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Background: Nematodes of the genus Steinernema and their Xenorhabdus bacterial symbionts are lethal entomopathogens that are useful in the biocontrol of insect pests, as sources of diverse natural products, and as research models for mutualism and parasitism. Xenorhabdus play a central role in all aspects of the Steinernema lifecycle, and a deeper understanding of their genomes therefore has the potential to spur advances in each of these applications., Results: Here, we report a comparative genomics analysis of Xenorhabdus griffiniae, including the symbiont of Steinernema hermaphroditum nematodes, for which genetic and genomic tools are being developed. We sequenced and assembled circularized genomes for three Xenorhabdus strains: HGB2511, ID10 and TH1. We then determined their relationships to other Xenorhabdus and delineated their species via phylogenomic analyses, concluding that HGB2511 and ID10 are Xenorhabdus griffiniae while TH1 is a novel species. These additions to the existing X. griffiniae landscape further allowed for the identification of two subspecies within the clade. Consistent with other Xenorhabdus, the analysed X. griffiniae genomes each encode a wide array of antimicrobials and virulence-related proteins. Comparative genomic analyses, including the creation of a pangenome, revealed that a large amount of the intraspecies variation in X. griffiniae is contained within the mobilome and attributable to prophage loci. In addition, CRISPR arrays, secondary metabolite potential and toxin genes all varied among strains within the X. griffiniae species., Conclusions: Our findings suggest that phage-related genes drive the genomic diversity in closely related Xenorhabdus symbionts, and that these may underlie some of the traits most associated with the lifestyle and survival of entomopathogenic nematodes and their bacteria: virulence and competition. This study establishes a broad knowledge base for further exploration of not only the relationships between X. griffiniae species and their nematode hosts but also the molecular mechanisms that underlie their entomopathogenic lifestyle., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Xenorhabdus and Photorhabdus bacteria, which live in mutualistic symbiosis with entomopathogenic nematodes, are currently recognised as an important source of bioactive compounds. During their extraordinary life cycle, these bacteria are capable of fine regulation of mutualism and pathogenesis towards two different hosts, a nematode and a wide range of insect species, respectively. Consequently, survival in a specific ecological niche favours the richness of biosynthetic gene clusters and respective metabolites with a specific structure and function, providing templates for uncovering new agrochemicals and therapeutics. To date, numerous studies have been published on the genetic ability of Xenorhabdus and Photorhabdus bacteria to produce biosynthetic novelty as well as distinctive classes of their metabolites with their activity and mechanism of action. Research shows diverse techniques and approaches that can lead to the discovery of new natural products, such as extract-based analysis, genetic engineering, and genomics linked with metabolomics. Importantly, the exploration of members of the Xenorhabdus and Photorhabdus genera has led to encouraging developments in compounds that exhibit pharmaceutically important properties, including antibiotics that act against Gram- bacteria, which are extremely difficult to find. This article focuses on recent advances in the discovery of natural products derived from these nematophilic bacteria, with special attention paid to new valuable leads for therapeutics.

We report the identification of 3,6-dihydroxy-1,2-benzisoxazole (DHB) in a screen of Photorhabdus and Xenorhabdus , whose symbiotic relationship with eukaryotic nematodes favors secondary metabolites that meet several requirements matching those for clinically useful antibiotics. DHB is produced by Photorhabdus laumondii and is selective against the Gram-negative species Escherichia coli, Enterobacter cloacae, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and Acinetobacter baumannii . It is inactive against anaerobic gut bacteria and nontoxic to human cells. Mutants resistant to DHB map to the ubiquinone biosynthesis pathway. DHB binds to 4-hydroxybenzoate octaprenyltransferase (UbiA) and prevents the formation of 4-hydroxy-3-octaprenylbenzoate. Remarkably, DHB itself is prenylated, forming an unusable chimeric product that likely contributes to the toxic effect of this antimicrobial. DHB appears to be both a competitive enzyme inhibitor and a prodrug; this dual mode of action is unusual for an antimicrobial compound., Importance: The spread of resistant pathogens has led to the antimicrobial resistance crisis, and the need for new compounds acting against Gram-negative pathogens is especially acute. From a screen of Photorhabdus symbionts of nematodes, we identified 3,6-dihydroxy-1,2-benzisoxazole (DHB) that acts against a range of Gram-negative bacteria, including Escherichia coli , Enterobacter cloacae , Klebsiella pneumoniae , and Acinetobacter baumannii . DHB had previously been isolated from other bacterial species, but its mechanism of action remained unknown. We show that DHB is unique among antimicrobials, with dual action as an inhibitor of an important enzyme, UbiA, in the biosynthesis pathway of ubiquinone and as a prodrug. DHB is a mimic of the natural substrate, and UbiA modifies it into a toxic product, contributing to the antimicrobial action of this unusual antibiotic. We also uncover the mechanism of DHB selectivity, which depends on a particular fold of the UbiA enzyme., Competing Interests: The authors declare no conflict of interest.

Currently, it is widely accepted that the type III secretion system (T3SS) serves as the transport platform for bacterial virulence factors, while flagella act as propulsion motors. However, there remains a noticeable dearth of comparative studies elucidating the functional disparities between these two mechanisms. Entomopathogenic nematode symbiotic bacteria (ENS), including Xenorhabdus and Photorhabdus , are Gram-negative bacteria transported into insect hosts by Steinernema or Heterorhabdus . Flagella are conserved in ENS, but the T3SS is only encoded in Photorhabdus . There are few reports on the function of flagella and the T3SS in ENS, and it is not known what role they play in the infection of ENS. Here, we clarified the function of the T3SS and flagella in ENS infection based on flagellar inactivation in X. stockiae ( flhDC deletion), T3SS inactivation in P. luminescens ( sctV deletion), and the heterologous synthesis of the T3SS of P. luminescens in X. stockiae . Consistent with the previous results, the swarming movement of the ENS and the formation of biofilms are dominated by the flagella. Both the T3SS and flagella facilitate ENS invasion and colonization within host cells, with minimal impact on secondary metabolite formation and secretion. Unexpectedly, a proteomic analysis reveals a negative feedback loop between the flagella/T3SS assembly and the type VI secretion system (T6SS). RT-PCR testing demonstrates the T3SS's inhibition of flagellar assembly, while flagellin expression promotes T3SS assembly. Furthermore, T3SS expression stimulates ribosome-associated protein expression.

Xenorhabdus nematophila is a Gram-negative bacterium, mutualistically associated with the soil nematode Steinernema carpocapsae , and this nemato-bacterial complex is parasitic for a broad spectrum of insects. The transcriptional regulator OxyR is widely conserved in bacteria and activates the transcription of a set of genes that influence cellular defence against oxidative stress. It is also involved in the virulence of several bacterial pathogens. The aim of this study was to identify the X. nematophila OxyR regulon and investigate its role in the bacterial life cycle. An oxyR mutant was constructed in X. nematophila and phenotypically characterized in vitro and in vivo after reassociation with its nematode partner. OxyR plays a major role during the X. nematophila resistance to oxidative stress in vitro . Transcriptome analysis allowed the identification of 59 genes differentially regulated in the oxyR mutant compared to the parental strain. In vivo , the oxyR mutant was able to reassociate with the nematode as efficiently as the control strain. These nemato-bacterial complexes harbouring the oxyR mutant symbiont were able to rapidly kill the insect larvae in less than 48 h after infestation, suggesting that factors other than OxyR could also allow X. nematophila to cope with oxidative stress encountered during this phase of infection in insect. The significantly increased number of offspring of the nemato-bacterial complex when reassociated with the X. nematophila oxyR mutant compared to the control strain revealed a potential role of OxyR during this symbiotic stage of the bacterial life cycle.

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., (© 2024. The Author(s).)

Entomopathogenic nematodes from the genus Steinernema (Nematoda: Steinernematidae) are capable of causing the rapid killing of insect hosts, facilitated by their association with symbiotic Gram-negative bacteria in the genus Xenorhabdus (Enterobacterales: Morganellaceae), positioning them as interesting candidate tools for the control of insect pests. In spite of this, only a limited number of species from this bacterial genus have been identified from their nematode hosts and their insecticidal properties documented. This study aimed to perform the genome sequence analysis of fourteen Xenorhabdus strains that were isolated from Steinernema nematodes in Argentina. All of the strains were found to be able of killing 7th instar larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). Their sequenced genomes harbour 110 putative insecticidal proteins including Tc, Txp, Mcf, Pra/Prb and App homologs, plus other virulence factors such as putative nematocidal proteins, chitinases and secondary metabolite gene clusters for the synthesis of different bioactive compounds. Maximum-likelihood phylogenetic analysis plus average nucleotide identity calculations strongly suggested that three strains should be considered novel species. The species name for strains PSL and Reich (same species according to % ANI) is proposed as Xenorhabdus littoralis sp. nov., whereas strain 12 is proposed as Xenorhabdus santafensis sp. nov. In this work, we present a dual insight into the biocidal potential and diversity of the Xenorhabdus genus, demonstrated by different numbers of putative insecticidal genes and biosynthetic gene clusters, along with a fresh exploration of the species within this genus.

