Photorhabdus: a tale of contrasting interactions.

Different model systems have, over the years, contributed to our current understanding of the molecular mechanisms underpinning the various types of interaction between bacteria and their animal hosts. The genus Photorhabdus comprises Gram-negative insect pathogenic bacteria that are normally found as symbionts that colonize the gut of the infective juvenile stage of soil-dwelling nematodes from the family Heterorhabditis. The nematodes infect susceptible insects and release the bacteria into the insect haemolymph where the bacteria grow, resulting in the death of the insect. At this stage the nematodes feed on the bacterial biomass and, following several rounds of reproduction, the nematodes develop into infective juveniles that leave the insect cadaver in search of new hosts. Therefore Photorhabdus has three distinct and obligate roles to play during this life-cycle: (1) Photorhabdus must kill the insect host; (2) Photorhabdus must be capable of supporting nematode growth and development; and (3) Photorhabdus must be able to colonize the gut of the next generation of infective juveniles before they leave the insect cadaver. In this review I will discuss how genetic analysis has identified key genes involved in mediating, and regulating, the interaction between Photorhabdus and each of its invertebrate hosts. These studies have resulted in the characterization of several new families of toxins and a novel inter-kingdom signalling molecule and have also uncovered an important role for phase variation in the regulation of these different roles.

Virulent secondary metabolites of entomopathogenic bacteria genera, Xenorhabdus and Photorhabdus, inhibit phospholipase A2 to suppress host insect immunity.

BACKGROUND: Xenorhabdus and Photorhabdus are entomopathogenic bacteria that cause septicemia and toxemia in insects. They produce secondary metabolites to induce host immunosuppression. Their metabolite compositions vary among bacterial species. Little is known about the relationship between metabolite compositions and the bacterial pathogenicity. The objective of this study was to compare pathogenicity and production of secondary metabolites of 14 bacterial isolates (species or strains) of Xenorhabdus and Photorhabdus. RESULTS: All bacterial isolates exhibited insecticidal activities after hemocoelic injection to Spodoptera exigua (a lepidopteran insect) larvae, with median lethal doses ranging from 168.8 to 641.3 CFU per larva. Bacterial infection also led to immunosuppression by inhibiting eicosanoid biosynthesis. Bacterial culture broth was fractionated into four different organic extracts. All four organic extracts of each bacterial species exhibited insecticidal activities and resulted in immunosuppression. These organic extracts were subjected to GC-MS analysis which predicted 182 compounds, showing differential compositions for 14 bacteria isolates. There were positive correlations between total number of secondary metabolites produced by each bacterial culture broth and its bacterial pathogenicity based on immunosuppression and insecticidal activity. From these correlation results, 70 virulent compounds were selected from secondary metabolites of high virulent bacterial isolates by deducting those of low virulent bacterial isolates. These selected virulent compounds exhibited significant immunosuppressive activities by inhibiting eicosanoid biosynthesis. They also exhibited relatively high insecticidal activities. CONCLUSION: Virulence variation between Xenorhabdus and Photorhabdus is determined by their different compositions of secondary metabolites, of which PLA2 inhibitors play a crucial role.

Phenotypic Heterogeneity of the Insect Pathogen Photorhabdus luminescens: Insights into the Fate of Secondary Cells.

Photorhabdus luminescens is a Gram-negative bacterium that lives in symbiosis with soil nematodes and is simultaneously highly pathogenic toward insects. The bacteria exist in two phenotypically different forms, designated primary (1°) and secondary (2°) cells. Yet unknown environmental stimuli as well as global stress conditions induce phenotypic switching of up to 50% of 1° cells to 2° cells. An important difference between the two phenotypic forms is that 2° cells are unable to live in symbiosis with nematodes and are therefore believed to remain in the soil after a successful infection cycle. In this work, we performed a transcriptomic analysis to highlight and better understand the role of 2° cells and their putative ability to adapt to living in soil. We could confirm that the major phenotypic differences between the two cell forms are mediated at the transcriptional level as the corresponding genes were downregulated in 2° cells. Furthermore, 2° cells seem to be adapted to another environment as we found several differentially expressed genes involved in the cells' metabolism, motility, and chemotaxis as well as stress resistance, which are either up- or downregulated in 2° cells. As 2° cells, in contrast to 1° cells, chemotactically responded to different attractants, including plant root exudates, there is evidence for the rhizosphere being an alternative environment for the 2° cells. Since P. luminescens is biotechnologically used as a bio-insecticide, investigation of a putative interaction of 2° cells with plants is also of great interest for agriculture.IMPORTANCE The biological function and the fate of P. luminescens 2° cells were unclear. Here, we performed comparative transcriptomics of P. luminescens 1° and 2° cultures and found several genes, not only those coding for known phenotypic differences of the two cell forms, that are up- or downregulated in 2° cells compared to levels in 1° cells. Our results suggest that when 1° cells convert to 2° cells, they drastically change their way of life. Thus, 2° cells could easily adapt to an alternative environment such as the rhizosphere and live freely, independent of a host, putatively utilizing plant-derived compounds as nutrient sources. Since 2° cells are not able to reassociate with the nematodes, an alternative lifestyle in the rhizosphere would be conceivable.

