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International audience; 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.

International audience; 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 in the genes pmrAB , mgrB , phoPQ , and crrB , only those bearing mutations in the 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 induces the restructuring of lipopolysaccharide on the outer membrane and thus leads to CST resistance. Moreover, crrB mutations also enhance the transcription of neighboring genes, such as H239_3063 , encoding an ABC transporter transmembrane region, H239_3064 , encoding a putative efflux pump also known as KexD, and H239_3065 , encoding an 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, or 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 not 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.

Lipid A is a potent endotoxin, and its fatty acids (lauric, myristic, and sometimes palmitic acid) anchors lipopolysaccharide (LPS) into the outer leaflet of the outer membrane of most Gram-negative bacteria. The highly anionic charge of the glucosamine lipid A moiety makes the LPS a powerful attractant for cationic antimicrobial peptides (AMPs). AMPs are major component of innate immunity that kill bacteria by permeabilization of lipid bilayers. Secondary lipid A acylation of Klebsiella pneumoniae, involving the acyltransferase LpxM (formally, msbB or WaaN) that acylates (KDO)2-(lauroyl)-lipid IV-A with myristate during lipid A biosynthesis, has been associated with bacterial resistance to AMPs contributing to virulence in animal models. We investigated here the role of the msbB gene of the entomopathogenic bacterium Photorhabdus luminescens in AMP resistance, by functional complementation of the AMP susceptible K. pneumoniae lpxM mutant with the P. luminescens msbB gene. We showed that msbB (lpxM) gene of P. luminescens is able to enhance polymyxin B, colistin and cecropin A resistance of K. pneumoniae lpxM mutant, compared to the non-complemented mutant. However, we could not obtain any msbB mutant of Photorhabdus by performing allelic exchange experiments based on positive selection of sucrose highly resistant mutants. We thus suggest that msbB-mediated Photorhabdus lipid A acylation is essential for outer membrane low-permeability and that modification of lipid A composition, fluidity and osmosis-resistance have an important role in the ability of Photorhabdus to grow in sucrose at high concentrations.

Photorhabdus luminescensis an entomopathogenic bacterium found in symbiosis with the nematodeHeterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, includingP. 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 inP. luminescenssymbiosis withH. bacteriophora. We constructed a strain overexpressingdamby inserting an additional copy of thedamgene under the control of a constitutive promoter in the chromosome ofP. luminescensand then achieved association between this recombinant strain and nematodes. Thedamoverexpressing strain was able to feed the nematodein vitroandin vivosimilarly 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 LT50was observed during insect infestation with the nematode associated with thedamoverexpressing strain. These results suggest that theP. luminescensDam plays a role in the pathogenicity of the nemato-bacterial complex.

International audience; 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.

International audience; 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.

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.

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. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent Xenorhabdus doucetiae strain FRM16. G6 had a smaller genome (500-700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (haemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, toxin-antitoxin systems). The genomes of all the X. poinarii strains analysed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.

Spodoptera littoralis, one of the major pests of many important crop plants, is more susceptible to Bacillus thuringiensis aizawai delta-endotoxins than to those of Bacillus thuringiensis kurstaki. Within the framework of the development of efficient bioinsecticides and the prevention against insect resistance, we tested the effect of mixing B. thuringiensis kurstaki delta-endotoxins and Photorhabdus luminescens cells on S. littoralis growth. The obtained results showed that the growth inhibition of this insect was more effective when B. thuringiensis kurstaki spore-crystal mixture and Photorhabdus luminescens cells were used in combination. Furthermore, this synergism is mainly due to the presence of Cry1Ac, which is one of the three delta-endotoxins that form the crystal of B. thuringiensis kurstaki strain BNS3 in addition to Cry1Aa and Cry2Aa. This work shows a possibility to use B. thuringiensis as a delivery means for Photorhabdus bacteria in order to infect the insect hemocoel and to reduce the risk of developing resistance in the target organism.

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.

There is a complex interplay between the regulation of flagellar motility and the expression of virulence factors in many bacterial pathogens. We investigated the role of FliZ in the regulation of flagellar and virulence genes in Xenorhabdus nematophila, an insect pathogen. The fliZ gene is the second gene in the fliAZ operon in X. nematophila. In vivo transcription analysis revealed a positive feedback loop of fliAZ transcription in which FliZ activates flhDC, the master operon of flagellar regulon in X. nematophila, leading to an increased transcription of the FlhDC-dependent promoter of fliAZ. We also showed that fliAZ and flhDC mutants lacked motility, had no haemolysin or Tween lipase activity and displayed an attenuated virulence phenotype in insects. Lipase activity is controlled by FliA, whereas haemolysin production and full virulence phenotype have been reported to be FliZ-dependent. Transcriptional analysis revealed that FliZ directly controlled expression of the xhlBA and xaxAB operons, which encode haemolysins from the two-partner secretion system and the binary XaxAB toxin family respectively. We suggest that this regulatory pathway may also occur in other pathogenic enterobacteria with genes encoding members of these two growing families of haemolysins.