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. The infective juveniles deliver the symbiotic bacteria to the hemocoel of target insects, in which the bacteria proliferate and help the development of the host nematode. The successful parasitism of the nematode-bacterial complex depends on host immunosuppression by the bacteria via their secondary metabolites. Leucine-responsive regulatory protein (Lrp) is a global bacterial transcriptional factor that plays a crucial role in parasitism. However, its regulatory targets to suppress insect immunity are not clearly understood. This study investigated the bacterial genes regulated by Lrp and the subsequent production of secondary metabolites in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage of the bacteria in a target insect, Spodoptera exigua. A preliminary in silico screening indicated that 3.7% genes among 4075 predicted genes encoded in X. hominickii had the Lrp-response element on their promoters, including two non-ribosomal peptide synthetases (NRPSs). Eight NRPS (NRPS1-NRPS8) genes were predicted in the bacterial genome, in which six NRPS (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites and the NRPS expression levels. The immunosuppressive activities of X. hominickii were dependent on the Lrp expression level. The metabolites produced by Lrp expression included the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (=cPF) was produced by the bacteria at high Lrp expression and inhibited the phospholipase A 2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays a crucial role in insect immunosuppression by modulating NRPS expression., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

To what extent are generalist species cohesive evolutionary units rather than a compilation of recently diverged lineages? We examine this question in the context of host specificity and geographic structure in the insect pathogen and nematode mutualist Xenorhabdus bovienii. This bacterial species partners with multiple nematode species across two clades in the genus Steinernema. We sequenced the genomes of 42 X. bovienii strains isolated from four different nematode species and three field sites within a 240-km 2 region and compared them to globally available reference genomes. We hypothesized that X. bovienii would comprise several host-specific lineages, such that bacterial and nematode phylogenies would be largely congruent. Alternatively, we hypothesized that spatial proximity might be a dominant signal, as increasing geographic distance might lower shared selective pressures and opportunities for gene flow. We found partial support for both hypotheses. Isolates clustered largely by nematode host species but did not strictly match the nematode phylogeny, indicating that shifts in symbiont associations across nematode species and clades have occurred. Furthermore, both genetic similarity and gene flow decreased with geographic distance across nematode species, suggesting differentiation and constraints on gene flow across both factors, although no absolute barriers to gene flow were observed across the regional isolates. Several genes associated with biotic interactions were found to be undergoing selective sweeps within this regional population. The interactions included several insect toxins and genes implicated in microbial competition. Thus, gene flow maintains cohesiveness across host associations in this symbiont and may facilitate adaptive responses to a multipartite selective environment. IMPORTANCE Microbial populations and species are notoriously hard to delineate. We used a population genomics approach to examine the population structure and the spatial scale of gene flow in Xenorhabdus bovienii, an intriguing species that is both a specialized mutualistic symbiont of nematodes and a broadly virulent insect pathogen. We found a strong signature of nematode host association, as well as evidence for gene flow connecting isolates associated with different nematode host species and collected from distinct study sites. Furthermore, we saw signatures of selective sweeps for genes involved with nematode host associations, insect pathogenicity, and microbial competition. Thus, X. bovienii exemplifies the growing consensus that recombination not only maintains cohesion but can also allow the spread of niche-beneficial alleles., Competing Interests: The authors declare no conflict of interest.

In bacteria, DNA-methyltransferase are responsible for DNA methylation of specific motifs in the genome. This methylation usually occurs at a very high rate. In the present study, we studied the MTases encoding genes found in the entomopathogenic bacteria Xenorhabdus. Only one persistent MTase was identified in the various species of this genus. This MTase, also broadly conserved in numerous Gram-negative bacteria, is called Dam: DNA-adenine MTase. Methylome analysis confirmed that the GATC motifs recognized by Dam were methylated at a rate of >99% in the studied strains. The observed enrichment of unmethylated motifs in putative promoter regions of the X. nematophila F1 strain suggests the possibility of epigenetic regulations. The overexpression of the Dam MTase responsible for additional motifs to be methylated was associated with impairment of two major phenotypes: motility, caused by a downregulation of flagellar genes, and hemolysis. However, our results suggest that dam overexpression did not modify the virulence properties of X. nematophila. This study increases the knowledge on the diverse roles played by MTases in bacteria.

Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity of Xenorhabdus and Photorhabdus against 15 antibiotic-resistant bacteria strains. Photorhabdus extracts had strong inhibitory the growth of Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion. The P. akhurstii s subsp. akhurstii (bNN168.5&#95;TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between either P. akhurstii subsp. akhurstii (bNN141.3&#95;TH) or P. akhurstii subsp. akhurstii (bNN168.5&#95;TH) or P. hainanensis (bNN163.3&#95;TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay for P. akhurstii subsp. akhurstii (bNN168.5&#95;TH) extract against S. aureus strain PB36 significantly decreased cell viability from 105 CFU/ml to 103 CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5&#95;TH extract caused treated S. aureus strain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5&#95;TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5&#95;TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent., Competing Interests: The authors have declared that no competing interests exist.

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex (Tc). The chi60 and chi70 chitinase genes are located on the gene cluster encoding Tc toxins. To clarify the insecticidal activity of chitinases and their relationship with Tc toxins, the insecticidal activity of the chitinases was assessed on Helicoverpa armigera . Then, the chi60 and chi70 genes of X. nematophila HB310 were knocked out by the pJQ200SK suicide plasmid knockout system. The insecticidal activity of Tc toxin from the wild-type strain (WT) and mutant strains was carried out. The results demonstrate that Chi60 and Chi70 had an obvious growth inhibition effect against the second instar larvae of H. armigera with growth-inhibiting rates of 81.99% and 90.51%, respectively. Chi70 had a synergistic effect with the insecticidal toxicity of Tc toxins, but Chi60 had no synergistic effect with Tc toxins. After feeding Chi60 and Chi70, the peritrophic membrane of H. armigera became inelastic, was easily broken and leaked blue dextran. The Δ chi60 , Δ chi70 and Δ chi60 - chi70 mutant strains were successfully screened. The toxicity of Tc toxins from the WT, Δ chi60 , Δ chi70 and Δ chi60 - chi70 was 196.11 μg/mL, 757.25 μg/mL, 885.74 μg/mL and 20,049.83 μg/mL, respectively. The insecticidal activity of Tc toxins from Δ chi60 and Δ chi70 was 3.861 and 4.517 times lower than that of Tc toxins from the WT, respectively, while the insecticidal activity of Tc toxins from the Δ chi60 - chi70 mutant strain almost disappeared. These results indicate that the presence of chi60 and chi70 is indispensable for the toxicity of Tc toxins.

Fungal colonization can severely damage artifacts. Nematode endosymbiotic bacteria exhibit good prospects in protecting artifacts from fungal damage. We previously found that supernatant from the fermentation of nematode endosymbiotic bacterium, Xenorhabdus bovienii , is effective in inhibiting the growth of Fusarium solani NK-NH1, the major disease fungus in the Nanhai No.1 Shipwreck. Further experiments proved that X. bovienii produces volatile organic compounds (VOCs) that inhibit NK-NH1. Here, using metabolomic analysis, GC-MS, and transcriptomic analysis, we explored the antifungal substances and VOCs produced by X. bovienii and investigated the mechanism underlying its inhibitory effect against NK-NH1. We show that X. bovienii produces several metabolites, mainly lipids and lipid-like molecules, organic acids and derivatives, and organoheterocyclic compounds. The VOCs produced by X. bovienii showed two specific absorption peaks, and based on the library ratio results, these were predicted to be of 2-pentanone, 3-(phenylmethylene) and 1-hexen-3-one, 5-methyl-1-phenyl. The inhibition of F. solani by VOCs resulted in upregulation of genes related to ribosome, ribosome biogenesis, and the oxidative phosphorylation and downregulation of many genes associated with cell cycle, meiosis, DNA replication, and autophagy. These results are significant for understanding the inhibitory mechanisms employed by nematode endosymbiotic bacteria and should serve as reference in the protection of artifacts.

With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups of bacteria. Teixobactin, the first antibiotic without detectable resistance that binds lipid II, comes from an uncultured Eleftheria terra , a betaproteobacterium; odilorhabdins, from Xenorhabdus , are broad-spectrum inhibitors of protein synthesis, and darobactins from Photorhabdus target BamA, the essential chaperone of the outer membrane of Gram-negative bacteria. Xenorhabdus and Photorhabdus are symbionts of the nematode gut microbiome and attractive producers of secondary metabolites. Only small portions of their biosynthetic gene clusters (BGC) are expressed in vitro. To access their silent operons, we first separated extracts from a small library of isolates into fractions, resulting in 200-fold concentrated material, and then screened them for antimicrobial activity. This resulted in a hit with selective activity against Escherichia coli, which we identified as a novel natural product antibiotic, 3'-amino 3'-deoxyguanosine (ADG). Mutants resistant to ADG mapped to gsk and gmk , kinases of guanosine. Biochemical analysis shows that ADG is a prodrug that is converted into an active ADG triphosphate (ADG-TP), a mimic of GTP. ADG incorporates into a growing RNA chain, interrupting transcription, and inhibits cell division, apparently by interfering with the GTPase activity of FtsZ. Gsk of the purine salvage pathway, which is the first kinase in the sequential phosphorylation of ADG, is restricted to E. coli and closely related species, explaining the selectivity of the compound. There are probably numerous targets of ADG-TP among GTP-dependent proteins. The discovery of ADG expands our knowledge of prodrugs, which are rare among natural compounds. IMPORTANCE Drug-resistant Gram-negative bacteria have become the major problem driving the antimicrobial resistance crisis. Searching outside the overmined actinomycetes, we focused on Photorhabdus , gut symbionts of enthomopathogenic nematodes that carry up to 40 biosynthetic gene clusters coding for secondary metabolites. Most of these are silent and do not express in vitro . To gain access to silent operons, we first fractionated supernatant from Photorhabdus and then tested 200-fold concentrated material for activity. This resulted in the isolation of a novel antimicrobial, 3'-amino 3'-deoxyguanosine (ADG), active against E. coli. ADG is an analog of guanosine and is converted into an active ADG-TP in the cell. ADG-TP inhibits transcription and probably numerous other GTP-dependent targets, such as FtsZ. Natural product prodrugs have been uncommon; discovery of ADG broadens our knowledge of this type of antibiotic.

