OxyR is required for oxidative stress resistance of the entomopathogenic bacterium Xenorhabdus nematophila and has a minor role during the bacterial interaction with its hosts.

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.

The AcrAB efflux pump confers self-resistance to stilbenes in Photorhabdus laumondii.

The Resistance-nodulation-division (RND)-type AcrAB-TolC efflux pump contributes to multidrug resistance in Gram-negative bacteria. Recently, the bacterium Photorhabdus laumondii TT01 has emerged as a goldmine for novel anti-infective drug discovery. Outside plants, Photorhabdus is the only Gram-negative known to produce stilbene-derivatives including 3,5-dihydroxy-4-ethyl-trans-stilbene and 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS). IPS is a bioactive polyketide which received considerable attention, mainly because of its antimicrobial properties, and is currently in late-stage clinical development as a topical treatment for psoriasis and dermatitis. To date, little is known about how Photorhabdus survives in the presence of stilbenes. We combined genetic and biochemical approaches to assess whether AcrAB efflux pump exports stilbenes in P. laumondii. We demonstrated that the wild-type (WT) exerts an antagonistic activity against its derivative ΔacrA mutant, and that is able to outcompete it in a dual-strain co-culture assay. The ΔacrA mutant also showed high sensitivity to 3,5-dihydroxy-4-ethyl-trans-stilbene and IPS as well as decreased IPS concentrations in its supernatant comparing to the WT. We report here a mechanism of self-resistance against stilbene derivatives of P. laumondii TT01, which enables these bacteria to survive under high concentrations of stilbenes by extruding them out via the AcrAB efflux pump.

Missense Mutations in the CrrB Protein Mediate Odilorhabdin Derivative Resistance in Klebsiella pneumoniae.

NOSO-502 is a preclinical antibiotic candidate of the Odilorhabdin class. This compound exhibits activity against Enterobacteriaceae pathogens, including carbapenemase-producing bacteria and most of the Colistin (CST)-resistant strains. Among a collection of CST-resistant Klebsiella pneumoniae strains harboring mutations on genes pmrAB, mgrB, phoPQ, and crrB, only those bearing mutations in gene crrB were found to be resistant to NOSO-502.CrrB is a histidine kinase which acts with the response regulator CrrA to modulate the PmrAB system, which finally induces the restructuring of the lipopolysaccharide present on the outer membrane and thus leading to CST resistance. Moreover, crrB mutations also enhance the transcription of neighboring genes such as H239_3063, an ABC transporter transmembrane region; H239_3064, a putative efflux pump also known as KexD; and H239_3065, a N-acetyltransferase.To elucidate the mechanism of resistance to NOSO-502 induced by CrrB missense mutations in K. pneumoniae, mutants of NCTC 13442 and ATCC BAA-2146 strains resistant to NOSO-502 and CST with single amino acid substitutions in CrrB (S8N, F33Y, Y34N, W140R, N141I, P151A, P151L, P151S, P151T, F303Y) were selected. Full susceptibility to NOSO-502 was restored in crrA or crrB deleted K. pneumoniae NCTC 13442 CrrB(P151L) mutants, confirming the role of CrrAB in controlling this resistance pathway. Deletion of kexD (but no other neighboring genes) in the same mutant also restored NOSO-502-susceptibility. Upregulation of the kexD gene expression was observed for all CrrB mutants. Finally, plasmid expression of kexD in a K. pneumoniae strain missing the locus crrABC and kexD significantly increased resistance to NOSO-502.

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.

AcrAB, the major RND-type efflux pump of Photorhabdus laumondii, confers intrinsic multidrug-resistance and contributes to virulence in insects.

