The Effects of Trehalose on the Bioluminescence and Pigmentation of the Phase I Variant of Photorhabdus luminescens

Photorhabdus luminescens is a Gram-negative, bioluminescent, pigment producing enteric bacterium, which is pathogenic to insects and has the capability to undergo phase variation. The phase I variant of P. luminescens exists as a mutualistic symbiont where it plays a critical role in the life-cycle of the soil-dwelling nematode, Heterorhabditis bacteriophora. Both the bacterium and the nematode receive their nutritional requirements from the bioconversion of the insect host which is rich in many macromolecules such as the disaccharide, trehalose. Trehalose is a non-reducing disaccharide of glucose that is formed by an a-1,1-glycosidic bond and is associated with the physiology of many bacteria, insects and nematodes. Trehalose has been shown to be the most abundant storage sugar found within insect hemolymph （1%-2%）. The physicochemical properties of trehalose allow this carbohydrate to act as a stress protectant where it has been implicated with thermal stress, dehydration, and osmotic protection of many microorganisms. Due to these properties, trehalose may allow culture stability of the phase I variant in vitro and in vivo. Traits of the phase I variant that were studied in this work include bioluminescence and the production of the red anthroquinone-derived pigment. The carbohydrates that were utilized in this study were glucose and trehalose; where shake flask cultures of the phase I variant were cultured at room temperature for up to six days in carbohydrate supplemented basal media with increasing carbohydrate concentrations of 0. 1%, 0.5% and 1.0% （v/v）. Relative luminosity, pigmentation and pH were graphed as a function of time, carbohydrate used, and carbohydrate concentration. Data obtained from this study suggests that the supplementation of 1.0% trehalose, when culturing the phase I variant ofP. luminescens, can maintain bioluminosity and pigmentation over extended periods of time （five days） as compared to basal media and basal media supplemented with 1.0% glucose.

Larvicidal activity of Xenorhabdus and Photorhabdus bacteria against Aedes aegypti and Aedes albopictus

Objective: To evaluate the efficacy of symbiotic bacteria, Xenorhabdus indica, Xenorhabdus stockiae, Photorhabdus luminescens subsp. akhurstii and Photorhabdus luminescens subsp. hainanensis as a larvicide against Aedes aegypti and Aedes albopictus. Methods: Larvae(L3-L4) of Aedes aegypti and Aedes albopictus were given 2 m L of a suspension 107-108 CFU/m L of each symbiotic bacterium. Distilled water and Escherichia coli ATCC襅25922 were used as the control. The mortality rate of the larval mosquitoes was observed at 24, 48, 72 and 96 h. The experiment was performed in triplicates. Results: The larvae of both Aedes species started to die at 24 h exposure. Aedes aegypti showed the highest mortality rate(87%-99%), 96 h after exposure to Xenorhabdus stockiae(b NBP22.2_TH). The mortality rate of Aedes albopictus was between 82% and 96% at 96 h after exposure to Xenorhabdus indica(b KK26.2_TH). Low effectiveness of distilled water and Escherichia coli ATCC襅25922 were observed in both Aedes larvae, with a mortality rate of 2% to 12%. Conclusions: The study confirms the oral toxicity of Xenorhabdus and Photorhabdus bacteria against Aedes spp. Xenorhabdus stockiae and Xenorhabdus indica may be an alternative agent for control Aedes spp. This is basic information for further study on the mechanism of action on Aedes larvae or application to control mosquito larvae in the community.

Characterization of Photorhabdus Virulence Cassette as a causative agent in the emerging pathogen Photorhabdus asymbiotica

The extracellular contractile injection systems(e CISs)are encoded in the genomes of a large number of bacteria and archaea.We have previously characterized the overall structure of Photorhabdus Virulence Cassette(PVC),a typical member of the e CIS family.PVC resembles the contractile tail of bacteriophages and exerts its action by the contraction of outer sheath and injection of inner tube plus central spike.Nevertheless,the biological function of PVC effectors and the mechanism of effector translocation are still lacking.By combining cryo-electron microscopy and functional experiments,here we show that the PVC effectors Pdp1(a new family of widespread d NTP pyrophosphatase effector in e CIS)and Pnf(a deamidase effector)are loaded inside the inner tube lumen in a"Peas in the Pod"mode.Moreover,we observe that Pdp1 and Pnf can be directly injected into J774 A.1 murine macrophage and kill the target cells by disrupting the d NTP pools and actin cytoskeleton formation,respectively.Our results provide direct evidence of how PVC cargoes are loaded and delivered directly into mammalian macrophages.

昆虫病原线虫共生细菌胞内晶体蛋白的研究进展

初生型发光杆菌属(Photorhabdus)及嗜线虫致病杆菌属(Xenorhabdus)细菌分别与异小杆线虫属(Heterorhabditis)和斯氏线虫属(Steinernema)昆虫病原线虫互惠共生。这类昆虫病原细菌在稳定生长期分别产生两种形态各异的胞内晶体蛋白。本文回顾了这类蛋白的研究历史和最新的研究进展,特别是胞内晶体蛋白的理化性质和生物学功能,同时讨论这种晶体蛋白的研究方法与技术。

A comparative analysis of entomoparasitic nematodes <i>Heterorhabditis bacteriophora</i>and <i>Steinernema carpocapsae</i>

Heterorhabditis bacteriophora and Steinernema carpocapsae are microscopic entomoparasitic nematodes (EPNs) that are attractive, organic alternatives for controlling a wide range of crop insect pests. EPNs evolved with parasitic adaptations that enable them to “feast” upon insect hosts. The infective juvenile, a non-feeding, developmentally arrested nematode stage, is destined to seek out insect hosts and initiates parasitism. After an insect host is located, EPNs enter the insect body through natural openings or by cuticle penetration. Upon access to the insect hemolymph, bacterial symbionts (Photorhabdus luminescens for H. bacteriophora and Xenorhabdus nematophila for S. carpocapsae) are regurgitated from the nematode gut and rapidly proliferate. During population growth, bacterial symbionts secrete numerous toxins and degradative enzymes that exterminate and bioconvert the host insect. During development and reproduction, EPNs obtain their nutrition by feeding upon both the bioconverted host and proliferated symbiont. Throughout the EPN life cycle, similar characteristics are seen. In general, EPNs are analogous to each other by the fact that their life cycle consists of five stages of development. Furthermore, reproduction is much more complex and varies between genera and species. In other words, infective juveniles of S. carpocapsae are destined to become males and females, whereas H. bacteriophora develop into hermaphrodites that produce subsequent generations of males and females. Other differences include insect host range, population growth rates, specificity of bacterial phase variants, etc. This review attempts to compare EPNs, their bacterial counterparts and symbiotic relationships for further enhancement of mass producing EPNs in liquid media.