The exbD gene of Photorhabdus temperata is required for full virulence in insects and symbiosis with the nematode Heterorhabditis

Photorhabdus are bacteria found colonizing the gut of a specialized stage of the nematode Heterorhabditis, called the infective juvenile (IJ). The IJ is a free-living stage of the nematode that seeks out and infects insect larvae. Once inside the insect the IJ release Photorhabdus into the haemolymph where the bacteria rapidly proliferate, killing the insect within 48-72 h. The nematodes grow and reproduce in the insect cadaver by feeding on the Photorhabdus biomass. In this study we use Photorhabdus temperata K122 to show that genes involved in iron acquisition play a key role during the course of the tripartite bacteria-nematode-insect interaction. We show that a strain carrying a mutation in a gene with homology to exbD, encoding a component of the TonB complex, is unable to grow well in conditions where iron is not freely available. In addition, this mutant, BMM417, requires a longer time to kill the insect larvae than the wild-type bacteria and this defect in pathogenicity is complemented by the co-injection of iron. Moreover, the increase in LT(50) observed with BMM417 is correlated with a significantly slower in vivo growth rate suggesting that iron is limiting in the insect. We also show that BMM417 is unable to support the growth and development of the Heterorhabditis nematode. Addition of exogenous iron to the growth media restores nematode growth and development on BMM417, suggesting that aspects of iron metaboism in Photorhabdus are important during the symbiosis with the nematode.     

Evidence of oral toxicity of Photorhabdus temperata strain K122 against Prays oleae and its improvement by heterologous expression of Bacillus thuringiensis cry1Aa and cry1Ia genes

Photorhabdus temperata strain K122 exhibited oral toxicity against Prays oleae with an LC50 of 58.1 x 10(6) cells ml(-1). Recombinant P. temperata strains expressing the cry1Aa and/or cry1Ia genes of Bacillus thuringiensis have been constructed. The two cry genes, encoding delta-endotoxins, were placed under the control of the lac promoter and IPTG dependent expression in P. temperata was demonstrated. The presence of the cry genes in K122 resulted in a clear improvement of oral toxicity. This improvement was of 6.2-, 6.6-, and 14.6-fold for the strains K122(pBCcry1Aa), K122(pBScry1Ia), and K122(pBCcry1Aa + pBScry1Ia), respectively. Furthermore, determination of the Synergistic Factor between Cry1Aa and Cry1Ia showed that they act synergistically. This work demonstrates that the heterologous expression of B. thuringiensis cry genes in P. temperata can be used to improve and broaden its host range for insect control.     

Isolation of insect pathogenic bacteria, Providencia rettgeri, from Heterorhabditis spp.

Nematodes of the genus Heterorhabditis carry bacteria of the genus Photorhabdus into insects including pests of horticultural crops. The bacteria kill the insect and provide conditions which allow for the growth and development of the nematodes. It is reported here that the majority of Heterorhabditis spp. strains tested contained a second bacterial species which was identified as Providencia rettgeri. Injection of the bacteria into waxmoth larvae showed that P. rettgeri was at least as pathogenic as Photorhabdus sp. K122. Both had LD₅₀ values of less than one bacterial cell/larva, but P. rettgeri killed the insects at a considerably faster rate than K122 at both 28°C and 9°C. Since Photorhabdus kills very slowly at low temperatures, it appeared that P. rettgeri might be a better pest control agent under these conditions. However, P. rettgeri was not pathogenic when carried into insect larvae by the nematode, indicating that the nematode suppressed either its release or pathogenicity. It will be necessary to find ways of bypassing or inhibiting this suppression for P. rettgeri to fulfil its potential in pest control.     

Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen

Pathogenicity and symbiosis are central to bacteria-host interactions. Although several human pathogens have been subjected to functional genomic analysis, we still understand little about bacteria-invertebrate interactions despite their ecological prevalence. Advances in our knowledge of this area are often hindered by the difficulty of isolating and working with invertebrate pathogenic bacteria and their hosts. Here we review studies on pathogenicity and symbiosis in an insect pathogenic bacterium Photorhabdus and its entomopathogenic nematode vector and model insect hosts. Whilst switching between these hosts, Photorhabdus changes from a state of symbiosis with its nematode vector to one of pathogenicity towards its new insect host and both the bacteria and the nematode then cooperatively exploit the dying insect. We examine candidate genes involved in symbiosis and pathogenicity, their secretion and expression patterns in culture and in the host, and begin to dissect the extent of their genetic coregulation. We describe the presence of several large genomic islands, putatively involved in pathogenicity or symbiosis, within the otherwise Yersinia-like backbone of the Photorhabdus genome. Finally, we examine the emerging comparative genomics of the Photorhabdus group and begin to describe the interrelationship between anti-invertebrate virulence factors and those used against vertebrates.     