A syringe-like injection mechanism in Photorhabdus luminescens toxins.

Photorhabdus luminescens is an insect pathogenic bacterium that is symbiotic with entomopathogenic nematodes. On invasion of insect larvae, P. luminescens is released from the nematodes and kills the insect through the action of a variety of virulence factors including large tripartite ABC-type toxin complexes (Tcs). Tcs are typically composed of TcA, TcB and TcC proteins and are biologically active only when complete. Functioning as ADP-ribosyltransferases, TcC proteins were identified as the actual functional components that induce actin-clustering, defects in phagocytosis and cell death. However, little is known about the translocation of TcC into the cell by the TcA and TcB components. Here we show that TcA in P. luminescens (TcdA1) forms a transmembrane pore and report its structure in the prepore and pore state determined by cryoelectron microscopy. We find that the TcdA1 prepore assembles as a pentamer forming an α-helical, vuvuzela-shaped channel less than 1.5 nanometres in diameter surrounded by a large outer shell. Membrane insertion is triggered not only at low pH as expected, but also at high pH, explaining Tc action directly through the midgut of insects. Comparisons with structures of the TcdA1 pore inserted into a membrane and in complex with TcdB2 and TccC3 reveal large conformational changes during membrane insertion, suggesting a novel syringe-like mechanism of protein translocation. Our results demonstrate how ABC-type toxin complexes bridge a membrane to insert their lethal components into the cytoplasm of the host cell. We believe that the proposed mechanism is characteristic of the whole ABC-type toxin family. This explanation of toxin translocation is a step towards understanding the host-pathogen interaction and the complex life cycle of P. luminescens and other pathogens, including human pathogenic bacteria, and serves as a strong foundation for the development of biopesticides.

Identifying Anti-host Effectors in Photorhabdus.

The death of the insect host is an essential part of the life cycle of Photorhabdus, and as a result, this bacterium comes equipped with a dazzlingly large array of toxins and virulence factors that ensure rapid insect death. Elucidation of the key players in insect infection and mortality has therefore proved difficult using traditional microbiological techniques such as individual gene knockouts due to the high level of functional redundancy displayed by Photorhabdus virulence factors. Thus, knockout of any individual toxin gene may serve to delay time to death but not to render the bacteria avirulent due to the continued presence of an array of other toxins and virulence factors in the single-gene mutant. This functional redundancy had led to the necessary development of an array of techniques and new model systems for identifying and dissecting apart the action of anti-insect effectors produced by Photorhabdus. These have been pivotal in both the identification of new toxins and virulence factors and in ascribing functions to them. These techniques have gone on to prove valuable in pathogenic bacteria other than Photorhabdus and are likely to be useful in many others.

Photorhabdus asymbiotica as an Insect and Human Pathogen.

Photorhabdus asymbiotica is a species of bacterium that is pathogenic to humans whilst retaining the ability to infect insect hosts. Currently, there are two recognised subspecies, P. asymbiotica subsp. asymbiotica and P. asymbiotica subsp. australis with strains isolated from various locations in the USA, Australia, Thailand, Nepal and Europe. Like other species of Photorhabdus, P. asymbiotica subsp. australis was shown to form a symbiotic relationship with a Heterorhabditis nematode. In contrast to most strains of Photorhabdus luminescens, P. asymbiotica can grow at 37 °C and this is a defining factor in its ability to cause human disease. Insights into other adaptations it has undergone that have enabled host switching to occur have come from whole genome sequencing and transcriptomic studies. P. asymbiotica has a smaller genome compared to P. luminenscens with a lower diversity of insecticidal toxins. However, it has acquired plasmids and several pathogenicity islands in its genome. These encode genes with similarity to effectors or systems found in other known human pathogens such as Salmonella and Yersinia and are therefore likely to contribute to human pathogenicity. Of crucial importance to virulence is the fact that P. asymbiotica undergoes a large metabolic shift at the human host temperature.

