Exploring the effects of entomopathogenic nematode symbiotic bacteria and their cell free filtrates on the tomato leafminer Tuta absoluta and its predator Nesidiocoris tenuis.

The use of biocontrol agents, such as predators and entomopathogenic nematodes, is a promising approach for the effective control of the tomato leafminer Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidaean), an oligophagous insect feeding mainly on Solanaceae species and a major pest of field- and greenhouse-grown tomatoes globally. In this context, the effects of two entomopathogenic nematode species Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora (Poinar) (Rhabditida: Heterorhabditidae), as well as their respective bacterial symbionts, Xenorhabdus nematophila and Photorhabdus luminescens (Enterobacterales: Morganelaceae), which were applied as bacterial cell suspensions and as crude cell-free liquid filtrates on T. absoluta larvae, were investigated. The results showed that of all treatments, the nematodes S. carpocapsae and H. bacteriophora were the most effective, causing up to 98 % mortality of T. absoluta larvae. Regarding bacteria and their filtrates, the bacterium X. nematophila was the most effective (69 % mortality in young larvae), while P. luminescens and both bacterial filtrates showed similar potency (ca. 48-55 % mortality in young larvae). To achieve a holistic approach of controlling this important pest, the impact of these factors on the beneficial predator Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) was also studied. The results demonstrated that although nematodes and especially S. carpocapsae, caused significant mortality on N. tenuis (87 %), the bacterial cell suspensions of X. nematophila and P. luminescens and crude cell-free liquid filtrates had minimum impact on this beneficial predator (∼11-30 % mortality).

Function and Global Regulation of Type III Secretion System and Flagella in Entomopathogenic Nematode Symbiotic Bacteria.

Currently, it is widely accepted that the type III secretion system (T3SS) serves as the transport platform for bacterial virulence factors, while flagella act as propulsion motors. However, there remains a noticeable dearth of comparative studies elucidating the functional disparities between these two mechanisms. Entomopathogenic nematode symbiotic bacteria (ENS), including Xenorhabdus and Photorhabdus, are Gram-negative bacteria transported into insect hosts by Steinernema or Heterorhabdus. Flagella are conserved in ENS, but the T3SS is only encoded in Photorhabdus. There are few reports on the function of flagella and the T3SS in ENS, and it is not known what role they play in the infection of ENS. Here, we clarified the function of the T3SS and flagella in ENS infection based on flagellar inactivation in X. stockiae (flhDC deletion), T3SS inactivation in P. luminescens (sctV deletion), and the heterologous synthesis of the T3SS of P. luminescens in X. stockiae. Consistent with the previous results, the swarming movement of the ENS and the formation of biofilms are dominated by the flagella. Both the T3SS and flagella facilitate ENS invasion and colonization within host cells, with minimal impact on secondary metabolite formation and secretion. Unexpectedly, a proteomic analysis reveals a negative feedback loop between the flagella/T3SS assembly and the type VI secretion system (T6SS). RT-PCR testing demonstrates the T3SS's inhibition of flagellar assembly, while flagellin expression promotes T3SS assembly. Furthermore, T3SS expression stimulates ribosome-associated protein expression.

Two novel XRE-like transcriptional regulators control phenotypic heterogeneity in Photorhabdus luminescens cell populations.

BACKGROUND: The insect pathogenic bacterium Photorhabdus luminescens exists in two phenotypically different forms, designated as primary (1°) and secondary (2°) cells. Upon yet unknown environmental stimuli up to 50% of the 1° cells convert to 2° cells. Among others, one important difference between the phenotypic forms is that 2° cells are unable to live in symbiosis with their partner nematodes, and therefore are not able to re-associate with them. As 100% switching of 1° to 2° cells of the population would lead to a break-down of the bacteria's life cycle the switching process must be tightly controlled. However, the regulation mechanism of phenotypic switching is still puzzling. RESULTS: Here we describe two novel XRE family transcriptional regulators, XreR1 and XreR2, that play a major role in the phenotypic switching process of P. luminescens. Deletion of xreR1 in 1° or xreR2 in 2° cells as well as insertion of extra copies of xreR1 into 2° or xreR2 into 1° cells, respectively, induced the opposite phenotype in either 1° or 2° cells. Furthermore, both regulators specifically bind to different promoter regions putatively fulfilling a positive autoregulation. We found initial evidence that XreR1 and XreR2 constitute an epigenetic switch, whereby XreR1 represses xreR2 expression and XreR2 self-reinforces its own gene by binding to XreR1. CONCLUSION: Regulation of gene expression by the two novel XRE-type regulators XreR1 and XreR2 as well as their interplay represents a major regulatory process in phenotypic switching of P. luminescens. A fine-tuning balance between both regulators might therefore define the fate of single cells to convert from the 1° to the 2° phenotype.

