Competition and Co-existence of Two Photorhabdus Symbionts with a Nematode Host.

Photorhabdus spp. (Enterobacteriales: Morganellaceae) occur exclusively as symbionts of Heterorhabditis nematodes for which they provide numerous services, including killing insects and providing nutrition and defence within the cadavers. Unusually, two species (Photorhabdus cinerea and Photorhabdus temperata) associate with a single population of Heterorhabditis downesi at a dune grassland site. Building on previous work, we investigated competition between these two Photorhabdus species both at the regional (between insects) and local (within insect) level by trait comparison and co-culture experiments. There was no difference between the species with respect to supporting nematode reproduction and protection of cadavers against invertebrate scavengers, but P. cinerea was superior to P. temperata in several traits: faster growth rate, greater antibacterial and antifungal activity and colonisation of a higher proportion of nematodes in co-culture. Moreover, where both bacterial symbionts colonised single nematode infective juveniles, P. cinerea tended to dominate in numbers. Differences between Photorhabdus species were detected in the suite of secondary metabolites produced: P. temperata produced several compounds not produced by P. cinerea including anthraquinone pigments. Bioluminescence emitted by P. temperata also tended to be brighter than that from P. cinerea. Bioluminescence and pigmentation may protect cadavers against scavengers that rely on sight. We conclude that while P. cinerea may show greater local level (within-cadaver) competitive success, co-existence of the two Photorhabdus species in the spatially heterogeneous environment of the dunes is favoured by differing specialisations in defence of the cadaver against differing locally important threats.

Insecticidal Activity of Photorhabdus luminescens 0805-P2R Against Plutella xylostella.

Photorhabdus luminescens is an entomopathogenic rod-shaped bacterium infected with insect nematodes of the Heterorhabditidae family. It kills insects through the secretion of high molecular weight toxin complexes. In this study, Plutella xylostella larvae were orally administered P. luminescens for bioassay. After incubation in Luria-Bertani (LB) medium for a sufficiently long period, the mortality rates of P. xylostella observed after diluting the fermentation broth 50 times and diluting the supernatant 5 times were 18.89% and 91.11%, respectively. Retentates measuring more than 70 kDa showed 88% mortality after ultrafiltration (UF) membrane treatment. Thus, the supernatant of P. luminescens had insecticidal activity, and the main insecticidal toxin complexes had a molecular weight exceeding 70 kDa. The L9 (34) Taguchi orthogonal experimental optimized medium mode-predicted insecticidal activity levels were 84% and 119% in the 50-fold diluted fermentation broth and 5-fold diluted supernatant, respectively. Moreover, the insecticidal activity was improved to 92.2% in the 100-fold diluted fermentation broth and to 97.8% in the 10-fold diluted supernatant in the experiments. All combinations tested showed clear indications of lethality, including swelling, vesicle formation, cytoplasm vacuolization, and brush border membrane lysis. Thus, these results promote the use of P. luminescens 0805-P2R as a potent biopesticide to effectively control P. xylostella.

Iso-propyl stilbene: a life cycle signal?

Members of the Gram-negative bacterial genus Photorhabdus are all highly insect pathogenic and exist in an obligate symbiosis with the entomopathogenic nematode worm Heterorhabditis. All members of the genus produce the small-molecule 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) as part of their secondary metabolism. IPS is a multi-potent compound that has antimicrobial, antifungal, immunomodulatory and anti-cancer activities and also plays an important role in symbiosis with the nematode. In this study we have examined the response of Photorhabdus itself to exogenous ectopic addition of IPS at physiologically relevant concentrations. We observed that the bacteria had a measureable phenotypic response, which included a decrease in bioluminescence and pigment production. This was reflected in changes in its transcriptomic response, in which we reveal a reduction in transcript levels of genes relating to many fundamental cellular processes, such as translation and oxidative phosphorylation. Our observations suggest that IPS plays an important role in the biology of Photorhabdus bacteria, fulfilling roles in quorum sensing, antibiotic-competition advantage and maintenance of the symbiotic developmental cycle.

Characterization of a Hybrid Nonribosomal Peptide-Carbohydrate Biosynthetic Pathway in Photorhabdus luminescens.

