Overcoming the production limitations of Photorhabdus temperata ssp. temperata strain K122 bioinsecticides in low-cost medium

For low-cost production of Photorhabdus temperata ssp. temperata strain K122 bioinsecticide, a cheap complex medium was optimized. Diluted seawater was used as the source of micronutrients, especially sodium chloride, involved in the improvement of cell density, culturability and oral toxicity of the bacterium P. temperata against Ephestia kuehniella larvae. Thus, the new formulated medium was composed only of 10 g/l of soya bean meal, used as the carbon and nitrogen main source, mixed in sevenfold diluted seawater. At such conditions, several limitations of P. temperata bioinsecticide productions were shown to be overcome. The appearance of variants small colony polymorphism was completely avoided. Thus, the strain K122 was maintained at the primary form even after prolonged incubation. Moreover, the viable but nonculturable state was partially overcome, since the ability of P. temperata cells to form colonies on the solid medium was prolonged until 78 h of incubation. In addition, when cultured in the complex medium, P. temperata cells were produced at high cell density of 12 × 10⁸ cells/ml and exhibited 81.48% improvement of oral toxicity compared to those produced in the optimized medium. With such medium, the large-scale bioinsecticides production into 3-l fully controlled fermenter improved the total cell counts, CFU counts and oral toxicity by 20, 5.81 and 16.73%, respectively. This should contribute to a significant reduction of production cost of highly potent P. temperata strain K122 cells, useful as a bioinsecticide.     

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 × 10⁹ 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.

     

Identification and sequence of an unstable DNA element in the entomopathogenic bacteria Photorhabdus temperata strain K122

AIMS: A search was conducted for a difference in genome composition between phenotypic variants of the insect pathogenic bacteria, Photorhabdus temperata. METHODS: An unstable 300 bp fragment of DNA was identified by amplified fragment length polymorphism (AFLP) analysis, which was not, however, associated with phenotypic variation. RESULTS: During prolonged culturing of the bacteria, one copy of the repeated fragment was deleted and a restriction site linked to one of the copies was lost or gained. The sequence did not show substantial identity to any in the database, but a 16-bp region was identical to part of the marR gene of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The work has implications for the understanding of genetic instability in this and other pathogenic species of bacteria. In addition, the complete unstable element may be useful as a genetic tool in Photorhabdus spp.     

Involvement of oxidative stress and growth at high cell density in the viable but nonculturable state of Photorhabdus temperata ssp. temperata strain K122

Photorhabdus temperata ssp. temperata strain K122 represents a promising source of bioinsecticide. When cultured in an optimized medium, P. temperata exhibited restricted survival in terms of colony-forming ability on solid medium, which remained lower than the total cell counts. Membrane integrity assessment by flow cytometry showed that almost 100% of P. temperata cells were viable indicating that this bacterium enters in the viable but nonculturable state (VBNC). According to the double staining results, hydrogen peroxide was demonstrated to be responsible of P. temperata VBNC state. Addition of catalase or sodium pyruvate upon the inoculation of P. temperata on agar plates promoted the recovery of nonculturable cells up to 24 h incubation. Further, growth at high cell density enhanced the VBNC state of this bacterium. This should evidenced extracellular signals accumulation involved in quorum sensing mechanism. Elucidation of this state is interesting for both toxicity study and production of P. temperata useful as bioinsecticide.     

Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection

During insect infection Photorhabdus luminescens emits light and expresses virulence factors, including insecticidal toxin complexes (Tcs) and an RTX-like metalloprotease (Prt). Using quantitative PCR and protein assays, we describe the expression patterns of these factors both in culture and during insect infection and compare them to the associated bacterial growth curves. In culture, light and active Prt protease are produced in stationary phase. Tca also appears in stationary phase, whereas Tcd is expressed earlier. These patterns seen in a culture flask are strikingly similar to those observed during insect infection. Thus, in an infected insect, bacteria grow exponentially until the time of insect death at approximately 48 h, when both light and the virulence factors Prt protease and Tca are produced. In contrast, Tcd appears much earlier in insect infection. However, at present, the biological significance of this difference in timing of the production of the two toxins in unclear. This is the first documentation of the expression of Tcs and Prt in an insect and highlights the malleability of Photorhabdus as a model system for bacterial infection.     

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

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

Effect of the insect pathogenic bacterium Photorhabdus on insect phagocytes

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

Differential pathogenicity of two entomopathogenic bacteria,Photorhabdus temperata subsp. temperata and Xenorhabdus nematophila against the red flour beetle,Tribolium castaneum

Two entomopathogenic bacteria, Photorhabdus temperata subsp. temperata (Ptt) and Xenorhabdus nematophila (Xn), are symbiotically associated with the nematodes, Heterorhabdis megidis and Steinernema carpocapsae, respectively. There is little information on natural host ranges of the nematodes, but a significant difference in pathogenicity was observed between these two bacteria against the red flour beetle, Tribolium castaneum, in which Ptt exhibited more than six times higher pathogenicity than Xn. The pathogenic difference was not due to their inhibitory effect on phospholipase A₂ activity that is required for expression of immune response of T. castaneum. The culture broths of both bacterial species had insecticidal activities when injected into the hemocoel. When the bacterial culture broths were fractionated into aqueous and organic extracts, most insecticidal activity remained in the aqueous extracts. The aqueous extracts of two bacteria contained proteins which showed different profiles.     