Microbial symbiosis drives physiological processes of higher-order systems, including the acquisition and consumption of nutrients that support symbiotic partner reproduction. Metabolic analytics provide new avenues to examine how chemical ecology, or the conversion of existing biomass to new forms, changes over a symbiotic life cycle. We applied these approaches to the nematode Steinernema carpocapsae, its mutualist bacterium, Xenorhabdus nematophila, and the insects they infect. The nematode-bacterium pair infects, kills, and reproduces in an insect until nutrients are depleted. To understand the conversion of insect biomass over time into either nematode or bacterium biomass, we integrated information from trophic, metabolomic, and gene regulation analyses. Trophic analysis established bacteria as meso-predators and primary insect consumers. Nematodes hold a trophic position of 4.6, indicative of an apex predator, consuming bacteria and likely other nematodes. Metabolic changes associated with Galleria mellonella insect bioconversion were assessed using multivariate statistical analyses of metabolomics data sets derived from sampling over an infection time course. Statistically significant, discrete phases were detected, indicating the insect chemical environment changes reproducibly during bioconversion. A novel hierarchical clustering method was designed to probe molecular abundance fluctuation patterns over time, revealing distinct metabolite clusters that exhibit similar abundance shifts across the time course. Composite data suggest bacterial tryptophan and nematode kynurenine pathways are coordinated for reciprocal exchange of tryptophan and NAD + and for synthesis of intermediates that can have complex effects on bacterial phenotypes and nematode behaviors. Our analysis of pathways and metabolites reveals the chemistry underlying the recycling of organic material during carnivory. IMPORTANCE The processes by which organic life is consumed and reborn in a complex ecosystem were investigated through a multiomics approach applied to the tripartite Xenorhabdus bacterium- Steinernema nematode- Galleria insect symbiosis. Trophic analyses demonstrate the primary consumers of the insect are the bacteria, and the nematode in turn consumes the bacteria. This suggests the Steinernema-Xenorhabdus mutualism is a form of agriculture in which the nematode cultivates the bacterial food sources by inoculating them into insect hosts. Metabolomics analysis revealed a shift in biological material throughout progression of the life cycle: active infection, insect death, and conversion of cadaver tissues into bacterial biomass and nematode tissue. We show that each phase of the life cycle is metabolically distinct, with significant differences including those in the tricarboxylic acid cycle and amino acid pathways. Our findings demonstrate that symbiotic life cycles can be defined by reproducible stage-specific chemical signatures, enhancing our broad understanding of metabolic processes that underpin a three-way symbiosis.

Antibiotic resistance is an increasing threat to human health. A direct link has been established between antimicrobial self-resistance determinants of antibiotic producers, environmental bacteria, and clinical pathogens. Natural odilorhabdins (ODLs) constitute a new family of 10-mer linear cationic peptide antibiotics inhibiting bacterial translation by binding to the 30S subunit of the ribosome. These bioactive secondary metabolites are produced by entomopathogenic bacterial symbiont Xenorhabdus ( Morganellaceae ), vectored by the soil-dwelling nematodes. ODL-producing Xenorhabdus nematophila symbionts have mechanisms of self-protection. In this study, we cloned the 44.5-kb odl biosynthetic gene cluster ( odl -BGC) of the symbiont by recombineering and showed that the N -acetyltransferase-encoding gene, oatA , is responsible for ODL resistance. In vitro acetylation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses showed that OatA targeted the side chain amino group of ODL rare amino acids, leading to a loss of translation inhibition and antibacterial properties. Functional, genomic, and phylogenetic analyses of oatA revealed an exclusive cis -link to the odilorhabdin BGC, found only in X. nematophila and a specific phylogenetic clade of Photorhabdus . This work highlights the coevolution of antibiotic production and self-resistance as ancient features of this unique tripartite complex of host-vector-symbiont interactions without odl -BGC dissemination by lateral gene transfer. IMPORTANCE Odilorhabdins (ODLs) constitute a novel antibiotic family with promising properties for treating problematic multidrug-resistant Gram-negative bacterial infections. ODLs are 10-mer linear cationic peptides inhibiting bacterial translation by binding to the small subunit of the ribosome. These natural peptides are produced by Xenorhabdus nematophila, a bacterial symbiont of entomopathogenic nematodes well known to produce large amounts of specialized secondary metabolites. Like other antimicrobial producers, ODL-producing Xenorhabdus nematophila has mechanisms of self-protection. In this study, we cloned the ODL-biosynthetic gene cluster of the symbiont by recombineering and showed that the N -acetyltransferase-encoding gene, oatA , is responsible for ODL resistance. In vitro acetylation and LC-MS/MS analyses showed that OatA targeted the side chain amino group of ODL rare amino acids, leading to a loss of translation inhibition and antibacterial properties. Functional, genomic, and phylogenetic analyses of oatA revealed the coevolution of antibiotic production and self-resistance as ancient feature of this particular niche in soil invertebrates without resistance dissemination.

Background: Entomopathogenic Xenorhabdus bacteria are endosymbionts of Steinernema nematodes and together they form an insecticidal mutualistic association that infects a wide range of insect species. Xenorhabdus produce an arsenal of toxins and secondary metabolites that kill the insect host. In addition, they can induce the production of diverse phage particles. A few studies have focused on one integrated phage responsible for producing a phage tail-like bacteriocin, associated with an antimicrobial activity against other Xenorhabdus species. However, very little is known about the diversity of prophage regions in Xenorhabdus species., Methods: In the present study, we identified several prophage regions in the genome of Xenorhabdus nematophila AN6/1. We performed a preliminary study on the relative expression of genes in these prophage regions. We also investigated some genes (not contained in prophage region) known to be involved in SOS bacterial response ( recA and lexA ) associated with mitomycin C and UV exposure., Results: We described two integrated prophage regions (designated Xnp3 and Xnp4) not previously described in the genome of Xenorhabdus nematophila AN6/1. The Xnp3 prophage region appears very similar to complete Mu-like bacteriophage. These prophages regions are not unique to X. nematophila species, although they appear less conserved among Xenorhabdus species when compared to the previously described p1 prophage region. Our results showed that mitomycin C exposure induced an up-regulation of recA and lexA suggesting activation of SOS response. In addition, mitomycin C and UV exposure seems to lead to up-regulation of genes in three of the four integrated prophages regions., Competing Interests: The authors declare that they have no competing interests., (© 2022 Lefoulon et al.)

Entomopathogenic nematodes (EPNs), including Heterorhabditis and Steinernema, are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines (Photorhabdus and Xenorhabdus, respectively) and therefore offer opportunities to study both mutualistic and parasitic symbiosis. The establishment of genetic tools in EPNs has been impeded by limited genetic tractability, inconsistent growth in vitro, variable cryopreservation, and low mating efficiency. We obtained the recently described Steinernema hermaphroditum strain CS34 and optimized its in vitro growth, with a rapid generation time on a lawn of its native symbiotic bacteria Xenorhabdus griffiniae. We developed a simple and efficient cryopreservation method. Previously, S. hermaphroditum isolated from insect hosts was described as producing hermaphrodites in the first generation. We discovered that CS34, when grown in vitro, produced consecutive generations of autonomously reproducing hermaphrodites accompanied by rare males. We performed mutagenesis screens in S. hermaphroditum that produced mutant lines with visible and heritable phenotypes. Genetic analysis of the mutants demonstrated that this species reproduces by self-fertilization rather than parthenogenesis and that its sex is determined chromosomally. Genetic mapping has thus far identified markers on the X chromosome and three of four autosomes. We report that S. hermaphroditum CS34 is the first consistently hermaphroditic EPN and is suitable for genetic model development to study naturally occurring mutualistic symbiosis and insect parasitism., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Xenorhabdus budapestensis strain C72 isolated from the entomopathogenic nematode of Steinernema bicornutum possesses an excellent biocontrol effect on southern corn leaf blight. However, its genomic information is lacking. Here, we report a high-quality complete and annotated genome sequence of X. budapestensis strain C72. Fifteen secondary metabolite biosynthetic gene clusters are identified in the genome, which are responsible for the production of a diverse group of antimicrobial compounds to help host plants against agricultural pathogenic diseases. This genome sequence could contribute to investigations of the molecular basis underlying the biocontrol activity of this Xenorhabdus strain.