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.

The Biocontrol Agent and Insect Pathogen Photorhabdus luminescens Interacts with Plant Roots.

The number of sustainable agriculture techniques to improve pest management and environmental safety is rising, as biological control agents are used to enhance disease resistance and abiotic stress tolerance in crops. Here, we investigated the capacity of the Photorhabdus luminescens secondary variant to react to plant root exudates and their behavior toward microorganisms in the rhizosphere. P. luminescens is known to live in symbiosis with entomopathogenic nematodes (EPNs) and to be highly pathogenic toward insects. The P. luminescens-EPN relationship has been widely studied, and this combination has been used as a biological control agent; however, not much attention has been paid to the putative lifestyle of P. luminescens in the rhizosphere. We performed transcriptome analysis to show how P. luminescens responds to plant root exudates. The analysis highlighted genes involved in chitin degradation, biofilm regulation, formation of flagella, and type VI secretion system. Furthermore, we provide evidence that P. luminescens can inhibit growth of phytopathogenic fungi. Finally, we demonstrated a specific interaction of P. luminescens with plant roots. Understanding the role and the function of this bacterium in the rhizosphere might accelerate the progress in biocontrol manipulation and elucidate the peculiar mechanisms adopted by plant growth-promoting rhizobacteria in plant root interactions.IMPORTANCE Insect-pathogenic Photorhabdus luminescens bacteria are widely used in biocontrol strategies against pests. Very little is known about the life of these bacteria in the rhizosphere. Here, we show that P. luminescens can specifically react to and interact with plant roots. Understanding the adaptation of P. luminescens in the rhizosphere is highly important for the biotechnological application of entomopathogenic bacteria and could improve future sustainable pest management in agriculture.

A survey of entomopathogenic nematodes and their symbiotic bacteria in agricultural areas of northern Thailand.

Entomopathogenic nematodes (EPNs) Steinernema and Heterorhabditis and their symbiotic bacteria, Xenorhabdus and Photorhabdus, have been successfully used for the control of insect pests. The objectives of this study were to survey the EPNs and symbiotic bacteria in the agricultural areas of the Phitsanulok province, Thailand, and to study the association between the soil parameters and presence of EPNs. We collected 200 soil samples from 40 soil sites in agricultural areas (field crops, horticulture crops and forest). The prevalence of EPNs was 8.0% (16/200). Fifteen of the EPN isolates were molecularly identified (based on 28S ribosomal DNA and internal transcribed spacer regions) as Steinernema siamkayai. Seven isolates of Xenorhabdus stockiae were identified using recombinase A sequencing. Phylogenetic analysis revealed that all the Steinernema and Xenorhabdus isolates were closely related to S. siamkayai (Indian strain) and X. stockiae (Thai strain), respectively. Significantly more EPNs were recovered from loam than from clay. Although the association between soil parameters (pH, temperature and moisture) and the presence of EPNs was not statistically significant, the elevation levels of the soil sites with and without EPNs were found to be different. Moreover, statistical comparisons between the agricultural areas revealed no significant differences. Therefore, we concluded that S. siamkayai is associated with X. stockiae in agricultural areas and that there is no association between the soil parameters of agricultural areas and presence of EPNs, except for soil texture and the elevation. Steinernema siamkayai may be applied as a biocontrol agent in agricultural areas.

Chemical language and warfare of bacterial natural products in bacteria-nematode-insect interactions.

