Production and Targeting of the Brucella abortus Antigen L7/L12 in Lactococcus lactis: a First Step towards Food-Grade Live Vaccines against Brucellosis

Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.     

Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml⁻¹ was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l⁻¹ h⁻¹, and in reactor, 104,000 U l⁻¹ h⁻¹ was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l⁻¹ h⁻¹, compared to shake-flask fermentations, 12,000 U l⁻¹ h⁻¹. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.     

Congruence between the sexes in preexisting receiver responses

Preexisting receiver biases have been shown to affect how femalesdetect and respond to new conspecific traits in a mate choicecontext. Although preexisting biases have often been discussedin the context of female mate choice, these biases need notbe sex limited. In the genus Xiphophorus, swordtail males possessa sexually selected trait, the sword. Here I consider evidencethat the state of a bias favoring sworded conspecifics maybe generally shared by the sexes in taxa in which the sword has not arisen. In three unsworded species of poeciliid fishes,both males and females prefer members of the other sex withswords. In a fourth species, males and females share the absenceof a response to a sword. This congruity between the sexessuggests that response biases may not be sex limited and thatthe sexes could historically share common mechanisms producingshared mating responses. Alternatively, selection may tendto result in parallel changes in biases in the sexes. Thiswork expands our understanding of receiver biases by usinga phylogenetic approach to examine whether biases are historicallyshared by the sexes and suggests that there can be general congruence between the sexes in such biases.     

Distribution of an 18 kDa-selenoprotein in several tissues of the rat.

By combining methods for trace element analysis, tracer techniques and various biochemical and electrophoretical procedures, information on the characteristics of an 18 kDa-selenoprotein was obtained. By labeling of rats in vivo with [75Se]-selenite and gel electrophoretic separation of the proteins in tissues and subcellular fractions, a larger number of selenium-containing proteins could be distinguished. In most of the tissues investigated a labeled 18 kDa-band was present. After co-electrophoresis of the 18 kDa-bands from kidney, liver and brain we found that they all migrated in the same way. Using ultracentrifugational fractionation the 18 kDa-band was localized in the mitochondrial and microsomal membranes. Two-dimensional electrophoresis showed that it consists of a single selenium-containing protein with an isoelectric point of about 4.9-5.0. By means of proteolytic cleavage of the 18 kDa-protein and separation of its peptides by tricine-SDS-PAGE six selenium-containing peptides with molecular masses of 17, 16, 14, 12, 10, and 8 kDa were detected. After electrophoretic separation of the mitochondrial and/or microsomal proteins and acid hydrolysis of the electroeluted protein its amino acid composition was analyzed by RP-HPLC. In this way it was shown that selenium is present in the 18 kDa-protein in form of selenocysteine which is a characteristic of a genetically encoded selenoprotein.     

Tree shape, forest structure and diversity of drosophilid community: Comparison between boreal and temperate birch forests

Some models, based on the latitudinal variation in sun angle distribution, predict that trees at high latitudes have narrowly conical crowns and constitute simple-layered forests, whereas trees at low latitudes have shallowly dome-shaped and form more structurally complex multilayered forests. There is a hypothesis that structurally complex habitats can harbor potentially more species than simple ones. In this study, we examined latitudinal correlations between tree shape, forest structure and diversity in drosophilid communities, comparing boreal and cool-temperate forests. We selected secondary birch forests with a common canopy tree species, white birch (Betula platyphylla Sukatchev), as study sites. The crown shape of white birch tended to be spherical in the cool-temperate forest, but narrowly conical in the boreal forest. The foliage structure differed between the two forests. The cool-temperate forest was characterized by a clearly two-layered structure, whereas foliage in the boreal forest was less clearly stratified, being distributed somewhat continuously from the ground to the canopy at lower densities. The structural complexity expressed by foliage height diversity was greater in the cool-temperate forest than in the boreal forest. Various measures of drosophilid diversity were higher in the cool-temperate forest than in the boreal forest, probably resulting from the impoverishment of the canopy subcommunity in the boreal forest. Thus, a physical environmental factor (i.e. the angle of solar inclination) could be a potentially important factor in structuring latitudinal patterns of sylvan animal communities through changes in plant structure at the individual and community levels.     

Do plants have rhodopsin after all? A mystery of plant G protein-coupled signalling

Considerable physiological and biochemical evidence suggests that plants, like animals, widely use intracellular signalling coupled to heterotrimeric G proteins. Yet, the molecular components of this machinery remained elusive until recently. We overview the work carried out during the last two decades, aimed at identification of the plant proteins involved in G protein-coupled signalling. The completion of the sequencing of the Arabidopsis genome now permits to assess whether plants possess signalling and regulatory components of this machinery corresponding to those known from animals.     

An Unexpected Diversity of Photoreceptor Classes in the Longfin Squid,Doryteuthis pealeii

Cephalopods are famous for their ability to change color and pattern rapidly for signaling and camouflage. They have keen eyes and remarkable vision, made possible by photoreceptors in their retinas. External to the eyes, photoreceptors also exist in parolfactory vesicles and some light organs, where they function using a rhodopsin protein that is identical to that expressed in the retina. Furthermore, dermal chromatophore organs contain rhodopsin and other components of phototransduction (including retinochrome, a photoisomerase first found in the retina), suggesting that they are photoreceptive. In this study, we used a modified whole-mount immunohistochemical technique to explore rhodopsin and retinochrome expression in a number of tissues and organs in the longfin squid, Doryteuthis pealeii. We found that fin central muscles, hair cells (epithelial primary sensory neurons), arm axial ganglia, and sucker peduncle nerves all express rhodopsin and retinochrome proteins. Our findings indicate that these animals possess an unexpected diversity of extraocular photoreceptors and suggest that extraocular photoreception using visual opsins and visual phototransduction machinery is far more widespread throughout cephalopod tissues than previously recognized.