Xenorhabdus and Photorhabdus are gram negative bacteria that can produce several secondary metabolites, including antimicrobial compounds. They have a symbiotic association with entomopathogenic nematodes (EPNs). The aim of this study was to isolate and identify Xenorhabdus and Photorhabdus species and their associated nematode symbionts from Northeastern region of Thailand. We also evaluated the antibacterial activity of these symbiotic bacteria. The recovery rate of EPNs was 7.82% (113/1445). A total of 62 Xenorhabdus and 51 Photorhabdus strains were isolated from the EPNs. Based on recA sequencing and phylogeny, Xenorhabdus isolates were identified as X. stockiae (n = 60), X. indica (n = 1) and X. eapokensis (n = 1). Photorhabdus isolates were identified as P. luminescens subsp. akhurstii (n = 29), P. luminescens subsp. hainanensis (n = 18), P. luminescens subsp. laumondii (n = 2), and P. asymbiotica subsp. australis (n = 2). The EPNs based on 28S rDNA and internal transcribed spacer (ITS) analysis were identified as Steinernema surkhetense (n = 35), S. sangi (n = 1), unidentified Steinernema (n = 1), Heterorhabditis indica (n = 39), H. baujardi (n = 1), and Heterorhabditis sp. SGmg3 (n = 3). Antibacterial activity showed that X. stockiae (bMSK7.5&#95;TH) extract inhibited several antibiotic-resistant bacterial strains. To the best of our knowledge, this is the first report on mutualistic association between P. luminescens subsp. laumondii and Heterorhabditis sp. SGmg3. This study could act as a platform for future studies focusing on the discovery of novel antimicrobial compounds from these bacterial isolates., Competing Interests: The authors have declared that no competing interests exist.

Polyamine moieties have been described as part of the fabclavine and zeamine family of natural products. While the corresponding biosynthetic gene clusters have been found in many different proteobacteria, a unique BGC was identified in the entomopathogenic bacterium Xenorhabdus bovienii. Mass spectrometric analysis of a X. bovienii mutant strain revealed a new deoxy-polyamine. The corresponding biosynthesis includes two additional reductive steps, initiated by an additional dehydratase (DH) domain, which was not found in any other Xenorhabdus strain. Moreover, this DH domain could be successfully integrated into homologous biosynthesis pathways, leading to the formation of other deoxy-polyamines. Additional heterologous production experiments revealed that the DH domain could act in cis as well as in trans., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Background: Steinernema feltiae is an entomopathogenic nematode used in biological control programs with a global distribution. Populations of this species show phenotypic plasticity derived from local adaptation and vary in different traits, such as location and host penetration. The aim of this work was to describe a Chilean isolate of this nematode species, using integrative approaches., Methods: Nematode morphological and morphometric studies were conducted along with molecular analysis of nuclear genes. The symbiotic bacterium was also identified by sequencing the 16S rRNA gene. Some ecological characteristics were described, including the temperature requirements for the nematode life cycle and the effect of soil water content for optimal reproduction., Results: Morphometric characterization revealed a large intra-specific variability. The isolate identity was also corroborated with the analysis of nuclear genes. Based on the 16S gene, its symbiont bacteria, Xenorhabdus bovienii, was identified. The lowest, optimal and highest temperatures found to limit the infestation and reproduction on Galleria mellonella were 10, 20 and 30 °C, respectively; the emergence from the host larvae occurred approximately 10 days after inoculation. Differences were observed in offspring, and 120 infective juveniles (IJ)/larva was the most prolific dose at 20 °C. The soil water content did not affect the number of IJ invaders, penetration efficacy and IJ emergence time or offspring per larva, but it caused a delay in achieving full mortality at the permanent wilting point with respect to saturation and field capacity., Conclusions: For the first time, a Chilean isolate of S. feltiae is described in detail considering morphological, molecular and ecological aspects. The isolate was shown to be efficient in soil containing water, with optimal temperatures ranging from 15 to 25 °C for host infestation and production of an abundant offspring; these characteristics would allow its potential use as control agents in a wide geographical area of the country.

During an infection, parasites face a succession of challenges, each decisive for disease outcome. The diversity of challenges requires a series of parasite adaptations to successfully multiply and transmit from host to host. Thus, the pathogen genotypes that succeed during one step might be counterselected in later stages of the infection. Using the bacterium Xenorhabdus nematophila and adult Drosophila melanogaster flies as hosts, we showed that such step-specific adaptations, here linked to GASP (i.e., growth advantage in stationary phase) mutations in the X. nematophila master gene regulator lrp , exist and can trade off with each other. We found that nonsense lrp mutations had lowered the ability to resist the host immune response, while all classes of mutations in lrp were associated with a decrease in the ability to proliferate during early infection. We demonstrate that reduced proliferation of X. nematophila best explains diminished virulence in this infection model. Finally, decreased proliferation during the first step of infection is accompanied by improved proliferation during late infection, suggesting a trade-off between the adaptations to each step. Step-specific adaptations could play a crucial role in the chronic phase of infections in any disease organisms that show similar small colony variants (SCVs) to X. nematophila IMPORTANCE Within-host evolution has been described in many bacterial diseases, and the genetic basis behind the adaptations has stimulated a lot of interest. Yet, the studied adaptations are generally focused on antibiotic resistance and rarely on the adaptation to the environment given by the host, and the potential trade-offs hindering adaptations to each step of the infection are rarely considered. Those trade-offs are key to understanding intrahost evolution and thus the dynamics of the infection. However, understanding these trade-offs supposes a detailed study of host-pathogen interactions at each step of the infection process, with an adapted methodology for each step. Using Drosophila melanogaster as the host and the bacterium Xenorhabdus nematophila , we investigated the bacterial adaptations resulting from GASP mutations known to induce the small colony variant (SCV) phenotype positively selected within the host over the course of an infection, as well as the trade-off between step-specific adaptations., (Copyright © 2021 Faucher et al.)

Xenorhabdus species are bacterial symbionts of Steinernema nematodes and pathogens of susceptible insects. Different species of Steinernema nematodes carrying specific species of Xenorhabdus can invade the same insect, thereby setting up competition for nutrients within the insect environment. While Xenorhabdus species produce both diverse antibiotic compounds and prophage-derived R-type bacteriocins (xenorhabdicins), the functions of these molecules during competition in a host are not well understood. Xenorhabdus bovienii ( Xb-Sj ), the symbiont of Steinernema jollieti, possesses a remnant P2-like phage tail cluster, xbp 1, that encodes genes for xenorhabdicin production. We show that inactivation of either tail sheath ( xbpS1 ) or tail fibre ( xbpH1 ) genes eliminated xenorhabdicin production. Preparations of Xb-Sj xenorhabdicin displayed a narrow spectrum of activity towards other Xenorhabdus and Photorhabdus species. One species, Xenorhabdus szentirmaii ( Xsz-Sr ), was highly sensitive to Xb-Sj xenorhabdicin but did not produce xenorhabdicin that was active against Xb-Sj . Instead, Xsz-Sr produced high-level antibiotic activity against Xb-Sj when grown in complex medium and lower levels when grown in defined medium (Grace's medium). Conversely, Xb-Sj did not produce detectable levels of antibiotic activity against Xsz-Sr . To study the relative contributions of Xb-Sj xenorhabdicin and Xsz-Sr antibiotics in interspecies competition in which the respective Xenorhabdus species produce antagonistic activities against each other, we co-inoculated cultures with both Xenorhabdus species. In both types of media Xsz-Sr outcompeted Xb-Sj , suggesting that antibiotics produced by Xsz-Sr determined the outcome of the competition. In contrast, Xb-Sj outcompeted Xsz-Sr in competitions performed by co-injection in the insect Manduca sexta , while in competition with the xenorhabdicin-deficient strain ( Xb-Sj:S1 ), Xsz-Sr was dominant. Thus, xenorhabdicin was required for Xb-Sj to outcompete Xsz-Sr in a natural host environment. These results highlight the importance of studying the role of antagonistic compounds under natural biological conditions.

Xenorhabdus bovienii strain jolietti (XBJ) is a Gram-negative bacterium that interacts with several organisms as a part of its life cycle. It is a beneficial symbiont of nematodes, a potent pathogen of a wide range of soil-dwelling insects and also has the ability to kill soil- and insect-associated microbes. Entomopathogenic Steinernema nematodes vector XBJ into insects, releasing the bacteria into the insect body cavity. There, XBJ produce a variety of insecticidal toxins and antimicrobials. XBJ's genome also encodes two separate Type Six Secretion Systems (T6SSs), structures that allow bacteria to inject specific proteins directly into other cells, but their roles in the XBJ life cycle are mostly unknown. To probe the function of these T6SSs, we generated mutant strains lacking the key structural protein Hcp from each T6SS and assessed phenotypes related to different parts of XBJ's life cycle. Here we demonstrate that one of the T6SSs is more highly expressed in in vitro growth conditions and has antibacterial activity against other Xenorhabdus strains, and that the two T6SSs have a redundant role in biofilm formation., (© FEMS 2020.)

The control of insects of medical importance, such as Aedes aegypti and Aedes albopictus are still the only effective way to prevent the transmission of diseases, such as dengue, chikungunya and Zika. Their control is performed mainly using chemical products; however, they often have low specificity to non-target organisms, including humans. Also, studies have reported resistance to the most commonly used insecticides, such as the organophosphate and pyrethroids. Biological control is an ecological and sustainable method since it has a slow rate of insect resistance development. Bacterial species of the genera Xenorhabdus and Photorhabdus have been the target of several research groups worldwide, aiming at their use in agricultural, pharmaceutical and industrial products. This review highlights articles referring to the use of Xenorhabdus and Photorhabdus for insects and especially for mosquito control proposing future ways for their biotechnological applicability. Approximately 24 species of Xenorhabdus and five species of Photorhabdus have been described to have insecticidal properties. These studies have shown genes that are capable of encoding low molecular weight proteins, secondary toxin complexes and metabolites with insecticide activities, as well as antibiotic, fungicidal and antiparasitic molecules. In addition, several species of Xenorhabdus and Photorhabdus showed insecticidal properties against mosquitoes. Therefore, these biological agents can be used in new control methods, and must be, urgently considered in short term, in studies and applications, especially in mosquito control.