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.

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-CT (FRM16)-mediated growth inhibition was specifically neutralized by CdiI(FRM16). Moreover, the cdi(FRM16) locus encodes an ortholog of toxin-activating proteins C that we named CdiC(FRM16). 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.

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.

International audience; Comparison of the proteomes of wild-type Photorhabdus luminescens and its hcaR derivative, grown in insect hemolymph, showed that hcaR disruption decreased the production of toxins (tcdA1, mcf and pirAB) and proteins involved in oxidative stress response (SodA, AhpC, Gor). The disruption of hcaR did not affect growth rate in insects, but did delay the virulence of P. luminescens in Bombyx mori and Spodoptera littoralis larvae. This delayed virulence was associated with a lower toxemia rather than delay in bacteremia. The disruption of hcaR also increased bacterial sensitivity to hydrogen peroxide. A sodA mutant and an hcaR mutant had similar phenotypes in terms of sensitivity to hydrogen peroxide, virulence, toxin gene expression, and growth rate in insects. Thus, the two processes affected by hcaR disruption - toxemia and oxidative stress response - appear to be related. Besides, expression of toxin genes tcdA1, mcf, and pirAB was decreased by paraquat challenge. We provide here the first demonstration of the importance of toxemia for P. luminescens virulence. Our results also highlight the power of proteomic analysis for detecting unexpected links between different, concomitant processes in bacteria

Entomopathogenic bacteria of the genus Photorhabdu s harbor a type III secretion system. This system was probably acquired prior to the separation of the species within this genus. Furthermore, the core components of the secretion machinery are highly conserved but the predicted effectors differ between Photorhabdus luminescens and P. asymbiotica , two highly related species with different hosts.

Analysis of the Photorhabdus luminescens genome sequence revealed that the pts region is related to the tail synthesis gene core of the P2 phage. The pts locus encodes a DNA invertase homologue. PCR-RFLP analysis showed the two potential tail fiber regions of the pts locus present DNA inversions. Electron microscopy revealed a phage tail-like particle, related to the R-type family and named R-photorhabdicin, in the culture supernatant of P. luminescens. Mass spectrometry analysis of two sub-units of R-photorhabdicin revealed that they are encoded by the pts locus. The role of this P2-related prophage remnant in the Photorhabdus genome is discussed.

We report the genome sequence of Xenorhabdus szentirmaii DSM16338 (4.84 Mb), a symbiont of the entomopathogenic nematode Steinernema rarum . This strain produces antimicrobial activity.

