Metabolites from a global regulator engineered strain of Pseudomonas lurida and their inducement of trap formation in Arthrobotrys oligospora

Background Plant parasitic nematodes (PPNs) cause serious harm to agricultural production. Nematode‑trapping fungi (NTF) can produce traps to capture nematodes and are the main resource for controlling nematodes. The number of traps determines the capturing ability of NTF. Results Pseudomonas lurida is widely existed in different habitats, which produces active metabolites to induce trap formation of Arthrobotrys oligospora , a famous NTF. To further identify the active substances, metabolic regulation was carried out in the strain by molecular biological methods. A mutant strain P. lurida araC ‑PoprL with abundant secondary metabolites was constructed, and 19 metabolites ( 1 – 19 ) including a new compound, 1,1‑dimethyl‑ 1,3,4,9‑tetrahydropyrano[3,4‑ b ]indole‑3‑carboxylic acid ( 1 ), were isolated and identified. The activity assay showed that 1‑methylhydantoin ( 9 ) could effectively induce A. oligospora to produce traps. Conclusions P. lurida and the metabolite 1‑methylhydantoin effectively induced trap formation in A. oligospora . Both provide sources for the screening of inducing active materials and show potential use in controlling plant parasitic nematodes.


Background
Nematodes are lower multicellular animals that are numerous and widely distributed [1].Among them, plant parasitic nematodes (PPNs) not only lead to damage to plant tissues, but also promote the infection of plants by pathogenic microorganisms in soil, thus causing or exacerbating other diseases [2].Almost all crops in the world are infected by PPNs, which is one of the reasons for the enormous economic losses of crops.According to a literature, a global loss of 170 billion dollars annually is caused by PPNs [3].
At present, the main control methods for hazard caused by PPNs can be divided into chemical, physical, agricultural and biological control.The biocontrol of PPNs has been extensively researched because it has few negative environmental impacts [4].Nematode-trapping fungi (NTF) form diverse special traps to capture nematodes through mycelium specialization, such as threedimensional networks, adhesive knobs, and constricting rings etc. [4].It is one of the ideal materials for studying the biocontrol of nematodes.It can kill nematodes directly depending on the number of traps.Many factors can induce the formation of traps, among which natural metabolite is an important factor.Many amino acids and small peptides have been reported to have induced activity [5].Interestingly, nematodes themselves can induce fungi to produce traps, and their products ascarosides, can also induce traps formation [6,7].
Recently, bacteria and their metabolites had been found to can induce trap formation in NTF.The research found that the coculture of Chryseobacterium sp. and Arthrobotrys oligospora could induce traps production [8].Dipiperazines (DKPs), the metabolites produced by Chryseobacterium sp., can enhance the activity of bacteria to promote the formation of traps [9].Ammonia produced by bacteria can induce the production of traps [10].The NTF Arthrobotrys conoides and A. oligospora produced traps after coculture with several bacteria for 48 h [11].Bacteria and their secondary metabolites are factors that cannot be ignored in the study of trap formation in NTF.
The AraC family of transcriptional regulator is representative of globally regulated genes that have been studied early and are widely distributed in a variety of bacteria [12].The members of this family can regulate the metabolism of bacteria, for example, the gapR gene could regulate glucose metabolism in Streptomyces aureofaciens by controlling the expression of glyceraldehyde-3-phosphate dehydrogenase [13]; transcriptional regulator GliR is related to the regulation of glycerolipid metabolism in Pseudomonas aeruginosa [14]; the global regulator SAV742 negatively regulates avermectin production in S. avermitilis [15]; the regulator MsmR1 is involved in production of polymyxin synthesis in Paenibacillus polymyxa SC2 [16].Promoters play an important role in gene transcription and expression [17].In metabolic engineering, the selection of a stably expressed promoter has a great impact on metabolites generation.PoprL, a promoter found in Pseudomonas putida [18], has been shown to have a strong enhancement effect on P. aeruginosa to increase production of rhamnoolipids [19].
Pseudomonas lurida is a gram-negative bacterium that is widely distributed; it was first isolated from strawberry leaves [20], and then obtained from soil in cold regions [21], plant rhizospheres [22], nematode [23], and milk [24].It has the characteristics of cold tolerance, phosphorus dissolution and plant growth promotion [25].In the present study, P. lurida can induce the formation of traps in A. oligospora, but it is not clear which substance plays a role.Up to now, only the compound massetolide E has been reported from the species [26], and further study of its metabolites is needed.Therefore, the study on the secondary metabolites of P. lurida aims to tap the active substances and obtain more resources for nematode control.