Effect of the insect pathogenic bacterium Photorhabdus on insect phagocytes

Photorhabdus are insect pathogenic bacteria that replicate within the insect haemocoel following release from their entomopathogenic nematode symbionts. To investigate how they escape the cellular immune response we examined the effects of two strains of Photorhabdus, W14 and K122, on Manduca sexta phagocytes (haemocytes), in vitro and in vivo. Following injection of Esherichia coli into Manduca larvae, these non-pathogenic bacteria are rapidly cleared from the haemolymph and the number of free haemocytes transiently increases. In contrast, following injection of either strain of pathogenic Photorhabdus, the bacteria grow rapidly while the number of haemocytes decreases dramatically. In vitro incubation of haemocytes with either Photorhabdus supernatant reduced haemocyte viability, and the W14 supernatant caused distinct changes in the actin cytoskeleton morphology of different haemocyte cell types. In phagocytosis assays both Photorhabdus strains can inhibit their own phagocytosis whether the bacterial cells are alive or dead. Further, the supernatant of W14 also contains a factor capable of inhibiting the phagocytosis of labelled E. coli. Together these results suggest that Photorhabdus evades the cellular immune response by killing haemocytes and suppressing phagocytosis by mechanisms that differ between strains.     

Purification and characterization of two distinct metalloproteases secreted by the entomopathogenic bacterium Photorhabdus sp. strain Az29

Photorhabdus sp. strain Az29 is symbiotic with an Azorean nematode of the genus Heterorhabditis in a complex that is highly virulent to insects even at low temperatures. The virulence of the bacteria is mainly attributed to toxins and bacterial enzymes secreted during parasitism. The bacteria secrete proteases during growth, with a peak at the end of the exponential growth phase. Protease secretion was higher in cultures growing at lower temperatures. At 10 degrees C the activity was highest and remained constant for over 7 days, whereas at 23 and 28 degrees C it showed a steady decrease. Two proteases, PrtA and PrtS, that are produced in the growth medium were purified by liquid chromatography. PrtA was inhibited by 1,10-phenantroline and by EDTA and had a molecular mass of 56 kDa and an optimal activity at pH 9 and 50 degrees C. Sequences of three peptides of PrtA showed strong homologies with alkaline metalloproteases from Photorhabdus temperata K122 and Photorhabdus luminescens W14. Peptide PrtA-36 contained the residues characteristic of metzincins, known to be involved in bacterial virulence. In vitro, PrtA inhibited antibacterial factors of inoculated Lepidoptera and of cecropins A and B. PrtS had a molecular mass of 38 kDa and was inhibited by 1,10-phenanthroline but not by EDTA. Its activity ranged between 10 and 80 degrees C and was optimal at pH 7 and 50 degrees C. PrtS also destroyed insect antibacterial factors. Three fragments of PrtS showed homology with a putative metalloprotease of P. luminescens TTO1. Polyclonal antibody raised against PrtA did not recognize PrtS, showing they are distinct molecules.     

Insect pathogenicity islands in the insect pathogenic bacterium Photorhabdus

Abstract. Genomic islands are regions of the bacterial genome responsible for unique aspects of bacterial behaviour, such as host symbiosis and pathogenicity. Where such regions are involved in pathogenesis, they are termed pathogenicity islands (PAIs). Photorhabdus luminescens is an insect pathogen that spends part of its life in symbiosis with a nematode and part of its life as an insect pathogen. Here, several novel PAIs from P. luminescens ssp. akhurstii strain W14 are described that encode factors involved apparently in both nematode symbiosis and insect pathogenicity. The structures of these islands are compared with those found in mammalian pathogens, and the potential cross‐talk between virulence factors used against invertebrates and those used against vertebrates is discussed.     