Advances in genome sequencing and analysis have afforded a trove of "orphan" bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the potential for these hypothetical proteins to carry out novel chemistry, orphan pathways serve as a rich reservoir for the discovery of new enzymes responsible for the production of metabolites with both fascinating chemistries and biological functions. We previously identified a rare hybrid nonribosomal peptide synthetase (NRPS)-carbohydrate genomic island in the entomopathogen Photorhabdus luminescens. Heterologous expression of the pathway led to the characterization of oligosaccharides harboring a 1,6-anhydro-ß-d- N-acetyl-glucosamine moiety, but these new metabolites lacked modification by the NRPS machinery. Here, through the application of top-down protein mass spectrometry, pathway-targeted molecular networking, stable isotope labeling, and in vitro protein biochemistry, we complete the characterization of this biosynthetic pathway and identify the hybrid product of the pathway, a new "glycoamino acid" metabolite termed photolose. Intriguingly, a hypothetical protein served as a bridge to condense a glycyl unit derived from the NRPS machinery onto the free 1,6-anhydro-ß-d- N-acetyl-glucosamine core. We further demonstrate that the gene cluster confers a growth advantage to antimicrobial peptide challenge.

Phenotypic and genomic comparison of Photorhabdus luminescens subsp. laumondii TT01 and a widely used rifampicin-resistant Photorhabdus luminescens laboratory strain.

BACKGROUND: Photorhabdus luminescens is an enteric bacterium, which lives in mutualistic association with soil nematodes and is highly pathogenic for a broad spectrum of insects. A complete genome sequence for the type strain P. luminescens subsp. laumondii TT01, which was originally isolated in Trinidad and Tobago, has been described earlier. Subsequently, a rifampicin resistant P. luminescens strain has been generated with superior possibilities for experimental characterization. This strain, which is widely used in research, was described as a spontaneous rifampicin resistant mutant of TT01 and is known as TT01-RifR. RESULTS: Unexpectedly, upon phenotypic comparison between the rifampicin resistant strain and its presumed parent TT01, major differences were found with respect to bioluminescence, pigmentation, biofilm formation, haemolysis as well as growth. Therefore, we renamed the strain TT01-RifR to DJC. To unravel the genomic basis of the observed differences, we generated a complete genome sequence for strain DJC using the PacBio long read technology. As strain DJC was supposed to be a spontaneous mutant, only few sequence differences were expected. In order to distinguish these from potential sequencing errors in the published TT01 genome, we re-sequenced a derivative of strain TT01 in parallel, also using the PacBio technology. The two TT01 genomes differed at only 30 positions. In contrast, the genome of strain DJC varied extensively from TT01, showing 13,000 point mutations, 330 frameshifts, and 220 strain-specific regions with a total length of more than 300 kb in each of the compared genomes. CONCLUSIONS: According to the major phenotypic and genotypic differences, the rifampicin resistant P. luminescens strain, now named strain DJC, has to be considered as an independent isolate rather than a derivative of strain TT01. Strains TT01 and DJC both belong to P. luminescens subsp. laumondii.

Combinatorial effect of Photorhabdus luminescens TT01 and Bacillus thuringiensis Vip3Aa16 toxin against Agrotis segetum.

The entomopathogenic Photorhabdus luminescens TT01 is a promoting bacterium that controls effectively many insect pests. Indeed, it exhibited a mortality rate of 32.36% against the first instar larvae of the turnip moth Agrotis segetum, when it was used at a concentration of 5 × 107 cells/ml but no toxicity against the second instar larvae in the same condition. P. luminescens TT01 oral toxicity is associated to septicaemia since cells fraction exhibited the highest mortality rate of 34%. In order to enhance P. luminescens TT01 insecticidal potential, combination with Bacillus thuringiensis Vip3Aa16 toxin was tested. An improvement of insecticidal activity was shown. Indeed, 100% mortality of A. segetum first instar larvae was obtained after 2 days of treatment, when using TT01 cells and Vip3Aa16 toxin at a concentration of 5 × 107 cells/ml and 9.025 ng/cm2, respectively. Moreover, growth inhibition rate of 45% of the second instar larvae was observed, when using the same combination. A. segetum mortality could be the result of several alterations in the midgut epithelium caused by Vip3Aa16 toxin, allowing a rapid invasion of the hemocoel by TT01 cells as demonstrated by histopathological study. Clear symptoms of intoxication were observed for all combinations tested, including swelling, vesicle formation, cytoplasm vacuolization and brush border membrane lysis. Taken together, these results promote the use of P. luminescens TT01 as a potent bioinsecticide to control effectively A. segetum by oral treatment in a mixture with Vip3Aa16 toxin.

