Improving the Yield of Xenocoumacin 1 by PBAD Promoter Replacement in Xenorhabdus nematophila CB6

Xenocoumacin 1 (Xcn1), which is produced by Xenorhabdus nematophila CB6, exhibits strong inhibition activity against plant pathogens, especially fungi and oomycetes. Therefore, it has attracted interest in developing it into a novel biofungicide applicable for plant protection. However, its low yield with concomitant high cost during the fermentation process limits its widespread application. In this study, we replaced the native promoter of xcnA with the arabinose-inducible araBAD promoter (PBAD), a well-known and widely used promoter for expressing heterologous genes, to evaluate its effects on Xcn1 yield and antimicrobial activity. Compared with wildtype strain, the fermentation yield of Xcn1 was improved from 68.5 mg/L to 249.7 mg/L (3.6-fold) and 234.9 mg/L (3.4-fold) at 0.5% and 1.0% L-arabinose concentration, respectively. We further explored the transcription level of the biosynthesis related genes of Xcn1 and found that their upregulation resulted in the yield improvement of Xcn1. Moreover, the antimicrobial activity of Xcn1 against Bacillus subtilis and Phytophthora capsici was determined by agar diffusion plate and growth inhibition assay, as expected, it was also found to be enhanced. The promoter-replacement strategy utilized here improves the yield of Xcn1 efficiently, which provides a basis for the industrial production of Xcn1.

Drosophila suzukii Susceptibility to the Oral Administration of Bacillus thuringiensis, Xenorhabdus nematophila and Its Secondary Metabolites

Drosophila suzukii, Spotted Wing Drosophila (SWD), is a serious economic issue for thin-skinned fruit farmers. The invasion of this dipteran is mainly counteracted by chemical control methods; however, it would be desirable to replace them with biological control. All assays were performed with Bacillus thuringiensis (Bt), Xenorhabdus nematophila (Xn), and Xn secretions, administered orally in single or combination, then larval lethality was assessed at different times. Gut damage caused by Bt and the influence on Xn into the hemocoelic cavity was also evaluated. In addition, the hemolymph cell population was analyzed after treatments. The data obtained show that the combined use of Bt plus Xn secretions on larvae, compared to single administration of bacteria, significantly improved the efficacy and reduced the time of treatments. The results confirm the destructive action of Bt on the gut of SWD larvae, and that Bt-induced alteration promotes the passage of Xn to the hemocoel cavity. Furthermore, hemocytes decrease after bioinsecticides treatments. Our study demonstrates that combining bioinsecticides can improve the efficacy of biocontrol and such combinations should be tested in greenhouse and in field in the near future.

Increasing the Production of Xenocoumacin 1 by Optimizing the Fermentation Process of Xenorhabdus Nematophila

Background: Xenocoumacin 1 (Xcn 1), a kind of water-soluble antibiotic discovered from the cell-free broth of Xenorhabdus nematophila YL001, has exhibited excellent activity against bacteria, oomycetes and fungi. However, the low yield limits the development and utilization of Xcn1. In order to increase the yield of Xcn1, the fermentation process was optimized in this study. Results: Maltose and proteose peptone were identified as the best carbon and nitrogen sources that significantly affected Xcn1 production using a-factor-at-a-time approach. Response surface methodology was applied to optimize the medium constituents for Xcn1 production by X. nematophila YL001. Higher Xcn1-content (113.65 μg/mL) was obtained after optimizing medium components. The optimal levels of medium components were (g/L): proteose peptone 20.83, maltose 12.74, K2HPO4 3.77. Fermentation conditions, such as initial pH, inoculum size, temperature, rotation speed, liquid volume and the length of fermentation, were also investigated by using a-factor-at-a-time method to get a higher production of Xcn1. X. nematophila YL001 was able to produce higher Xcn1 (153.56 μg/mL) at 25°C, initial pH 7.0, inoculum size 10%, culture medium 75 mL in a 250 mL shake flash with an agitation rate of 150 rpm for 48h. Additionally, kinds, concentrations and adding time of the precursor were also investigated. X. nematophila YL001 was able to produce the highest Xcn1 (173.99 μg/mL) when the arginine was added to the broth with 3 mmol/L at the 12th hour. An overall 243.38% increase in Xcn1 content was obtained as compared with mean observed response at TSB medium.Conclusions: To the best of our knowledge, there are no reports on optimization of fermentation process for Xcn1 production quantified by HPLC. The results show that nutrition, precursors and fermentation conditions had a highly influence on the production of Xcn1 by X. nematophila YL001. The optimized medium and fermentation conditions resulted in a 243.38% increase in Xcn1 production. This work will be helpful for the development of X. nematophila YL001 cultivation process for efficient Xcn1 production and lay a foundation for its industrial production.

