Xenorhabdus aichiensis sp. nov., Xenorhabdus anantnagensis sp. nov., and Xenorhabdus yunnanensis sp. nov., Isolated from Steinernema Entomopathogenic Nematodes.

Three bacterial strains, XENO-2T, XENO-7T, and XENO-10T, isolated from Steinernema entomopathogenic nematodes, were found to represent novel Xenorhabdus species. In this study, we describe these new species by whole-genome and whole-proteome phylogenomic reconstructions, by calculating sequence identity scores using core genome sequences, and by phenotypic characterization. Phylogenomic reconstructions using ribosomal and house-keeping genes, and whole-genome and whole-proteome sequences show that XENO-2T and XENO-10T are closely related to Xenorhabdus japonica DSM 16522T and that XENO-7T is closely related to Xenorhabdus bovienii subsp. africana XENO-1T and to X. bovienii subsp. bovienii T228T. The dDDH values between XENO-2T and XENO-10T and between XENO-2T and X. japonica DSM 16522T are 56.4 and 51.8%, respectively. The dDDH value between XENO-10T and X. japonica DSM 16522T is 53.4%. The dDDH values between XENO-7T and X. bovienii subsp. africana XENO-1T and between XENO-7T and X. bovienii subsp. bovienii T228T are 63.6 and 69.4%, respectively. These dDDH values are below the 70% divergence threshold for prokaryotic species delineation. The newly described species are highly pathogenic to G. mellonella larvae, grow at pH between 5 and 9 (optimum 5-7), at salt concentrations of 1-3% (optimum 1-2%), and temperatures between 20 and 37 °C (optimum 28-30 °C). Biochemical tests such as lysine decarboxylase, ornithine decarboxylase, urease, gelatinase, citrate utilization, indole and acetoin production, and cytochrome oxidase tests allow to differentiate the novel species from their more closely related species. Considering these genetic and phenotypic divergencies, we propose the following new species: Xenorhabdus aichiensis sp. nov. with XENO-7T (= CCM 9233T = CCOS 2024T) as the type strain, Xenorhabdus anantnagensis sp. nov., with XENO-2T (= CCM 9237T = CCOS 2023T) as the type strain, and Xenorhabdus yunnanensis sp. nov., with XENO-10T (= CCM 9322T = CCOS 2071T) as the type strain. Our study contributes to a better understanding of the biodiversity and phylogenetic relationships of entomopathogenic bacteria associated with insect parasitic nematodes.

Diverse Roles for a Conserved DNA-Methyltransferase in the Entomopathogenic Bacterium Xenorhabdus.

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.

Photorhabdus heterorhabditis subsp. aluminescens subsp. nov., Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov., Photorhabdus australis subsp. thailandensis subsp. nov., Photorhabdus australis subsp. australis subsp. nov., and Photorhabdus aegyptia sp. nov. isolated from Heterorhabditis entomopathogenic nematodes.

Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria, BA1T, Q614T and PB68.1T, isolated from the digestive system of Heterorhabditis entomopathogenic nematodes, were biochemically and molecularly characterized to clarify their taxonomic affiliations. The 16S rRNA gene sequences of these strains suggest that they belong to the Gammaproteobacteria, to the family Morganellacea, and to the genus Photorhabdus. Deeper analyses using whole genome-based phylogenetic reconstructions suggest that BA1T is closely related to Photorhabdus akhursti, that Q614T is closely related to Photorhabdus heterorhabditis, and that PB68.1T is closely related to Photorhabdus australis. In silico genomic comparisons confirm these observations: BA1T and P. akhursti 15138T share 68.8 % digital DNA-DNA hybridization (dDDH), Q614T and P. heterorhabditis SF41T share 75.4 % dDDH, and PB68.1T and P. australis DSM 17609T share 76.6  % dDDH. Physiological and biochemical characterizations reveal that these three strains also differ from all validly described Photorhabdus species and from their more closely related taxa, contrary to what was previously suggested. We therefore propose to classify BA1T as a new species within the genus Photorhabdus, Q614T as a new subspecies within P. heterorhabditis, and PB68.1T as a new subspecies within P. australis. Hence, the following names are proposed for these strains: Photorhabdus aegyptia sp. nov. with the type strain BA1T(=DSM 111180T=CCOS 1943T=LMG 31957T), Photorhabdus heterorhabditis subsp. aluminescens subsp. nov. with the type strain Q614T (=DSM 111144T=CCOS 1944T=LMG 31959T) and Photorhabdus australis subsp. thailandensis subsp. nov. with the type strain PB68.1T (=DSM 111145T=CCOS 1942T). These propositions automatically create Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov. with SF41T as the type strain (currently classified as P. heterorhabditis) and Photorhabdus australis subsp. australis subsp. nov. with DSM17609T as the type strain (currently classified as P. australis).

rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing.

BACKGROUND: Microbiome composition is frequently studied by the amplification and high-throughput sequencing of specific molecular markers (metabarcoding). Various hypervariable regions of the 16S rRNA gene are classically used to estimate bacterial diversity, but other universal bacterial markers with a finer taxonomic resolution could be employed. We compared specificity and sensitivity between a portion of the rpoB gene and the V3 V4 hypervariable region of the 16S rRNA gene. RESULTS: We first designed universal primers for rpoB suitable for use with Illumina sequencing-based technology and constructed a reference rpoB database of 45,000 sequences. The rpoB and V3 V4 markers were amplified and sequenced from (i) a mock community of 19 bacterial strains from both Gram-negative and Gram-positive lineages; (ii) bacterial assemblages associated with entomopathogenic nematodes. In metabarcoding analyses of mock communities with two analytical pipelines (FROGS and DADA2), the estimated diversity captured with the rpoB marker resembled the expected composition of these mock communities more closely than that captured with V3 V4. The rpoB marker had a higher level of taxonomic affiliation, a higher sensitivity (detection of all the species present in the mock communities), and a higher specificity (low rates of spurious OTU detection) than V3 V4. We compared the performance of the rpoB and V3 V4 markers in an animal ecosystem model, the infective juveniles of the entomopathogenic nematode Steinernema glaseri carrying the symbiotic bacteria Xenorhabdus poinarii. Both markers showed the bacterial community associated with this nematode to be of low diversity (< 50 OTUs), but only rpoB reliably detected the symbiotic bacterium X. poinarii. CONCLUSIONS: Our results confirm that different microbiota composition data may be obtained with different markers. We found that rpoB was a highly appropriate marker for assessing the taxonomic structure of mock communities and the nematode microbiota. Further studies on other ecosystems should be considered to evaluate the universal usefulness of the rpoB marker. Our data highlight two crucial elements that should be taken into account to ensure more reliable and accurate descriptions of microbial diversity in high-throughput amplicon sequencing analyses: i) the need to include mock communities as controls; ii) the advantages of using a multigenic approach including at least one housekeeping gene (rpoB is a good candidate) and one variable region of the 16S rRNA gene. This study will be useful to the growing scientific community describing bacterial communities by metabarcoding in diverse ecosystems.

Variable virulence phenotype of Xenorhabdus bovienii (γ-Proteobacteria: Enterobacteriaceae) in the absence of their vector hosts.

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 bovienii CS03, the bacterial symbiont of the entomopathogenic nematode Steinernema weiseri, is a non-virulent strain against lepidopteran insects.

Xenorhabdus bacteria (γ-proteobacteria: Enterobacteriaceae) have dual lifestyles. They have a mutualistic relationship with Steinernema nematodes (Nematoda: Steinernematidae) and are pathogenic to a wide range of insects. Each Steinernema nematode associates with a specific Xenorhabdus species. However, a Xenorhabdus species can have multiple nematode hosts. For example, Xenorhabdus bovienii (Xb) colonizes at least nine Steinernema species from two different phylogenetic clades. The Steinernema-Xb partnership has been found in association with different insect hosts. Biological and molecular data on the Steinernema jollieti-Xb strain SS-2004 pair have recently been described. In particular, the Xb SS-2004 bacteria are virulent alone after direct injection into insect, making this strain a model for studying Xb virulence. In this study, we searched for Xb strains attenuated in virulence. For this purpose, we underwent infection assays with five Steinernema spp.-Xb pairs with two insects, Galleria mellonella (Lepidoptera: Pyralidae) and Spodoptera littoralis (Lepidoptera: Noctuidae). The S. weiseri-Xb CS03 pair showed attenuated virulence and lower fitness in S. littoralis in comparison to the other nematode-bacteria pairs. Furthermore, when injected alone into the hemolymph of G. mellonella or S. littoralis, the Xb CS03 bacterial strain was the only non-virulent strain. By comparison with the virulent Xb SS-2004 strain, Xb CS03 showed an increased sensitivity to the insect antimicrobial peptides, suggesting an attenuated response to the insect humoral immunity. To our current knowledge, Xb CS03 is the first non-virulent Xb strain identified. We propose this strain as a new model for studying the Xenorhabdus virulence.

