昆虫病原斯氏线虫SY-5对重组Cip晶体蛋白的营养利用（英文稿）

【目的】初生型Photorhabdus luminescens细菌产生两种胞内晶体蛋白CipA和CipB,为其共生的昆虫病原异小杆线虫提供营养。探索非共生的斯氏线虫对Cip蛋白的营养利用情况。【方法】在已构建重组Cip蛋白大肠杆菌表达体系的基础上,建立重组菌细胞与无菌斯氏SY-5线虫共培养系统,检测线虫的生长发育情况。【结果】Cip蛋白对目标线虫生长有显著支持作用:发育为成虫的比例达到65%-82%,雌虫的怀卵率为80%-95%,平均怀卵量为30-50粒,并显著降低各虫态的死亡率。【结论】Cip蛋白不仅为共生的异小杆线虫提供营养,亦能为斯氏线虫所利用。     

携带cip基因的酿酒酵母对自由生活线虫Panagrellus redivivus生长繁殖的影响

Photorhabdus luminescens细菌与昆虫病原异小杆属Heterorhabditis线虫专性共生。初生型共生细菌产生两种胞内晶体蛋白CipA and CipB,为共生线虫提供营养。为探索Cip蛋白是否对自由生活的全齿复活线虫Panagrellus redivivus具有类似的营养功能,建立了Cip蛋白的重组酿酒酵母表达体系,并用于饲喂无菌的P. redivivus线虫J1幼虫。重组酿酒酵母表达的Cip蛋白能为线虫所利用,表现为营养支持作用,体现为线虫生长发育速度的加快以及繁殖能力的提高,说明Cip蛋白能为此种自由生活线虫提供营养来源。     

重组昆虫病原细菌Cip蛋白基因工程菌发酵条件的初步研究

昆虫病原细菌产生两种胞内晶体蛋白CipA和CipB,为新型的生物农药——昆虫病原线虫提供必需的营养。在已构建原核表达载体pET-15b-cipA和pET-15b-cipB的基础上,研究了这两种重组载体在不同宿主菌中的表达情况,重组菌的生长特性,摇瓶培养时表达重组Cip蛋白工程菌的发酵条件。结果显示:以E.coli BL21(DE3)为宿主菌,在LB培养基中培养至OD600为0.8～1.0时,1 mmol/L IPTG诱导8 h,CipA和CipB的表达量可达30.9%和32.6%,重组质粒具有良好的分离稳定性和结构稳定性。     

昆虫病原斯氏和异小杆线虫对各种非共生微生物的信息反应(英文)

非线虫共生细菌 (Bacillussubtilis ,B .thuringiensis,Pseudomonasfluorescens ,Micromonosporapur purea,Rhizopusdelemar ,Pseudomonasaeruginosa ,Streptomycesvenezuelae ,Streptomycesantibioticus ,Penicilliumcitrnum ,Ganodermalucidum ,Agaricusbisporus,Pleurotusostreatus,Rhizobiumlegumi unosarum和Photobacteriumphosphoreum)的培养液以及其上清液、斜纹夜蛾 (Spodopteralitura)昆虫细胞系用于引诱无菌SteinernemacarpocapsaeA2 4和HeterorhabditisbacteriophoraH0 6发育。上述培养物均未能诱导H .bacteriophoraH0 6发育。虽然P .phosphoreum菌液可致死A2 4线虫 ,但是其上清液可诱导线虫发育。无菌S .carpocapsaeA2 4线虫可利用斜纹夜蛾昆虫细胞繁殖 ,产生下一代感染期线虫。结果进一步说明 ,引诱H .bacteriophoraH0 6发育的化学信息物质比S .carpocapsaeA2 4的专一。     

Pathogenicity, development, and reproduction of Heterorhabditis bacteriophora and Steinernema carpocapsae under axenic in vivo conditions