The pbgPE operon in Photorhabdus luminescens is required for pathogenicity and symbiosis

Photorhabdus is a genus of gram-negative Enterobacteriaceae that is pathogenic to insect larvae while also maintaining a mutualistic relationship with nematodes from the family Heterorhabditis, where the bacteria occupy the gut of the infective juvenile (IJ) stage of the nematode. In this study we describe the identification and characterization of a mutation in the pbgE1 gene of Photorhabdus luminescens TT01, predicted to be the fifth gene in the pbgPE operon. We show that this mutant, BMM305, is strongly attenuated in virulence against larvae of the greater wax moth, Galleria mellonella, and we report that BMM305 is more sensitive to the cationic antimicrobial peptide, polymyxin B, and growth in mildly acidic pH than the parental strain of P. luminescens. Moreover, we also show that the lipopolysaccharide (LPS) present on the surface of BMM305 does not appear to contain any O antigen. Complementation studies reveal that the increased sensitivity to polymyxin B and growth in mildly acidic pH can be rescued by the in trans expression of pbgE1, while the defects in O-antigen assembly and pathogenicity require the in trans expression of pbgE1 and the downstream genes pbgE2 and pbgE3. Finally, we show that BMM305 is defective in symbiosis as this mutant is unable to colonize the gut of the IJ stage of the nematode. Therefore, we conclude that the pbgPE operon is required for both pathogenicity and symbiosis in P. luminescens.     

Heterologous Expression of Bacillus thuringiensis Vegetative Insecticidal Protein-Encoding Gene vip3LB in Photorhabdus temperata Strain K122 and Oral Toxicity against the Lepidoptera Ephestia kuehniella and Spodoptera littoralis

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.     

Swarming motility by Photorhabdus temperata is influenced by environmental conditions and uses the same flagella as that used in swimming motility

Photorhabdus temperata, an insect pathogen and nematode symbiont, is motile in liquid medium by swimming. We found that P.?temperata was capable of surface movement, termed swarming behavior. Several lines of evidence indicate that P. temperata use the same flagella for both swimming and swarming motility. Both motility types required additional NaCl or KCl in the medium and had peritrichous flagella, which were composed of the same flagellin as detected by immunoblotting experiments. Mutants defective in flagellar structural proteins were nonmotile for both motility types. Unlike swimming, we observed swarming behavior to be a social form of movement in which the cells coordinately formed intricate channels covering a surface. The constituents of the swarm media affected motility. Swarming was optimal on low agar concentrations; as agar concentrations increased, swarm ring diameters decreased.     

昆虫病原线虫共生细菌共生性的分子生物学研究进展

嗜线虫致病杆菌属Xenorhabdus和发光杆菌属Photorhabdus细菌隶属肠杆菌科Enterobacteriaceae,对多种害虫致病能力强,分别与斯氏属Steinernema和异小杆属Heterorhabditis昆虫病原线虫互惠共生。该两属共生细菌既存在对昆虫寄主的病原性,又存在与线虫寄主的共生性。共生细菌与其线虫寄主的共生性主要表现以下4方面：（1）细菌产生食物信号诱导滞育不取食的感染期线虫恢复;（2）细菌为线虫生长与繁殖提供营养;（3）细菌能于感染期线虫的肠道定殖与生长;（4）细菌产生杀线虫毒素杀死非共生线虫。本文综述了共生菌以上4方面的共生性及其相关的分子机制。     

Regulation of Phenotypic Switching and Heterogeneity in Photorhabdus luminescens Cell Populations

Phenotypic heterogeneity in bacterial cell populations allows genetically identical organisms to different behavior under similar environmental conditions. The Gram‐negative bacterium Photorhabdus luminescens is an excellent organism to study phenotypic heterogeneity since their life cycle involves a symbiotic interaction with soil nematodes as well as a pathogenic association with insect larvae. Phenotypic heterogeneity is highly distinct in P. luminescens. The bacteria exist in two phenotypic forms that differ in various morphologic and phenotypic traits and are therefore distinguished as primary (1°) and secondary (2°) cells. The 1 cells are bioluminescent, pigmented, produce several secondary metabolites and exo-enzymes, and support nematode growth and development. The 2° cells lack all these 1°-specific phenotypes. The entomopathogenic nematodes carry 1° cells in their upper gut and release them into an insect's body after slipping inside. During insect infection, up to the half number of 1° cells undergo phenotypic switching and convert to 2° cells. Since the 2° cells are not able to live in nematode symbiosis any more, they cannot re-associate with their symbiosis partners after the infection and remain in the soil. Phenotypic switching in P. luminescens has to be tightly regulated since a high switching frequency would lead to a complete break-down of the nematode-bacteria life cycle. Here, we present the main regulatory mechanisms known to-date that are important for phenotypic switching in P. luminescens cell populations and discuss the biological reason as well as the fate of the 2° cells in the soil.     