The resistance-nodulation-division (RND)-type efflux pumps AcrAB and MdtABC contribute to multidrug-resistance (MDR) in Gram-negative bacteria. Photorhabdus is a symbiotic bacterium of soil nematodes that also produces virulence factors killing insects by septicaemia. We previously showed that mdtA deletion in Photorhabdus laumondii TT01 resulted in no detrimental phenotypes. Here, we investigated the roles of the last two putative RND transporters in TT01 genome, AcrAB and AcrAB-like (Plu0759-Plu0758). Only ΔacrA and ΔmdtAΔacrA mutants were multidrug sensitive, even to triphenyltetrazolium chloride and bromothymol blue used for Photorhabdus isolation from nematodes on the nutrient bromothymol blue-triphenyltetrazolium chloride agar (NBTA) medium. Both mutants also displayed slightly attenuated virulence after injection into Spodoptera littoralis. Transcriptional analysis revealed intermediate levels of acrAB expression in vitro, in vivo and post-mortem, whereas its putative transcriptional repressor acrR was weakly expressed. Yet, plasmid-mediated acrR overexpression did not decrease acrAB transcript levels neither MDR in TT01 WT. While no pertinent mutations were detected in acrR of the same P. laumondii strain grown either on NBTA or nutrient agar, we suggest that AcrR-mediated repression of acrAB is not physiologically required under conditions tested. Finally, we propose that AcrAB is the primary RND-efflux pump, which is essential for MDR in Photorhabdus and may confer adaptive advantages during insect infection.

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.

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.

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.

Spatiotemporal expression of the putative MdtABC efflux pump of Phtotorhabdus luminescens occurs in a protease-dependent manner during insect infection.

Photorhabdus luminescens is an enterobacterium establishing a mutualistic symbiosis with nematodes, that also kills insects after septicaemia and connective tissue colonization. The role of the bacterial mdtABC genes encoding a putative multidrug efflux system from the resistance/nodulation/cell division family was investigated. We showed that a mdtA mutant and the wild type had similar levels of resistance to antibiotics, antimicrobial peptides, metals, detergents and bile salts. The mdtA mutant was also as pathogenic as the wild-type following intrahaemocoel injection in Locusta migratoria, but had a slightly attenuated phenotype in Spodoptera littoralis. A transcriptional fusion of the mdtA promoter (PmdtA) and the green fluorescent protein (gfp) encoding gene was induced by copper in bacteria cultured in vitro. The PmdtA-gfp fusion was strongly induced within bacterial aggregates in the haematopoietic organ during late stages of infection in L. migratoria, whereas it was only weakly expressed in insect plasma throughout infection. A medium supplemented with haematopoietic organ extracts induced the PmdtA-gfp fusion ex vivo, suggesting that site-specific mdtABC expression resulted from insect signals from the haematopoietic organ. Finally, we showed that protease inhibitors abolished ex vivo activity of the PmdtA-gfp fusion in the presence of haematopoietic organ extracts, suggesting that proteolysis by-products play a key role in upregulating the putative MdtABC efflux pump during insect infection with P. luminescens.

Odilorhabdins, Antibacterial Agents that Cause Miscoding by Binding at a New Ribosomal Site.

Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome.

DNA Adenine Methyltransferase (Dam) Overexpression Impairs Photorhabdus luminescens Motility and Virulence.

Dam, the most described bacterial DNA-methyltransferase, is widespread in gamma-proteobacteria. Dam DNA methylation can play a role in various genes expression and is involved in pathogenicity of several bacterial species. The purpose of this study was to determine the role played by the dam ortholog identified in the entomopathogenic bacterium Photorhabdus luminescens. Complementation assays of an Escherichia coli dam mutant showed the restoration of the DNA methylation state of the parental strain. Overexpression of dam in P. luminescens did not impair growth ability in vitro. In contrast, compared to a control strain harboring an empty plasmid, a significant decrease in motility was observed in the dam-overexpressing strain. A transcriptome analysis revealed the differential expression of 208 genes between the two strains. In particular, the downregulation of flagellar genes was observed in the dam-overexpressing strain. In the closely related bacterium Xenorhabdus nematophila, dam overexpression also impaired motility. In addition, the dam-overexpressing P. luminescens strain showed a delayed virulence compared to that of the control strain after injection in larvae of the lepidopteran Spodoptera littoralis. These results reveal that Dam plays a major role during P. luminescens insect infection.

An antimicrobial peptide-resistant minor subpopulation of Photorhabdus luminescens is responsible for virulence.

