Expression and activity of a probable toxin from Photorhabdus luminescens

As an insect pathogen, Photorhabdus luminescens possesses an arsenal of toxins. Here we cloned and expressed a probable toxin from P. luminescens subsp. akhurstii YNd185, designated as Photorhabdus insecticidal toxin (Pit). The pit gene shares 94% nucleotide and 98% predicted amino acid sequence identity with plu1537, a predicted ORF from P. luminescens subsp. laumondii TT01 and 30% predicted amino acid sequence similarity to a fragment of a 13.6 kDa insecticidal crystal protein gene of Bacillus thuringiensis (Bt). The pit was expressed as a GST-Pit fusion protein in E. coli, most of which was insoluble and sequestered into inclusion bodies. The inclusion bodies were harvested and dissolved. The resultant protein was purified and the Pit was cleaved from the fusion protein by thrombin and purified from GST then used for bioassay. Pit killed Galleria mellonella (LD₅₀, 30 ng/larva) and Spodoptera litura (LD₅₀, 191 ng/larva) via hemocoel injection. Relative to a control that lacked toxin, Pit did not significantly increase mortality of S. litura and Helicoverpa armigera when introduced orally, but the treatment did inhibit growth of the insects. The present study demonstrated that Pit possessed insecticidal activity.     

Generation of thermostable monomeric luciferases from Photorhabdus luminescens

Bacterial luciferases and the genes encoding these light-emitting enzymes have an increasing number of applications in biological sciences. Temperature lability and the heterodimeric nature of these luciferases have been the major obstacles for their widespread use, for instance, as genetic reporters. Escherichia coli expressing wild-type Photorhabdus luminescens luciferase was found to produce eight times more light than the corresponding Vibrio harveyi luciferase clone in vivo at 37 degrees C. Three monomeric luciferases were created by translationally fusing the two genes encoding luxA and luxB proteins of P. luminescens. These clones were equally active in producing light in vivo when cultivated at 37 degrees C compared to cultivation at 30 degrees C. The fusion containing the longest linker showed the highest activity. In vitro, the monomeric luciferases were less active having at best 20% of activity of the wild-type enzyme due to the partial formation of insoluble aggregates. The results suggest that P. luminescens luciferase and monomeric derivatives thereof should be more suitable than the corresponding V. harveyi enzyme to be used as reporters in cell types which need cultivation at elevated temperatures.     

Identification of two pigments and a hydroxystilbene antibiotic from Photorhabdus luminescens.

Two yellow pigments were isolated for the first time from the entomopathogenic bacterium Photorhabdus luminescens in liquid culture and were identified as the anthraquinone derivatives 3,8-dimethoxy-1-hydroxy-9,10-anthraquinone (minor) and 1,3-dimethoxy-8-hydroxy-9,10-anthraquinone (major). A known antibiotic, 3,5-dihydroxy-4-isopropylstilbene, was also detected and for the first time showed strong fungicidal activity against several fungi of medical and agricultural importance.     

Two new subspecies of Photorhabdus luminescens, isolated from Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae): Photorhabdus luminescens subsp. kayaii subsp. nov. and Photorhabdus luminescens subsp. thracensis subsp. nov

Bacterial isolates from nematodes from Turkish soil samples were initially characterized by molecular methods and seven members of the genus Photorhabdus identified to the species level, using riboprint analyses and metabolic properties. Strain 07-5 (DSM 15195) was highly related to the type strain of Photorhabdus luminescens subsp. laumondii DSM 15139T, and was regarded a strain of this subspecies. Strains 1121T (DSM 15194T), 68-3 (DSM 15198) and 47-10 (DSM 15197) formed one, strain 39-8T (DSM 15199T), 39-7 (DSM 15196) and 01-12 (DSM 15193) formed a second cluster that branched intermediate the three subspecies of Photorhabdus luminescens. Based upon moderate 16S rRNA gene sequence similarities and differences in metabolic properties among themselves and with type strains of the three subspecies we consider the two clusters to represent two new subspecies of Photorhabdus luminescens for which the names Photorhabdus luminescens subsp. kayaii, type strain 1121T (DSM 15194T, NCIMB 13951T), and Photorhabdus luminescens subsp. thracensis subsp. nov., type strain 39-8T (DSM 15199T, NCIMB 13952T) are proposed.     

Triggering the production of the cryptic blue pigment indigoidine from Photorhabdus luminescens

The production of the blue pigment indigoidine has been achieved in the entomopathogenic bacterium Photorhabdus luminescens by a promoter exchange and in Escherichia coli following heterologous expression of the biosynthesis gene indC. Moreover, genes involved in the regulation of this previously “silent” biosynthesis gene cluster have been identified in P. luminescens.     