Neuroprotective and Anti-Neuroinflammatory Activities of Anthraquinones Isolated from Photorhabdus temperata Culture Broth.

Photorhabdus temperata (PT), a gram-negative bacterium, lives symbiotically within entomopathogenic nematodes. The insecticidal compounds derived from Photorhabdus are used as biopesticides in agriculture. However, the physiological properties are not well characterized. In the course of our screening for neuroprotective and anti-neuroinflammatory substances from natural products, the culture broth of PT showed considerable activities. By activity-guided purification, five anthraquinones, namely, 3-methoxychrysazine (1), 1,3-dimethoxy-8-hydroxy-9,10-anthraquinone (2), 1,3,8-trihydroxy-9,10-anthraquinone (3), 3,8-dihydroxy-1-methoxy-9,10-anthraquinone (4), and 1,3,4-trimethoxy-8-hydroxy-9,10-anthraquinone (5), were isolated from the ethyl acetate fraction of the PT culture broth. Among the isolated compounds, 75 µM 3 significantly protected mouse hippocampal neuronal cells (HT22) against 5 mM glutamate-induced cell death via the inhibition of reactive oxygen species production, Ca²âº influx, and lipid peroxidation. Additionally, 3 and 4 effectively suppressed the interferon-γ-induced neuroinflammation of mouse-derived microglial cells (BV2) at 10 ng/ml, via the reduction of nitric oxide, interleukin-6, and tumor necrosis factor-α. Anthraquinones 3 and 4 derived from the PT culture broth are a potential starting point to discover neuroprotective and anti-neuroinflammatory drug leads. The novel compound 5 is reported for the first time in this study.

Rapid in situ toxicity testing with luminescent bacteria Photorhabdus luminescens and Vibrio fischeri adapted to a small portable luminometer.

The present study demonstrates development of a rapid testing protocol based on a small portable luminometer using flash kinetic assessment of bacterial bioluminescence. The laboratory comparisons based on six model organic toxicants and two metals showed significant correlations between responses of freshwater bacteria Photorhabdus luminescens and standard marine bacterial species Vibrio fisheri. While P. luminescens was less sensitive in standard arrangements, the responses of both organisms were comparable in the newly introduced portable luminometer setup. The applicability and reproducibility of the portable luminometer protocol was further demonstrated in the assessment of 43 European wastewater effluents that were simultaneously tested for toxicity and analysed for 150 organic and 20 inorganic contaminants grouped into 13 major chemical classes. Clear association between the toxic responses in both compared bacterial species and the elevated levels of inorganic compounds (toxic metals), chlorophenols and benzotriazole anticorrosives was observed. The new protocol with a portable luminometer provides a fast (30 s) response and may be used as a tool for rapid in situ toxicity evaluation of freshwater environmental samples such as effluents.

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.

Evolution of virulence in Photorhabdus spp., entomopathogenic nematode symbionts.

Photorhabdus is a genus of Gram-negative bacteria belonging to the Enterobacteriaceae family. In addition to forming a mutualistic relationship with the Heterorhabditidae family of nematodes, these bacteria are the causal agent of insect mortality during nematode infection, and are commonly used as biocontrol agents against pest insects in managed ecosystems. There are three described species of Photorhabdus; Photorhabdus luminescens and Photorhabdus temperata, which are strictly entomopathogens, and Photorhabdus asymbiotica, which has been isolated from wound infections in humans. While there has been extensive research on its virulence mechanisms, the evolution of virulence in Photorhabdus has not previously been investigated within a phylogenetic context. To investigate how virulence has evolved in this genus, we first reconstructed the phylogenetic relationships among 18 strains representing each of the main taxonomic lineages in the genus. Bacterial cells were injected into Galleria mellonella and Tenebrio molitor larvae, and the LT50 was calculated for each strain. These values were mapped onto the phylogeny using ancestral character reconstruction methods. With few exceptions, we found that the general trend of Photorhabdus evolution is one of increasing virulence. We also explored the relationship between virulence and Photorhabdus cell types and growth rates. Although we found no correlation between cell type and virulence, there was a strong correlation between virulence and growth rates in T. molitor. A better understanding of the origin and maintenance of virulence in this bacterium will aid in unraveling the mechanisms of the Heterorhabditis-Photorhabdus complex, resulting in the selection of more effective nematode-bacterium complexes for biocontrol.

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.

A culture-based method for determining the production of secreted protease inhibitors.

