Project Module
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 (Figure 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 (Figure 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 (Figure 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(Figure 4-a.). As for the control groups, both of the two small circular filters are added 5μL DMSO (Figure 4-b.) to eliminate the influence of the position of the nematodes. We also add 5 μL DMSO and M9 saline respectively (Figure 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
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Bacillus nematocida
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The processes for B. nematocida to kill nematodes
Backgrounds
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.
(A) Both the intestine and cuticle of nematodes were intact when treated with E. coli .(B) Structures of pharynx, muscle, and intestine were disorganized when treated with B. nematocidastra in B16. (C) Nematodes in the E. coli-treated control group had smooth undisturbed surfaces with a healthy cuticle structure that included the regular striae and lateral lines. (D) Nematodes infected with B. nematocidastrain B16 showed a lightly exfoliated cuticle. (E) The cross-section of an untreated, healthy nematode showed a highly ordered and compact intestinal structure. (F)Thecross-section of a nematode infected with B. nematocida strain B16 showed numerous defects in cluding fusion, vesiculation , and loosening of various organs. (G)Low-magnification TEM of the midgut of the control nematode showed ordered, densely arrayed, and normal-looking microvilli. (H) Microvilli in strain B16-infected nematodes appeared destroyed with significant membrane-tethering defects. Arrows indicate healthy (G) and damaged (H) and microvilli.
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 cor