A Review of Clinical Cases of Infection with Photorhabdus Asymbiotica.

The three recognised Photorhabdus species are bioluminescent Gram-negative bacilli of the family Enterobacteriaceae. They are all pathogenic to insects and form a symbiotic relationship with nematodes of the genus Heterorhabditis. P. luminescens and P. temperata are both harmless to humans whilst P. asymbiotica, on the other hand, is a human pathogen that is a symbiont of the newly described nematode vector, Heterorhabditis gerrardi. In this chapter, we review the epidemiological and clinical features of eighteen human cases of P. asymbiotica infection including fifteen from the published literature and three previously unreported cases. Human infection has been reported in the USA and Australia and probably occurs in other parts of Asia where it remains undocumented. Infection occurs most commonly in warmer months particularly after rainfall. Patients may have a history of recent exposure to sand or sandy soil. P. asymbiotica causes both locally invasive soft tissue infection and disseminated disease with bacteraemia. Soft tissue infection may be multifocal with involvement of more than one limb and the trunk. The organism is sensitive to a number of antibiotics in vitro, but treatment failures have been associated with the use of beta-lactams and aminoglycosides. We suggest treatment with a four-week course of an oral fluoroquinolone such as ciprofloxacin. The organism grows readily on standard media from specimens such as wound swabs, pus, blood and even sputum and can be identified in a clinical microbiology laboratory but the diagnosis needs to be considered. The correct diagnosis is most likely to be made where there is close cooperation between clinician and microbiologist.

Entomopathogenic bacteria Photorhabdus luminescens as drug source against Leishmania amazonensis.

Leishmaniasis is a widely spread and zoonotic disease with serious problems as low effectiveness of drugs, emergence of parasite resistance and severe adverse reactions. In recent years, considerable attention has been given to secondary metabolites produced by Photorhabdus luminescens, an entomopathogenic bacterium. Here, we assessed the leishmanicidal activity of P. luminescens culture fluids. Initially, promastigotes of Leishmania amazonensis were incubated with cell free conditioned medium of P. luminescens and parasite survival was monitored. Different pre-treatments of the conditioned medium revealed that the leishmanicidal activity is due to a secreted peptide smaller than 3 kDa. The Photorhabdus-derived leishmanicidal toxin (PLT) was enriched from conditioned medium and its effect on mitochondrial membrane potential of promastigotes, was determined. Moreover, the biological activity of PLT against amastigotes was evaluated. PLT inhibited the parasite growth and showed significant leishmanicidal activity against promastigote and amastigotes of L. amazonensis. PLT also caused mitochondrial dysfunction in parasites, but low toxicity to mammalian cell and human erythrocytes. Moreover, the anti-amastigote activity was independent of nitric oxide production. In summary, our results highlight that P. luminescens secretes Leishmania-toxic peptide(s) that are promising novel drugs for therapy against leishmaniasis.

Differential immunosuppression by inhibiting PLA2 affects virulence of Xenorhabdus hominickii and Photorhabdus temperata temperata.

Immunity negatively influences bacterial pathogenicity. Eicosanoids mediate both cellular and humoral immune responses in insects. This study tested a hypothesis that differential bacterial virulence of Xenorhabdus/Photorhabdus is dependent on their inhibitory activity against phospholipase A2 (PLA2) activity. P. temperata subsp. temperata ('Ptt') was more than 40 times more potent than X. hominickii ('Xh'). Although both bacteria suppressed cellular immune responses, Ptt infection suppressed hemocyte nodule formation much more than Xh infection. Their differential immunosuppression appeared to be induced by their secondary metabolites because organic extracts of Ptt-cultured broth exhibited higher inhibitory activities against cellular immune responses than Xn-cultured broth extracts. Humoral immune responses were analyzed by measuring expression levels of 11 antimicrobial peptide (AMP) genes. Among inducible AMPs in hemocytes and fat body, higher number and more kinds of AMPs exhibited lower expression levels in Ptt infection than those in Xh infection. Suppressed immune responses induced by Ptt or Xh infection were significantly rescued by the addition of a catalytic product of PLA2, suggesting that PLA2 was a common inhibitory target. In fact, Ptt infection inhibited PLA2 activity more strongly than Xh infection. RNA interference of a PLA2 gene decreased its expression and significantly increased bacterial virulence. Moreover, addition of PLA2 inhibitor to Xh infection enhanced its virulence, similar to virulence level of Ptt infection. These results suggest that variation in Xenorhabdus/Photorhabdus bacterial virulence can be explained by their differential inhibitory activities against host insect PLA2.

