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Microbial Cell Factories Apr 2024Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life...
BACKGROUND
Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work.
RESULTS
In the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743.
CONCLUSIONS
The results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.
Topics: Xenorhabdus; Photorhabdus; Gene Editing; Biological Products; Clustered Regularly Interspaced Short Palindromic Repeats
PubMed: 38561780
DOI: 10.1186/s12934-024-02363-8 -
Scientific Reports Nov 2023The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study,...
The discovery of novel bioactive compounds produced by microorganisms holds significant potential for the development of therapeutics and agrochemicals. In this study, we conducted genome mining to explore the biosynthetic potential of entomopathogenic bacteria belonging to the genera Xenorhabdus and Photorhabdus. By utilizing next-generation sequencing and bioinformatics tools, we identified novel biosynthetic gene clusters (BGCs) in the genomes of the bacteria, specifically plu00736 and plu00747. These clusters were identified as unidentified non-ribosomal peptide synthetase (NRPS) and unidentified type I polyketide synthase (T1PKS) clusters. These BGCs exhibited unique genetic architecture and encoded several putative enzymes and regulatory elements, suggesting its involvement in the synthesis of bioactive secondary metabolites. Furthermore, comparative genome analysis revealed that these BGCs were distinct from previously characterized gene clusters, indicating the potential for the production of novel compounds. Our findings highlighted the importance of genome mining as a powerful approach for the discovery of biosynthetic gene clusters and the identification of novel bioactive compounds. Further investigations involving expression studies and functional characterization of the identified BGCs will provide valuable insights into the biosynthesis and potential applications of these bioactive compounds.
Topics: Genome, Bacterial; Bacteria; Computational Biology; Multigene Family; Biosynthetic Pathways
PubMed: 38007490
DOI: 10.1038/s41598-023-47121-9 -
Scientific Reports Jun 2022Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce...
Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.
Topics: Antiprotozoal Agents; Entamoeba histolytica; Humans; Photorhabdus; Trypanosoma cruzi; Xenorhabdus
PubMed: 35750682
DOI: 10.1038/s41598-022-13722-z -
Journal of Invertebrate Pathology Feb 2022Recovery, yield, and dispersal are crucial developmental and behavioral indices for the infective juveniles of entomopathogenic nematodes, which are used as biocontrol...
Recovery, yield, and dispersal are crucial developmental and behavioral indices for the infective juveniles of entomopathogenic nematodes, which are used as biocontrol agents against a variety of agricultural pests. Ascarosides and isopropylstilbene (ISO) function as nematode pheromones with developmental and behavioral effects. In this study, 11 synthesized ascarosides identified from Caenorhabditis elegans, together with ISO identified from Photorhabdus luminescens, were used to determine their influence on the IJ recovery, growth on agar plates, and dispersal of S. carpocapsae All, H. bacteriophora H06 and H. indica LN2 nematodes. Compared with the controls, significant differences in IJ recovery of three nematode species were detected from the supernatants of their corresponding bacterial cultures with almost all ascarosides or isopropylstilbene (ISO) at 0.04 nM in 6 days. The highest IJ recovery percentages was obtained from ISO and ascr#3 for All strain, ascr#5 and ascr#6 for LN2 strain, and ISO and ascr#12 for H06 strain. The ISO detected from Photorhabdus bacteria also induced IJ recovery of S. carpocapsae All. IJ yields was significantly stimulated by all synthesized compounds for S. carpocapsae All, and by most compounds for H. bacteriophora H06. The higher IJ yields varied with ascarosides. Ascr#7 and DMSO was common for the improved IJ yields of both nematode species. The three nematode species showed marked differences in dispersal behavior. In response to the ascarosides or ISO, S. carpocapsae All IJs actively moved with different dispersal rates, H. indica LN2 IJs in very low dispersal rates, and H. bacteriophora H06 IJs in variable and even suppressed rates on the agar plates at least during the assay period. Based on the synthesized standards, ascr#1, ascr#9 and ascr#10 were detected from three nematode species, ascr#5 and ascr#11 also from S. carpocapsae All and H. bacteriophora H06, and ascr#12 also from H. bacteriophora H06 and H. indica LN2. Ascr#9 was most abundant in three nematode species. Compared with the sterile PBS, significantly more ascr#1, ascr#9 and ascr#10 were detected from S. carpocapsae All and H. indica LN2, but less ascr#5 and ascr#11 from S. carpocapsae All, ascr#1, ascr#5, ascr#11 and ascr#12 from H. bacteriophora H06, in the corresponding bacterial supernatant. It seems that the bacterial supernatants could regulate the ascaroside secretion by the three nematode species. These results will provide useful clues for selecting suitable ascarosides to induce the recovery, improve the yield, and enhance the dispersal of the IJs of these nematodes.
Topics: Agar; Animals; Nematoda; Pheromones; Photorhabdus
PubMed: 35031295
DOI: 10.1016/j.jip.2022.107717 -
Systematic and Applied Microbiology May 2016Photorhabdus is a genus of Gram-negative bacteria belonging to the Enterobacteriaceae family. In addition to forming a mutualistic relationship with the...