Covering: up to November 2017 Organismic interaction is one of the fundamental principles for survival in any ecosystem. Today, numerous examples show the interaction between microorganisms like bacteria and higher eukaryotes that can be anything between mutualistic to parasitic/pathogenic symbioses. There is also increasing evidence that microorganisms are used by higher eukaryotes not only for the supply of essential factors like vitamins but also as biological weapons to protect themselves or to kill other organisms. Excellent examples for such systems are entomopathogenic nematodes of the genera Heterorhabditis and Steinernema that live in mutualistic symbiosis with bacteria of the genera Photorhabdus and Xenorhabdus, respectively. Although these systems have been used successfully in organic farming on an industrial scale, it was only shown during the last 15 years that several different natural products (NPs) produced by the bacteria play key roles in the complex life cycle of the bacterial symbionts, the nematode host and the insect prey that is killed by and provides nutrients for the nematode-bacteria pair. Since the bacteria can switch from mutualistic to pathogenic lifestyle, interacting with two different types of higher eukaryotes, and since the full system with all players can be established in the lab, they are promising model systems to elucidate the natural function of microbial NPs. This review summarizes the current knowledge as well as open questions for NPs from Photorhabdus and Xenorhabdus and tries to assign their roles in the tritrophic relationship.

Quorum Sensing and LuxR Solos in Photorhabdus.

Bacterial communication via small diffusible molecules to mediate group-coordinated behaviour is commonly referred to as 'quorum sensing'. The prototypical quorum sensing system of Gram-negative bacteria consists of a LuxI-type autoinducer synthase that produces acyl-homoserine lactones (AHLs) as signals and a LuxR-type receptor that detects the AHLs to control expression of specific genes. However, many bacteria possess LuxR homologs but lack a cognate LuxI-type AHL-synthase. Those LuxR-type receptors are designated as 'LuxR orphans' or 'solos'. Entomopathogenic bacteria of the genus Photorhabdus all harbour a large number of LuxR solos, more than any other bacteria examined so far. Two novel quorum sensing systems were found to regulate cell clumping in Photorhabdus and therefore affect pathogenicity. In Photorhabdus luminescens and Photorhabdus temperata the LuxR solo PluR senses α-pyrones named 'photopyrones' instead of AHLs, which are produced by the pyrone synthase PpyS. In contrast, Photorhabdus asymbiotica, a closely related insect and human pathogen, has the PluR homolog PauR, which senses dialkylresorcinols produced by the DarABC pathway to regulate pathogenicity. All three Photorhabdus species harbour at least one LuxR solo with an intact AHL-binding motif, which might also allow sensing of exogenous AHLs. However, the majority of the LuxR solos in all Photorhabdus species have a PAS4 signal-binding domain. These receptors are assumed to detect eukaryotic compounds and are proposed to be involved in host sensing. Overall, because of the large number of LuxR solos they encode, bacteria of the genus Photorhabdus are ideal candidates to study and to identify novel bacterial communication networks.

Resistance and tolerance: The role of nutrients on pathogen dynamics and infection outcomes in an insect host.

Tolerance and resistance are the two ways in which hosts can lessen the effects of infection. Tolerance aims to minimize the fitness effects resulting from incumbent pathogen populations, whereas resistance aims to reduce the pathogen population size within the host. While environmental impacts on resistance have been extensively, recorded their impacts on variation in tolerance are virtually unexplored. Here, we ask how the environment, namely the host diet, influences the capacity of an organism to tolerate and resist infection, using a model host-parasite system, the burying beetle, Nicrophorus vespilloides and the entomopathogenic bacteria, Photorhabdus luminescens. We first considered dose-responses and pathogen dynamics within the host, and compared our findings to responses known from other host species. We then investigated how investment in tolerance and resistance changed under different nutritional regimes. Beetles were maintained on one of five diets that varied in their ratio of protein to fat for 48 hr and then injected with P. luminescens. Survival was monitored and the phenoloxidase (PO) response and bacterial load at 24-hr postinfection were ascertained. The dose required to kill 50% of individuals in this species was several magnitudes higher than in other species and the bacteria were shown to display massive decreases in population size, in contrast to patterns of proliferation found in other host species. Diet strongly modified host survival after infection, with those on the high fat/low protein diet showing 30% survival at 8 days, vs. almost 0% survival on the low-fat/high-protein diet. However, this was independent of bacterial load or variation in PO, providing evidence for diet-mediated tolerance mechanisms rather than immune-driven resistance. Evolutionary ecology has long focussed on immune resistance when investigating how organisms avoid succumbing to infection. Tolerance of infection has recently become a much more prominent concept and is suggested to be influential in disease dynamics. This is one of the first studies to find diet-mediated tolerance.

Eicosanoid mediation of immune responses at early bacterial infection stage and its inhibition by Photorhabdus temperata subsp. temperata, an entomopathogenic bacterium.