The mechanisms of action of the complex including entomopathogenic nematodes of the genera Steinernema and Heterorhabditis and their mutualistic partners, i.e., bacteria Xenorhabdus and Photorhabdus , have been well explained, and the nematodes have been commercialized as biological control agents against many soil insect pests. However, little is known regarding the nature of the relationships between these bacteria and the gut microbiota of infected insects. In the present study, 900 bacterial isolates that were obtained from the midgut samples of Melolontha melolontha larvae were screened for their antagonistic activity against the selected species of the genera Xenorhabdus and Photorhabdus . Twelve strains exhibited significant antibacterial activity in the applied tests. They were identified based on 16S rRNA and rpoB , rpoD , or recA gene sequences as Pseudomonas chlororaphis , Citrobacter murliniae , Acinetobacter calcoaceticus , Chryseobacterium lathyri , Chryseobacterium sp., Serratia liquefaciens , and Serratia sp. The culture filtrate of the isolate P. chlororaphis MMC3 L3 04 exerted the strongest inhibitory effect on the tested bacteria. The results of the preliminary study that are presented here, which focused on interactions between the insect gut microbiota and mutualistic bacteria of entomopathogenic nematodes, show that bacteria inhabiting the gut of insects might play a key role in insect resistance to entomopathogenic nematode pressure.

Bacterial infections are often composed of cells with distinct phenotypes that can be produced by genetic or epigenetic mechanisms. This phenotypic heterogeneity has proved to be important in many pathogens, because it can alter both pathogenicity and transmission. We studied how and why it can emerge during infection in the bacterium Xenorhabdus nematophila , a pathogen that kills insects and multiplies in the cadaver before being transmitted by the soil nematode vector Steinernema carpocapsae We found that phenotypic variants cluster in three groups, one of which is composed of lrp defective mutants. These mutants, together with variants of another group, have in common that they maintain high survival during late stationary phase. This probably explains why they increase in frequency: variants of X. nematophila with a growth advantage in stationary phase (GASP) are under strong positive selection both in prolonged culture and in late infections. We also found that the within-host advantage of these variants seems to trade off against transmission by nematode vectors: the variants that reach the highest load in insects are those that are the least transmitted. IMPORTANCE Pathogens can evolve inside their host, and the importance of this mutation-fueled process is increasingly recognized. A disease outcome may indeed depend in part on pathogen adaptations that emerge during infection. It is therefore important to document these adaptations and the conditions that drive them. In our study, we took advantage of the possibility to monitor within-host evolution in the insect pathogen X. nematophila We demonstrated that selection occurring in aged infection favors lrp defective mutants, because these metabolic mutants benefit from a growth advantage in stationary phase (GASP). We also demonstrated that these mutants have reduced virulence and impaired transmission, modifying the infection outcome. Beyond the specific case of X. nematophila , we propose that metabolic mutants are to be found in other bacterial pathogens that stay for many generations inside their host., (Copyright © 2019 Cambon et al.)

High-strength polymers, such as aramid fibres, are important materials in space technology. To obtain these materials in remote locations, such as Mars, biological production is of interest. The aromatic polymer precursor para-aminobenzoic acid (pABA) can be derived from the shikimate pathway through metabolic engineering of Bacillus subtilis, an organism suited for space synthetic biology. Our engineering strategy included repair of the defective indole-3-glycerol phosphate synthase (trpC), knockout of one chorismate mutase isozyme (aroH) and overexpression of the aminodeoxychorismate synthase (pabAB) and aminodeoxychorismate lyase (pabC) from the bacteria Corynebacterium callunae and Xenorhabdus bovienii respectively. Further, a fusion-protein enzyme (pabABC) was created for channelling of the carbon flux. Using adaptive evolution, mutants of the production strain, able to metabolize xylose, were created, to explore and compare pABA production capacity from different carbon sources. Rather than the efficiency of the substrate or performance of the biochemical pathway, the product toxicity, which was strongly dependent on the pH, appeared to be the overall limiting factor. The highest titre achieved in shake flasks was 3.22 g l -1 with a carbon yield of 12.4% [C-mol/C-mol] from an amino sugar. This promises suitability of the system for in situ resource utilization (ISRU) in space biotechnology, where feedstocks that can be derived from cyanobacterial cell lysate play a role., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Background: Xenorhabdus spp. live in close symbiosis with nematodes of the Steinernema genus. Steinernema nematodes infect an insect larva and release their symbionts into the haemocoel of the insect. Once released into the haemocoel, the bacteria produce bioactive compounds to create a semi-exclusive environment by inhibiting the growth of bacteria, yeasts and molds. The antimicrobial compounds thus far identified are xenocoumacins, xenortides, xenorhabdins, indole derivatives, xenoamicins, bicornutin and a number of antimicrobial peptides. The latter may be linear peptides such as the bacteriocins xenocin and xenorhabdicin, rhabdopeptides and cabanillasin, or cyclic, such as PAX lipopeptides, taxlllaids, xenobactin and szentiamide. Thus far, production of antimicrobial compounds have been reported for Xenorhabdus nematophila, Xenorhabdus budapestensis, Xenorhabdus cabanillasii, Xenorhabdus kozodoii, Xenorhabdus szentirmaii, Xenorhabdus doucetiae, Xenorhabdus mauleonii, Xenorhabdus indica and Xenorhabdus bovienii. Here we describe, for the first time, PAX lipopeptides and xenocoumacin 2 produced by Xenorhabdus khoisanae. These compounds were identified using ultraperformance liquid chromatography, linked to high resolution electrospray ionisation mass spectrometry and tandem mass spectrometry., Results: Cell-free supernatants of X. khoisanae SB10 were heat stable and active against Bacillus subtilis subsp. subtilis, Escherichia coli and Candida albicans. Five lysine-rich lipopeptides from the PAX group were identified in HPLC fractions, with PAX1' and PAX7 present in the highest concentrations. Three novel PAX7 peptides with putative enoyl modifications and two linear analogues of PAX1' were also detected. A small antibiotic compound, yellow in colour and λ max of 314 nm, was recovered from the HPLC fractions and identified as xenocoumacin 2. The PAX lipopeptides and xenocoumacin 2 correlated with the genes and gene clusters in the genome of X. khoisanae SB10., Conclusion: With UPLC-MS and MS e analyses of compounds in the antimicrobial complex of X. khoisanae SB10, a number of PAX peptides and a xenocoumacin were identified. The combination of pure PAX1' peptide with xenocoumacin 2 resulted in high antimicrobial activity. Many of the fractions did, however, contain labile compounds and some fractions were difficult to resolve. It is thus possible that strain SB10 may produce more antimicrobial compounds than reported here, as suggested by the APE Ec biosynthetic complex. Further research is required to develop these broad-spectrum antimicrobial compounds into drugs that may be used in the fight against microbial infections.

CpxR is a global response regulator that negatively influences the antimicrobial activities of Xenorhabdus nematophila. Herein, the wildtype and ΔcpxR mutant of X. nematophila were cultured in a 5-l and 70-l bioreactor. The kinetic analysis showed that ΔcpxR significantly increased the cell biomass and antibiotic activity. The maximum dry cell weight (DCW) and antibiotic activity of ΔcpxR were 20.77 ± 1.56 g L -1 and 492.0 ± 31.2 U ml -1 and increased by 17.28 and 97.33% compared to the wildtype respectively. Xenocoumacin 1 (Xcn1), a major antimicrobial compound, was increased 3.07-fold, but nematophin was decreased by 48.7%. In 70-l bioreactor, DCW was increased by 18.97%, while antibiotic activity and Xcn1 were decreased by 27.71% and 11.0% compared to that in 5-l bioreactor respectively. Notably, pH had remarkable effects on the cell biomass and antibiotic activity of ΔcpxR, where ΔcpxR was sensitive to alkaline pH conditions. The optimal cell growth and antibiotic activity of ΔcpxR occurred at pH 7.0, while Xcn1 was increased 5.45- and 3.87-fold relative to that at pH 5.5 and 8.5 respectively. These findings confirmed that ΔcpxR considerably increased the biomass of X. nematophila at a late stage of fermentation. In addition, ΔcpxR significantly promoted the biosynthesis of Xcns but decreased the production of nematophin., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Xenorhabdus species are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. Nematodes enter insect hosts and perforate the midgut to invade the haemocoel where Xenorhabdus bacteria are released transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the haemocoel. Different species of nematodes carrying specific strains of Xenorhabdus can also invade the same insect. Xenorhabdus species thereby compete for nutrients and space with both related strains and non-related gut microbes. While Xenorhabdus species produce diverse antimicrobial compounds in complex media, their functions in insect hosts are not well understood. We show that Xenorhabdus szentirmaii produced ngrA-dependent antibiotics that were active against both gut-derived microbes and Xenorhabdus nematophila whereas antibiotics of X. nematophila were not active against X. szentirmaii. X. nematophila growth was inhibited in co-cultures with wild-type X. szentirmaii in medium that mimics insect haemolymph. An antibiotic-deficient strain of X. szentirmaii was created by inactivating the ngrA gene that encodes the enzyme that attaches the 4' phosphopantetheinyl moiety to non-ribosomal peptide synthetases involved in antibiotic biosynthesis. X. nematophila growth was not inhibited in co-cultures with the ngrA strain. The growth of X. nematophila was suppressed in Manduca sexta co-injected with wild-type X. szentirmaii and X. nematophila. In contrast, growth of X. nematophila was not suppressed in M. sexta co-injected with the ngrA strain. Two unique compounds were detected by MALDI-TOF MS analysis in haemolymph infected with the wild-type but not with the ngrA strain. Finally, killing of M. sexta was delayed in insects infected with the ngrA strain. These findings indicate that in the insect host X. szentirmaii produces ngrA-dependent products involved in both interspecies competition and virulence.

