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.     

Antibacterial Screening of Secreted Compounds Produced by the Phase I Variant of Photorhabdus luminescens

In this study, antibacterial activity of metabolites secreted by the phase I variant of Photorhabdus luminescens was investigated. Bioactivity of these metabolites was screened against 28 different bacterial species and strains. Bacterial sensitivity was determined by a modified-version of the Kirby–Bauer disk diffusion susceptibility method, whereas the phase I variant’s culture permeate was utilized as the “antibacterial” agent. This investigation demonstrates that 11 of the 28 bacterial species tested were sensitive to at least one of the secreted compounds or a combination thereof.     

Mass Production of the Beneficial Nematode Heterorhabditis bacteriophora and Its Bacterial Symbiont Photorhabdus luminescens

Entomoparasitic nematodes (EPNs) are being commercialized as a biocontrol measure for crop insect pests, as they provide advantages over common chemical insecticides. Mass production of these nematodes in liquid media has become a major challenge for commercialization. Producers are not willing to share the trade secrets of mass production and by doing so, have made culturing EPNs extremely difficult to advance existing technologies. Theoretically, mass production in liquid media is an ideal culturing method as it increases cost efficiency and nematode quantity. This paper will review current culturing methodologies and suggest basic culturing parameters for mass production. This review is focused on Heterorhabditis bacteriophora; however, this information can be useful for other nematode species.     

Characterization of Photorhabdus luminescens Growth for the Rearing of the Beneficial Nematode Heterorhabditis bacteriophora

Culturing the bioluminescent bacterium Photorhabdus luminescens in nutrient broth (NB) is used to recover phase I cells. These phase I cells were highly luminescent for up to 7 h in this media and the luminosity could also be seen with the naked eye after a 15 min eye adjustment period in a dark room. Red pigmentation is a known trait of phase I cells and was visually distinct within the culture media. The color shade of the red pigment varied on nutrient agar and in NB suggesting that the concentration of the pigment produced is dependent upon density of phase I cells within a specified area. The specific growth rate (μ) and doubling time (g) was determined during the logarithmic growth phase to be 0.36 h⁻¹ and 2.1 h, respectively in NB medium. The nematode-bacterium suspension was injected into larvae of Galleria mellonella to test for entomopathogencity. Within 24 h post-injection insect mortality was seen along with dark red pigmentation and extremely high luminosity indicating infection with P. luminescens.     

Sequencing and Comparative Analysis of the lux Operon of Photorhabdus luminescens Strain Zm1: ERIC Elements as Putative Recombination Hot Spots

The EcoRI chromosomal fragment (6782 bp) containing the lux operon of Photorhabdus luminescens was cloned in pUC18 and completely sequenced. Enteric repetitive intergenic consensus (ERIC), an imperfect palindrome (125–127 bp) characteristic of Enterobacteriaceae genomes, was found in three sites. Strain Zm1 proved to differ in the ERIC number and location from strains Hb, Hm, and Hw. Nucleotide substitution analysis showed that luxC and luxB, which are more than 1 kb away from ERIC, are similar to the corresponding Hb genes, whereas luxD, luxA, and luxE, which are close to ERIC, are intermediate between their Hb and Hw counterparts. The Hb/Hw nucleotide substitution ratio was 1:1 in regions adjacent to ERIC. Hence ERIC can be thought to be recombination hot spots in the bacterial genome.     

荧光发光杆菌TT01品系pirA2B2 基因的克隆表达与杀虫活性

【目的】Photorhabdus luminescens TT01基因组中的一对ORF plu4437-plu4436(简称pirA2B2)的预测氨基酸序列与另一对已证明编码产物有口服杀虫活性的ORF plu4093-plu4092(简称pirA1B1)有50%和45%的一致性,本文旨在研究pirA2B2基因座的表达产物是否也有杀虫活性。【方法】PCR扩增并克隆了pirA2,pirB2和pirA2B2基因,构建了重组表达载体pQE-pirA2,pQE-pirB2和pQE-pirA2B2并分别转入M15菌株表达,经SDS-PAGE和Western blot检测证明,3个重组菌株经IPTG诱导后,分别成功表达了可溶的PirA2,PirB2和PirA2B2蛋白。用亲和层析结合脱盐技术对3个重组菌株表达的外源蛋白分别进行纯化,并通过生物测定确定纯化蛋白的杀虫活性。【结果】生物测定结果显示联合表达的PirA2B2对大蜡螟和斜纹夜蛾五龄幼虫均有明显的血腔杀虫活性,LD50分别为每虫4.0和2.8μg,单独表达的PirA2或PirB2对上述2种害虫没有血腔杀虫活性,但两者的混合物具有与两者联合表达相似的杀虫活性;PirA2B2对大蜡螟和斜纹夜蛾初孵幼虫均无口服杀虫活性。【结论】pirA2B2是P.luminescens TT01菌株基因组中的另一个二元杀虫毒素基因。【意义】pirA2B2的成功克隆表达和杀虫功能的确定为进一步研究其与pirA1B1的关系以及该基因的表达调控等打下了基础。     

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。     

Expression and insecticidal activity of Yersinia pseudotuberculosis and Photorhabdus luminescens toxin complex proteins

Photorhabdus luminescens toxin complex (Tc) has been characterized as a potent three-component insecticidal protein complex. Homologues of genes encoding P. luminescens Tc components have been identified in several other enterobacteria and in Gram-positive bacteria, showing these genes are widespread in bacteria. In particular, tc gene homologues have been identified in Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis and may have a role in Y. pestis evolution. Y. enterocolitica tc genes have been shown to be active against Manduca sexta larvae. Here, we demonstrate that expression optimization is essential to obtain bioactive P. luminescens Tc proteins and demonstrate that TcaAB and TcdB + TccC are stand-alone toxins against a M. sexta insect model. Moreover, we report that Y. pseudotuberculosis IP32953 Tc proteins are also toxic to M. sexta larvae but do not cross-potentiate as P. luminescens Tc components.     