Photorhabdus is a genus of Gram-negative bacteria belonging to the Enterobacteriaceae family. In addition to forming a mutualistic relationship with the Heterorhabditidae family of nematodes, these bacteria are the causal agent of insect mortality during nematode infection, and are commonly used as biocontrol agents against pest insects in managed ecosystems. There are three described species of Photorhabdus; Photorhabdus luminescens and Photorhabdus temperata, which are strictly entomopathogens, and Photorhabdus asymbiotica, which has been isolated from wound infections in humans. While there has been extensive research on its virulence mechanisms, the evolution of virulence in Photorhabdus has not previously been investigated within a phylogenetic context. To investigate how virulence has evolved in this genus, we first reconstructed the phylogenetic relationships among 18 strains representing each of the main taxonomic lineages in the genus. Bacterial cells were injected into Galleria mellonella and Tenebrio molitor larvae, and the LT50 was calculated for each strain. These values were mapped onto the phylogeny using ancestral character reconstruction methods. With few exceptions, we found that the general trend of Photorhabdus evolution is one of increasing virulence. We also explored the relationship between virulence and Photorhabdus cell types and growth rates. Although we found no correlation between cell type and virulence, there was a strong correlation between virulence and growth rates in T. molitor. A better understanding of the origin and maintenance of virulence in this bacterium will aid in unraveling the mechanisms of the Heterorhabditis-Photorhabdus complex, resulting in the selection of more effective nematode-bacterium complexes for biocontrol.
Topics: Animals; Base Sequence; Biological Control Agents; DNA Gyrase; DNA, Bacterial; Moths; Photorhabdus; Phylogeny; RNA, Ribosomal, 16S; Rhabditoidea; Sequence Alignment; Sequence Analysis, DNA; Tenebrio; Virulence
PubMed: 27020955
DOI: 10.1016/j.syapm.2016.02.003 -
Insect Biochemistry and Molecular... Nov 2023PirAB binary toxin from Photorhabdus is toxic to the larvae of dipteran and lepidopteran insect pests. However, the 3-D structures and their toxicity mechanism are not...
PirAB binary toxin from Photorhabdus is toxic to the larvae of dipteran and lepidopteran insect pests. However, the 3-D structures and their toxicity mechanism are not yet fully understood. Here we report the crystal structures of PirA and PirB proteins from Photorhabdus akhurstii subsp. akhurstii K-1 at 1.6 and 2.1 Å, respectively. PirA comprises of eight β-strands depicting jelly-roll topology while PirB folds into two distinct domains, an N-terminal domain (PirB-N) made up of seven α-helices and a C-terminal domain (PirB-C) consists of ten β-strands. Despite the low sequence identity, PirA adopts similar architecture as domain III and PirB shared similar architecture as domain I/II of the Cry δ-endotoxin of Bacillus thuringiensis, respectively. However, PirA shows significant structural variations as compared to domain III of lepidopteran and dipteran specific Cry toxins (Cry1Aa and Cry11Ba) suggesting its role in virulence among range of insect pests and hence, in receptor binding. High structural resemblance between PirB-N and domain I of Cry toxin raises the possibility that the putative PirAB binary toxin may mimic the toxicity mechanism of the Cry protein, particularly its ability to perform pore formation. The mixture of independently purified PirA and PirB proteins are not toxic to insects. However, PirA-PirB protein complex purified from expression of pir operon with non-coding Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences found toxic to Galleria mellonella larvae with LD value of 1.62 μg/larva. This suggests that toxic conformation of PirA and PirB are achieved in-vivo with the help of ERIC sequences.
Topics: Animals; Photorhabdus; Bacterial Proteins; Endotoxins; Moths; Larva; Insecta; Hemolysin Proteins
PubMed: 37778713
DOI: 10.1016/j.ibmb.2023.104014 -
Applied and Environmental Microbiology Jun 2022Phytopathogens represent a large agricultural challenge. The use of chemical pesticides is harmful to the environment, animals, and humans. Therefore, new sustainable...
Phytopathogens represent a large agricultural challenge. The use of chemical pesticides is harmful to the environment, animals, and humans. Therefore, new sustainable and biological alternatives are urgently needed. The insect-pathogenic bacterium Photorhabdus luminescens, already used in combination with entomopathogenic nematodes (EPNs) as a biocontrol agent, is characterized by two different phenotypic cell forms, called primary (1°) and secondary (2°). The 1° cells are symbiotic with EPNs and are used for biocontrol, and the 2° cells are unable to undergo symbiosis with EPNs, remain in the soil after insect infection, and specifically interact with plant roots. A previous RNA sequencing (RNAseq) analysis showed that genes encoding the exochitinase Chi2A and chitin binding protein (CBP) are highly upregulated in 2° cells exposed to plant root exudates. Here, we investigate Chi2A and CBP functions and demonstrate that both are necessary for P. luminescens 2° cells to inhibit the growth of the phytopathogenic fungus Fusarium graminearum. We provide evidence that Chi2A digests chitin and thereby inhibits fungal growth. Furthermore, we show that 2° cells specifically colonize fungal hyphae as one of the first mechanisms to protect plants from fungal phytopathogens. Finally, soil pot bioassays proved plant protection from F. graminearum by 2° cells, where Chi2A and CPB were essential for this process. This work gives molecular insights into the new applicability of as a plant-growth-promoting and plant-protecting organism in agriculture. The enteric enterobacterium Photorhabdus luminescens is already being used as a bioinsecticide since it is highly pathogenic toward a broad range of insects. However, the bacteria exist in two phenotypically different cell types, called 1° and 2° cells. Whereas only 1° cells are symbiotic with their nematode partner to infect insects, 2° cells were shown to remain in the soil after an insect infection cycle. It was demonstrated that 2° cells specifically interact with plant roots. Here, we show that the bacteria are beneficial for the plants by protecting them from phytopathogenic fungi. Specific colonization of the fungus mycelium as well as chitin-degrading activity mediated by the chitin binding protein (CBP) and the chitinase Chi2A are essential for this process. Our data give evidence for the novel future applicability of as a plant-growth-promoting organism and biopesticide.