A Novel Insecticidal Toxin from Photorhabdus luminescens, Toxin Complex a (Tca), and Its Histopathological Effects on the Midgut of Manduca sexta

Photorhabdus luminescens is a bacterium which is mutualistic with entomophagous nematodes and which secretes high-molecular-weight toxin complexes following its release into the insect hemocoel upon nematode invasion. Thus, unlike other protein toxins from Bacillus thuringiensis (δ-endotoxins and Vip’s), P. luminescens toxin (Pht) normally acts from within the insect hemocoel. Unexpectedly, therefore, the toxin complex has both oral and injectable activities against a wide range of insects. We have recently fractionated the protein toxin and shown it to consist of several native complexes, the most abundant of which we have termed Toxin complex a (Tca). This complex is highly active against the lepidopteran Manduca sexta. In view of the difference in the normal mode of delivery of P. luminescens toxin and the apparent communality in the histopathological effects of other gut-active toxins from B. thuringiensis, as well as cholesterol oxidase, we were interested in investigating the effects of purified Tca protein on larvae of M. sexta. Here we report that the histopathology of the M. sexta midgut is similar to that for other novel midgut-active toxins. Following oral ingestion of Tca by M. sexta, we observed an acceleration in the blebbing of the midgut epithelium into the gut lumen and eventual lysis of the epithelium. The midgut shows a similar histopathology following injection of Tca into the insect hemocoel. These results not only show that Tca is a highly active oral insecticide but also confirm the similar histopathologies of a range of very different gut-active toxins, despite presumed differences in modes of action and/or delivery. The implications for the mode of action of Tca are discussed.     

Insecticidal Toxic Proteins Produced by Photorhabdus luminescens akhurstii,a Symbiont of Heterorhabditis indica

We describe the isolation and characterization of an insect pathogenic bacterium from the entomopathogenic nematode Heterorhabditis indica (Karnataka strain), an isolate from the southern regions of India. The strain has been identified and characterized by phenotypic, biochemical tests and PCR-RFLP analysis of the 16S rRNA gene as Photorhabdus luminescens subsp. akhurstii. The insecticidal toxin complex produced by this bacterium has been purified through a series of steps including ultrafiltration, anion exchange chromatography, and gel filtration chromatography. The toxin consists of two protein complexes of approximately 1,000 kD and was active against the larvae of Spodoptera litura and Galleria mellonella.     

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.     

Identification and Characterization of a Novel Gene Involved in the trans-Specific Nematicidal Activity of Photorhabdus luminescens LN2

Photorhabdus luminescens subsp. akhurstii LN2 from Heterorhabditis indica LN2 showed nematicidal activity against axenic Heterorhabditis bacteriophora H06 infective juveniles (IJs). Transposon mutagenesis identified an LN2 mutant that supports the growth of H06 nematodes. Tn5 disrupted the namA gene, encoding a novel 364-residue protein and involving the nematicidal activity. The green fluorescent protein-labeled namA mutant was unable to colonize the intestines of H06 IJs.Entomopathogenic Heterorhabditis and Steinernema nematodes are safe and effective bioinsecticides for the biological control of many economically important pests (9). The infective juveniles (IJs) of these nematodes harbor Photorhabdus or Xenorhabdus bacteria as symbionts in their intestines. The IJ nematodes properly maintain and carry the bacteria needed for killing insects and providing a suitable environment for the reproduction of new vectors (5, 8). Different bacterial isolates differ in their ability to support in vitro monoxenic cultures of nonhost nematodes (2, 7, 13) and to retain the bacterial cells in the IJ intestines (2, 8, 11).Strains of Photorhabdus and Xenorhabdus spp. not only show insecticidal activities toward different insects (3, 4, 21) but also exhibit nematicidal activities against nematodes (14, 16, 17). The trans-specific nematicidal activity of Photorhabdus luminescens subsp. akhurstii LN2, a normal symbiont of Heterorhabditis indica LN2 against Heterorhabditis bacteriophora H06, was previously observed (12). The LN2 bacteria may secrete unidentified toxic factors that are lethal to the H06 nematodes. However, the genes of these bacteria involved in the trans-specific nematicidal activities have not been reported.This paper describes the identification, through Tn5 mutagenesis and characterization, of a novel P. luminescens LN2 gene involved in nematicidal activity against the H06 IJs. The colonization of the green fluorescent protein (GFP)-labeled mutant cells in H06 IJ intestines was examined.     