Galleria mellonella larvae cultured axenically were treated with axenic dauer juveniles of Heterorhabditis bacteriophora and Steinernema carpocapsae. After 3 days S. carpocapsae had killed all insects, with 9.4 +/- 4.3 nematodes per larva. H. bacteriophora were unable to kill G. mellonella, although 13.3 +/- 6.4 nematodes per Galleria were found in the hemocoel. Invading nematodes of both strains recovered from the dauer stage. H. bacteriophora developed into hermaphrodites with eggs and J1 in the uterus and in the hemolymph of the living insects. Development beyond the J1 stage was not recorded. An injection of supernatants from different Photorhabdus luminescens cultures killed the insects but could not provide nutrients to support a further development. Only the injection of bacterial cells supported production of dauers in the axenic insects. Axenic S. carpocapsae developed to adults and produced offspring. After 3 weeks an average of 5275 nematodes per larva were counted, of which 6.7% were dauer juveniles, 39.2% other juvenile stages, 11.9% males, and 42.2% females. Compared to in vivo reproduction in the presence of the symbiotic bacterium Xenorhabdus nematophilus the dauer juvenile yields were low. Even after 5 weeks the percentage of dauer juveniles did not surpass 10%.     

Scavenger deterrent factor (SDF) from symbiotic bacteria of entomopathogenic nematodes

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis are symbiotically associated with bacteria in the genera Xenorhabdus and Photorhabdus, respectively. The symbiotic bacteria produce a chemical compound(s) that deterred ants from feeding on nematode-killed insects (i.e., cadavers) and has been previously referred to as an Ant Deterrent Factor (ADF). We studied the response of different arthropod scavenger species which included the ant Lepisiota frauenfeldi, cricket Gryllus bimaculatus, wasps Vespa orientalis and Paravespula sp., and calliphorid fly Chrysomya albiceps, to ADF. These scavengers (ants, crickets, and wasps) were exposed to cadavers with and without the nematode/bacterium complex or to Photorhabdus luminescens cultures of different ages on different substrates. The ant, cricket, and wasp species did not feed on nematode-killed insects containing the nematode/bacterium complex that were 2 days old and older but fed on 1-day-old nematode-killed and freeze -killed insects. Crickets consumed 2- to 7-day-old axenic nematode-killed insects, 1-, 4-, and 5-day-old insects killed by the bacterium, Serratia marcescens, and freeze-killed, putrid insects that were up to 10 days old. The crickets only partially consumed 2- and 3-day-old insects killed by S. marcescens which differed significantly from the 1-, 4-, and 5-day-old killed insects by this bacterium. Ants fed only on 5% sucrose solution (control) and 1- to 3- day old cultures of P. luminescens containing 5% sucrose but not on older cultures of P. luminescens. Wasps did not feed on meat treated with P. luminescens supernatant, whereas they fed on meat treated with Escherichia coli supernatant and control meat. Calliphorid flies did not oviposit on meat treated with P. luminescens supernatant but did oviposit on untreated meat. Based on the response of these scavengers, the chemical compound(s) responsible for this deterrent activity should be called "scavenger deterrent factor" (SDF).     

Effect of Photorhabdus luminescens phase variants on the in vivo and in vitro development and reproduction of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae

Photorhabdus luminescens (Enterobacteriaceae) is a symbiont of entomopathogenic nematodes Heterorhabditis spp. (Nematoda: Rhabditida) used for biological control of insect pests. For industrial mass production, the nematodes are produced in liquid media, pre-incubated with their bacterial symbiont, which provides nutrients essential for the nematode's development and reproduction. Particularly under in vitro conditions, P. luminescens produces phase variants, which do not allow normal nematode development. The phase variants were distinguished based on dye absorption, pigmentation, production of antibiotic substances, occurrence of crystalline inclusion proteins and bioluminescence. To understand the significance of the phase shift for the symbiotic interaction between the bacterium and the nematode, feeding experiments tested the effect of homologous and heterologous P. luminescens phase variants isolated from a Chinese Heterorhabditis bacteriophora (HO6), the Heterorhabditis megidis type strain from Ohio (HNA) and the type strain of Heterorhabditis indica (LN2) on the in vivo and in vitro development and reproduction of the nematode species H. bacteriophora (strain HO6) and another rhabditid and entomopathogenic nematode, Steinernema carpocapsae (A24). In axenically cultured insect larvae (Galleria mellonella) and in vitro in liquid media, H. bacteriophora produced offspring on phase I of its homologous symbiont and on the heterologous symbiont of H. megidis, but not on the two corresponding phase II variants. In solid media, nematode yields were much lower on phase II than on phase I variants. On the heterologous phase I symbiont isolated from H. indica the development of H. bacteriophora was not beyond the fourth juvenile stage of the nematode in any of the media tested, but further progressed on phase II with even a small amount of offspring recorded in solid media. Infective juveniles of S. carpocapsae did not develop beyond the J3 stage on all phase I P. luminescens. They died in phase I P. luminescens isolated from H. bacteriophora. Development to adults was recorded for S. carpocapsae on all phase II symbionts and offspring were produced in all media except in liquid. It is concluded that a lack of essential nutrients or the production of toxins is not responsible for the negative impact of homologous phase II symbiont cells on the development and reproduction of H. bacteriophora. The infective juveniles of H. bacteriophora retained cells of the homologous phase I symbiont, but not phase II cells and cells from heterologous symbionts, indicating that the transmission of the symbiont by the infective juvenile is selective for phase I cells and the homologous bacterial associate.     

AN IMPROVED METHOD FOR OBTAINING AXENIC CLONES OF PLANKTONIC BLUE-GREEN ALGAE1,2

Axenic clones from 5 isolates of Anabaena flosaquae, 1 isolate of Microcystis acruginosa, and 1 isolate of Aphanizomenon flos-aquae were obtained by a combination of steps that provided a 1000-fold reduction in the bacteria-algae ratio and permitted bacteria-free filaments or cells to be isolated and grown from agar pour plates. The first step consisted of the addition of phenol to a dark-treated culture to selectively reduce the numbers of actively growing bacteria while leaving the resting algal cells viable. The next steps involved washing the treated algal suspension on a Millipore filter pad or membrane followed by plating in washed agar containing buffered mineral medium plus vitamins and soil extract. The final steps consisted of incubating the agar pour plates, coring bacteria-free filaments or cells, culturing the agar cores in a buffered mineral medium, and rigorously testing the resulting cultures for bacteriological contamination. Between 50 and 90% of the cores grew, and of these about 50% were judged axenic. The method, with appropriate adaptations, should be suitable for obtaining axenic clones of other freshwater and marine algae.     

Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50-96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus, Photorhabdus luminescens W14 P. luminescens TTO1, and Yersinia pestis CO92. The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity.     

RECOVERY RESPONSE OF HETERORHABDITIS BACTERIOPHORA AND STEINERNEMA CARPOCAPSAE TO DIFFERENT NON‐SYMBIOTIC MICROORGANISMS*

Abstract Axenic Steinernema carpocapsae Agriotos (A24) and Heterorhabditis bacteriophora H06 dauer juveniles were exposed to Spodoptera litura insect cell cultures, and the cell‐free filtrates or cells of different non‐symbiotic microorganism cultures, including Bacillus subtilis, B. thuringiensis, Pseudomonas fluorescens, Micromonospora purpurea, Rhizopus delemar, Pseudomonas aeruginosa, Streptomyces venezuelae, Streptomyces antibioticus, Penicillium citrnum, Ganoderma lucidum, Agaricus bisporus, Pleurotus ostreatus, Rhizobium legumiunosarum, and Photobacterium phosphoreum. None of these cell‐free filtrates or cultures, or insect cell culture triggered recovery of H. bacteriophora H06. However, cell‐free filtrate of P. phosphoreum induced recovery of S. carpocapsae A24, although the cell culture of this bacterium kill the A24 dauer juveniles before recovery. S. litura insect cells provided the nutrients for axenic S. carpocapsae A24 nematode growth and next generation of dauer juveniles were observed. These results further demonstrated that food signals were much more specific to H. bacteriophora than to S. carpocapsae.     