Strains and nematode cultivation
Stock cultures of P. lurida YMF 3.02383, C. elegans, Escherichia coli DH5α and A. oligospora YMF1.01883 were preserved in Microbial Library of the Germplasm Bank of Wild Species from Southwest China.P. lurida was cultivated in liquid media [LB (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl), NB (3 g/L beef extract, 10 g/L peptone, 5 g/L NaCl) or KB (20 g/L tryptone, 0.685 g/L K 2 HPO 4 •3H 2 O, 1.5 g/L MgSO 4 •7H 2 O, 15 mL glycerol)] at 28 °C at 180 rpm for 2 days to prepare prepagula.1 mL of the prepagula was then inoculated into medium and cultured for 4 days at the above conditions.The fermentation broth was filtered and the broth was collected.The cultured bacterial liquid was used for activity experiments.A. oligospora was activated on PDA medium (200 g/L potato, 20 g/L glucose, 15 g/L agar) and inoculated on CMY solid medium (20 g/L corn, 5 g/L yeast extract, 15 g/L agar), and cultured at 28 ℃ for 8-12 d.The proper amount of sterile water and glass beads were added to the triangular flask, shaken to wash all hyphae, and filtered with six layers of Lens paper to obtain a spore suspension.C. elegans was cultured with Escherichia coli OP50 on NGM (3 g/L NaCl, 2.5 g/L peptone, 1 mL 5 mg/ mL cholesterol ethanol solution, 1 mL 1 M MgSO 4 , 1 mL 1 M CaCl 2 , 25 mL 1 M K 2 HPO 4 •3H 2 O, 15 g/L agar) plates at 20 ℃.

Determination of induced activity of fermentation broth and extract
Spore suspensions of A. oligospora at approximately 3000 spores and 100 μL fermentation broth were thoroughly mixed in a 1.5 mL centrifuge tube.The control was 100 μL of culture medium mixing with spore suspensions.The broth was extracted by n-butanol and dissolved in methanol to prepare a 30 mg/mL mother liquor.Then, 97 μL of spore liquid and 3 μL of mother liquor were fully mixed in a 1.5 mL centrifuge tube.The mixed solutions (broth and spores, extracts and spores) were coated on a 1.5% water agar plate, and three parallel plates were set up in each experiment.The mixture was cultured at 28 ℃ for 2-3 d.After 24 h of culture, the traps were observed under a microscope.The experiment was repeated three times.

Overexpression of the araC gene in P. lurida
The araC gene in P. lurida was overexpressed in our experiment.The upstream and downstream homologous arm primers of araC gene were designed, and the fragments between homologous arms were replaced by strong promoters.The suicide plasmid pK18 mobsacB was used as the carrier, and the plasmid was linearized by enzyme digestion.Four fragments of the upstream homologous arm, PoprL, screening marker fragment gmR and the downstream homologous arm were sequentially connected by overlapping PCR.The fragment was connected to the pK18 mobsacB vector by In-Fusion ligase, transformed into E. coli competent DH5α cells and screened to obtain the recombinant plasmid pYUZ180.The recombinant plasmid pYUZ180 was transformed into P. lurida for homologous recombination screening to obtain the transformant P. lurida araC-PoprL.

Weight and LC-MS detection of metabolites from P. lurida and P. lurida araC-PoprL
Two culture media, NB and KB were selected to culture WT and P. lurida araC-PoprL.Then, the cultured fermentation broth was extracted with the same volume of n-butanol three times.The solvent was evaporated with a rotary evaporator and the amount of the extract was weighed.The concentration of sample was prepared as 10 mg/mL with chromatography-grade methanol.The sample was filtered and put into a bottle, left overnight at 4 ℃ to ensure that there was no precipitation, and detected by LC-MS.LC-MS was performed on a Dionex UltiMate 3000 LC system coupled with a Q-Exactive Orbitrap mass spectrometer.Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in methanol.The 30 min gradient for positive ESI mode was set as follows: 0-3 min, 5% solvent B; 3-22 min, 5-95% solvent B; 22-25 min, 95% solvent B; and 25-30 min, 5% solvent B.

Extraction and isolation of secondary metabolites from P. lurida araC-PoprL
P. lurida araC-PoprL was fermented in KB medium and cultured in a shake flask at 28 ℃ and 180 rpm for 4 d, with a total fermentation of 100 L. The fermentation broth was concentrated under reduced pressure and extracted with ethyl acetate to obtain 108.49g of extract.

Determination of induced activity of isolated metabolites
The isolated compounds were dissolved in methanol and diluted with sterile water to different concentrations.The sample solution was mixed with A. oligospora spore solution.After fully mixing, it was evenly coated on a 60 mm WA (water agar, 15 g/L agar) plate, and cultured at 28 ℃.Methanol with the same concentration was used as a control.After 24 h, it was observed under a microscope and then observed every 12 h, and the number of traps was counted.The experiment was set up in three parallels and repeated three times.The differences of traps numbers among the different concentrations were compared in order to find statistically significant correlations by using the F (ANOVA) test (significance p < 0.05).