For the insect pathogen Photorhabdus luminescens,which end of a nematode is out?

The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae. The dauer juvenile (DJ) stage nematode selectively retains P. luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host. We report the results of studying the transmission of the bacteria by its nematode vector. Cells of P. luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus. Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria. Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth. This regurgitation reaction was induced by a low-molecular-weight, heat- and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures. Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph. The ability to visualize P. luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae. This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study.     

Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads

Actively growing cultures of Photorhabdus luminescens were encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads, containing approximately 2.5 x 10(7)Photorhabdus cells per bead, when mixed with sterilized soil and exposed to Spodoptera litura larvae resulted in 100% mortality in 48 h, while the use of alginate encapsulated Heterorhabditis nematode resulted in 40% mortality after 72 h. The bacteria were reisolated from the dead insect thus proving Koch's postulates and demonstrating the ability of P. luminescens to kill the insect host on their own, independent of the symbiont nematode. The LC(50) dose of Photorhabdus cells was estimated at 1010 cells per larva for killing S. litura 6th instar larvae in 48 h.     

Effect of growth conditions on the motility of<Emphasis Type="Italic"> Photorhabdus temperata</Emphasis>

Photorhabdus temperata is a bioluminescent bacterium that lives in mutualistic association with entomopathogenic nematodes of the genus Heterorhabditis. The bacterium exists in two morphologically distinguishable phases (primary and secondary). The swimming behavior of P. temperata was investigated. Both the primary and secondary variants were able to swim in liquid or semisolid media under appropriate conditions. Variation in the oxygen levels had little affect on the chemotaxis and motility of the primary form, but greatly influenced the behavior of the secondary form. Under oxic conditions the secondary form was nonmotile, but motility was induced under anoxic conditions. Several phenotypic traits of the primary form were not expressed under anoxic conditions. The constituents of the growth media affected the motility of both variants. P. temperata required additional NaCl or KCl for optimum motility and chemotaxis. Optimal chemotactic behavior required the presence of bacto-peptone and yeast extract in the swim-migration medium. A mutant that was isolated from the secondary form was able to swim under oxic conditions and possessed an altered salt requirement for motility.     

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.     

Phylogenetic and cophylogenetic relationships of entomopathogenic nematodes (Heterorhabditis: Rhabditida) and their symbiotic bacteria (Photorhabdus: Enterobacteriaceae)

Mutualistic association between entomopathogenic Photorhabdus bacteria and Heterorhabditis nematodes represents one of the emerging model systems in symbiosis studies, yet little is known about this partnership from a coevolutionary perspective. Herein, we investigated phylogenetic and cophylogenetic relationships of Heterorhabditis and Photorhabdus strains using molecular markers Internal Transcribed Spacer and gyrase B gene sequences, respectively. The phylogenies presented consistent, well supported, monophyletic groups in the parsimonious and likelihood analyses for both the nematode and bacterial strains and supported the placement of currently recognized taxa, from which a potentially new Heterorhabditis species represented by a Thailand strain MP68 was identified. While the nematode strains with distant geographic distributions showed no detectable phylogenetic divergence within H. bacteriophora or H. georgiana monophyletic groups, their respective symbiotic bacteria speciated into two Photorhabdus species: P. luminescens and P. temperata, indicating the occurrence of duplication. Although such evolutionary process reduces the phylogenetic congruence between Heterorhabditis nematodes and Photorhabdus bacteria, global cophylogenetic tests using ParaFit detected a highly significant correlation between the two phylogenies (ParaFitGlobal = 0.001). Further, the associations between H. zealandica, H. indica and H. megidis strains and their symbiotic bacteria exhibited significant contribution to the overall cophylogenetic structure. Overall, this study reveals evidence of coevolution between Photorhabdus bacteria and Heterorhabditis nematodes and provides a framework for further examination of the evolution of these associations.