The genus Xenorhabdus consists of the specific bacterial symbionts of the entomopathogenic nematodes of the family Steinernematidae (40) and was separated from the genus Photorhabdus (11), which contains the symbionts of the entomopathogenic nematodes of the family Heterorhabditidae. Both genera are entomopathogenic gram-negative bacteria belonging to the Enterobacteriaceae. The nematodes carry their bacterial symbionts monoxenically in a special vesicle of the infective stage (L3 juveniles) in Steinernematidae (8) and throughout the whole intestine of Heterorhabditidae (20). These bacteria are transported by their nematode hosts into the hemocoel of the insect prey, which is killed, probably via a combination of toxin action and septicemia. The bacterial symbionts also contribute to the symbiotic relationship by establishing and maintaining suitable conditions for nematode reproduction (31). Recently, isolation of some Photorhabdus strains from infected humans in Australia and the United States was reported (21, 30), and the strains from the United States were classified as Photorhabdus asymbiotica (23). The form of the bacterium that is normally isolated from symbiotic infective-stage nematodes is referred to as phase I. Like many pathogenic bacteria, Xenorhabdus and Photorhabdus strains spontaneously produce colonial variants which have been called phase II variants (10). The two variants of the bacteria have generally been shown to be equally pathogenic for the larvae of the greater wax moth, Galleria mellonella (3). However, Volgyi et al. (42) described for the first time a phase II variant that showed reduced virulence in the tobacco hornworm, Manduca sexta. Xenorhabdus nematophila and Photorhabdus luminescens are highly pathogenic to insects, and 50% insect mortality has been reported with direct infection with fewer than 20 bacteria per larva (5). The bacterial factors involved in killing of the insect or in overcoming the insect immune reactions are still under investigation. Following invasion of the insect host by the nematodes, both bacteria produce potential virulence factors, including lipase, protease, lecithinase, and lipopolysaccharides (LPSs), in the hemocoel (for a review, see reference 24). It was shown that purified LPS, Photorhabdus protease fractions, or Xenorhabdus lecithinase isomers showed no toxic effect following injection into insect hemocoel (12, 16, 39). Recently, a novel toxin complex with both oral and injectable activities against a wide range of insects was identified in a supernatant of P. luminescens (13). Purified toxin complex a (Tca) has specific effects on the midgut epithelium of the insect (9). In order to study Xenorhabdus and Photorhabdus virulence in insects, a genetic approach was also used. Avirulent mutants of X. nematophila have been isolated by transposon mutagenesis (Tn5). These mutants were pleiotropic, but all five mutants that tested as avirulent in G. mellonella were nonmotile and partially impaired in blood hemolysis (43). It was also shown that a homoserine lactone autoinducer restored virulence to one avirulent X. nematophila strain and stimulated the level of bacterial lipase activity (17). Recently we reported that flhDC, the flagellar master operon of X. nematophila, controls flagellin expression. Furthermore we revealed that lipolytic and extracellular hemolysin activity is flhD dependent. We also showed that the flhD null mutant displayed an attenuated virulence phenotype in the common cutworm, Spodoptera littoralis, compared to the wild-type strain (25). The recently published partial genome sequence of P. luminescens (22) revealed a diverse array of genes that putatively encodes potential virulence factors. These factors include exoenzymes (proteases, lipases, and chitinases), a type III secretion system (Yop homolog), and several classes of toxins (insecticidal toxin complex, Rtx-like toxins, and hemolysin and cytotoxin homologs) (22). Until now, studies examining hemolytic activity of both genera have not been reported. Cytolysins are proteins which cause lysis of red blood cells (RBC) as well as nucleated cell types by hydrolysis (lipases, phospholipases, or proteases) or by forming pores in the plasma membrane. Surfactants may also cause cytolysis by solubilization of the target cell membrane. Bacterial cytolysins are usually recognized as hemolysin on blood agar where a transparent zone appears around colonies. The production by a few strains of Xenorhabdus and Photorhabdus of hemolysin has been detected on agar supplemented with sheep blood (6, 21, 25). Apart from their phoretic location inside infective juvenile nematodes, these bacteria are only observed in insect hemolymph, where they enter their growth cycle. Here the bacteria are in contact with hemocytes which achieve defense reactions in insects. Some of these cells are immunocompetent cells able to engulf (phagocytosis) or to isolate and kill (nodule formation) bacteria. We hypothesize that hemolytic activities could target the immunocompetent cells in insect hemolymph. In this study, we report that different cytolytic activities were found in supernatants of Xenorhabdus whereas none was detected in supernatants of various strains of Photorhabdus. We have studied the kinetics of the production of cytolytic activities over the course of in vitro bacterial growth. We also provide evidence on the characteristics and on the specificity of each of these cytolytic activities against mammalian RBC and insect hemocyte types.

International audience; 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.

This study investigates if acclimatization to residual pesticide contamination in agricultural soils is reflected in detoxification, antioxidant enzyme activities and energy budget of earthworms. Five fields within a joint agricultural area exhibited different chemical and farming histories from conventional cultivation to organic pasture. Soil multiresidual pesticide analysis revealed up to 9 molecules including atrazine up to 2.4 ng g -1 dry soil. Exposure history of endogeic Aporrectodea caliginosa and Allolobophora chlorotica modified their responses to pesticides. In the field, activities of soluble glutathione-S-transferases (sGST) and catalase increased with soil pesticide contamination in A. caliginosa. Pesticide stress was reflected in depletion of energy reserves in A. chlorotica. Acute exposure of pre-adapted and naïve A. caliginosa to pesticides (fungicide Opus ®, 0.1 μg active ingredient epoxiconazole g-1 dry soil, RoundUp Flash®, 2.5 μg active ingredient glyphosate g-1 dry soil, and their mixture), revealed that environmental pre-exposure accelerated activation of the detoxification enzyme sGST towards epoxiconazole.

International audience; Terrestrial isopods can be killed by some entomopathogenic bacteria among Xenorhabdus and Photorhabdus species even with no or very limited multiplication. This suggests that toxemia and not septicemia is the major cause of entomopathogenic bacteria pathogenicity against these crustaceans. In this paper, we revealed that the injection of stationary phase culture supernatant of P. luminescens TT01, in which toxins can be accumulated, led alone to a rapid decrease in the number of host immune cells and killed most of the Armadillidium vulgare individuals within 48 h. The pathogenicity was strongly attenuated when supernatant was heated and totally suppressed after 100-kDa filtration suggesting that the toxin responsible for killing A. vulgare would be a protein above this size. Additionally, we tested the culture supernatant of two TT01 mutants that have been previously shown as being altered in their pathogenicity against lepidopteran insects one of them being known as exhibiting lower expression of some toxins. However, the supernatants of the mutants was as pathogenic for A. vulgare as the wild type strains suggesting that the toxins involved in killing A. vulgare may be different than previously described ones.