Topics: Animals; Chitin; Fusarium; Insecta; Nematoda; Photorhabdus; Soil; Symbiosis
PubMed: 35604230
DOI: 10.1128/aem.00645-22 -
Pathogens (Basel, Switzerland) Aug 2023Vector-borne diseases pose a severe threat to human and animal health. L. (Diptera: Culicidae) is a widespread mosquito species and serves as a vector for the...
Vector-borne diseases pose a severe threat to human and animal health. L. (Diptera: Culicidae) is a widespread mosquito species and serves as a vector for the transmission of infectious diseases such as West Nile disease and Lymphatic Filariasis. Synthetic insecticides have been the prime control method for many years to suppress populations. However, recently, the use of insecticides has begun to be questioned due to the detrimental impact on human health and the natural environment. Therefore, many authorities urge the development of eco-friendly control methods that are nontoxic to humans. The bacterial associates [ and spp. (Enterobacterales: Morganellaceae)] of entomopathogenic nematodes (EPNs) ( spp. and spp.) (Rhabditida: Heterorhabditidae and Steinernematidae) are one of the green approaches to combat a variety of insect pests. In the present study, the mosquitocidal activity of the cell-free supernatants and cell suspension (4 × 10 cells mL) of four different symbiotic bacteria (, , , and subsp. ) was assessed against different development stages of (The 1st/2nd and 3rd/4th instar larvae and pupa) under laboratory conditions. The bacterial symbionts were able to kill all the development stages with varying levels of mortality. The 1st/2nd instar larvae exhibited the highest susceptibility to the cell-free supernatants and cell suspensions of symbiotic bacteria and the efficacy of the cell-free supernatants and cell suspensions gradually declined with increasing phases of growth. The highest effectiveness was achieved by the KCS-4S strain inducing 95% mortality to the 1st/2nd instar larvae. The results indicate that tested bacterial symbionts have great potential as an eco-friendly alternative to insecticides.
PubMed: 37764903
DOI: 10.3390/pathogens12091095 -
Applied Microbiology and Biotechnology Apr 2022In this study, we evaluated a new biopesticide containing different combinations of Photorhabdus luminescens (ATCC 29,999; Pl) and Bacillus...
In this study, we evaluated a new biopesticide containing different combinations of Photorhabdus luminescens (ATCC 29,999; Pl) and Bacillus thuringiensis subsp. aizawai (Bt) to leverage their insecticidal activity against Plutella xylostella. Mixtures containing proteins of various sizes were assayed to determine which combination of the two bacteria would yield the maximum insecticidal activity. A histopathologic slide revealed vacuole formations and rifts near the apical membrane (a symptom of Bt) and severe thinning of the intestinal wall (a symptom of Pl). When the two bacteria were cultured separately and then mixed, the insecticidal activity of the treatment reached 83.33% ± 8.82%. The insecticidal activity was elevated and significantly accelerated when Bt was mixed with both the Pl supernatant and the isolated protein with a molecular mass [Formula: see text] 100 kDa of Pl. These results highlight the potential of Pl as a potent bioinsecticide to economically and sustainably control Pl. xylostella and other lepidopteran pests. KEY POINTS: • Growth inhibition by Bacillus thuringiensis exerted a significant effect on insecticidal activity. • Large Photorhabdus luminescens proteins can accelerate the synergistic insecticidal effect on Plutella xylostella.
Topics: Animals; Bacillus thuringiensis; Bacterial Proteins; Endotoxins; Hemolysin Proteins; Insecticides; Lepidoptera; Moths; Photorhabdus
PubMed: 35384447
DOI: 10.1007/s00253-022-11905-2 -
PloS One 2014Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological...
Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1Pl and ail2Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1Pl expression was directly controlled by PhoPQ and low external Mg2+ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2Pl and pagCPl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.
Topics: Antimicrobial Cationic Peptides; Bacterial Outer Membrane Proteins; Bacterial Proteins; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Genome, Bacterial; Humans; Magnesium Sulfate; Phenotype; Photorhabdus; Phylogeny
PubMed: 25333642
DOI: 10.1371/journal.pone.0110060