Effect of temperature on the development of <Emphasis Type="Italic">Steinernema carpocapsae</Emphasis> and <Emphasis Type="Italic">Steinernema feltiae</Emphasis> (Nematoda: Rhabditida) in liquid culture

For commercial use of the entomopathogenic nematodes Steinernema carpocapsae and Steinernema feltiae in biological control of insect pests, they are produced in liquid culture on artificial media pre-incubated with their symbiotic bacteria Xenorhabdus nematophila and Xenorhabdus bovienii, respectively. After 1 day of the bacterial culture, nematode dauer juveniles (DJs) are inoculated, which recover development. The adult nematodes produce DJ offspring, which are harvested and can be sprayed. This study determined optimal temperatures to obtain high DJ progeny within a short process time. Temperatures assessed were 23°C, 25°C, 27°C, and 29°C for S. carpocapsae and 20°C, 23°C, 25°C, and 27°C for S. feltiae. The recovery of inoculated DJs was hardly affected and was reduced only in S. carpocapsae at 29°C. The fecundity (eggs in uterus) in S. carpocapsae reached a maximum at 27°C; whereas, maximum yields were recorded at 25°C. For both Steinernema spp., highest DJ densities were obtained after 15 days incubation at 25°C. Optimal culture temperature for both nematode species is 25°C. S. carpocapsae was more sensible to suboptimal temperature than S. feltiae. Results on total DJ density and DJ proportion of the total nematode population were more variable at non-optimal temperature condition for S. carpocapsae than for S. feltiae. Suboptimal culture temperature also reduced DJ infectivity.     

The effect of intraspecific competition on the development and quality of Phasmarhabditis hermaphrodita (Rhabditida: Rhabditidae)

The effect of intraspecific competition on the development and reproduction of the slug parasitic nematode Phasmarhabditis hermaphrodita was studied in a series of laboratory experiments. Different inocula concentrations (1, 10, 25, 50, 100, 250, 500 and 1000) of P. hermaphrodita dauer juveniles (DJ) were applied to 0.02 g of homogenised slug Deroceras reticulatum on agar in multi-well plates and the development time, yield, lipid content, and body length of females and DJs were recorded. There was an inverse relationship between dose and recorded female length or lipid content. In newly emerged DJs the body length and lipid content were relatively invariable up to a dose of 250 DJs (ranging between 850 and 925 µm, 6.8 and 7.4, respectively), and decreased at higher doses (500 and 1000 DJs). The yield increased with increasing dosage to its optimum at a dose of 100 (200,000 DJs per 1 g of substrate) DJs and thereafter decreased. In the treatment with the highest dose (1000 DJs), only a minor portion of the inoculum developed to adult stage and completed their reproduction cycle while the rest remained at the larval stage. It might, therefore, be concluded that P. hermaphrodita tolerates intraspecific competition to a certain level at which crowding becomes too severe and a part of the inoculum do not develop and probably leave the site to avoid the fatal overcrowding. In conclusion, we show that intraspecific competition negatively affects the yield and quality of DJs of P. hermaphrodita, but it seems that this nematode can partly prevent overcrowding by avoiding occupied sites.     

Desiccation tolerance among different isolates of the entomopathogenic nematode Steinernema feltiae (Fillipjev)