Thirteen bacterial strains of Xenorhabdus and 14 strains of Photorhabdus originating from a wide range of geographical and nematode host sources were typed by analyzing 16S rRNA gene (rDNA) restriction patterns obtained after digestion of PCR-amplified 16S rDNAs. Eight tetrameric restriction endonucleases were examined. A total of 17 genotypes were identified, forming two heterogeneous main clusters after analysis by the unweighted pair-group method using arithmetic averages: group I included all Xenorhabdus species and strains, symbionts of Steinernema, whereas group II encompassed the Photorhabdus strains, symbionts of Heterorhabditis. To identify the four valid species of Xenorhabdus and unclassified strains and all the genotypes of Photorhabdus luminescens, three restriction enzymes are required: CfoI, AluI, and HaeIII. Our results, in substantial agreement with DNA-DNA pairing and 16S rDNA sequence data, indicate that amplified 16S rDNA restriction analysis is a simple and accurate tool for identifying entomopathogenic nematode bacterial symbionts.

International audience; Since the early 1980s, fungi have emerged as a major cause of human disease. Fungal infections are associated with high levels of morbidity and mortality, and are now recognized as an important public health problem. Gram-negative bacterial strains of genus Xenorhabdus are known to form symbiotic associations with soil-dwelling nematodes of the Steinernematidae family. We describe here the discovery of a new antifungal metabolite, cabanillasin, produced by Xenorhabdus cabanillasii. We purified this molecule by cation-exchange chromatography and reverse-phase chromatography. We then determined the chemical structure of cabanillasin by homo- and heteronuclear NMR and MS-MS. Cabanillasin was found to be active against yeasts and filamentous fungi involved in opportunistic infections.

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.

International audience; Due to the importance of young-of-the-year (YOY) perch in the peri-alpine regions where they are consumed, the microcystin (MC) contamination of YOY perch was analysed both in field (Lake Bourget, France) and experimentally using force-feeding protocols with pure MCs. In-situ, schools of YOY perch present in the epilimnion of the lake were never found in direct contact with the P. rubescens blooms that were present in the metalimnion. However, MCs were detected in the muscles and liver of the fish and were thus assumed to reach YOY perch through dietary routes, particularly via the consumption of MC-containing Daphnia. Force-feeding experiment demonstrates the existence of MC detoxification/ excretion processes and suggests that in situ, YOY perch could partly detoxify and excrete ingested MCs, thereby limiting the potential negative effects on perch populations under bloom conditions. However, because of chronic exposure these processes could not allow for the complete elimination of MCs. In both experimental and in situ studies, no histological change was observed in YOY perch, indicating that MC concentrations that occurred in Lake Bourget in 2009 were too low to cause histological damage prone to induce mortality. However, Deoxyribonucleic acid (DNA) damages were observed for both the high and low experimental MC doses, suggesting that similar effects could occur in situ and potentially result in perch population disturbance during cyanobacterial blooms. Our results indicate the presence of MCs in wild perch, the consumption of this species coming from Lake Bourget is not contested but more analyses are needed to quantify the risk.

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., Author Summary Heterogeneity in the expression of bacterial genes may result in the presence of cells with different phenotypes in an isogenic population. The existence of such “non-genetic individuality” was the first described many years ago for the flagellum-driven swimming behavior of bacteria. In this study, we identified a new bimodal switch controlling the expression of genes involved in flagellum biosynthesis and host interactions in the insect pathogen Xenorhabdus nematophila. This switch is modulated by a transcriptional regulator called FliZ. In addition to identifying all the specific genes up- and downregulated by FliZ, we showed that the concentration of FliZ fine-tuned the expression of FliZ target genes, resulting in individuals with different potentials for bacterial locomotion, host colonization and virulence.

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the family Steinernematidae, occur spontaneously in two phases. Only the phase I variants of Xenorhabdus nematophilus F1 expressed fimbriae when the bacteria were grown on a solid medium (nutrient agar; 24 and 48 h of growth). These appendages were purified and characterized. They were rigid, with a diameter of 6.4 (plusmn) 0.3 nm, and were composed of 16-kDa pilin subunits. The latter were synthesized and assembled during the first 24 h of growth. Phase II variants of X. nematophilus did not possess fimbriae and apparently did not synthesize pilin. Phase I variants of X. nematophilus have an agglutinating activity with sheep, rabbit, and human erythrocytes and with hemocytes of the insect Galleria mellonella. The purified fimbriae agglutinated sheep and rabbit erythrocytes. The hemagglutination by bacteria and purified fimbriae was mannose resistant and was inhibited by porcine gastric mucin and N-acetyl-lactosamine. The last sugar seems to be a specific inhibitor of hemagglutination by X. nematophilus.

Insects are the largest group of animals on earth. Like mammals, virus, fungi, bacteria and parasites infect them. Several tissue barriers and defense mechanisms are common for vertebrates and invertebrates. Therefore some insects, notably the fly Drosophila and the caterpillar Galleria mellonella, have been used as models to study host-pathogen interactions for several insect and mammal pathogens. They are excellent tools to identify pathogen determinants and host tissue cell responses. We focus here on the comparison of effectors used by two different groups of bacterial insect pathogens to accomplish the infection process in their lepidopteran larval host: Bacillus thuringiensis and the nematode-associated bacteria, Photorhabdus and Xenorhabdus. The comparison reveals similarities in function and expression profiles for some genes, which suggest that such factors are conserved during evolution in order to attack the tissue encountered during the infection process.