Alarmone (p)ppGpp regulates the transition from pathogenicity to mutualism in Photorhabdus luminescens.

The enteric gamma-proteobacterium Photorhabdus luminescens kills a wide range of insects, whilst also maintaining a mutualistic relationship with soil nematodes from the family Heterorhabditis. Pathogenicity is associated with bacterial exponential growth, whilst mutualism is associated with post-exponential (stationary) phase. During post-exponential growth, P. luminescens also elaborates an extensive secondary metabolism, including production of bioluminescence, antibiotics and pigment. However, the regulatory network that controls the expression of this secondary metabolism is not well understood. The stringent response is a well-described global regulatory system in bacteria and mediated by the alarmone (p)ppGpp. In this study, we disrupted the genes relA and spoT, encoding the two predicted (p)ppGpp synthases of P. luminescens TTO1, and we showed that (p)ppGpp is required for secondary metabolism. Moreover, we found the (p)ppGpp is not required for pathogenicity of P. luminescens, but is required for bacterial survival within the insect cadaver. Finally, we showed that (p)ppGpp is required for P. luminescens to support normal nematode growth and development. Therefore, the regulatory network that controls the transition from pathogenicity to mutualism in P. luminescens requires (p)ppGpp. This is the first report outlining a role for (p)ppGpp in controlling the outcome of an interaction between a bacteria and its host.

Identification and occurrence of the hydroxamate siderophores aerobactin, putrebactin, avaroferrin and ochrobactin C as virulence factors from entomopathogenic bacteria.

Effective iron acquisition and fine-tuned intracellular iron storage systems are the main prerequisites for a successful host invasion by a pathogen. Bacteria have developed several different strategies to sequester this essential element from their environment, one relies on the secretion of low molecular weight compounds with high affinity for ferric iron, the so-called siderophores. Here, we report hydroxamate siderophore structures produced by entomopathogenic bacteria of the species Xenorhabdus and Photorhabdus, which are known for their potential to produce bioactive natural products, required for their role as nematode symbiont and insect pathogen. Four siderophores could be identified, namely aerobactin, putrebactin, avaroferrin and ochrobactin C, which was found previously only in marine bacteria. While the putrebactin and avaroferrin producing biosynthesis gene cluster (BGC) is more widespread and most likely was present in a common ancestor of these bacteria, the aerobactin and ochrobactin producing BGC was probably taken up by a few strains individually. For aerobactin a role in virulence towards Galleria mellonella larvae is shown.

Involvement of Vitamin B6 Biosynthesis Pathways in the Insecticidal Activity of Photorhabdus luminescens.

UNLABELLED: Photorhabdus luminescens is a Gram-negative entomopathogenic bacterium which symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans To understand the virulence mechanisms of P. luminescens, we obtained virulence-deficient and -attenuated mutants against C. elegans through a transposon-mutagenized library. From the genetic screening, we identified the pdxB gene, encoding erythronate-4-phosphate dehydrogenase, as required for de novo vitamin B6 biosynthesis. Mutation in pdxB caused growth deficiency of P. luminescens in nutrient-poor medium, which was restored under nutrient-rich conditions or by supplementation with pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 Supplementation with three other B6 vitamers (pyridoxal, pyridoxine, and pyridoxamine) also restored the growth of the pdxB mutant, suggesting the existence of a salvage pathway for vitamin B6 biosynthesis in P. luminescens Moreover, supplementation with PLP restored the virulence-deficient phenotype against C. elegans Combining these results with the fact that pdxB mutation also caused attenuation of insecticidal activity, we concluded that the production of appropriate amounts of vitamin B6 is critical for P. luminescens pathogenicity. IMPORTANCE: The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans We have obtained several virulence-deficient and -attenuated P. luminescens mutants against C. elegans through genetic screening. From the genetic analysis, we present the vitamin B6 biosynthetic pathways in P. luminescens that are important for its insecticidal activity. Mutation in pdxB, encoding erythronate-4-phosphate dehydrogenase and required for the de novo vitamin B6 biosynthesis pathway, caused virulence deficiency against C. elegans and growth deficiency of P. luminescens in nutrient-poor medium. Because such phenotypes were restored under nutrient-rich conditions or by supplementation with B6 vitamers, we showed the presence of the two vitamin B6 synthetic pathways (de novo and salvage) in P. luminescens and also showed that the ability to produce an appropriate amount of vitamin B6 is critical for P. luminescens pathogenicity.

