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Plasmids containing a Pseudomonas sp. strain 109 extracellular lipase gene (lipL) lacking NH 2 -terminal sequence and a lipase modulator gene (limL) lacking the NH 2 -terminal hydrophobic region were constructed and expressed independently in Escherichia coli by using the T7 promoter expression vector system. Recombinant LipL (rLipL) was produced as inclusion bodies, whereas recombinant LimL (rLimL) was present as a soluble protein. During in vitro renaturation of the purified rLipL inclusion bodies after they had been dissolved in 8 M urea, addition of rLimL was essential to solubilize and modulate rLipL. The solubility and activity of rLipL were influenced by the rLimL/rLipL molar ratio; the highest level of solubility was obtained at an rLimL/rLipL ratio of 4:5, whereas the highest activity level was obtained at an rLimL/rLipL ratio of 4:1. After renaturation, rLipL and rLimL were coprecipitated with anti-rLipL antibody, indicating the formation of an rLipL-rLimL complex. Activity of the native lipase purified from Pseudomonas sp. strain 109 was also inhibited by rLimL. By Western blotting (immunoblotting) with anti-rLimL antibody, native LimL was detected in Pseudomonas cells solubilized by sarcosyl treatment. LimL was purified from Pseudomonas sp. strain 109, and the NH 2 -terminal amino acid sequence was determined to be NH 2 -Leu-Glu-Pro-Ser-Pro-AlaPro-. We propose that to prevent membrane degradation, LimL weakens lipase activity inside the cell, especially in the periplasm, in addition to modulating lipase folding.

Analysis of fermentation broth of the entomopathogenic fungus Metarhizium anisopliae has led to isolation of aurovertin D (1) and three new aurovertin-type metabolites, aurovertin F (2), aurovertin G (3), and aurovertin H (4). Their structures were determined on the basis of spectroscopic analyses and chemical conversions.Aurovertins are secondary metabolites initially identified as toxic substances in fermentation broth of the fungus Calcarisporium arbuscula. 2 Aurovertins are of polyketide origin and are characterized by a 2,6-dioxabicyclo[3.2.1]octane ring system with a conjugated R-pyrone moiety. 3 There have been five aurovertins (A-E) structurally elucidated to date. Relative and absolute configurations of aurovertin B were determined by spectroscopic analysis and total synthesis, respectively. 4,5 Structures of aurovertins A, C, and D were determined on the basis of spectroscopic comparison with aurovertin B. 3 Aurovertin E was also isolated from the fungus Albatrellus confluens. 6 Aurovertin B binds to F1-ATPase and thus inhibits ATP synthesis and hydrolysis in mitochondorial enzyme systems. 7 During our continuing investigation of bioactive microbial secondary metabolites, three new aurovertins, aurovertin F (2), aurovertin G (3), and aurovertin H (4), were isolated from the culture broth of the entomopathogenic fungus Metarizhium anisopliae HF260 along with previously reported aurovertin D (1). The structures of these compounds were determined by combined spectroscopic and chemical methods.The producing strain M. anisopliae HF260 was isolated from a soil sample collected in Okinawa, Japan, by the baiting method. 8 In the course of screening for plant growth regulating substances, the 1-butanol extract of strain HF260 inhibited seed germination of barnyard millet. Investigation of active principles led to the isolation of aurovertin D (1) and three new aurovertins. The major aurovertin produced by strain HF260 was aurovertin D (1). Compound 1 has an additional OH group at C-2 of aurovertin B, but the relative and absolute configurations are obscure. Therefore, the structural analysis of a series of aurovertins from our strain was started from 1. The 1 H and 13 C NMR data of 1 were identical to those reported in the literature. 3 The relative configuration of 1 was elucidated by analyzing the NOESY spectrum of its acetate (1a). A NOESY correlation between H-2 and H-8 indicated the anti-relationship of H-2 and H-3, which was consistent with the large coupling constant between H-2 and H-3 (9.0 Hz). The key NOESY correlation was observed between the methyl of 2-OAc and the angular methyl at C-20, illustrating the preferable conformation where the 2-OAc group is oriented on the same side as Me-20 while Me-1 is on the opposite side. Accordingly, the relative configuration at C-2 was determined as depicted in Figure 1. Since the optical rotation of 1 shows a negative value of [R] 23 D -55.7 (c 0.5, EtOH) similar to aurovertin B {natural compound: [R] 20 D -50.6 (EtOH); 4 synthetic compound: [R] 27 D -57.9 (c 0....

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We examined whether stimuli among one another are involved in the short-range locations of the Halyomorpha halys adult, which often forms aggregations when overwintering. Ten diapausing adults were released into a clear container at 10°C. After a certain time period, their distribution was compared with a random distribution, and a tendency for a strong concentrated distribution with crowding was observed under both light and dark conditions. However, antennectomy prevented bugs from aggregating and this clarified the role of antenna in short-range locations. This finding suggests that overwintering aggregation in H. halys is guided by a chemical or tactile interaction among individuals, at least within a narrow range. In addition, the relationship between this aggregation habit and temperature was examined. Although temperatures of 15°C and 20°C caused bugs to become active, the habit of settling close to one another was not reduced. Based on these results, we inferred that the aggregation habit of H. halys adults itself is not temperature dependent, and that the overwintering aggregation is caused by an increase in opportunity for contact with each other, such as from a mutual preference for the overwintering environment, and a decrease in behavioral activity resulting from a decrease in temperature.