International audience; An antimicrobial peptide (AMP) of the cecropin family was isolated by HPLC from plasma of the insect pest, Spodoptera frugiperda. Its molecular mass is 3910.9 Da as determined by mass spectrometry. Thanks to the EST database Spodobase, we were able to describe 13 cDNAs encoding six different cecropins which belong to the sub-families CecA, CecB, CecC and CecD. The purified peptide identified as CecB1 was chemically synthesized (syCecB1). It was shown to be active against Gram-positive and Gram-negative bacteria as well as fungi. Two closely related entomopathogenic bacteria, Xenorhabdus nematophila F1 and Xenorhabdus mauleonii VC01(T) showed different susceptibility to syCecB1. Indeed, X. nematophila was sensitive to syCecB1 whereas X. mauleonii had a minimal inhibitory concentration (MIC) eight times higher. Interestingly, injection of live X. nematophila into insects did not induce the expression of AMPs in hemolymph. This effect was not observed when this bacterium was heat-killed before injection. On the opposite, both live and heat-killed X. mauleonii induced the expression of AMPs in the hemolymph of S. frugiperda. The same phenomenon was observed for another immune-related protein lacking antimicrobial activity. Altogether, our data suggest that Xenorhabdus strains have developed different strategies to supplant the humoral defense mechanisms of S. frugiperda, either by increasing their resistance to AMPs or by preventing their expression during such host-pathogen interaction. (C) 2012 Elsevier Ltd. All rights reserved.

Background Flexible genomes facilitate bacterial evolution and are classically organized into polymorphic strain-specific segments called regions of genomic plasticity (RGPs). Using a new web tool, RGPFinder, we investigated plasticity units in bacterial genomes, by exhaustive description of the RGPs in two Photorhabdus and two Xenorhabdus strains, belonging to the Enterobacteriaceae and interacting with invertebrates (insects and nematodes). Results RGPs account for about 60% of the genome in each of the four genomes studied. We classified RGPs into genomic islands (GIs), prophages and two new classes of RGP without the features of classical mobile genetic elements (MGEs) but harboring genes encoding enzymes catalyzing DNA recombination (RGPmob), or with no remarkable feature (RGPnone). These new classes accounted for most of the RGPs and are probably hypervariable regions, ancient MGEs with degraded mobilization machinery or non canonical MGEs for which the mobility mechanism has yet to be described. We provide evidence that not only the GIs and the prophages, but also RGPmob and RGPnone, have a mosaic structure consisting of modules. A module is a block of genes, 0.5 to 60 kb in length, displaying a conserved genomic organization among the different Enterobacteriaceae. Modules are functional units involved in host/environment interactions (22-31%), metabolism (22-27%), intracellular or intercellular DNA mobility (13-30%), drug resistance (4-5%) and antibiotic synthesis (3-6%). Finally, in silico comparisons and PCR multiplex analysis indicated that these modules served as plasticity units within the bacterial genome during genome speciation and as deletion units in clonal variants of Photorhabdus. Conclusions This led us to consider the modules, rather than the entire RGP, as the true unit of plasticity in bacterial genomes, during both short-term and long-term genome evolution.

International audience; 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.

International audience; The Subantarctic Kerguelen Islands (49°S, 70°E) contain freshwater ecosystems among the most isolated in the world. Concentrations of polychlorinated biphenyls (PCBs) were assessed in the muscle of 48 brook trout and 38 brown trout caught during summer and spring 2006 in the rivers, lakes and ponds of Kerguelen. The sum of 29 PCBs averaged 404 and 358 ng g-1 lipid, and dioxin-like PCB was 19 and 69 ng g-1 lipid, in brook and brown trout, respectively. The values showed a high variability and some fish accumulated PCBs at levels similar to those of fish from impacted areas. While inter-sex differences were limited, the season and the morphotype appeared to have the most influence. Fish captured in summer had muscle PCB concentrations about three times higher than those caught in spring and the 'river' morphotype of brook trout showed the highest PCB levels.

In non-motile forms of Azospirillum lipoferum isolated from the rhizosphere of rice, polyphenol oxidase activity was observed which correlated with production of a dark-brown pigment. Using a combination of substrate/inhibitor specificity tests, intracellular enzyme extracts of non-motile strains were clearly demonstrated to have a laccase activity by oxidising various o- and p-diphenols. This work is the first report on laccase activity in Azospirillum.

