Stimulation of n-alkane conversion to dicarboxylic acid by organic-solvent- and detergent-treated microbes

Summary A wild-type strain of Cryptococcus neoformans and Pseudomonas aeruginosa were used to convert n-pentadecane to the corresponding dioic acid, tridecane 1,13-dicarboxylic acid (DC-15). Altering the cell permeability by treating C. neoformans with 1% (v/v) toluene or 7% (v/v) Triton X-100 stimulated production of DC-15 by 1.5-fold and fourfold, respectively. Furthermore, DC-15 productivity was increased from 2.5 mg/l per hour to 18 or 30 mg/l per hour, respectively. If 10% (v/v) hexane was used to treat the yeast culture, stimulation of DC-15 production could reach 200% and more viable cells remained compared to the toluene-treated culture. Data from the organic solvent treatment experiment indicated that the solvent with a higher polarity showed a more adverse effect on DC-15 production. P. aeruginosa was vulnerable to most organic solvents; however, Tween 80 could greatly stimulate the conversion of n-pentadecane to DC-15. Although organic solvents and non-ionic detergents could enhance DC-15 formation by microbial conversion, it was inhibited by elevated levels of DC-15.Offprint requests to: E.-C. Chan     

Chaperonins GroEL and GroES: views from atomic force microscopy.

The Escherichia coli chaperonins, GroEL and GroES, as well as their complexes in the presence of a nonhydrolyzable nucleotide AMP-PNP, have been imaged with the atomic force microscope (AFM). We demonstrate that both GroEL and GroES that have been adsorbed to a mica surface can be resolved directly by the AFM in aqueous solution at room temperature. However, with glutaraldehyde fixation of already adsorbed molecules, the resolution of both GroEL and GroES was further improved, as all seven subunits were well resolved without any image processing. We also found that chemical fixation was necessary for the contact mode AFM to image GroEL/ES complexes, and in the AFM images. GroEL with GroES bound can be clearly distinguished from those without. The GroEL/ES complex was about 5 nm higher than GroEL alone, indicating a 2 nm upward movement of the apical domains of GroEL. Using a slightly larger probe force, unfixed GroEL could be dissected: the upper heptamer was removed to expose the contact surface of the two heptamers. These results clearly demonstrate the usefulness of cross-linking agents for the determination of molecular structures with the AFM. They also pave the way for using the AFM to study the structural basis for the function of GroE system and other molecular chaperones.     

The Puccinia striiformis effector Hasp98 facilitates pathogenicity by blocking the kinase activity of wheat TaMAPK4

The obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst) employs virulence effectors to disturb host immunity and causes devastating stripe rust disease. However, our understanding of how Pst effectors regulate host defense responses remains limited. In this study, we determined that the Pst effector Hasp98, which is highly expressed in Pst haustoria, inhibits plant immune responses triggered by flg22 or nonpathogenic bacteria. Overexpression of Hasp98 in wheat (Triticum aestivum) suppressed avirulent Pst-triggered immunity, leading to decreased H₂O₂ accumulation and promoting P. striiformis infection, whereas stable silencing of Hasp98 impaired P. striiformis pathogenicity. Hasp98 interacts with the wheat mitogen-activated protein kinase TaMAPK4, a positive regulator of plant resistance to stripe rust. The conserved TEY motif of TaMAPK4 is important for its kinase activity, which is required for the resistance function. We demonstrate that Hasp98 inhibits the kinase activity of TaMAPK4 and that the stable silencing of TaMAPK4 compromises wheat resistance against P. striiformis. These results suggest that Hasp98 acts as a virulence effector to interfere with the MAPK signaling pathway in wheat, thereby promoting P. striiformis infection.     

Exchanging sequence domains between S-RNases from Nicotiana alata disrupts pollen recognition

In self-incompatible plants of the Solanaceae, the specificity of pollen rejection is controlled by a single multiallelic S-locus. Pollen tube growth is inhibited in the style when its single S-allele matches either S-allele present in the diploid pistil. Each S-allele encodes an S-RNase with a unique sequence. S-RNases are secreted into the extracellular matrix of the transmitting tract which guides pollen tubes toward the ovary. Although it is known that S-RNases are the determinants of S-allele specificity in the pistil, it is not known how allele-specific information is encoded in the sequence. Therefore, we exchanged domains between S-RNases with different recognition specificities and expressed the chimeric proteins in transgenic plants to determine their effects on pollination behavior. Nine chimeric constructs were prepared in which domains from Nicotiana alata S_A2- and S_C10-RNases were exchanged. Among these nine constructs, the entire S-RNase sequence was sampled by exchanging single variable domains as well as larger blocks of contiguous sequences. The chimeric S-RNases retained enzymatic activity and were expressed at levels comparable to control transformants expressing S_A2- and S_C10-RNase. However, none of the chimeric S-RNases caused rejection of either S_A2- or S_C10-pollen. We conclude that the recognition function of S-RNases can be disrupted by alterations in many parts of the sequence. It appears that the recognition function of S-RNase is not localized to a specific domain.     

新型平均势函数在蛋白质反向折叠中的应用

利用蛋白质主链的极性分数及主链二面角为参量,构建了一种基于蛋白质结构数据库的势函数。将该势函数应用于蛋白质反向折叠研究中,发现该函数可成功地将蛋白质分子的天然构象从构建的构象库中识别出来;将一目标序列与构象库的每一可能的构象匹配,并用该势函数计算相应的能量,结果表明对绝大多数蛋白质分子来说,天然的构象的能量值总是最低。此外,该函数还将一些序列相似性较低,而结构相似性较高的蛋白质分子识别出来。我们认     

High-density Escherichia coli cultivation process for hyperexpression of recombinant porcine growth hormone.

Fermentation studies were performed on an Escherichia coli culture that carries a recombinant plasmid composed of an ampicillin-resistant gene, a temperature-regulated pL promoter, and a porcine pituitary cDNA sequence coding for growth hormone. The objective was to achieve high cell density while maintaining the specific expression level of recombinant porcine growth hormone (r-pGH) observed in shake flasks. At a specific expression level of 20% of total cell protein, the cell density of a glucose-limited fed-batch process reached 38 units of OD600 in 14 h, compared to flask cultivation, which resulted in only 1.4 units of OD600 in the same period. The observed critical fermentation conditions for maximal expression included (1) limiting glucose concentration below 1 g l-1 throughout the fed-batch growth and induction phases, (2) keeping postinduction temperature at 42 degrees C for 5-7 h, and (3) maintaining a postinduction growth rate around 0.17-0.21 h-1.     

Fine structure and histochemistry of the gastric shield in the lamellibranch Donax trunculus L.

The histochemical observations performed by the authors on the stomach wall of Donax trunculus are not wholly in agreement with the results of workers on other bivalves. The ultrastructure of the gastric shield and underlying cells was therefore, studied. It was confirmed that the epithelial cells contain glycogen granules at the base. In addition two distanct P.A.S. positive inclusions were identified: lysosomal residual bodies and neutral mucopolysaccharidic inclusions. The gastric shield is composed largely of long narrow microvilli embedded in a chitinous glycocalyx but is nevertheless easily removable. Hence, there is no causal relationship between abundance of microvilli and the possibility of removing the shield.