光杆菌(NLK-1) Txp40毒蛋白基因克隆表达及预测

【背景】光杆菌存在于嗜菌异小杆线虫肠道内,并与其互惠共生,其能够产生多种高效、广谱的杀虫蛋白及毒素,是近年来继苏云金芽胞杆菌(Bt)之后挖掘新型杀虫蛋白及杀虫基因的热点研究对象。【目的】克隆Photorhabdus luminescens(NLK-1)Txp40毒蛋白基因,分析其与已知其他同属共生菌相似毒蛋白在基因序列、蛋白组成、理化性质及构象的区别,构建原核表达载体并转化大肠杆菌进行诱导表达,初步测定其杀虫活性。【方法】采用侵染的大蜡螟幼虫血腔直接分离初生型共生细菌,根据已报道的序列经比对分析设计引物,扩增目的基因,连接克隆质粒p MD19-T后测序,利用Expasy在线Prot Param tool预测其基本理化特性参数,NPS@-Network Protein Sequence Analysis在线工具进行二级结构预测。通过克隆、酶切、连接目的基因在p ET28a原核表达载体上,转化大肠杆菌BL21中,利用蓝白斑筛选阳性克隆,测序验证后进行IPTG诱导表达;菌体超声破碎离心,以毒蛋白含量较高的上清溶液对大蜡螟幼虫进行饲喂和血腔注射毒性测定。【结果】Photorhabdus luminescens(NLK-1)Txp40毒蛋白基因全长为1 008 bp,与已知相关基因的序列相似性为94%,与已知40 k D相关蛋白的氨基酸相似性达到99%,分子量37.9 k D,p I 8.37,二级结构预测表明其主要由α螺旋35.71%,无规卷曲54.46%,延伸链9.52%组成,跨膜区域与已知蛋白基本相似,克隆构建了原核表达载体p ET28a-(NLK-1)Txp40,SDS-PAGE分析其在38 k D处有特异条带,蛋白分子量与预测值基本一致,且表达相对单一,表达量较高。Photorhabdus luminescens(NLK-1)Txp40蛋白对大蜡螟幼虫具有较高的血腔毒性,大蜡螟幼虫注射5μL蛋白粗提液剂量下48 h内致死率达100%,未发现胃毒活性。【结论】获得Photorhabdus luminescens(NLK-1)Txp40毒蛋白基因,比对、分析了与已知基因在序列组成、蛋白基本理化性质和二级结构的异同,构建了原核表达载体并成功诱导表达,验证了Photorhabdus luminescens(NLK-1)Txp40毒蛋白具有较高的大蜡螟幼虫血腔毒性,为进一步发掘Photorhabdus luminescens(NLK-1)中的杀虫功能基因和蛋白奠定基础。     

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.     

Influence of Osmolarity on Phase Shift in Photorhabdus luminescens

The influence of osmolarity and other environmental factors like low oxygen levels, light, extreme pH values, and temperatures on phase variation of Photorhabdus luminescens, the symbiotic bacterium of entomopathogenic nematodes of the genus Heterorhabditis, was investigated. Only subculturing in low-osmolarity medium triggered a phase shift to secondary phase reliably.     

Photorhabdus luminescens LN2转座突变菌株的研究

发光杆菌属Photorhabdus细菌与异小杆属Heterorhabditis昆虫病原线虫的共生关系是这类生物杀虫剂产业化生产和田间应用的基础。本文采用Tn5转座方法构建了共生细菌P. luminescens LN2突变体库;从中筛选出一个对其共生线虫H. indica LN2的生长繁殖有显著促进作用的突变菌株（LN2-M2716）;测定了该突变菌株的菌落特征、对大蜡螟Galleria mellonella及非特异共生线虫H. bacteriophora H06的毒性、对线虫产量的影响。结果显示,LN2-M2716菌株在菌落形态、色素分泌、过氧化氢酶反应、荧光、食物信息作用以及对大蜡螟毒力等方面与野生型菌株差异不明显;但对非特异共生线虫H. bacteriophora H06的毒性及对特异共生线虫H. indica LN2生长繁殖的促进作用方面均明显高于野生型菌株。论文结果为构建支持线虫高产的菌株提供了关键技术。     

发光杆菌碳端截短的Mcf毒素杀虫活性研究

发光杆菌能产生很多毒素杀死昆虫宿主.这些毒素中有一种叫Mcf致软因子.可以在大肠杆菌中表达并毒杀毛虫.通过同源性比对分析.在Mcf氨基酸序列中N端有一个BH3的区域,拥有BH3结构域的蛋白具有细胞凋亡的功能,推测保留N端BH3结构域的碳端截短Mcf毒素具有杀虫毒力.从发光杆菌属(Photorhabdus luminescens subsp.laumondii)中克隆了杀虫毒素基因mcf的5'端3 960 bp的核酸序列(包含BH3结构域).将该基因连接到E.coli表达载体pET28a上,并转入BL21(DE3).经IPTG诱导后,在SDS-PAGE上发现145 kD的目的蛋白条带.利用亲和层析纯化得到纯毒素,分别对甜菜夜蛾一龄幼虫喂食生测和对甜菜夜蛾四龄幼虫(Spodoptera exigua)注射生测,显示该毒素具有喂食毒力和血腔毒力,证明含有BH3区域的碳端截短Mcf毒素有杀虫的生物活性.     