Photorhabdus luminescens lives in a mutualistic association with entomopathogenic nematodes and is pathogenic for insects. Variants of Photorhabdus frequently arise irreversibly and are studied because they have altered phenotypic traits that are potentially important for the host interaction. VAR* is a colonial and phenotypic variant displaying delayed pathogenicity when directly injected into the insect, Spodoptera littoralis . In this study, we evaluated the role of transcriptomic modulation in determining the phenotypic variation and delayed pathogenicity of VAR* with respect to the corresponding wild-type form, TT01α. A P. luminescens microarray identified 148 genes as differentially transcribed between VAR* and TT01α. The net regulator status of VAR* was found to be significantly modified. We also observed in VAR* a decrease in the transcription of genes supporting certain phenotypic traits, such as pigmentation, crystalline inclusion, antibiosis, and protease and lipase activities. Three genes encoding insecticidal toxins ( pit and pirB ) or putative insecticidal toxins ( xnp2 ) were less transcribed in VAR* than in the TT01α. The overexpression of these genes was not sufficient to restore the virulence of VAR* to the levels of ΤΤ01α, which suggests that the lower virulence of VAR* does not result from impaired toxemia in insects. Three loci involved in oxidative stress responses ( sodA , katE , and the hca operon) were found to be downregulated in VAR*. This is consistent with the greater sensitivity of VAR* to H 2 O 2 and may account for the impaired bacteremia in the hemolymph of S. littoralis larvae observed with VAR*. In conclusion, we demonstrate here that some phenotypic traits of VAR* are regulated transcriptionally and highlight the multifactorial nature of pathogenicity in insects.

International audience; Cycle inhibiting factors (Cif) constitute a broad family of cyclomodulins present in bacterial pathogens of invertebrates and mammals. Cif proteins are thought to be type III effectors capable of arresting the cell cycle at G2/M phase transition in human cell lines. We report here the first direct functional analysis of CifPl, from the entomopathogenic bacterium Photorhabdus luminescens, in its insect host. The cifPl gene was expressed in P. luminescens cultures in vitro. The resulting protein was released into the culture medium, unlike the well characterized type III effector LopT. During locust infection, cifPl was expressed in both the hemolymph and the hematopoietic organ, but was not essential for P. luminescens virulence. CifPl inhibited proliferation of the insect cell line Sf9, by blocking the cell cycle at the G2/M phase transition. It also triggered host cell death by apoptosis. The integrity of the CifPl catalytic triad is essential for the cell cycle arrest and pro-apoptotic activities of this protein. These results highlight, for the first time, the dual role of Cif in the control of host cell proliferation and apoptotic death in a non-mammalian cell line.

International audience; Xenorhabdus nematophila/Steinernema carpocapsae and Photorhabdus luminescens/Heterorhabditis bacteriophora are nemato-bacterial complexes highly pathogenic for insects. Using a syringe as artificial vector, we have analyzed the effects of the two bacteria, X. nematophila and P. luminescens on the genetic tool insect, Drosophila melanogaster. Both bacteria were found to kill adult flies in a dose dependent manner with X. nematophila being the fastest. On the other hand, when an injection of non-pathogenic bacteria, Escherichia coli, is performed 1 day before challenge with the entomopathogenic bacteria, then the survival of Drosophila flies was prolonged by at least 20 h. After injection of entomopathogenic bacteria, Drosophila mutant Dif1, affected on the Toll pathway, showed a similar phenotype than wild-type flies whereas Drosophila mutant DreddD55, affected on the imd pathway, was not protected by a prior injection of E. coli. This suggested that members of the imd pathway might be targets of these entomopathogenic bacteria albeit synthesis of antimicrobial peptides through this signaling pathway was induced by X. nematophila as well as P. luminescens. Finally, P. luminescens phoP mutant, an avirulent mutant in the Lepidopteran insect, Spodoptera littoralis, was found poorly virulent for D. melanogaster. phoP mutant partially protected D. melanogaster flies if injected 1 day before the injection of P. luminescens wild-type TT01 to the same extent than the E. coli-induced protection. However, phoP recovered a level of pathogenicity comparable to P. luminescens wild-type TT01 when injected to Drosophila flies affected on the imd pathway.

Correspondance auteur: R. Zumbihl E-mail: zumbihl@univ-montp2.fr; International audience; Photorhabdus asymbiotica is unique among the entomopathogenic bacteria of this genus in also being able to infect humans, leading to its isolation from some clinical samples. Recent comparative genomics data and the results of studies of interactions between bacteria and cells provide insight into the adaptation of this bacterium to its new niche, the human body