Intracellular plasma membrane guidance of Photorhabdus asymbiotica toxin is crucial for cell toxicity.

The bacterial toxin Photorhabdus asymbiotica toxin (PaTox) modifies Rho proteins by tyrosine GlcNAcylation and heterotrimeric Gα proteins by deamidation. Inactivation of Rho proteins results in F-actin disassembly in host cells. Here, we analyzed the subcellular distribution of PaTox and show that the glycosyltransferase domain of PaTox associates with the negatively charged inner surface of the plasma membrane. Localization studies with site-directed mutants, liposome precipitation analysis, lipid overlay assays, and confocal time-lapse microscopy revealed that a patch of positively charged lysine and arginine residues located on helix α1 of the glycosyltransferase is essential for membrane attachment. Using a helix1 deletion mutant, we show that plasma membrane localization of PaTox is essential for cytotoxicity and proved this by substitution of helix1 by an N-terminal myristoylation signal peptide, which restored plasma membrane localization and cytotoxicity. Furthermore, we also show that the intracellular deamidase activity of PaTox depends on the presence of the membrane localization domain. Comparison of PaTox membrane-binding domain with the 4-helix-bundle membrane-binding domain of Pasteurella multocida toxin, Vibrio cholerae multifunctional autoprocessing repeats-in-toxin, and clostridial glucosylating toxins revealed similar spatial geometry and charge distribution but different structural topology, indicating convergent evolution of toxin domains for optimized host target interaction.

Identification and characterization of Photorhabdus temperata mutants altered in hemolysis and virulence.

Photorhabdus temperata is a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora and an insect pathogen. This bacterium produces a wide variety of virulence factors and hemolytic activity. The goal of this study was to identify hemolysin-defective mutants and test their virulence. A genetic approach was used to identify mutants with altered hemolytic activity by screening a library of 10 000 P. temperata transposon mutants. Three classes of mutants were identified: (i) defective (no hemolytic activity), (ii) delayed (delayed initiation of hemolytic activity), and (iii) early (early initiation of hemolytic activity). The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis and motility. The hemolysin-defective mutants, P10A-C11, P10A-H12, and P79-B5, had inserts in genes involved in RNA turnover (RNase II and 5'-pentaphospho-5'-adenosine pyrophosphohydrolase) and showed reduced virulence and production of extracellular factors. These data support the role of RNA turnover in insect pathogenesis and other physiological functions.

One-shot NMR analysis of microbial secretions identifies highly potent proteasome inhibitor.

Natural products represent valuable lead structures for drug discovery. However, for most bioactive compounds no cellular target is yet identified and many substances predicted from genome analysis are inaccessible due to their life stage-dependent biosynthesis, which is not reflected in common isolation procedures. In response to these issues, an NMR-based and target-directed protease assay for inhibitor detection of the proteasome was developed. The methodology is suitable for one-shot identification of inhibitors in conglomerates and crude culture broths. The technique was applied for analysis of the different life stages of the bacterium Photorhabdus luminescens, which resulted in the isolation and characterization of cepafungin I (CepI), the strongest proteasome inhibitor described to date. Its biosynthesis is strictly regulated and solely induced by the specific environmental conditions determined by our methodology. The transferability of the developed technique to other drug targets may disclose an abundance of novel compounds applicable for drug development.

A Novel Formulation of Bacillus thuringiensis for the Control of Brassica Leaf Beetle, Phaedon brassicae (Coleoptera: Chrysomelidae).