We have developed a culture-based method for determining the production of secreted protease inhibitors. The assay utilizes standard proteolysis detection plates to support microbial growth followed by infiltrating the plate with a protease and subsequently detecting the remaining protein by trichloroacetic acid (TCA) precipitation, or by bromocreosol green (BCG) or Ponseau S (PS) staining. The presence of a protease inhibitor can be observed in the form of a protected zone of protein around the protease inhibitor-producing strain. Using the protease inhibitors α-2-macroglobulin, aprotinin, leupeptin, and bestatin and the primary and secondary forms of Photorhabdus luminescens in combination with the protease trypsin, we were able to demonstrate that the assay is specific for the cognate inhibitor of the protease and for bacteria secreting protease inhibitors. In addition, when casein-containing plates were used, the size of the diffusion zone was inversely correlated with the molecular weight of the inhibitor allowing a relative estimation of the protease inhibitor molecular weight. This assay is useful for detecting the presence of microbial secreted protease inhibitors and may reveal their production by microorganisms that were not previously recognized to produce them.

Combinatorial effect of Bacillus thuringiensis kurstaki and Photorhabdus luminescens against Spodoptera littoralis (Lepidoptera: Noctuidae).

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.

The expression of stlA in Photorhabdus luminescens is controlled by nutrient limitation.

Photorhabdus is a genus of Gram-negative entomopathogenic bacteria that also maintain a mutualistic association with nematodes from the family Heterorhabditis. Photorhabdus has an extensive secondary metabolism that is required for the interaction between the bacteria and the nematode. A major component of this secondary metabolism is a stilbene molecule, called ST. The first step in ST biosynthesis is the non-oxidative deamination of phenylalanine resulting in the production of cinnamic acid. This reaction is catalyzed by phenylalanine-ammonium lyase, an enzyme encoded by the stlA gene. In this study we show, using a stlA-gfp transcriptional fusion, that the expression of stlA is regulated by nutrient limitation through a regulatory network that involves at least 3 regulators. We show that TyrR, a LysR-type transcriptional regulator that regulates gene expression in response to aromatic amino acids in E. coli, is absolutely required for stlA expression. We also show that stlA expression is modulated by σ(S) and Lrp, regulators that are implicated in the regulation of the response to nutrient limitation in other bacteria. This work is the first that describes pathway-specific regulation of secondary metabolism in Photorhabdus and, therefore, our study provides an initial insight into the complex regulatory network that controls secondary metabolism, and therefore mutualism, in this model organism.

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.

Phospholipase A2 inhibitors in bacterial culture broth enhance pathogenicity of a fungus Nomuraea rileyi.

An entomopathogenic fungus, Nomuraea rileyi, was isolated and its identity was confirmed by its internal transcribed spacer DNA sequence. The isolated N. rileyi exhibited a specific pathogenicity to lepidopteran species. This study was focused on enhancing the fungal pathogenicity by using immunosuppressive agents. In response to infection of N. rileyi, Spodoptera exigua larvae significantly induced catalytic activity of phospholipase A(2) (PLA(2)) in three immune-associated tissues, namely hemocytes, fat body, and hemolymph plasma. Furthermore, the infected S. exigua larvae induced transcription of several antimicrobial peptide (AMP) genes. Two entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata subsp. temperata (Ptt), possessed specific PLA(2)-inhibitory activities and their culture broths significantly inhibited the enzyme activities in hemocytes, fat body, and plasma of S. exigua. In addition, the bacterial metabolites inhibited transcription of AMP genes in S. exigua that would normally respond to the immune challenge by N. rileyi. The immunosuppressive effect of Xn or Ptt bacterial broth resulted in significant enhancement of the fungal pathogenicity against late instar larvae of S. exigua and Plutella xylostella. The effect of such a mixture was confirmed by field assay against two lepidopteran species. These results suggest that the bacterial and fungal mixture can be applied to develop a novel biopesticide to control lepidopteran species.

Improvement of Photorhabdus temperata bioinsecticides production in low-cost media through adequate fermentation technology.