Fabclavines, unusual peptide-polyketide-polyamine hybrids, show broad-spectrum bioactivity against a variety of different organism like Gram-positive and -negative bacteria, fungi and protozoa. We elucidated the biosynthesis of these NRPS-PKS hybrids in Xenorhabdus szentirmaii by deletion of most genes encoded in the fabclavine BGC and subsequent analysis of produced fabclavine or polyamine intermediates. Thereby, we identified shortened fabclavines similar to the bioactive zeamines. Furthermore, we analyzed the thioester reductase FclG and the free-standing condensation domain-like protein FclL in detail and observed low substrate specificity for both enzymes.

We evaluated the impact of bacterial rhabduscin synthesis on bacterial virulence and phenoloxidase inhibition in a Spodoptera model. We first showed that the rhabduscin cluster of the entomopathogenic bacterium Xenorhabdus nematophila was not necessary for virulence in the larvae of Spodoptera littoralis and Spodoptera frugiperda. Bacteria with mutations affecting the rhabduscin synthesis cluster (ΔisnAB and ΔGT mutants) were as virulent as the wild-type strain. We then developed an assay for measuring phenoloxidase activity in S. frugiperda and assessed the ability of bacterial culture supernatants to inhibit the insect phenoloxidase. Our findings confirm that the X. nematophila rhabduscin cluster is required for the inhibition of S. frugiperda phenoloxidase activity. The X. nematophila ΔisnAB mutant was unable to inhibit phenoloxidase, whereas ΔGT mutants displayed intermediate levels of phenoloxidase inhibition relative to the wild-type strain. The culture supernatants of Escherichia coli and of two entomopathogenic bacteria, Serratia entomophila and Xenorhabdus poinarii, were unable to inhibit S. frugiperda phenoloxidase activity. Heterologous expression of the X. nematophila rhabduscin cluster in these three strains was sufficient to restore inhibition. Interestingly, we observed pseudogenization of the X. poinarii rhabduscin gene cluster via the insertion of a 120 bp element into the isnA promoter. The inhibition of phenoloxidase activity by X. poinarii culture supernatants was restored by expression of the X. poinarii rhabduscin cluster under the control of an inducible Ptet promoter, consistent with recent pseudogenization. This study paves the way for advances in our understanding of the virulence of several entomopathogenic bacteria in non-model insects, such as the new invasive S. frugiperda species in Africa., Competing Interests: The authors have declared that no competing interests exist.

Xenocoumacin 1 (Xcn1), a major antimicrobial compound produced by Xenorhabdus nematophila, has great potential for use in agricultural productions. In this study, we evaluated the effects of CpxR, a global response regulator associated with the mutualism and pathogenesis of X. nematophila, on the antimicrobial activity and Xcn1 production. The mutation of cpxR could promote the production of Xcn1 significantly with its level in ΔcpxR mutant being 3.07 times higher than that in the wild type. Additionally, the expression levels of xcnA-L genes, which are responsible for the production of Xcn1, were increased in ΔcpxR mutant while the expression levels of xcnMN, which are required for the conversion of Xcn1 into Xcn2 was reduced. Noticeably, Xcn2 was also enhanced on account of the conversion of excessive Xcn1 in spite of low expression levels of xcnM and xcnN in ΔcpxR mutant. The transcriptional levels of ompR and lrp, encoding the global response regulators OmpR and Lrp which negatively and positively regulate the production of Xcn1 were concurrently decreased and increased, respectively. Correspondingly, ΔcpxR mutant also exhibited increased antimicrobial activities in vitro and in vivo. Together, these findings suggest that CpxR negatively regulates xcnA-L genes expression while positively regulating xcnMN expression in X. nematophila YL001, which led to a high yield of Xcn1 in ΔcpxR mutant., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by specific docking domains (DDs), whose structures are not known yet, to synthesize well-defined peptide products. In contrast to classical NRPSs, single-module NRPS subunits responsible for the generation of rhabdopeptide/xenortide-like peptides (RXPs) can act in different order depending on subunit stoichiometry thereby producing peptide libraries. To define the basis for their unusual interaction patterns, we determine the structures of all N-terminal DDs ( N DDs) as well as of an N DD- C DD complex and characterize all putative DD interactions thermodynamically for such a system. Key amino acid residues for DD interactions are identified that upon their exchange change the DD affinity and result in predictable changes in peptide production. Recognition rules for DD interactions are identified that also operate in other megasynthase complexes.

Xenorhabdus nematophila are Gram-negative bacteria that engage in mutualistic associations with entomopathogenic nematodes. To reproduce, the nematodes invade insects and release X. nematophila into the haemolymph where it functions as an insect pathogen. In complex medium, X. nematophila cells produce two distinct types of intracellular crystalline inclusions, one composed of the methionine-rich PixA protein and the other composed of the PixB protein. Here we show that PixB crystalline inclusions were neither apparent in X. nematophila cells grown in medium that mimics insect haemolymph (Grace's medium) nor in cells grown directly in the insect haemocoel. The identified pixB gene was regulated by a conserved σ70 promoter while the pixA promoter was less well conserved. Expression of pixA and pixB under biological conditions was analysed using GFP promoter reporters. Microplate fluorescence detection and flow cytometry analyses revealed that pixB was expressed at high levels in Grace's medium and in insect haemolymph and at lower levels in complex medium, while pixA was expressed at lower levels under all conditions. Although pixB was highly expressed in Grace's medium, PixB crystalline inclusions were not present, suggesting that under biological conditions PixB production may be controlled post-transcriptionally. Although a pixB-minus strain was constructed, the function of PixB remains unresolved. The pixB gene was present in few Xenorhabdus species and pixB-type genes were identified in some Proteobacteria and Gram-positive species, while pixA was only present in Xenorhabdus species. Two conserved sequences were identified in PixB-type proteins that characterize this previously unrecognized gene family.

Background: Xenorhabdus innexi is a bacterial symbiont of Steinernema scapterisci nematodes, which is a cricket-specialist parasite and together the nematode and bacteria infect and kill crickets. Curiously, X. innexi expresses a potent extracellular mosquitocidal toxin activity in culture supernatants. We sequenced a draft genome of X. innexi and compared it to the genomes of related pathogens to elucidate the nature of specialization., Results: Using green fluorescent protein-expressing X. innexi we confirm previous reports using culture-dependent techniques that X. innexi colonizes its nematode host at low levels (~3-8 cells per nematode), relative to other Xenorhabdus-Steinernema associations. We found that compared to the well-characterized entomopathogenic nematode symbiont X. nematophila, X. innexi fails to suppress the insect phenoloxidase immune pathway and is attenuated for virulence and reproduction in the Lepidoptera Galleria mellonella and Manduca sexta, as well as the dipteran Drosophila melanogaster. To assess if, compared to other Xenorhabdus spp., X. innexi has a reduced capacity to synthesize virulence determinants, we obtained and analyzed a draft genome sequence. We found no evidence for several hallmarks of Xenorhabdus spp. toxicity, including Tc and Mcf toxins. Similar to other Xenorhabdus genomes, we found numerous loci predicted to encode non-ribosomal peptide/polyketide synthetases. Anti-SMASH predictions of these loci revealed one, related to the fcl locus that encodes fabclavines and zmn locus that encodes zeamines, as a likely candidate to encode the X. innexi mosquitocidal toxin biosynthetic machinery, which we designated Xlt. In support of this hypothesis, two mutants each with an insertion in an Xlt biosynthesis gene cluster lacked the mosquitocidal compound based on HPLC/MS analysis and neither produced toxin to the levels of the wild type parent., Conclusions: The X. innexi genome will be a valuable resource in identifying loci encoding new metabolites of interest, but also in future comparative studies of nematode-bacterial symbiosis and niche partitioning among bacterial pathogens.

Background: Xenocoumacin 1 (Xcn1) and Xenocoumacin 2 (Xcn2) are the main antimicrobial compounds produced by Xenorhabdus nematophila. Culture conditions, including pH, had remarkably distinct effects on the antimicrobial activity of X. nematophila. However, the regulatory mechanism of pH on the antimicrobial activity and antibiotic production of this bacterium is still lacking., Results: With the increase of initial pH, the antimicrobial activity of X. nematophila YL001 was improved. The levels of Xcn1 and nematophin at pH 8.5 were significantly (P < 0.05) higher than that at pH 5.5 and 7.0. In addition, the expression of xcnA-L, which are responsible for the production of Xcn1 was increased and the expression of xcnMN, which are required for the conversion of Xcn1 to Xcn2 was reduced at pH 8.5. Also, the expression of ompR and cpxR were decreased at pH 8.5., Conclusion: The alkaline pH environment was found to be beneficial for the production of Xcn1 and nematophin, which in turn led to high antimicrobial activity of X. nematophila at pH 8.5.