Correspondance auteur: E. Oswald E-mail: e.oswald@envt.fr Numéro article: e4855; International audience; The cycle inhibiting factor (Cif) produced by enteropathogenic and enterohemorrhagic Escherichia coli was the first cyclomodulin to be identified that is injected into host cells via the type III secretion machinery. Cif provokes cytopathic effects characterized by G(1) and G(2) cell cycle arrests, accumulation of the cyclin-dependent kinase inhibitors (CKIs) p21(waf1/cip1) and p27(kip1) and formation of actin stress fibres. The X-ray crystal structure of Cif revealed it to be a divergent member of a superfamily of enzymes including cysteine proteases and acetyltransferases that share a conserved catalytic triad. Here we report the discovery and characterization of four Cif homologs encoded by different pathogenic or symbiotic bacteria isolated from vertebrates or invertebrates. Cif homologs from the enterobacteria Yersinia pseudotuberculosis, Photorhabdus luminescens, Photorhabdus asymbiotica and the beta-proteobacterium Burkholderia pseudomallei all induce cytopathic effects identical to those observed with Cif from pathogenic E. coli. Although these Cif homologs are remarkably divergent in primary sequence, the catalytic triad is strictly conserved and was shown to be crucial for cell cycle arrest, cytoskeleton reorganization and CKIs accumulation. These results reveal that Cif proteins form a growing family of cyclomodulins in bacteria that interact with very distinct hosts including insects, nematodes and humans

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus , which is both an opportunistic human pathogen and an insect pathogen. The Δ dlt Bc mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda . Δ dlt Bc was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis . We detected the dlt operon in three gram-negative genera: Erwinia ( Erwinia carotovora ), Bordetella ( Bordetella pertussis , Bordetella parapertussis , and Bordetella bronchiseptica ), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Δ dlt Bc ). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.

Correspondance auteur: S. Jaoua E-mail: samir.jaoua@cbs.rnrt.tn; International audience; Photorhabdus temperata and Bacillus thuringiensis are entomopathogenic bacteria exhibiting toxicities against different insect larvae. Vegetative Insecticidal Protein Vip3LB is a Bacillus thuringiensis insecticidal protein secreted during the vegetative growth stage exhibiting lepidopteran specificity. In this study, we focused for the first time on the heterologous expression of vip3LB gene in Photorhabdus temperata strain K122. Firstly, Western blot analyses of whole cultures of recombinant Photorhabdus temperata showed that Vip3LB was produced and appeared lightly proteolysed. Cellular fractionation and proteinase K proteolysis showed that in vitro-cultured recombinant Photorhabdus temperata K122 accumulated Vip3LB in the cell and appeared not to secrete this protein. Oral toxicity of whole cultures of recombinant Photorhabdus temperata K122 strains was assayed on second-instar larvae of Ephestia kuehniella, a laboratory model insect, and the cutworm Spodoptera littoralis, one of the major pests of many important crop plants. Unlike the wild strain K122, which has no effect on the larval growth, the recombinant bacteria expressing vip3LB gene reduced or stopped the larval growth. These results demonstrate that the heterologous expression of Bacillus thuringiensis vegetative insecticidal protein-encoding gene vip3LB in Photorhabdus temperata could be considered as an excellent tool for improving Photorhabdus insecticidal activities

The nitrogen-fixing bacteria, Azospirillum lipoferum and Azospirillum brasilense , contain several large plasmids. Cryptic plasmids of about 150 MDa have only been detected in A. lipoferum strains. The 150 MDa plasmid of A. lipoferum 4B used as probe hybridized with plasmids of comparable size in all A. lipoferum strains. Total DNA of six A. lipoferum strains were digested and hybridized with the p150 probe. Restriction fragment length polymorphism (RFLP) patterns differ from that of strain 4B except for one strain isolated from the same rhizosphere. However, a high number of hybridizing bands in each strain was close to 150 MDa, showing a high degree of similarity between 150 MDa plasmids. This strongly suggests that A. lipoferum 150 MDa plasmids, though not identical, have evolved from a common ancestor and likely constitute a family of plasmids sharing unidentified common functions.

The reliability of Tn 5 as labelling tool was investigated in soil microcosm. The occurence of a selective in soil microcosm. The occurence of resistances encoded by Tn 5 nptII gene was assesed by kanamycin and neomycin amendment. The bioassay developed to monitor the persistence of the soil-added kanamycin did not detect the antibiotic activity in soil extract. A nptII-engineered Escherichia coli strain showed no enhanced survival in aminoglycoside amended soil. Tn 5-marker properties were investigated within indigenous bacteria to determine the specificity of labelling to follow the fate of recombinant DNA. Kanamycin and neomycin resistant population levels made Tn 5 aminoglycoside-resistance phenotype non-sensitive enough to select a soil dissemination of the labelled DNA. The unexpected occurrence of homologous sequences among soil organisms also prevented Tn 5 from being a specific DNA marker. By contrast, colony hybridization did not reveal homology to nptII suggesting its use as a reliable gene transfer indicator.