Effects of entomopathogenic bacterium Photorhabdus temperata infection on the digestive enzymes of Diatraea saccharalis (Lepidoptera: Crambidae) larvae

This study investigated the effects of Photorhabdus temperata infection on the activities of digestive enzymes of the sugarcane stalk borer Diatraea saccharalis. Non-infected D. saccharalis larvae present a major alpha-amylase, several proteinases, three sucrose hydrolases and two alpha-glucosidases in their midgut. Analysis of these hydrolases by electrophoresis and "in gel" assays showed that the activities of all enzymes decreased following infection, with an initial decline observed 12 h after infection. The activities of alpha-glucosidases decreased by 50% twelve hours after infection, whereas, at this time, the alpha-galactosidase activities decreased by 70%. Interestingly, the animals died 48 h after infection, but approximately 5% of all the enzymes tested remained active in the midgut following host death. At this time, most of the cultivable native intestinal bacteria had died.     

Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta

Invertebrates, including insects, are being developed as model systems for the study of bacterial virulence. However, we understand little of the interaction between bacteria and specific invertebrate tissues or the immune system. To establish an infection model for Photorhabdus, which is released directly into the insect blood system by its nematode symbiont, we document the number and location of recoverable bacteria found during infection of Manduca sexta. After injection into the insect larva, P. luminescens multiplies in both the midgut and haemolymph, only later colonizing the fat body and the remaining tissues of the cadaver. Bacteria persist by suppressing haemocyte-mediated phagocytosis and culture supernatants grown in vitro, as well as plasma from infected insects, suppress phagocytosis of P. luminescens. Using GFP-labelled bacteria, we show that colonization of the gut begins at the anterior of the midgut and proceeds posteriorly. Within the midgut, P. luminescens occupies a specific niche between the extracellular matrix and basal membrane (lamina) of the folded midgut epithelium. Here, the bacteria express the gut-active Toxin complex A (Tca) and an RTX-like metalloprotease PrtA. This close association of the bacteria with the gut, and the production of toxins and protease, triggers a massive programmed cell death of the midgut epithelium.     

Effects of entomopathogenic bacterium Photorhabdus temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis (Lepidoptera: Crambidae)

Photorhabdus temperata is an entomopathogenic bacterium that is associated with nematodes of the Heterorhabditidae family in a symbiotic relationship. This study investigated the effects of P. temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis. Histopathology of the infection was also investigated using scanning electron microscopy. Groups of 20 larvae were infected by injection of approximately 50 bacterial cells directly into the hemocoel. After different periods of infection, larvae were dissected and different tissues were used for bacterial cell quantification. P. temperata was highly virulent with an LD₅₀ of 16.2 bacterial cells at 48 h post-infection. Infected larvae started dying as soon as 30 h post-infection with a LT₅₀ value of 33.8 h (confidence limits 32.2–35.6) and an LT₉₀ value of 44.8 h (CL 40.8–51.4). Following death of the larvae, bacteria from the midgut did not invade the hemocoel. In the midgut epithelium, P. temperata occupied the space underneath the basal lamina. The cultivable intestinal bacterial populations decreased as soon as 1 h post-infection and at 48 h post-infection, 90% of the gut microbiota had died. The role of P. temperata in control of the midgut microbiota was discussed.     

Site-specific antiphagocytic function of the Photorhabdus luminescens type III secretion system during insect colonization

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

In vivo impairment of neutrophil recruitment during lentivirus infection

Evidence indicates that the lentivirus, HIV, infection affects neutrophil response to bacteria and bacterial products in vitro. We used a novel model of rapid onset immunosuppression following infection with a similar lentivirus, feline immunodeficiency virus (FIV), in cats to examine neutrophil function within the microvasculature in vivo and to determine the steps that are impaired in the neutrophil recruitment cascade. In uninfected cats and cats infected neonatally with FIV, the mesentery was exteriorized, but remained autoperfused during intravital microscopy for 4 h. When the tissue was superfused with 10 micro g/ml of LPS for 4 h, intravital microscopy displayed a profound increase in neutrophil rolling at both 8 and 12 wk of age in uninfected cats. At 12 wk of age, FIV-infected animals showed a profound decrease in the number of rolling neutrophils. In vitro studies revealed that neutrophils from infected and uninfected animals rolled equally well on surrogate selectin substrata. In addition, in vivo neutrophil adhesion and emigration out of the vasculature were severely reduced, and in vitro neutrophil chemotaxis from FIV-infected animals was significantly impaired in response to fMLP or IL-8. However, FIV infection of neutrophils could not be detected. In summary, in vivo lentivirus infection with immunosuppression leads to a severe impairment in neutrophil rolling, adhesion, and emigration in response to bacterial stimulants potentially involving both endothelial and neutrophil dysfunction. These in vivo studies also indicate that neutrophil dysfunction should be taken into account when treating infections and tissue injury.     