Background: Aedes aegypti is a potential vector of West Nile, Japanese encephalitis, chikungunya, dengue and Zika viruses. Alternative control measurements of the vector are needed to overcome the problems of environmental contamination and chemical resistance. Xenorhabdus and Photorhabdus are symbionts in the intestine of entomopathogenic nematodes (EPNs) Steinernema spp. and Heterorhabditis spp. These bacteria are able to produce a broad range of bioactive compounds including antimicrobial, antiparasitic, cytotoxic and insecticidal compounds. The objectives of this study were to identify Xenorhabdus and Photorhabdus isolated from EPNs in upper northern Thailand and to study their larvicidal activity against Ae. aegypti larvae., Results: A total of 60 isolates of symbiotic bacteria isolated from EPNs consisted of Xenorhabdus (32 isolates) and Photorhabdus (28 isolates). Based on recA gene sequencing, BLASTN and phylogenetic analysis, 27 isolates of Xenorhabdus were identical and closely related to X. stockiae, 4 isolates were identical to X. miraniensis, and one isolate was identical to X. ehlersii. Twenty-seven isolates of Photorhabdus were closely related to P. luminescens akhurstii and P. luminescens hainanensis, and only one isolate was identical and closely related to P. luminescens laumondii. Xenorhabdus and Photorhabdus were lethal to Ae aegypti larvae. Xenorhabdus ehlersii bMH9.2&#95;TH showed 100% efficiency for killing larvae of both fed and unfed conditions, the highest for control of Ae. aegypti larvae and X. stockiae (bLPA18.4&#95;TH) was likely to be effective in killing Ae. aegypti larvae given the mortality rates above 60% at 72 h and 96 h., Conclusions: The common species in the study area are X. stockiae, P. luminescens akhurstii, and P. luminescens hainanensis. Three symbiotic associations identified included P. luminescens akhurstii-H. gerrardi, P. luminescens hainanensis-H. gerrardi and X. ehlersii-S. Scarabaei which are new observations of importance to our knowledge of the biodiversity of, and relationships between, EPNs and their symbiotic bacteria. Based on the biological assay, X. ehlersii bMH9.2&#95;TH begins to kill Ae. aegypti larvae within 48 h and has the most potential as a pathogen to the larvae. These data indicate that X. ehlersii may be an alternative biological control agent for Ae. aegypti and other mosquitoes.

In mutually beneficial and pathogenic symbiotic associations, microbes must adapt to the host environment for optimal fitness. Both within an individual host and during transmission between hosts, microbes are exposed to temporal and spatial variation in environmental conditions. The phenomenon of phenotypic variation, in which different subpopulations of cells express distinctive and potentially adaptive characteristics, can contribute to microbial adaptation to a lifestyle that includes rapidly changing environments. The environments experienced by a symbiotic microbe during its life history can be erratic or predictable, and each can impact the evolution of adaptive responses. In particular, the predictability of a rhythmic or cyclical series of environments may promote the evolution of signal transduction cascades that allow preadaptive responses to environments that are likely to be encountered in the future, a phenomenon known as adaptive prediction. In this review, we summarize environmental variations known to occur in some well-studied models of symbiosis and how these may contribute to the evolution of microbial population heterogeneity and anticipatory behavior. We provide details about the symbiosis between Xenorhabdus bacteria and Steinernema nematodes as a model to investigate the concept of environmental adaptation and adaptive prediction in a microbial symbiosis., (Copyright © 2017 American Society for Microbiology.)

Xenorhabdus nematophila bacteria are mutualistic symbionts of Steinernema carpocapsae nematodes and pathogens of insects. The X. nematophila global regulator Lrp controls the expression of many genes involved in both mutualism and pathogenic activities, suggesting a role in the transition between the two host organisms. We previously reported that natural populations of X. nematophila exhibit various levels of Lrp expression and that cells expressing relatively low levels of Lrp are optimized for virulence in the insect Manduca sexta The adaptive advantage of the high-Lrp-expressing state was not established. Here we used strains engineered to express constitutively high or low levels of Lrp to test the model in which high-Lrp-expressing cells are adapted for mutualistic activities with the nematode host. We demonstrate that high-Lrp cells form more robust biofilms in laboratory media than do low-Lrp cells, which may reflect adherence to host tissues. Also, our data showed that nematodes cultivated with high-Lrp strains are more frequently colonized than are those associated with low-Lrp strains. Taken together, these data support the idea that high-Lrp cells have an advantage in tissue adherence and colonization initiation. Furthermore, our data show that high-Lrp-expressing strains better support nematode reproduction than do their low-Lrp counterparts under both in vitro and in vivo conditions. Our data indicate that heterogeneity of Lrp expression in X. nematophila populations provides diverse cell populations adapted to both pathogenic (low-Lrp) and mutualistic (high-Lrp) states. IMPORTANCE Host-associated bacteria experience fluctuating conditions during both residence within an individual host and transmission between hosts. For bacteria that engage in evolutionarily stable, long-term relationships with particular hosts, these fluctuations provide selective pressure for the emergence of adaptive regulatory mechanisms. Here we present evidence that the bacterium Xenorhabdus nematophila uses various levels of the transcription factor Lrp to optimize its association with its two animal hosts, nematodes and insects, with which it behaves as a mutualist and a pathogen, respectively. Building on our previous finding that relatively low cellular levels of Lrp are optimal for pathogenesis, we demonstrate that, conversely, high levels of Lrp promote mutualistic activities with the Steinernema carpocapsae nematode host. These data suggest that X. nematophila has evolved to utilize phenotypic variation between high- and low-Lrp-expression states to optimize its alternating behaviors as a mutualist and a pathogen., (Copyright © 2017 American Society for Microbiology.)

In the entomopathogenic bacterium Xenorhabdus nematophila , cell-to-cell variation in the abundance of the Lrp transcription factor leads to virulence modulation; low Lrp levels are associated with a virulent phenotype and suppression of antimicrobial peptides (AMPs) in Manduca sexta insects, while cells that lack lrp or express high Lrp levels are virulence attenuated and elicit AMP expression. To better understand the basis of these phenotypes, we examined X. nematophila strains expressing fixed Lrp levels. Unlike the lrp -null mutant, the high- lrp strain is fully virulent in Drosophila melanogaster , suggesting that these two strains have distinct underlying causes of virulence attenuation in M. sexta Indeed, the lrp -null mutant was defective in cytotoxicity against M. sexta hemocytes relative to that in the high- lrp and low- lrp strains. Further, supernatant derived from the lrp -null mutant but not from the high- lrp strain was defective in inhibiting weight gain when fed to 1st instar M. sexta These data suggest that contributors to the lrp -null mutant virulence attenuation phenotype are the lack of Lrp-dependent cytotoxic and extracellular oral growth inhibitory activities, which may be particularly important for virulence in D. melanogaster In contrast, the high-Lrp strain was sensitive to the antimicrobial peptide cecropin, had a transient survival defect in M. sexta , and had reduced extracellular levels of insecticidal activity, measured by injection of supernatant into 4th instar M. sexta Thus, high- lrp strain virulence attenuation may be explained by its hypersensitivity to M. sexta host immunity and its inability to secrete one or more insecticidal factors. IMPORTANCE Adaptation of a bacterial pathogen to host environments can be achieved through the coordinated regulation of virulence factors that can optimize success under prevailing conditions. In the insect pathogen Xenorhabdus nematophila , the global transcription factor Lrp is necessary for virulence when injected into Manduca sexta or Drosophila melanogaster insect hosts. However, high levels of Lrp, either naturally occurring or artificially induced, cause attenuation of X. nematophila virulence in M. sexta but not D. melanogaster Here, we present evidence suggesting that the underlying cause of high-Lrp-dependent virulence attenuation in M. sexta is hypersensitivity to host immune responses and decreased insecticidal activity and that high-Lrp virulence phenotypes are insect host specific. This knowledge suggests that X. nematophila faces varied challenges depending on the type of insect host it infects and that its success in these environments depends on Lrp-dependent control of a multifactorial virulence repertoire., (Copyright © 2017 American Society for Microbiology.)

Background: Steinernematid nematodes form obligate symbioses with bacteria from the genus Xenorhabdus. Together Steinernema nematodes and their bacterial symbionts successfully infect, kill, utilize, and exit their insect hosts. During this process the nematodes and bacteria disassociate requiring them to re-associate before emerging from the host. This interaction can be complicated when two different nematodes co-infect an insect host., Results: Non-cognate nematode-bacteria pairings result in reductions for multiple measures of success, including total progeny production and virulence. Additionally, nematode infective juveniles carry fewer bacterial cells when colonized by a non-cognate symbiont. Finally, we show that Steinernema nematodes can distinguish heterospecific and some conspecific non-cognate symbionts in behavioral choice assays., Conclusions: Steinernema-Xenorhabdus symbioses are tightly governed by partner recognition and fidelity. Association with non-cognates resulted in decreased fitness, virulence, and bacterial carriage of the nematode-bacterial pairings. Entomopathogenic nematodes and their bacterial symbionts are a useful, tractable, and reliable model for testing hypotheses regarding the evolution, maintenance, persistence, and fate of mutualisms.