Photorhabdus heterorhabditis sp. nov., a symbiont of the entomopathogenic nematode Heterorhabditis zealandica.

The bacterial symbionts SF41T and SF783 were isolated from populations of the insect pathogenic nematode Heterorhabditis zealandica collected in South Africa. Both strains were closely related to strain Q614 isolated from a population of Heterorhabditis sp. collected from soil in Australia in the 1980s. Sequence analysis based on a multigene approach, DNA-DNA hybridization data and phenotypic traits showed that strains SF41T, SF783 and Q614 belong to the same species of the genus Photorhabdus with Photorhabdus temperata subsp. cinerea as the most closely related taxon (DNA-DNA hybridization value of 68%). Moreover, the phylogenetic position of Photorhabdus temperata subsp. cinerea DSM 19724T initially determined using the gyrB sequences, was reconsidered in the light of the data obtained by our multigene approach and DNA-DNA hybridization experiments. Strains SF41T, SF783 and Q614 represent a novel species of the genus Photorhabdus, for which the name Photorhabdus heterorhabditis sp. nov. is proposed (type strain SF41T=ATCC BAA-2479T=DSM 25263T).

Photorhabdus luminescens subsp. noenieputensis subsp. nov., a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica.

The bacterial symbiont AM7(T), isolated from a novel entomopathogenic nematode species of the genus Heterorhabditis, displays the main phenotypic traits of the genus Photorhabdus and is highly pathogenic to Galleria mellonella. Phylogenetic analysis based on a multigene approach (16S rRNA, recA, gyrB, dnaN, gltX and infB) confirmed the classification of isolate AM7(T) within the species Photorhabdus luminescens and revealed its close relatedness to Photorhabdus luminescens subsp. caribbeanensis, P. luminescens subsp. akhurstii and P. luminescens subsp. hainanensis. The five concatenated protein-encoding sequences (4197 nt) of strain AM7(T) revealed 95.8, 95.4 and 94.9 % nucleotide identity to sequences of P. luminescens subsp. caribbeanensis HG29(T), P. luminescens subsp. akhurstii FRG04(T) and P. luminescens subsp. hainanensis C8404(T), respectively. These identity values are less than the threshold of 97 % proposed for classification within one of the existing subspecies of P. luminescens. Unlike other strains described for P. luminescens, strain AM7(T) produces acid from adonitol, sorbitol and xylitol, assimilates xylitol and has no lipase activity on medium containing Tween 20 or 60. Strain AM7(T) is differentiated from P. luminescens subsp. caribbeanensis by the assimilation of N-acetylglucosamine and the absence of haemolytic activity. Unlike P. luminescens subsp. akhurstii, strain AM7(T) does not assimilate mannitol, and it is distinguished from P. luminescens subsp. hainanensis by the assimilation of trehalose and citrate, the inability to produce indole from tryptophan and the presence of acetoin production and urease activity. Strain AM7(T) ( = ATCC BAA-2407(T)  = DSM 25462(T)) belongs to a novel subspecies, and is proposed as the type strain of Photorhabdus luminescens subsp. noenieputensis sp. nov.

Description of Xenorhabdus magdalenensis sp. nov., the symbiotic bacterium associated with Steinernema australe.