Stability of entomopathogenic bacteria, <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis> and <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis>, during in vitro culture

The entomopathogenic nematode–bacteria complexes Heterorhabditis bacteriophora/Photorhabdus luminescens and Steinernema carpocapsae/Xenorhabdus nematophila are mass produced for use as biological insecticides. Stability of the bacterial partner in culture is essential for maintaining traits important for both biological control and production. Two geographically distinct strains of each bacterial species were isolated from their nematode partners and serially subcultured on in vitro media to assess trait stability. Subculturing resulted in a shift to secondary cell production in one P. luminescens strain and both X. nematophila strains within ten in vitro culture cycles. However, when cell phenotypic variation was controlled in X. nematophila strains by regular selection for primary variants, no trait change was detected in the primary variant after prolonged subculture. When P. luminescens cell phenotypic variation was controlled by selection for primary variants, changes in the primary variant of both strains were noted including reductions in cell and inclusion body size and inclusion body prevalence. Bacterial ability to cause lethal infections following injection into the hemocoel of Tenebrio molitor larvae declined by more than half in primary variants of one P. luminescens strain. Conversely, yield was enhanced, with the subcultured P. luminescens strains showing 53.5 and 75.8% increases in primary cell density. Field adapted traits of primary variant P. luminescens strains tend to deteriorate during in vitro culture as tradeoffs for gains in yield. In vitro producers of the P. luminescens/H. bacteriophora complex must weigh the need for superior bacterial yield against the need to preserve traits important for biological control.     

Mutualistic association of Photorhabdus asymbiotica with Japanese heterorhabditid entomopathogenic nematodes

Gram-negative bacteria, Photorhabdus luminescens and P. temperata, form a mutualistic association with entomopathogenic heterorhabditid nematodes while P. asymbiotica is known as an opportunistic human pathogen that causes disseminated bacteremic spread on two continents, the United States and Australia. In the course of our phylogenetic study of Photorhabdus bacteria associated with Japanese Heterorhabditis nematodes, we found two Photorhabdus isolates (Photorhabdus sp. Cbkj163 and OnIr40) whose partial 16S rRNA gene sequence showed high similarities to clinical isolates of this pathogen from Heterorhabditis indica. The phylogenetic study, based upon the gyrase subunit B gene sequences of the two isolates, revealed clustering with these clinical isolates of P. asymbiotica from both the United States and Australia but not with other Photorhabdus bacteria associated with nematodes. The two bacterial isolates were also found to share microbiological and biochemical characteristics with clinical and entomopathogenic Photorhabdus strains. Moreover, not only the two novel Photorhabdus isolates but also an Australian clinical isolate of P. asymbiotica formed mutualistic association with H. indica isolates. These data suggest that the bacteria isolated from H. indica CbKj163 and OnIr40 are a novel subspecies of P. asymbiotica, and that some clinical isolates of P. asymbiotica could have originated from bacteria associated with entomopathogenic nematodes.     

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.     

Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.     

Expression and characterization of inhA gene from Bacillus thuringiensis 8010

InhA, a zinc metalloprotease secreted by Bacillus thuringiensis, specifically hydrolyzes antibacterial peptides produced by insect hosts. In this study, the inhA gene was cloned from B. thuringiensis 8010 using a pair of degenerate primers and the deduced 796 amino acid sequence showed a high degree of similarity with other InhA proteins in the Bacillus cereus group. The deduced amino acid sequence contained the zinc-binding motif (HEXXH), which is characteristic of the zinc-metalloprotease family. Additionally, the inhA gene was expressed in Escherichia coli BL21 (DE3). The expressed InhA protein was shown to be toxic to the third larvae of Plutella xylostella, contrary to preliminary study concerning the effect of InhA on Bombyx mori. This study provided insights into the potential of InhA for the biological control of certain lepidopteran insects.     

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.     

Expression, purification and characterization of pectin methylesterase inhibitor from kiwi fruit in Escherichia coli

A significant problem in production of fruit juices for human consumption is auto-clarification, where enzyme catalyzes pectin demethylation resulting in loss of the ‘‘natural” cloudy appearance of juices. To overcome this problem, a plant inhibitor protein which blocks the action of pectin methylesterase has been used. In this paper, expression of recombinant kiwi pectin methylesterase inhibitor (PMEI) was carried out in Escherichia coli, and the target protein was expressed in the form of inclusion bodies. The expression level reached 46% of total cell protein. Then the fusion protein was purified by nickel ion metal affinity chromatography, and the purity was finally up to 98%. After refolding in GSH/GSSG redox system, recombinant PMEI not only could efficiently inhibit PMEs from eight different plants, but could remain effective inhibitor activity in the pH 3.0–10.0 and 20–40 °C. Thus, recombinant PMEI has potential application in the production of fruit juices product industry.     

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus

Background

Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling.

Results

Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster.

Conclusions

Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1763-2) contains supplementary material, which is available to authorized users.     

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.