Inducement trap formation in A. oligospora by P. lurida
The bacterial broth was mixed with spores of A. oligospora, and the traps were observed.The result showed that the fermentation broth of P. lurida had a good induction effect, traps began to form at 48 h, a large number of traps formed at 60 h (Fig. 1A, B), and the control group treated with medium had no traps (Fig. 1C).To further verify whether the active substances that can induce A. oligospora to produce traps are secondary metabolites, the organic compounds in the fermentation broth were extracted with n-butanol, and the induced activity of the extracts was also tested.The results showed that the n-butanol extract from the fermentation broth of P. lurida still had obvious induction activity, and when the concentration was 0.9 mg/mL, it could induce A. oligospora to produce a large number of traps (Fig. 1D, E), but the methanol treatment control group had no activity (Fig. 1F).

Overexpression of the araC gene in P. lurida and comparison of metabolites of P. lurida araC-PoprL with WT
The araC gene is a conserved regulatory gene in bacteria [27].A strong promoter, PoprL, was inserted to overexpress the araC gene and the transformant P. lurida araC-PoprL was obtained.WT and P. lurida araC-PoprL were cultured in 100 mL of NB and KB media, and extracted with n-butanol, and the extracts of the two strains were weighed.In the two media, the amount of extracts cultured in KB (141.6 and 236.4 mg for WT and P. lurida araC-PoprL) was much higher than that cultured in NB (39.1 and 66.1 mg for WT and P. lurida araC-PoprL).Compared with WT, the weight of extract from P. lurida araC-PoprL was increased by nearly 2 times, so the transformant was selected for subsequent amplification fermentation and fermented on KB medium.In addition, by analyzing the detection results P. lurida araC-PoprL and WT using LC-MS, metabolites of transformant P. lurida araC-PoprL were more abundant.Some distinct peaks were found in the chromatogram of Base Peak, and their retention times were 3.81, 7.36, 12.54, 12.83, 13.07 and 14.95 min (Fig. 2).Therefore, the yield and types of metabolites of P. lurida araC-PoprL are more abundant than those of WT.

Determination of induced activity of isolated metabolites
All isolated compounds were tested their inducing activity for trap formation in A. oligospora.The results showed that compound 1-methylhydantoin (9) had good induction activity, but other metabolites had no obvious activity.In a further assay, 1-methylhydantoin (9) showed induction activity in a concentration-dependent manner, and the induction effect was the best at 0.1 mg/mL (Fig. 5).

Discussion
NTF, as natural enemies of nematodes, are ideal materials for controlling of nematodes.Many researchers have focused on inducing NTF to produce traps through external factors, thus enhancing their ability to capture nematodes.Most NTF need to be induced by specific external signals to form traps [7,46,47]; among them, bacteria and their metabolites are one of the potential resources to induce the production of traps.Our previous research reported a new mechanism by which bacteria resist nematodes.Bacteria are food for nematodes, and when facing nematodes, they mobilize the nematode's natural enemies, NTF, to prey on nematodes.That is, bacterium induced NTF produce traps to kill nematodes to maintain their own population [48].
The fermentation broth and its organic extracts of P. lurida can effectively induce A. oligospora to produce traps.To further explore active substances, we studied the secondary metabolites of P. lurida.First, we used molecular biology methods to regulate the metabolism of the bacterium, overexpressed the gene of araC global regulation, and obtained P. lurida araC-PoprL.The metabolite amount of P. lurida araC-PoprL was much higher than that of the WT strain.LC-MS detection showed that the metabolites of P. lurida araC-PoprL were significantly abundant compared with those of WT.This result shows that the strong promoter PoprL can enhance the synthesis of secondary metabolites in P. lurida, whose araC expression is positively regulated.Next, nineteen compounds were identified from the extract of P. lurida araC-PoprL, among which, 1,1-dimethyl-1,3,4,9-tetrahydropyrano [3,4-b] indole-3-carboxylic acid (1) was a new compound.
Soil is a complex ecological environment, and metabolites are important communication tools among organisms living in soil.NTF, as aboriginal organisms in soil, play a significant ecological role in regulating nematode dynamics in soils.With the in-depth research on control of nematodes, integrated control of nematodes is receiving increasing attention.In addition to searching for metabolites that are directly active against nematodes, the factors that can mobilize other organisms in the soil to control nematodes are also worth paying attention to.Therefore, the bacteria and their metabolites with inducing activity in traps formation of NTF provide resources for the development of new biocontrol agents.

Conclusions
In the present study, P. lurida can induce A. oligospora to produce traps.The transformant P. lurida araC-PoprL was constructed.Nineteen metabolites, including one new compound, were identified from P. lurida araC-PoprL.Compound 1-methylhydantoin (9) exhibited obvious inducement activity of trap formation in A. oligospora.Bacteria-nematodes-fungi share living spaces in the natural environment, and the mutual cooperation and restriction among them is the result of long-term nutrition competition and selection pressure.It was found that P. lurida plays an important role between nematodes and nematode-trapping fungi.The study of the interaction among bacteria, nematodes and fungi is beneficial to better control the diseases caused by nematodes.

Fig. 1
Fig. 1 Induction activity of fermentation broth and extract of P. lurida to A. oligospora.A and B Fermentation broth treatment; C Medium control; D and E Extract treatment; F Methanol control