A symbiotic bacterium, strain IMI 397775(T), was isolated from the insect-pathogenic nematode Steinernema australe. On the basis of 16S rRNA gene sequence similarity, this bacterial isolate was shown to belong to the genus Xenorhabdus, in agreement with the genus of its nematode host. The accurate phylogenetic position of this new isolate was defined using a multigene approach and showed that isolate IMI 397775(T) shares a common ancestor with Xenorhabdus doucetiae FRM16(T) and Xenorhabdus romanii PR06-A(T), the symbiotic bacteria associated with Steinernema diaprepesi and Steinernema puertoricense, respectively. The nucleotide identity (less than 97%) between isolate IMI 397775(T), X. doucetiae FRM16(T) and X. romanii PR06-A(T) calculated for the concatenated sequences of five gene fragments encompassing 4275 nt, several phenotypic traits and the difference between the upper temperatures that limit growth of these three bacteria allowed genetic and phenotypic differentiation of isolate IMI 397775(T) from the two closely related species. Strain IMI 397775(T) therefore represents a novel species, for which the name Xenorhabdus magdalenensis sp. nov. is proposed, with the type strain IMI 397775(T) ( = DSM 24915(T)).

Mutagenesis of both prophenoloxidases in the fall armyworm induces major defects in metamorphosis.

Upon infection, the phenoloxidase system in arthropods is rapidly mobilized and constitutes a major defense system against invaders. The activation of the key enzymes prophenoloxidase (PPO) and their action in immunity through melanization and encapsulation of foreign bodies in hemolymph has been described in many insects. On the other hand, little is known about PPOs involvement in other essential functions related to insect development. In this paper, we investigated the function of the two PPOs of the crop pest, Spodoptera frugiperda (PPO1 and PPO2). We show that PPOs are mainly expressed in hemocytes with the PPO2 expressed at higher levels than the PPO1. In addition, these two genes are expressed in the same tissue and at the same stages of insect development. Through the generation of loss-of-function mutants by CRISPR/Cas9 method, we show that the presence of PPOs is essential for the normal development of the pupa and the survival of the insect.

Studies of the dynamic expression of the Xenorhabdus FliAZ regulon reveal atypical iron-dependent regulation of the flagellin and haemolysin genes during insect infection.

Xenorhabdus nematophila engages in complex interactions with invertebrates, through its symbiosis with soil nematodes and its pathogenicity to a broad range of insect larvae. Among the regulatory proteins of Xenorhabdus involved in host interactions, the sigma factor FliA and the regulator FliZ, expressed from the fliAZ operon, play a key role in mediating the production of exoenzymes, motility and full virulence in insects (Lanois et al., 2008). In this study, we investigated the dynamics of the FliA-dependent flagellin gene fliC and FliZ-dependent haemolysin genes xaxAB during insect infection and nematode association by carrying out real-time expression analysis using an unstable GFP monitoring system. We showed that expression of the FliAZ-dependent genes in infected insects is not restricted to a specific tissue but increases significantly just prior to host death and reaches a maximal level in larvae cadaver. Using an iron availability reporter construct, we also showed that iron starvation conditions inhibit expression of FliAZ-dependent genes in vitro, as well as during the first steps of the infectious process. These findings shed further light on the role of the FliAZ regulon in the Xenorhabdus life cycle and suggest that iron may constitute a signal governing Xenorhabdus adaptation to shifting host environments.

The first record of entomopathogenic nematodes (Rhabiditiae: Steinernematidae and Heterorhabditidae) in natural ecosystems in Lebanon: A biogeographic approach in the Mediterranean region.

A survey of entomopathogenic nematodes in Lebanon was conducted for the first time during 2008-2009. Samples were collected on the coastal strip and in nine vegetation types extending from the coastal line to 3088m above sea level. Wooded and herbaceous ecosystems were considered for sampling purposes. A total of 570 samples were taken, out of which 1% were positive for entomopathogenic nematodes. Approximately, 15.8% out of the 19 sites sampled revealed entomopathogenic nematodes presence (representing three samples). Two entomopathogenic nematodes species Heterorhabditis bacteriophora and Steinernema feltiae were recovered, and identification of their symbiotic bacteria revealed the presence of a Xenorhabdus bovienii, Photorhabdus temperata subsp. thracensis, Photorhabdus luminescens subsp. kayaii and Photorhabdus luminescens subsp. Laumondii.

A novel semi-selective medium for Pseudomonas protegens isolation from soil samples.