An entomopathogenic bacterium Photorhabdus temperata subsp. temperata (Ptt) infects insect hemocoel by the vectoring activity of its symbiotic nematode, Heterorhabditis megidis. The bacterium induces host immunosuppression by inhibiting eicosanoid biosynthesis. This study investigated the role of eicosanoids in immune responses of the beet armyworm, Spodoptera exigua, in the early bacterial infection stage (first 3 hr postinfection [PI]). After infection with the nonpathogenic Escherichia coli (Ec), the bacterium maintained its population for the first 3 hr PI, then rapidly decreased in numbers. During the 3 hr PI of Ptt, this pathogenic bacterium also did not show any significant change in bacterial population. However, Ptt rapidly increased its population size after the initial lag phase, inducing fatal septicemia. This study further analyzed cellular and humoral immune responses of the beet armyworm during the initial 3 hr PI. During this early stage, challenge with Ec stimulated hemocyte-spreading behavior along with extensive F-actin growth. However, Ptt infection suppressed hemocyte spreading. Expression levels of three antimicrobial peptides (lysozyme, gloverin, and gallerimycin) were significantly inhibited during Ptt infection. Phospholipase A2 activity was significantly induced during the early infection stage of Ec, but not during Ptt infection. Addition of eicosanoid biosynthesis inhibitors significantly reversed the initial immunosuppression. These results suggest that, during the early infection stage, Ptt can shutdown eicosanoid biosynthesis which can prevent acute immune responses of host insects.

Photorhabdus-nematode symbiosis is dependent on hfq-mediated regulation of secondary metabolites.

Photorhabdus luminescens maintains a symbiotic relationship with the nematodes Heterorhabditis bacteriophora and together they infect and kill insect larvae. To maintain this symbiotic relationship, the bacteria must produce an array of secondary metabolites to assist in the development and replication of nematodes. The regulatory mechanisms surrounding production of these compounds are mostly unknown. The global post-transcriptional regulator, Hfq, is widespread in bacteria and performs many functions, one of which is the facilitation of sRNA binding to target mRNAs, with recent research thoroughly exploring its various pleiotropic effects. Here we generate and characterize an hfq deletion mutant and show that in the absence of hfq, the bacteria are no longer able to maintain a healthy symbiosis with nematodes due to the abolishment of the production of all known secondary metabolites. RNAseq led us to produce a second deletion of a known repressor, HexA, in the same strain, which restored both metabolite production and symbiosis.

Dynamics of transcriptomic response to infection by the nematode Heterorhabditis bacteriophora and its bacterial symbiont Photorhabdus temperata in Heliothis virescens larvae.

Entomopathogenic nematodes in the Heterorhabditis genus and their symbiotic Photorhabdus bacteria are important biocontrol agents of insect pests and models for the study of microbe-host interactions. In this work, we used larvae of the tobacco budworm (Heliothis virescens) as a model to study its defensive mechanisms against Heterorhabditis bacteriophora nematodes carrying symbiotic Photorhabdus temperata. We first determined time points of initial nematode entry and release of bacteria into the haemolymph to perform transcriptional analysis of insect gene expression during these steps in the infective process. RNA-Sequencing analyses were then performed to profile differential gene expression in the insect during nematode invasion, bacterial release and final steps of infection, relative to the untreated controls. Our results support the theory that insect immune response genes are induced upon nematode invasion, but the majority of these genes are suppressed upon the release of bacteria by the nematodes into the haemolymph. Overall, these findings provide information on the dynamics of the insect's response to a progressing infection by this entomopathogenic nematode-bacteria complex and facilitate development of Hel. virescens as a pest model for future functional studies of the key insect defence factors.

An Entomopathogenic Nematode Extends Its Niche by Associating with Different Symbionts.