To develop a cost effective process for bioinsecticides production by Photorhabdus temperata, dissolved oxygen (DO) requirements were investigated in both the complex and the optimized media using diluted seawater as a source of micronutrients. By varying DO concentrations, tolerance to hydrogen peroxide was shown to be medium dependant. Indeed, P. temperata cells grown in the complex medium, exhibited higher tolerance than cells grown in the optimized medium (OM). Tolerance to H(2)O(2) was shown to be related to intracellular reactive oxygen species (ROS) accumulation during soya bean meal or glucose assimilation, as shown by flow cytometry analysis. To avoid oxidative stress damages in P. temperata cells cultured in the OM, DO concentration should be constant 50% saturation throughout the fermentation. However, a DO-shift control strategy was demonstrated to be beneficial for P. temperata bioinsecticide production in the complex medium. By using such a strategy biomass, culturability, and oral toxicity reached 16.5 × 10(8), 1.15 × 10(8) cells/mL and 64.2%, respectively, thus was 16.19, 26.37, and 12.2% more than in the cultures carried out at a constant 50% saturation.

A single promoter inversion switches Photorhabdus between pathogenic and mutualistic states.

Microbial populations stochastically generate variants with strikingly different properties, such as virulence or avirulence and antibiotic tolerance or sensitivity. Photorhabdus luminescens bacteria have a variable life history in which they alternate between pathogens to a wide variety of insects and mutualists to their specific host nematodes. Here, we show that the P. luminescens pathogenic variant (P form) switches to a smaller-cell variant (M form) to initiate mutualism in host nematode intestines. A stochastic promoter inversion causes the switch between the two distinct forms. M-form cells are much smaller (one-seventh the volume), slower growing, and less bioluminescent than P-form cells; they are also avirulent and produce fewer secondary metabolites. Observations of form switching by individual cells in nematodes revealed that the M form persisted in maternal nematode intestines, were the first cells to colonize infective juvenile (IJ) offspring, and then switched to P form in the IJ intestine, which armed these nematodes for the next cycle of insect infection.

Improvement of Photorhabdus temperata strain K122 bioinsecticide production by batch and fed-batch fermentations optimization.

Optimization of a fermentation process for bioinsecticides production by Photorhabdus temperata strain K122 was investigated into fully controlled 3-L fermenter using an optimized medium (OM). Development of large-scale inocula showed that the composition of the growth medium greatly influenced the physiological state of P. temperata cells. The effect of pH, agitation and dissolved oxygen concentration (DO) on the growth, culturability and oral toxicity of P. temperata cells were also investigated. Indeed, maintaining the pH at 7 and controlling DO concentration at 50 % saturation throughout the fermentation process, improved biomass production, CFU counts and oral toxicity by 41.1, 35 and 32.1 %, respectively, as compared to cultures carried out in 500 mL shake flasks. At such conditions, 8 g/L glucose fed-batch fermentation, enhanced cell lysis and variants small colony (Vsm) polymorphism appearance. To overcome such limitations, glucose concentration should be maintained at 4 g/L. In this case, P. temperata cells were produced at high cell density and culturability reaching 4.5 and 1.2 × 10(9) cells/mL, respectively. In addition, the stability of the primary form was maintained for a long period in the stationary growth phase and Vsm polymorphism was completely avoided that can be crucial for scale-up the bioprocess of P. temperata bioinsecticide.

Phospholipase A2 inhibitors synthesized by two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata.

The entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata suppress insect immune responses by inhibiting the catalytic activity of phospholipase A(2) (PLA(2)), which results in preventing biosynthesis of immune-mediating eicosanoids. This study identified PLA(2) inhibitors derived from culture broths of these two bacteria. Both X. nematophila and P. temperata subsp. temperata culture broths possessed significant PLA(2)-inhibitory activities. Fractionation of these bacterial metabolites in the culture broths using organic solvent and subsequent chromatography purified seven potent PLA(2) inhibitors, three of which (benzylideneacetone [BZA], proline-tyrosine [PY], and acetylated phenylalanine-glycine-valine [FGV]) were reported in a previous study. Four other compounds (indole, oxindole, cis-cyclo-PY, and p-hydroxyphenyl propionic acid) were identified and shown to significantly inhibit PLA(2). X. nematophila culture broth contained these seven compounds, while P. temperata subsp. temperata culture broth contained three compounds (BZA, acetylated FGV, and cis-cyclo-PY). BZA was detected in the largest amount among these PLA(2) compounds in both bacterial culture broths. All seven bacterial metabolites also showed significant inhibitory activities against immune responses, such as phenoloxidase activity and hemocytic nodulation; BZA was the most potent. Finally, this study characterized these seven compounds for their insecticidal activities against the diamondback moth, Plutella xylostella. Even though these compounds showed relatively low toxicities to larvae, they significantly enhanced the pathogenicity of Bacillus thuringiensis. This study reports bacterial-origin PLA(2) inhibitors, which would be applicable for developing novel insecticides.