Three N2-fixing yellow bacteria (5AJ, 5ATR, and 29AJ) isolated from the rhizosphere of rice, previously found to be related to Flavobacterium capsulatum by DNA–rRNA hybridization, were compared on the basis of 115 morphological, physiological, biochemical, and nutritional characters. The bacteria were nonfermentative, irregular motile rods with polar flagella. They exhibited a gram-negative reaction and yellow pigmentation. A numerical analysis using these phenotypic characteristics showed that the bacteria were more closely related to Pseudomonas paucimobilis than to F. capsulatum. A relationship to the P. paucimobilis cluster was also supported by the presence of the pigment nostoxanthin. This pigment was also found in F. capsulatum and could be used as a distinctive marker for this group of yellow bacteria. Their phenotypic properties, protein patterns, and serological tests (ELISA) showed that two of the isolates (5AJ and 5ATR) were identical and had originated from the same parental strain. On the other hand, the serological tests indicated that both N2-fixing yellow bacteria (5AJ or 5ATR and 29AJ) differed from the reference strains, F. capsulatum and P. paucimobilis. Key words: N2-fixing bacteria, taxonomic study, nostoxanthin, Pseudomonas.

International audience; Xenorhabdus nematophila, a member of the Enterobacteriaceae, kills many species of insects by strongly depressing the immune system and colonizing the entire body. A peptide cytotoxin has been purified from X. nematophila broth growth, and the cytolytic effect on insect immunocytes and hemolytic effect on mammalian red blood cells of this toxin have been described (Ribeiro, C., Vignes, M., and Brehelin, M. (2003) J. Mal. Chem. 278, 3030-3039). We show here that this toxin, Xenorhabdus alpha-xenorhabdolysin (Xax), triggers apoptosis in both insect and mammalian cells. We also report the cloning and sequencing of two genes, xaxAB, encoding this toxin in X. nematophila. The expression of both genes in recombinant Escherichia coli led to the production of active cytotoxin/hemolysin. However, hemolytic activity was observed only if the two peptides were added in the appropriate order. Furthermore, we report here that inactivation of xaxAB genes in X. nematophila abolished the major cytotoxic activity present in broth growth, called C I. We also show that these genes are present in various entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus, in Pseudomonas entomophila, in the human pathogens Yersinia enterocolitica and Proteus mirabilis, and in the plant pathogen Pseudomonas syringae. This toxin cannot be classified in any known family of cytotoxins on the basis of amino acid sequences, locus organization, and activity features. It is, therefore, probably the prototype of a new family of binary toxins.

Bacterial virulence is an integrative process that may involve quorum sensing. In this work, we compared by global expression profiling the wild-type entomopathogenic Photorhabdus luminescens subsp. laumondii TT01 to a luxS- deficient mutant unable to synthesize the type 2 quorum-sensing inducer AI-2. AI-2 was shown to regulate more than 300 targets involved in most compartments and metabolic pathways of the cell. AI-2 is located high in the hierarchy, as it controls the expression of several transcriptional regulators. The regulatory effect of AI-2 appeared to be dose dependent. The luxS- deficient strain exhibited decreased biofilm formation and increased type IV/V pilus-dependent twitching motility. AI-2 activated its own synthesis and transport. It also modulated bioluminescence by regulating the synthesis of spermidine. AI-2 was further shown to increase oxidative stress resistance, which is necessary to overcome part of the innate immune response of the host insect involving reactive oxygen species. Finally, we showed that the luxS- deficient strain had attenuated virulence against the lepidopteran Spodoptera littoralis . We concluded that AI-2 is involved mainly in early steps of insect invasion in P. luminescens .

The bacterium Photorhabdus establishes a highly specific association with Heterorhabditis , its nematode host. Photorhabdus strains associated with Heterorhabditis bacteriophora or Heterorhabditis megidis were compared using a Photorhabdus DNA microarray. We describe 31 regions belonging to the Photorhabdus flexible gene pool. Distribution analysis of regions among the Photorhabdus genus identified loci possibly involved in nematode specificity.

International audience; Photorhabdus is an entomopathogenic bacterium belonging to the Enterobacteriaceae. The genome of the TT01 strain of Photorhabdus luminescens was recently sequenced and a large number of toxin-encoding genes were found. Genomic analysis predicted the presence on the chromosome of genes encoding a type three secretion system (TTSS), the main role of which is the delivery of effector proteins directly into eukaryotic host cells. We report here the functional characterization of the TTSS. The locus identified encodes the secretion/translocation apparatus, gene expression regulators and an effector protein - LopT - homologous to the Yersinia cysteine protease cytotoxin YopT. Heterologous expression in Yersinia demonstrated that LopT was translocated into mammal cells in an active form, as shown by the appearance of a form of the RhoA GTPase with modified electrophoretic mobility. In vitro study showed that recombinant LopT was able to release RhoA and Rac from human and insect cell membrane. In vivo assays of infection of the cutworm Spodoptera littoralis and the locust Locusta migratoria with a TT01 strain carrying a translational fusion of the lopT gene with the gfp reporter gene revealed that the lopT gene was switched on only at sites of cellular defence reactions, such as nodulation, in insects. TTSS-mutant did not induce nodule formation and underwent phagocytosis by insect macrophage cells, suggesting that the LopT effector plays an essential role in preventing phagocytosis and indicating an unexpected link between TTSS expression and the nodule reaction in insects

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus . When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria , also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.

50 ref.; International audience; Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.