Xenorhabdus bovienii bacteria have a dual lifestyle: they are mutualistic symbionts to many species of Steinernema nematodes and are pathogens to a wide array of insects. Previous studies have shown that virulence of X.bovienii-Steinernema spp. pairs decreases when the nematodes associate with non-cognate bacterial strains. However, the virulence of the X. bovienii strains alone has not been fully investigated. In this study, we characterized the virulence of nine X. bovienii strains in Galleria mellonella and Spodoptera littoralis and performed a comparative genomic analysis to correlate observed phenotypes with strain genotypes. Two X. bovienii strains were found to be highly virulent against the tested insect hosts, while three strains displayed attenuated insect virulence. Comparative genomic analyses revealed the presence of several clusters present only in virulent strains, including a predicted type VI secretion system (T6SS). We performed intra-species-competition assays, and showed that the virulent T6SS+ strains generally outcompeted the less virulent T6SS- strains. Thus, we speculate that the T6SS in X. bovienii may be another addition to the arsenal of antibacterial mechanisms expressed by these bacteria in an insect, where it could potentially play three key roles: (1) competition against the insect host microbiota; (2) protection of the insect cadaver from necrotrophic microbial competitors; and (3) outcompeting other Xenorhabdus species and/or strains when co-infections occur.

Xenorhabdus is a bacterial symbiont of entomopathogenic Steinernema nematodes and is pathogenic for insects. Its life cycle involves a stage inside the insect cadaver, in which it competes for environmental resources with microorganisms from soil and the insect gut. Xenorhabdus is, thus, a useful model for identifying new interbacterial competition systems. For the first time, in an entomopathogenic bacterium, Xenorhabdus doucetiae strain FRM16, we identified a cdi-like locus. The cdi loci encode contact-dependent inhibition (CDI) systems composed of proteins from the two-partner secretion (TPS) family. CdiB is the outer membrane protein and CdiA is the toxic exoprotein. An immunity protein, CdiI, protects bacteria against inhibition. We describe here the growth inhibition effect of the toxic C-terminus of CdiA from X. doucetiae FRM16, CdiA-CTFRM16, following its production in closely and distantly related enterobacterial species. CdiA-CTFRM16 displayed Mg2+-dependent DNase activity, in vitro. CdiA-CTFRM16-mediated growth inhibition was specifically neutralized by CdiIFRM16. Moreover, the cdi FRM16 locus encodes an ortholog of toxin-activating proteins C that we named CdiCFRM16. In addition to E. coli, the cdiBCAI-type locus was found to be widespread in environmental bacteria interacting with insects, plants, rhizospheres and soils. Phylogenetic tree comparisons for CdiB, CdiA and CdiC suggested that the genes encoding these proteins had co-evolved. By contrast, the considerable variability of CdiI protein sequences suggests that the cdiI gene is an independent evolutionary unit. These findings further characterize the sparsely described cdiBCAI-type locus., Competing Interests: The authors have declared that no competing interests exist.

The amicoumacins belong to a class of dihydroisocoumarin natural products and display antibacterial, antifungal, anticancer, and anti-inflammatory activities. Amicoumacins are the pro-drug activation products of a bacterial nonribosomal peptide-polyketide hybrid biosynthetic pathway and have been isolated from Gram-positive Bacillus and Nocardia species. Here, we report the stimulation of a "cryptic" amicoumacin pathway in the entomopathogenic Gram-negative bacterium Xenorhabdus bovienii, a strain not previously known to produce amicoumacins. X. bovienii participates in a multi-lateral symbiosis where it is pathogenic to insects and mutualistic to its Steinernema nematode host. Waxmoth larvae are common prey of the X. bovienii-Steinernema pair. Employing a medium designed to mimic the amino acid content of the waxmoth circulatory fluid led to the detection and characterization of amicoumacins in X. bovienii. The chemical structures of the amicoumacins were supported by 2D-NMR, HR-ESI-QTOF-MS, tandem MS, and polarimeter spectral data. A comparative gene cluster analysis of the identified X. bovienii amicoumacin pathway to that of the Bacillus subtilis amicoumacin pathway and the structurally-related Xenorhabdus nematophila xenocoumacin pathway is presented. The X. bovienii pathway encodes an acetyltransferase not found in the other reported pathways, which leads to a series of N-acetyl-amicoumacins that lack antibacterial activity. N-acetylation of amicoumacin was validated through in vitro protein biochemical studies, and the impact of N-acylation on amicoumacin's mode of action was examined through ribosomal structural analyses., Competing Interests: The authors declare no conflict of interest.

Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Within Xenorhabdus bovienii species, the X. bovienii CS03 strain (Xb CS03) is nonvirulent when directly injected into lepidopteran insects, and displays a low virulence when associated with its Steinernema symbiont. The genome of Xb CS03 was sequenced and compared with the genome of a virulent strain, X. bovienii SS-2004 (Xb SS-2004). The genome size and content widely differed between the two strains. Indeed, Xb CS03 had a large genome containing several specific loci involved in the inhibition of competitors, including a few NRPS-PKS loci (nonribosomal peptide synthetases and polyketide synthases) producing antimicrobial molecules. Consistently, Xb CS03 had a greater antimicrobial activity than Xb SS-2004. The Xb CS03 strain contained more pseudogenes than Xb SS-2004. Decay of genes involved in the host invasion and exploitation (toxins, invasins, or extracellular enzymes) was particularly important in Xb CS03. This may provide an explanation for the nonvirulence of the strain when injected into an insect host. We suggest that Xb CS03 and Xb SS-2004 followed divergent evolutionary scenarios to cope with their peculiar life cycle. The fitness strategy of Xb CS03 would involve competitor inhibition, whereas Xb SS-2004 would quickly and efficiently kill the insect host. Hence, Xenorhabdus strains would have widely divergent host exploitation strategies, which impact their genome structure., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Species of Xenorhabdus and Photorhabdus bacteria form mutualistic associations with Steinernema and Heterorhabditis nematodes, respectively and serve as model systems for studying microbe-animal symbioses. Here, we profiled gene expression of Xenorhabdus koppenhoeferi during their symbiotic persistence in the newly formed infective juveniles of the host nematode Steinernema scarabaei through the selective capture of transcribed sequences (SCOTS). The obtained gene expression profile was then compared with other nematode-bacteria partnerships represented by Steinernema carpocapsae-Xenorhabdus nematophila and Heterorhabditis bacteriophora-Photorhabdus temperata. A total of 29 distinct genes were identified to be up-regulated and 53 were down-regulated in X. koppenhoeferi while in S. scarabaei infective juveniles. Of the identified genes, 8 of the up-regulated and 14 of the down-regulated genes were similarly expressed in X. nematophila during persistence in its host nematode S. carpocapsae. However, only one from each of these up- and down-regulated genes was common to the mutualistic partnership between the bacterium P. temperata and the nematode H. bacteriophora. Interactive network analysis of the shared genes between X. koppenhoeferi and X. nematophila demonstrated that the up-regulated genes were mainly involved in bacterial survival and the down-regulated genes were more related to bacterial virulence and active growth. Disruption of two selected genes pta (coding phosphotransacetylase) and acnB (coding aconitate hydratase) in X. nematophila with shared expression signature with X. koppenhoeferi confirmed that these genes are important for bacterial persistence in the nematode host. The results of our comparative analyses show that the two Xenorhabdus species share a little more than a quarter of the transcriptional mechanisms during persistence in their nematode hosts but these features are quite different from those used by P. temperata bacteria in their nematode host H. bacteriophora.

Background: Xenorhabdus bacteria engage in a beneficial symbiosis with Steinernema nematodes, in part by providing activities that help kill and degrade insect hosts for nutrition. Xenorhabdus strains (members of a single species) can display wide variation in host-interaction phenotypes and genetic potential indicating that strains may differ in their encoded symbiosis factors, including secreted metabolites., Methods: To discern strain-level variation among symbiosis factors, and facilitate the identification of novel compounds, we performed a comparative analysis of the genomes of 10 Xenorhabdus bovienii bacterial strains., Results: The analyzed X. bovienii draft genomes are broadly similar in structure (e.g. size, GC content, number of coding sequences). Genome content analysis revealed that general classes of putative host-microbe interaction functions, such as secretion systems and toxin classes, were identified in all bacterial strains. In contrast, we observed diversity of individual genes within families (e.g. non-ribosomal peptide synthetase clusters and insecticidal toxin components), indicating the specific molecules secreted by each strain can vary. Additionally, phenotypic analysis indicates that regulation of activities (e.g. enzymes and motility) differs among strains., Conclusions: The analyses presented here demonstrate that while general mechanisms by which X. bovienii bacterial strains interact with their invertebrate hosts are similar, the specific molecules mediating these interactions differ. Our data support that adaptation of individual bacterial strains to distinct hosts or niches has occurred. For example, diverse metabolic profiles among bacterial symbionts may have been selected by dissimilarities in nutritional requirements of their different nematode hosts. Similarly, factors involved in parasitism (e.g. immune suppression and microbial competition factors), likely differ based on evolution in response to naturally encountered organisms, such as insect hosts, competitors, predators or pathogens. This study provides insight into effectors of a symbiotic lifestyle, and also highlights that when mining Xenorhabdus species for novel natural products, including antibiotics and insecticidal toxins, analysis of multiple bacterial strains likely will increase the potential for the discovery of novel molecules.