio fischeri which is small and diffusible. Luxl, which is the expression product of the luxl gene, can catalyze the synthesis of the AHL inside the cell at a certain rate. AHL then diffuses outside the cell. And the expression product of luxR is LuxR. At sufficiently high concentrations, it binds with AHL to become a LuxR-AHL complex and it can also activate the LuxR transcriptional regulator, which in turn induces the expression of a killer gene (E) under the control of a luxI promoter (pluxI)15). But lux pR does not have promoter activity without LuxR-AHL complex.

The toxic protein MazF expressed by mazF gene is a sequence-specific endoribonuclease. It can freely and specifically shear the ACA sequence of mRNA to inhibit the synthesis of the protein. So the growth of cells will stop.

Design

Upstream: The Production of LuxR and AHL

We link the RBS, the constitutive promoter(BBa_J23100), LuxR(BBa_C0062) and LuxI(BBa_C0061) in order. So during the growth of the bacteria, they will producce the AHL and LuxR constantly.

Downstream: The Starting of the luxpR and Expression of the Toxic Protein MazF

When the concentration of the AHL reaches a specific threshold, it will bind with LuxR to become a LuxR-AHL complex to activate the luxpR promoter. And then the expression of the mazF gene in downstream will start. The MazF will lead to the apoptosis. So the population density will decrease.



• [1] Jared G. Ali, Hans T. Alborn and Lukasz L. Stelinski, 2011. Constitutive and induced subterranean plant volatiles attract both entomopathogenic and plant parasitic nematodes. Journal of Ecology. 99, 26-35.
• [2]Ali J G, Alborn H T, Stelinski L L. Subterranean Herbivore-induced Volatiles Released by Citrus Roots upon Feeding by Diaprepes abbreviatus Recruit Entomopathogenic Nematodes[J]. Journal of Chemical Ecology, 2010, 36(4):361-8.
• [3] Du et al.: Enhanced limonene production by optimizing the expression of limonene biosynthesis and MEP pathway genes in E. coli. Bioresources and Bioprocessing 2014 1:10.
• [4] Jorge Alonso-Gutierrez, Rossana Chan, Tanveer S. Batth, Paul D. Adams, Jay D. Keasling, Christopher J. Petzold, Taek Soon Lee, 2013. Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metabolic Engineering. 19, 33-41
• [5] Wolfgang Eisenreich, Felix Rohdich and Adelbert Bacher, 2001. Deoxyxylulose phosphate pathway to terpenoids. Trends in Plant Science. 6, 78-84.
• [6] Margie O, Palmer C, Chin-Sang I. C. elegans chemotaxis assay.[J]. Journal of Visualized Experiments, 2013, (74):e50069-e50069.
• [7] Huang X W, Niu Q H, Zhou W, et al. Bacillus nematocida sp. nov., a novel bacterial strain with nematotoxic activity isolated from soil in Yunnan, China[J]. Systematic and applied microbiology, 2005, 28(4): 323-327.
• [8] Maizels R M, Blaxter M L, Selkirk M E. Forms and functions of nematode surfaces[J]. Experimental parasitology, 1993, 77(3): 380-384.
• [9] Niu Q, Huang X, Zhang L, et al. Functional identification of the gene bace16 from nematophagous bacterium Bacillus nematocida[J]. Applied microbiology and biotechnology, 2007, 75(1): 141-148.
• [10] Day R M, Thalhauser C J, Sudmeier J L, et al. Tautomerism, acid‐base equilibria, and H‐bonding of the six histidines in subtilisin BPN′ by NMR[J]. Protein Science, 2003, 12(4): 794-810.
• [11] Qiuhong N, Xiaowei H, Baoyu T, et al. Bacillus sp. B16 kills nematodes with a serine protease identified as a pathogenic factor[J]. Applied microbiology and biotechnology, 2006, 69(6): 722-730.
• [12] You L, Cox R S, Weiss R, et al. Programmed population control by cell–cell communication and regulated killing[J]. Nature, 2004, 428(6985): 868-871.
• [13] Zhang C, Ye B C. Real-time measurement of quorum-sensing signal autoinducer 3OC6HSL by a FRET-based nanosensor[J]. Bioprocess and biosystems engineering, 2014, 37(5): 849-855.
• [14] Zhang Y, Zhang J, Hoeflich K P, et al. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli[J]. Molecular cell, 2003, 12(4): 913-923.