Poor storage capacity is a major constraint limiting further expansion of the use of entomopathogenic nematodes. In order to prolong shelf life, a quiescent state of the dauer juveniles (DJs) should be induced. This can be attained by means of desiccation of DJs. In this study, 24 natural isolations of Steinernema feltiae were exposed to desiccation stress in non-ionic polyethylene glycol 600. The dehydrating conditions were measured as water activity, a(w)-value. Non-adapted and adapted DJs were tested separately under a series of dehydrating conditions. The mean tolerated a(w)-value (MW50) ranged from 0.85 for the isolate NEP1 to 0.95 for FIN1, ISR5 and ITA2 when not adapted to desiccation stress and from MW50 of 0.822 for CR1 to 0.98 for ISR6 when adapted to the stress conditions. CR1 tolerated the lowest desiccation stress at an a(w)-value for the most tolerant 10% of the population (MW10) at 0.65 when DJs had been adapted to stress. No significant differences were recorded between all isolates in non-adapted DJs populations MW10 was compared. No correlation between tolerance under non-adapted and adapted conditions were found. Most tolerant isolates will now be used for cross-breeding and subsequent genetic selection to enhance desiccation tolerance.     

Screening Isolates of Hirsutella Species for Biocontrol of Heterodera glycines

The fungi, Hirsutella rhossiliensis and Hirsutella minnesotensis, are two endoparasites of second-stage juveniles (J2) of the soybean cyst nematode (SCN), Heterodera glycines. The objective of this study was to screen for effective fungal isolates to control the nematode in laboratory and greenhouse assays. A total of 93 isolates of H. rhossiliensis and 25 isolates of H. minnesotensis were evaluated for parasitism of SCN J2 on cornmeal agar. Percentage of SCN J2 parasitized by the fungi varied among the fungal isolates. Most H. rhossiliensis isolates parasitized a high percentage of J2. The isolates of H. rhossiliensis obtained from bacteria-feeding nematodes, however, generally did not parasitize J2 on agar. H. minnesotensis parasitized fewer J2 on agar than did H. rhossiliensis . Forty isolates of H. rhossiliensis and four isolates of H. minnesotensis that parasitized a high percentage of J2 on agar were evaluated for their biocontrol potential in soil treated with microwave heating. Most isolates selected from the agar assay also parasitized a high percentage of J2 in the soil but there was variation among isolates. Pathogenicity of 14 isolates of H. rhossiliensis and four isolates of H. minnesotensis to the SCN was also investigated in the greenhouse using untreated field soil. Most isolates of H. rhossiliensis reduced SCN population density and increased plant growth when compared with 1% corn-grits control (culture media). One isolate (OWVT-1) of H. rhossiliensis reduced the SCN egg density by 95% and J2 density by 98% when compared with the control. Isolates of H. minnesotensis, however, neither reduced SCN density nor increased plant growth in the greenhouse.     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida)

The rhabditid nematodes Steinernema carpocapsae and Steinernema feltiae are used in biological control of insect pests. Mass production is done in liquid culture media pre-incubated with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, before nematode dauer juveniles (DJs) are inoculated. As a response to food signals produced by the bacterial symbionts, the DJs exit from the developmentally arrested dauer stage (they recover development) and grow to adults, which produce DJ offspring. Variable DJ recovery after inoculation often causes process failure due to non-synchronous population development and low numbers of adult nematodes. This contribution investigated the influence of the bacterial cell density on DJ recovery and development to adults. At higher density of 10¹⁰ bacterial cells ml⁻¹, a higher percentage of DJ recovery was induced, and adults occurred earlier in both Steinernema spp. than at lower density of 10⁹ and 10⁸ cells ml⁻¹. Xenorhabdus symbionts produce phase variants. Recovery in bacteria-free supernatants was lower than in supernatants containing bacterial cells for both primary and secondary phase Xenorhabdus spp. and lower in secondary than in primary phase supernatants or cell suspensions. In general, recovery was lower for Steinernema feltiae and the time at which 50% of the population had recovered after exposure to the food signal was longer (RT₅₀ = 17.1 h) than for Steinernema carpocapsae (RT₅₀ = 6.6 h). Whereas >90% S. carpocapsae DJs recovered in hemolymph serum of the lepidopteran insect Galleria mellonella, recovery of S. feltiae only reached 31%. Penetration into a host insect prior to exposure to the insect’s food signal did not enhance DJ recovery. Consequences for liquid culture mass production of the nematodes and differences between species of the genera Steinernema and Heterorhabditis are discussed.     