Some of the bacterial cells in isogenic populations behave differently from others. We describe here how a new type of phenotypic heterogeneity relating to resistance to cationic antimicrobial peptides (CAMPs) is determinant for the pathogenic infection process of the entomopathogenic bacterium Photorhabdus luminescens. We demonstrate that the resistant subpopulation, which accounts for only 0.5% of the wild-type population, causes septicemia in insects. Bacterial heterogeneity is driven by the PhoPQ two-component regulatory system and expression of pbgPE, an operon encoding proteins involved in lipopolysaccharide (LPS) modifications. We also report the characterization of a core regulon controlled by the DNA-binding PhoP protein, which governs virulence in P. luminescens. Comparative RNAseq analysis revealed an upregulation of marker genes for resistance, virulence and bacterial antagonism in the pre-existing resistant subpopulation, suggesting a greater ability to infect insect prey and to survive in cadavers. Finally, we suggest that the infection process of P. luminescens is based on a bet-hedging strategy to cope with the diverse environmental conditions experienced during the lifecycle.

Flagellar Regulation and Virulence in the Entomopathogenic Bacteria-Xenorhabdus nematophila and Photorhabdus luminescens.

There is a complex interplay between the regulation of flagellar motility and the expression of virulence factors in many bacterial pathogens. Here, we review the literature on the direct and indirect roles of flagellar motility in mediating the tripartite interaction between entomopathogenic bacteria (Photorhabdus and Xenorhabdus), their nematode hosts, and their insect targets. First, we describe the swimming and swarming motility of insect pathogenic bacteria and its impact on insect colonization. Then, we describe the coupling between the expression of flagellar and virulence genes and the dynamic of expression of the flagellar regulon during invertebrate infection. We show that the flagellar type 3 secretion system (T3SS) is also an export apparatus for virulence proteins in X. nematophila. Finally, we demonstrate that phenotypic variation, a common property of the bacterial symbionts of nematodes, also alters flagellar motility in Photorhabdus and Xenorhabdus. Finally, the so-called phenotypic heterogeneity phenomenon in the flagellar gene expression network will be also discussed. As the main molecular studies were performed in X. nematophila, future perspectives for the study of the interplay between flagellum and invertebrate interactions in Photorhabdus will be discussed.

A New Member of the Growing Family of Contact-Dependent Growth Inhibition Systems in Xenorhabdus doucetiae.

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.

Ail and PagC-related proteins in the entomopathogenic bacteria of Photorhabdus genus.

Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1Pl and ail2Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1Pl expression was directly controlled by PhoPQ and low external Mg2+ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2Pl and pagCPl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.

FliZ is a global regulatory protein affecting the expression of flagellar and virulence genes in individual Xenorhabdus nematophila bacterial cells.

Heterogeneity in the expression of various bacterial genes has been shown to result in the presence of individuals with different phenotypes within clonal bacterial populations. The genes specifying motility and flagellar functions are coordinately regulated and form a complex regulon, the flagellar regulon. Complex interplay has recently been demonstrated in the regulation of flagellar and virulence gene expression in many bacterial pathogens. We show here that FliZ, a DNA-binding protein, plays a key role in the insect pathogen, Xenorhabdus nematophila, affecting not only hemolysin production and virulence in insects, but efficient swimming motility. RNA-Seq analysis identified FliZ as a global regulatory protein controlling the expression of 278 Xenorhabdus genes either directly or indirectly. FliZ is required for the efficient expression of all flagellar genes, probably through its positive feedback loop, which controls expression of the flhDC operon, the master regulator of the flagellar circuit. FliZ also up- or downregulates the expression of numerous genes encoding non-flagellar proteins potentially involved in key steps of the Xenorhabdus lifecycle. Single-cell analysis revealed the bimodal expression of six identified markers of the FliZ regulon during exponential growth of the bacterial population. In addition, a combination of fluorescence-activated cell sorting and RT-qPCR quantification showed that this bimodality generated a mixed population of cells either expressing ("ON state") or not expressing ("OFF state") FliZ-dependent genes. Moreover, studies of a bacterial population exposed to a graded series of FliZ concentrations showed that FliZ functioned as a rheostat, controlling the rate of transition between the "OFF" and "ON" states in individuals. FliZ thus plays a key role in cell fate decisions, by transiently creating individuals with different potentials for motility and host interactions.

Draft Genome Sequence and Annotation of the Entomopathogenic Bacterium Xenorhabdus nematophila Strain F1.

We report the 4.3-Mb genome sequence of Xenorhabdus nematophila strain F1, a Gram-negative bacterium that is a symbiont of the entomopathogenic nematode Steinernema carpocapsae and pathogenic by direct injection for a wide variety of insects.

The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus: convergent lifestyles from divergent genomes.

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.

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.