Bacterial symbionts are increasingly recognised as mediators of ecologically important traits of their animal hosts, with acquisition of new traits possible by uptake of novel symbionts. The entomopathogenic nematode Heterorhabditis downesi associates with two bacterial symbionts, Photorhabdus temperata subsp. temperata and P. temperata subsp. cinerea. At one intensively studied coastal dune site, P. temperata subsp. cinerea is consistently more frequently isolated than P. temperata subsp. temperata in H. downesi recovered from under the bare sand/Ammophila arrenaria of the front dunes (where harsh conditions, including drought, prevail). This is not the case in the more permissive closed dune grassland further from the sea. No differences were detected in ITS1 (internal transcribed spacer) sequence between nematode lines carrying either of the two symbiont subspecies, nor did they differ in their ability to utilise insects from three orders. The two symbionts could be readily swapped between lines, and both were carried in equal numbers within infective juveniles. In laboratory experiments, we tested whether the symbionts differentially affected nematode survival in insect cadavers that were allowed to dry. We assessed numbers of nematode infective juveniles emerging from insects that had been infected with H. downesi carrying either symbiont subspecies and then allowed to desiccate for up to 62 days. In moist conditions, cadavers produced similar numbers of nematodes, irrespective of the symbiont subspecies present, while under desiccating conditions, P. temperata subsp. cinerea cadavers yielded more nematode progeny than P. temperata subsp. temperata cadavers. Desiccating cadavers with the same nematode isolates, carrying either one or the other symbiont subspecies, confirmed that the symbiont was responsible for differences in nematode survival. Moreover, cadavers harbouring P. temperata subsp. cinerea had a reduced rate of drying relative to cadavers harbouring P. temperata subsp. temperata. Our experiments support the hypothesis that H. downesi can extend its niche into harsher conditions by associating with P. temperata subsp. cinerea.

Death Becomes Them: Bacterial Community Dynamics and Stilbene Antibiotic Production in Cadavers of Galleria mellonella Killed by Heterorhabditis and Photorhabdus spp.

UNLABELLED: Insect larvae killed by entomopathogenic nematodes are thought to contain bacterial communities dominated by a single bacterial genus, that of the nematode's bacterial symbiont. In this study, we used next-generation sequencing to profile bacterial community dynamics in greater wax moth (Galleria mellonella) larvae cadavers killed by Heterorhabditis nematodes and their Photorhabdus symbionts. We found that, although Photorhabdus strains did initially displace an Enterococcus-dominated community present in uninfected G. mellonella insect larvae, the cadaver community was not static. Twelve days postinfection, Photorhabdus shared the cadaver with Stenotrophomonas species. Consistent with this result, Stenotrophomonas strains isolated from infected cadavers were resistant to Photorhabdus-mediated toxicity in solid coculture assays. We isolated and characterized a Photorhabdus-produced antibiotic from G. mellonella cadavers, produced it synthetically, and demonstrated that both the natural and synthetic compounds decreased G. mellonella-associated Enterococcus growth, but not Stenotrophomonas growth, in vitro Finally, we showed that the Stenotrophomonas strains described here negatively affected Photorhabdus growth in vitro Our results add an important dimension to a broader understanding of Heterorhabditis-Photorhabdus biology and also demonstrate that interspecific bacterial competition likely characterizes even a theoretically monoxenic environment, such as a Heterorhabditis-Photorhabdus-parasitized insect cadaver. IMPORTANCE: Understanding, and eventually manipulating, both human and environmental health depends on a complete accounting of the forces that act on and shape microbial communities. One of these underlying forces is hypothesized to be resource competition. A resource that has received little attention in the general microbiological literature, but likely has ecological and evolutionary importance, is dead/decaying multicellular organisms. Metazoan cadavers, including those of insects, are ephemeral and nutrient-rich environments, where resource competition might shape interspecific macrobiotic and microbiotic interactions. This study is the first to use a next-generation sequencing approach to study the community dynamics of bacteria within a model insect cadaver system: insect larvae parasitized by entomopathogenic nematodes and their bacterial symbionts. By integrating bioinformatic, biochemical, and classic in vitro microbiological approaches, we have provided mechanistic insight into how antibiotic-mediated bacterial interactions may shape community dynamics within insect cadavers.

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.

First Report of the Isolation of the Symbiotic Bacterium Photorhabdus luminescens subsp. laumondii Associated with Heterorhabditis safricana from South Africa.

Photorhabdus luminescens subsp. laumondii is closely associated with the entomopathogenic nematode Heterorhabditis bacteriophora and has, to date, not been isolated from other nematode species. This study is the first report of P. luminescens subsp. laumondii from two South African isolates of entomopathogenic nematodes, Heterorhabditis safricana SF281 and H. bacteriophora SF351. Both symbiotic bacterial strains are phenotypically closely related to P. luminescens subsp. laumondii previously isolated and described from H. bacteriophora. The genetic relatedness between P. luminescens subsp. laumondii strains SF281B and SF351B was confirmed by comparing 16S rDNA, recA, gyrB and gltX sequences with sequences of P. luminescens subsp. laumondii, including the type strain (TT01T) and strain E21.