Influence of inoculum density on population dynamics and dauer juvenile yields in liquid culture of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida)

For improvement of mass production of the rhabditid biocontrol nematodes Steinernema carpocapsae and Steinernema feltiae in monoxenic liquid culture with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, the effect of the initial nematode inoculum density on population development and final concentration of dauer juveniles (DJs) was investigated. Symbiotic bacterial cultures are pre-incubated for 1 day prior to inoculation of DJs. DJs are developmentally arrested and recover development as a reaction to food signals provided by their symbionts. After development to adults, the nematodes produce DJ offspring. Inoculum density ranged from 1 to 10 × 10³ DJ per milliliter for S. carpocapsae and 1 to 8 × 10³ DJs per milliliter for S. feltiae. No significant influence of the inoculum density on the final DJ yields in both nematode species was recorded, except for S. carpocapsae cultures with a parental female density <2 × 10³ DJs per milliliter, in which the yields increased with increasing inoculation density. A strong negative response of the parental female fecundity to increasing DJ inoculum densities was recorded for both species with a maximum offspring number per female of >300 for S. carpocapsae and almost 200 for S. feltiae. The compensative adaptation of fecundity to nematode population density is responsible for the lack of an inoculum (or parental female) density effect on DJ yields. At optimal inoculation density of S. carpocapsae, offspring were produced by the parental female population, whereas S. feltiae always developed a F1 female population, which contributed to the DJ yields and was the reason for a more scattered distribution of the yields. The F1 female generation was accompanied by a second peak in X. bovienii density. The optimal DJ inoculum density for S. carpocapsae is 3–6 × 10³ DJs per milliliter in order to obtain >10³ parental females per milliliter. Density-dependent effects were neither observed on the DJ recovery nor on the sex ratio in the parental adult generation. As recovery varied between different batches, assessment of the recovery of inoculum DJ batches is recommended. S. feltiae was less variable in DJ recovery usually reaching >90%. The recommended DJ inoculum density is >5 × 10³ DJs per milliliter to reach >2 × 10³ parental females per milliliter. The mean yield recorded for S. carpocapsae was 135 × 10³ and 105 × 10³ per mililiter for S. feltiae.     

Response of Steinernema carpocapsae Infective Juveniles to the Plasma of Three Insect Species

Exsheathed infective juveniles of Steinernema carpocapsae All strain were attracted to the plasma of three species of insects in agar plate bioassays. Plasma of Pieris rapae crucivora, Spodoptera litura, and Agrotis segetum attracted 88.6%, 80.4%, and 64.4%, respectively, of Steinernema carpocapsae juveniles added to plates. Autoclaved plasma of S. litura larvae attracted more juveniles than saline controls, but less than nonautoclaved plasma. The active agent passed through a 14,000 MW dialysis membrane.     

Optimization of Bacteriocin Release Protein (BRP)-Mediated Protein Release by Escherichia coli: Random Mutagenesis of the pCloDF13-Derived BRP Gene To Uncouple Lethality and Quasi-Lysis from Protein Release

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli periplasm into the culture medium. However, high-level expression of BRP causes apparent lysis of the host cells in liquid cultures (quasi-lysis) and inhibition of growth on broth agar plates (lethality). To optimize BRP-mediated protein release, the pCloDF13 BRP gene was subjected to random mutagenesis by using PCR techniques. Mutated BRPs with a strongly reduced capacity to cause growth inhibition on broth agar plates were selected, analyzed by nucleotide sequencing, and further characterized by performing growth and release experiments in liquid cultures. A subset of these BRP derivatives did not cause quasi-lysis and had only a small effect on growth but still functioned in the release of the periplasmic protein β-lactamase and the periplasmic K88 molecular chaperone FaeE and in the release of the bacteriocin cloacin DF13 into the culture medium. These BRP derivatives can be more efficiently used for extracellular production of proteins by E. coli than can the original BRP.