Pseudomonas protegens is a rhizosphere pseudomonad with a high agronomical potential (entomopathogenic and beneficial to plants) and bio-catalytic activities, but no selective medium has been described for its isolation. We developed a semi-selective minimum agar medium for the specific isolation and growth of P. protegens. We searched for both (i) a carbon source allowing the growth of P. protegens but potentially inhibiting the growth of other pseudomonads and (ii) an antimicrobial agent suppressing other members of the bacterial rhizosphere community. The M9-PP-agar medium consists of M9 base agar with adipic acid as the only carbon source and Irgasan® as an anti-bacterial agent. We tested the selectivity and sensitivity of M9-PP-agar by measuring the growth of 68 bacterial strains from 36 different species on this medium. Ten of the species tested were able to grow on M9-PP-agar medium: four species from the Pseudomonadaceae (Pseudomonas aeruginosa, Pseudomonas protegens, Pseudomonas putida, Stenotrophomonas maltophilia) as well as Achromobacter xylosoxidans, Agrobacterium tumefaciens, Brevundimonas sp., Serratia liquefaciens, Serratia marcescens and Variovorax paradoxus. All colonies were white, except for those of P. protegens (12 strains), which were typically brown. We demonstrated the efficiency of the M9-PP agar medium for P. protegens isolation, by inoculating two soils with the reference strain P. protegens CHAOT and then reisolating them. We also developed a fitF-PCR test targeting a regulator gene of the insecticidal P. protegens fit locus, for the rapid molecular detection of P. protegens colonies. We, therefore, developed a highly specific process for the routine isolation of new P. protegens strains from the soil environment, based on the use of a semi-selective medium and the specific color of colonies.

Entomopathogenic nematode-associated microbiota: from monoxenic paradigm to pathobiome.

BACKGROUND: The holistic view of bacterial symbiosis, incorporating both host and microbial environment, constitutes a major conceptual shift in studies deciphering host-microbe interactions. Interactions between Steinernema entomopathogenic nematodes and their bacterial symbionts, Xenorhabdus, have long been considered monoxenic two partner associations responsible for the killing of the insects and therefore widely used in insect pest biocontrol. We investigated this "monoxenic paradigm" by profiling the microbiota of infective juveniles (IJs), the soil-dwelling form responsible for transmitting Steinernema-Xenorhabdus between insect hosts in the parasitic lifecycle. RESULTS: Multigenic metabarcoding (16S and rpoB markers) showed that the bacterial community associated with laboratory-reared IJs from Steinernema carpocapsae, S. feltiae, S. glaseri and S. weiseri species consisted of several Proteobacteria. The association with Xenorhabdus was never monoxenic. We showed that the laboratory-reared IJs of S. carpocapsae bore a bacterial community composed of the core symbiont (Xenorhabdus nematophila) together with a frequently associated microbiota (FAM) consisting of about a dozen of Proteobacteria (Pseudomonas, Stenotrophomonas, Alcaligenes, Achromobacter, Pseudochrobactrum, Ochrobactrum, Brevundimonas, Deftia, etc.). We validated this set of bacteria by metabarcoding analysis on freshly sampled IJs from natural conditions. We isolated diverse bacterial taxa, validating the profile of the Steinernema FAM. We explored the functions of the FAM members potentially involved in the parasitic lifecycle of Steinernema. Two species, Pseudomonas protegens and P. chlororaphis, displayed entomopathogenic properties suggestive of a role in Steinernema virulence and membership of the Steinernema pathobiome. CONCLUSIONS: Our study validates a shift from monoxenic paradigm to pathobiome view in the case of the Steinernema ecology. The microbial communities of low complexity associated with EPNs will permit future microbiota manipulation experiments to decipher overall microbiota functioning in the infectious process triggered by EPN in insects and, more generally, in EPN ecology.

Bacterial community profile after the lethal infection of Steinernema-Xenorhabdus pairs into soil-reared Tenebrio molitor larvae.