Growth of Photorhabdus luminescens in batch and glucose fed-batch culture

Photorhabdus luminescens, a bacterial symbiont of entomopathogenic biocontrol nematodes, was grown in batch and glucose fed-batch culture. The cell density, bioluminescence, production of antibiotic substances, number of cells with inclusion bodies, glucose concentration and oxygen uptake rate were recorded. The addition of 12.4 g l⁻¹ glucose prolonged the growth, and the yield almost doubled, from 6.85 g l⁻¹ to 12.45 g l⁻¹ dry mass. The production of antibiotic substances increased by 140%. Bioluminescence was higher in the batch culture. A shift of P. luminescens to phase II variants was not detected. Received: 21 January 2000 / Received revision: 3 April 2000 / Accepted: 7 April 2000     

Antimicrobial properties and morphological characteristics of two Photorhabdus luminescens strains.

The biological properties of two Photorhabdus luminescens isolates (MU1 and MU2) of environmental source and the activity of antimicrobial agar diffusible agents (AADA) produced by the same are reported. With regard to cultural features, two variant forms for P. luminescens MU1 and three for P. luminescens MU2 (including an intermediate phase I-like form) have been found. These three forms differ in biological and biochemical properties: beta-lactamase, urease, bioluminescence and antimicrobial agar diffusible substance production associated with the phase I form, were less evident in the intermediate phase I-like MU2 and were absent in phase II form. Antimicrobial activity was present in both strains, with the production of a large amount of a diffusible compound with a wide spectrum of action against bacteria of other genera; a reduced activity against correlated species was also observed. Examination by electron microscopy of MU1 and MU2 purified broth cultures revealed the presence of particles belonging to the class of the phage tail-like bacteriocins, described in recent studies as responsible for antibacterial activity against correlated bacteria, a result never confirmed "in vitro". A plasmid of 21 Mdal was observed in all the form variants of P. luminescens MU2, suggesting that plasmids are not involved in the transition from primary to secondary phase; no plasmid was detected in P. luminescens MU1.     

Anaerobic metabolism of primary and secondary forms of Photorhabdus luminescens

Abstract A Vibrio cholerae O1 strain (1150) of the EITor biotype and Ogawa serotype with haemagglutination (HA) activity was subjected to TnphoA mutagenesis. Out of several mutants isolated, one HA⁻ and another HA⁺ mutant were further characterised. The HA⁻ mutant showed about 50% reduction in its intestinal adherence capacity in vitro and about 9-fold decrease of its colonisation ability in vivo, as compared to the wild-type strain. Subsequent studies showed that the HA activity of strain 1150 was mediated by a mannose-sensitive haemagglutinin (MSHA). Thus, the phenotypic expression of MSHA appears to be partly responsible for the intestinal adherence and colonisation properties of strain 1150.     

Putative toxins from the entomopathogenic bacterium Photorhabdus luminescens kill Armadillidium vulgare (Terrestrial isopod)

Terrestrial isopods can be killed by some entomopathogenic bacteria among Xenorhabdus and Photorhabdus species even with no or very limited multiplication. This suggests that toxemia and not septicemia is the major cause of entomopathogenic bacteria pathogenicity against these crustaceans. In this paper, we revealed that the injection of stationary phase culture supernatant of P. luminescens TT01, in which toxins can be accumulated, led alone to a rapid decrease in the number of host immune cells and killed most of the Armadillidium vulgare individuals within 48 h. The pathogenicity was strongly attenuated when supernatant was heated and totally suppressed after 100-kDa filtration suggesting that the toxin responsible for killing A. vulgare would be a protein above this size. Additionally, we tested the culture supernatant of two TT01 mutants that have been previously shown as being altered in their pathogenicity against lepidopteran insects one of them being known as exhibiting lower expression of some toxins. However, the supernatants of the mutants was as pathogenic for A. vulgare as the wild type strains suggesting that the toxins involved in killing A. vulgare may be different than previously described ones.