Enteric bacteria of field-collected Colorado potato beetle larvae inhibit growth of the entomopathogens Photorhabdus temperata and Beauveria bassiana

The nematode Heterorhabditis marelatus fails to reproduce in the Colorado potato beetle, Leptinotarsa decemlineata, possibly due to interference from the enteric bacteria of the beetle. Specifically, the enteric bacteria inhibit the growth of Photorhabdus temperata, the enteric symbiont of the nematode, in vitro. However, previous work was based on a laboratory culture of L. decemlineata, and we wished to determine if similar bacteria were present in the field. Therefore, we cultured the enteric bacteria of fourth-instar larvae collected from the field at two locations in Maryland and Virginia. Representatives of the genera Pantoea, Enterobacter, Pseudomonas, Acinetobacter, Serratia, Stenotrophomonas, Curtobacterium, Bacillus, Lactococcus and Enterococcus were identified by sequencing of their 16S rDNA. Isolates belonging to the genera Pantoea, Enterobacter, Pseudomonas, Serratia and Bacillus inhibited the growth of P. temperata. A number of these isolates also inhibited the entomopathogenic fungus Beauveria bassiana in vitro.     

Cloning and sequence of the gene encoding the major structural component of mannose-resistant fimbriae of Serratia marcescens.

Serratia marcescens US46, a human urinary tract isolate, exhibits mannose-resistant hemagglutination and agglutinates yeast cells, thereby indicating that it has two types of adhesins. We constructed a cosmid library for the DNA of this organism and isolated DNA clones carrying genes for mannose-sensitive (MS) and mannose-resistant (MR) fimbriae. On introduction of the cloned genes into Escherichia coli K-12, MS and MR fimbriae were formed. These fimbriae were functionally and morphologically indistinguishable from those of S. marcescens. Subcloning of these gene clusters revealed that the genes encoding MS fimbriae reside on a 9-kilobase (kb) DNA fragment, while those encoding MR fimbriae are present on a 12-kb fragment. Transposon insertion and maxicell analyses revealed that formation of MR fimbriae is controlled by several genes which reside on the 9-kb fragment. The nucleotide sequence of smfA, the gene encoding the major structural component of MR fimbriae, revealed that this gene encodes a 174-amino-acid polypeptide with a typical procaryotic signal peptide. The primary structure of the smfA product showed significant homology with the primary structure of the E. coli fimbrial subunit.     

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

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

Gibberellins synthesized by the entomopathogenic bacterium,Photorhabdus temperata M1021 as one of the factors of rice plant growth promotion

In the present study, different types of gibberellins (GAs) in the culture filtrate (CF) of Photorhabdus temperata M1021 were quantified. The analysis of CF helped in profiling various bioactive GAs: GA₁, GA₃, GA₄, and GA₇. Several physiologically inactive GAs: GA₉, GA₁₂, and GA₂₀ were detected as well. Siderophore production was also investigated by growing P. temperata M1021 on chrome azurol-S blue agar plates. Furthermore, the strain was inoculated into ‘Waito-C’ (Oryza sativa L.) rice plants, which significantly (P < 0.05) increased plant growth attributes such as plant length, chlorophyll content, and fresh and dry biomass compared with those in controls. In a separate experiment, canola (Brassica napus L.) seeds treated with CF of M1021 were significantly (P < 0.05) accelerated germination rate as well as biomass production. Findings of the present study suggest that the strain M1021 contributes an important role in the plant growth by synthesizing a wide array of bioactive metabolites.     

In vivo inactivation of the insect neuropeptide proctolin in Periplaneta americana

Supranormal levels (0.13–31 μg) of proctolin injected into the cockroach, Periplaneta americana, disappear within minutes from the haemolymph. Treated insects exhibit a short-term catatonic state followed by full recovery. Using [¹⁴C-Tyr²]proctolin, it was shown that inactivation results from the removal of arginine yielding H-Tyr-Leu-Pro-Thr-OH. Tyrosine was produced in a subsequent step. The aminopeptidase responsible for this in vivo hydrolysis of proctolin differed from leucine aminopeptidase of porcine kidney in the relative rates of hydrolysis of proctolin and H-Tyr-Leu-Pro-Thr-OH. The latter was not detected as an intermediate in proctolin degradation by leucine aminopeptidase. This study indicates that proctolin cannot be transported via the haemolymph unless it is protected in some manner from enzymatic degradation.