Cabbage is a major vegetable crop over the world. Various insect pests can affect cabbage production. Excessive spray of chemical insecticides can lead to the development of insecticide resistance with various adverse effects on the environment and humans. Brassica leaf beetle, Phaedon brassicae Baly, is a coleopteran pest. Both larvae and adults cause damages to cabbage. The objective of this study was to develop an effective microbial insecticide against P. brassicae by adding an immunosuppressive agent to Bacillus thuringiensis (Bt). The immunosuppressive agent was chosen from bacterial cultured broth of Photorhabdus temperata subsp. temperata (Ptt). Reverse phase HPLC revealed that Ptt-cultured broth possessed at least two eicosanoid biosynthesis inhibitors (oxindole and indole) in its hexane extract. The bacterial cultured broth exhibited potent immunosuppressive activity against P. brassicae. Based on toxicity results, B. thuringiensis subsp. tenebrionis (BtT) was selected from four strains of Bts. When Ptt-cultured broth was added to spore-producing BtT cells, the insecticidal activities of BtT against both larvae and adults of P. brassicae were significantly increased. This bacterial mixture applied to develop a "Bt-Plus," which was formulated by mixing BtT cells (10(11) spores per ml) and 48-h Ptt-cultured broth along with additives (surfactant and preservative). When Bt-Plus was sprayed to cabbage infested by P. brassicae at 1,000-fold dilution, the mixture exhibited much higher control efficacy than BtT treatment alone, suggesting it could be used as a novel Bt insecticide for the control of P. brassicae.

Pdl1 is a putative lipase that enhances Photorhabdus toxin complex secretion.

The Toxin Complex (TC) is a large multi-subunit toxin first characterized in the insect pathogens Photorhabdus and Xenorhabdus, but now seen in a range of pathogens, including those of humans. These complexes comprise three protein subunits, A, B and C which in the Xenorhabdus toxin are found in a 4:1:1 stoichiometry. Some TCs have been demonstrated to exhibit oral toxicity to insects and have the potential to be developed as a pest control technology. The lack of recognisable signal sequences in the three large component proteins hinders an understanding of their mode of secretion. Nevertheless, we have shown the Photorhabdus luminescens (Pl) Tcd complex has been shown to associate with the bacteria's surface, although some strains can also release it into the surrounding milieu. The large number of tc gene homologues in Pl make study of the export process difficult and as such we have developed and validated a heterologous Escherichia coli expression model to study the release of these important toxins. In addition to this model, we have used comparative genomics between a strain that releases high levels of Tcd into the supernatant and one that retains the toxin on its surface, to identify a protein responsible for enhancing secretion and release of these toxins. This protein is a putative lipase (Pdl1) which is regulated by a small tightly linked antagonist protein (Orf53). The identification of homologues of these in other bacteria, linked to other virulence factor operons, such as type VI secretion systems, suggests that these genes represent a general and widespread mechanism for enhancing toxin release in gram negative pathogens.

Photorhabdus and a host of hosts.

Photorhabdus is a member of the family Enterobacteriaceae that lives in a mutualistic association with a Heterorhabditis nematode worm. The nematode worm burrows into insect prey and regurgitates Photorhabdus, which goes on to kill the insect. The nematode feeds off the growing bacteria until the insect tissues are exhausted, whereupon they reassociate and leave the cadaver in search of new prey. This highly efficient partnership has been used for many years as a biological crop protection agent. The dual nature of Photorhabdus as a pathogen and mutualist makes it a superb model for understanding these apparently exclusive activities. Furthermore, recently identified clinical isolates of Photorhabdus are helping us to understand how human pathogens can emerge from the enormous reservoir of invertebrate pathogens in the environment. As Photorhabdus has never been found outside a host animal, its niche represents an entirely biotic landscape. In this review we discuss what molecular adaptations allow this bacterium to complete this fascinating and complex life cycle.

The genetic basis of the symbiosis between Photorhabdus and its invertebrate hosts.

Photorhabdus is a pathogen of insects that also maintains a mutualistic association with nematodes from the family Heterorhabditis. Photorhabdus colonizes the gut of the infective juvenile (IJ) stage of the nematode. The IJ infects an insect and regurgitates the bacteria and the bacteria reproduce to kill the insect. The nematodes feed on the resulting bacterial biomass until a new generation of IJs emerges from the insect cadaver. Therefore, during its life cycle, Photorhabdus must (1) kill the insect host, (2) support nematode growth and development, and (3) be able to colonize the new generation of IJs. In this review, functional genomic studies that have been aimed at understanding the molecular mechanisms underpinning each of these roles will be discussed. These studies have begun to reveal that distinct gene sets may be required for each of these interactions, suggesting that there is only a minimal genetic overlap between pathogenicity and mutualism in Photorhabdus.

Daphnia magna, a host for evaluation of bacterial virulence.

We show that Daphnia magna can be used to assess acute virulence of pathogens relevant to human health, such as Pseudomonas aeruginosa or Photorhabdus asymbiotica. Analysis of bacterial mutants suggests that P. aeruginosa uses similar mechanisms to infect Daphnia and other hosts.