The host microbiota may have an impact on pathogens. This is often studied in laboratory-reared hosts but rarely in individuals whose microbiota looks like that of wild animals. In this study, we modified the gut microbiota of the insect Tenebrio molitor by rearing larvae in soil sampled from the field. We showed by high throughput sequencing methods that this treatment modifies the gut microbiota so that it is more diversified than that of laboratory-reared insects, and closely resembled the one of soil-dwelling insects. To describe what the entomopathogenic bacterial symbiont Xenorhabdus (Enterobacteriaceae), vectored by the soil-dwelling nematode Steinernema, might experience in natural conditions, we studied the infestation of the soil-reared T. molitor larvae with three Steinernema-Xenorhabdus pairs. We performed the infestation at 18°C, which delays the emergence of new infective juveniles (IJs), the soil-dwelling nematode forms, but which is a temperature compatible with natural infestation. We analyzed by high throughput sequencing methods the composition of the bacterial community within the insect cadavers before the first emergences of IJs. These bacterial communities were generally characterized by one or two non-symbiont taxa. Even for highly lethal Steinernema-Xenorhabdus pairs, the symbiont does not dominate the bacterial community within the insect cadaver.

Partner-specific induction of Spodoptera frugiperda immune genes in response to the entomopathogenic nematobacterial complex Steinernema carpocapsae-Xenorhabdus nematophila.

The Steinernema carpocapsae-Xenorhabdus nematophila association is a nematobacterial complex used in biological control of insect crop pests. The infection success of this dual pathogen strongly depends on its interactions with the host's immune system. Here, we used the lepidopteran pest Spodoptera frugiperda to analyze the respective impact of each partner in the induction of its immune responses. First, we used previously obtained RNAseq data to construct the immunome of S. frugiperda and analyze its induction. We then selected representative genes to study by RT-qPCR their induction kinetics and specificity after independent injections of each partner. We showed that both X. nematophila and S. carpocapsae participate in the induction of stable immune responses to the complex. While X. nematophila mainly induces genes classically involved in antibacterial responses, S. carpocapsae induces lectins and genes involved in melanization and encapsulation. We discuss putative relationships between these differential inductions and the pathogen immunosuppressive strategies.

Selection of Bacterial Mutants in Late Infections: When Vector Transmission Trades Off against Growth Advantage in Stationary Phase.

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.

Inhibition of Spodoptera frugiperda phenoloxidase activity by the products of the Xenorhabdus rhabduscin gene cluster.

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.

Role of the Photorhabdus Dam methyltransferase during interactions with its invertebrate hosts.

Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens during the whole bacterial life cycle including during symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that during the life cycle of P. luminescens, Dam is not involved the bacterial symbiosis with the nematode H. bacteriophora, but it contributes to the pathogenicity of the nemato-bacterial complex.

The population structure of Ochrobactrum isolated from entomopathogenic nematodes indicates interactions with the symbiotic system.

Entomopathogenic nematodes (EPNs) form specific mutualistic associations with bioluminescent enterobacteria. In Heterorhabditidis indica, Ochrobactrum spp. was identified beside the symbiont Photorhabdus luminescens but its involvement in the symbiotic association in the EPNs remains unclear. This study describe the population structure and the diversity in Ochrobactrum natural populations isolated from EPNs in the Caribbean basin in order to question the existence of EPN-specialized clones and to gain a better insight into Ochrobactrum-EPNs relationships. EPN-associated Ochrobactrum and Photorhabdus strains were characterized by multi-locus sequence typing, Pulsed-Field Gel Electrophoresis fingerprinting and phenotypic traits. Population study showed the absence of EPN-specialized clones in O. intermedium and O. anthropi but suggested the success of some particular lineages. A low level of genetic and genomic diversification of Ochrobactrum isolated from the natural population of Caribbean nematodes was observed comparatively to the diversity of human-associated Ochrobactrum strains. Correspondences between Ochrobactrum and P. luminescens PFGE clusters have been observed, particularly in the case of nematodes from Dominican Republic and Puerto Rico. O. intermedium and O. anthropi associated to EPNs formed less biofilm than human-associated strains. These results evoke interactions between Ochrobactrum and the EPN symbiotic system rather than transient contamination. The main hypothesis to investigate is a toxic/antitoxic relationship because of the ability of Ochrobactrum to resist to antimicrobial and toxic compounds produced by Photorhabdus.