An unbiased method for clustering bacterial effectors using host cellular phenotypes.

We present a novel method implementing unbiased high-content morphometric cell analysis to classify bacterial effector phenotypes. This clustering methodology represents a significant advance over more qualitative visual approaches and can also be used to classify, and therefore predict the likely function of, unknown effector genes from any microbial genome. As a proof of concept, we use this approach to investigate 23 genetic regions predicted to encode antimacrophage effectors located across the genome of the insect and human pathogen Photorhabdus asymbiotica. Statistical cluster analysis using multiple cellular measures categorized treated macrophage phenotypes into three major groups relating to their putative functionality: (i) adhesins, (ii) cytolethal toxins, and (iii) cytomodulating toxins. Further investigation into their effects on phagocytosis revealed that several effectors also modulate this function and that the nature of this modulation (increased or decreased phagocytosis) is linked to the phenotype cluster group. Categorizing potential functionalities in this way allows rapid functional follow-up of key candidates for more-directed cell biological or biochemical investigation. Such an unbiased approach to the classification of candidate effectors will be useful for describing virulence-related regions in a wide range of genomes and will be useful in assigning putative functions to the growing number of microbial genes whose function remains unclear from homology searching.

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.

Merging chemical ecology with bacterial genome mining for secondary metabolite discovery.

The integration of chemical ecology and bacterial genome mining can enhance the discovery of structurally diverse natural products in functional contexts. By examining bacterial secondary metabolism in the framework of its ecological niche, insights into the upregulation of orphan biosynthetic pathways and the enhancement of the enzyme substrate supply can be obtained, leading to the discovery of new secondary metabolic pathways that would otherwise be silent or undetected under typical laboratory cultivation conditions. Access to these new natural products (i.e., the chemotypes) facilitates experimental genotype-to-phenotype linkages. Here, we describe certain functional natural products produced by Xenorhabdus and Photorhabdus bacteria with experimentally linked biosynthetic gene clusters as illustrative examples of the synergy between chemical ecology and bacterial genome mining in connecting genotypes to phenotypes through chemotype characterization. These Gammaproteobacteria share a mutualistic relationship with nematodes and a pathogenic relationship with insects and, in select cases, humans. The natural products encoded by these bacteria distinguish their interactions with their animal hosts and other microorganisms in their multipartite symbiotic lifestyles. Though both genera have similar lifestyles, their genetic, chemical, and physiological attributes are distinct. Both undergo phenotypic variation and produce a profuse number of bioactive secondary metabolites. We provide further detail in the context of regulation, production, processing, and function for these genetically encoded small molecules with respect to their roles in mutualism and pathogenicity. These collective insights more widely promote the discovery of atypical orphan biosynthetic pathways encoding novel small molecules in symbiotic systems, which could open up new avenues for investigating and exploiting microbial chemical signaling in host-bacteria interactions.

Priming Galleria mellonella (Lepidoptera: Pyralidae) larvae with heat-killed bacterial cells induced an enhanced immune protection against Photorhabdus luminescens TT01 and the role of innate immunity in the process.

The current study investigated the characteristics and mechanism of the invertebrate immune priming using Galleria mellonella (L.) (Lepidoptera: Pyralidae) larvae (host) and Photorhabdus luminescens TT01 (pathogen) as a model. The following parameters of the G. mellonella larvae primed by hemocoel injection of heat-killed cells of TT01 or Bacillus thuringiensis HD-1 were determined at designated times after priming and then compared and analyzed systematically: mortality of the primed larvae against TT01 infection (immune protection level), hemocyte density, phagocytosis and encapsulation abilities ofhemocyte, and antibacterial activity of cell free hemolymph (major innate parameters). The results showed that 1) immune priming increased survival of the larvae against a lethal infection of TT01 and the levels and periods of protection correlated positively to the priming dose; 2) the changes on the levels of protection and the major innate parameters of the larvae primed with either TT01 or HD-1 followed a similar pattern of the convex curve, although the levels and the timing of changes differed significantly among the four innate immune parameters and between two priming bacteria; and 3) the immune protection level at a time after priming was correlated to the overall level of four innate immune parameters of the primed larvae. The current study demonstrated that the immune priming phenomenon of G. mellonella larvae has low level of specificity, and it was achieved mainly by the regulation on the quantity and activity of major innate immune parameters, such as hemocytes, antimicrobial peptides, and enzymes.