Functional and Transcriptional Analysis of Chromosomal Encoded hipBAXn2 Type II Toxin-Antitoxin (TA) Module From Xenorhabdus Nematophila

Xenorhabdus nematophila is an entomopathogenic bacterium that synthesizes numerous toxins and kills its larval host. The genome of this bacterium also encodes a total of 39 putative toxin-antitoxin (TA) systems. These systems are also associated with maintaining the bacterial genomic stability and survival of bacteria under adverse environmental conditions. Three hipBA TA homologs were identified on the chromosome of X. nematophila, among them first hipBAXn TA has been studied, second hipBAXn2 TA is still unexplored while third hipBAXn3 TA has been reported as a pseudo-type TA system. Thus, for the first time, here, we are exploring the functionality of the type II hipBAXn2 TA system. This TA system was identified in the genome of X. nematophila ATCC 19061 (NCBI Refseq NC_014228) at position 3774379–3775635 bp, which consists of hipAXn2 toxin gene encoding 270 amino acid residues protein and hipBXn2 encoding antitoxin of 135 amino acid residues protein. It was observed that the overexpression of HipAXn2 toxin inhibits the growth of Escherichia coli cells in a bacteriostatic manner and amino-acids G8, H164, N167, and S169 were key residues for its toxicity. Promoter activity and expression profiling of messenger RNA from the hipBAXn2 TA system was also studied and showed that it was activated in both E. coli as well as X. nematophila upon exposure to different stress conditions. Further, we have exhibited the binding features of HipAXn2 toxin and HipBXn2 antitoxin to their promoter. This study provides the first evidence for the presence of a functional and active hipBAXn2 TA system in X. nematophila.

Chemical ecology of an apex predator life cycle

Microbial symbiotic interactions, mediated by small molecule signaling, drive physiological processes of higher order systems. Metabolic analytic technologies advancements provide new avenues to examine how chemical ecology, or conversion of existing biomass to new forms, changes over a symbiotic lifecycle. We examine such processes using the tripartite relationship between nematode host Steinernema carpocapsae, its obligate mutualist bacterium, Xenorhabdus nematophila, and the insects they infect together. We integrate trophic, metabolomics, and gene regulation analyses to understand insect biomass conversion to nematode or bacterium biomass. Trophic analysis established bacteria as the primary insect consumers, with nematodes at trophic position 4.37, indicating consumption of bacteria and likely other nematodes. Significant, discrete metabolic phases were distinguishable from each other, indicating the insect chemical environment changes reproducibly during bioconversion. Tricarboxylic acid cycle components and amino acids were significantly affected throughout infection. These findings contribute to an ongoing understanding of how symbiont associations shape chemical environments.TeaserEntomopathogenic nematodes act as an apex predator in some ecosystems through altering chemical environments of their prey.

Novel Toxin-antitoxin System Xn-mazEF from Xenorhabdus nematophi-la: Identification, Characterization and Functional Exploration

Background: Xenorhabdus nematophila maintains species-specific mutual interaction with nematodes of Steinernema genus. Type II Toxin Antitoxin (TA) systems, the mazEF TA system controls stress and programmed cell death in bacteria. Objective: This study elucidates the functional characterization of Xn-mazEF, a mazEF homolog in X. nematophila by computational and in vitro approaches. Methods: 3 D- structural models for Xn-MazE toxin and Xn-MazF antitoxin were generated, validated and characterized for protein - RNA interaction analysis. Further biological and cellular functions of Xn-MazF toxin were also predicted. Molecular dynamics simulations of 50ns for Xn-MazF toxin complexed with nucleic acid units (DU, RU, RC, and RU) were performed. The MazF toxin and complete MazEF operon were endogenously expressed and monitored for the killing of Escherichia coli host cells under arabinose induced tightly regulated system. Results: Upon induction, E. coli expressing toxin showed rapid killing within four hours and attained up to 65% growth inhibition, while the expression of the entire operon did not show significant killing. The observation suggests that the Xn-mazEF TA system control transcriptional regulation in X. nematophila and helps to manage stress or cause toxicity leading to programmed death of cells. Conclusion: The study provides insights into structural and functional features of novel toxin, XnMazF and provides an initial inference on control of X. nematophila growth regulated by TA systems.