A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)₄ repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.     

The paired box gene pox neuro: a determinant of poly-innervated sense organs in Drosophila.

This study describes the structure and function of pox neuro (poxn), a gene previously isolated by virtue of a conserved domain, the paired box, which it shares with the segmentation genes paired and gooseberry. Its expression pattern has been analyzed, particularly during development of the PNS. We propose that poxn is a "neuroblast identity" gene acting in both the PNS and the CNS on the basis of the following evidence. Its expression is restricted to four neuronal precursors in each hemisegment: two neuronal stem cells (neuroblasts) in the CNS, and two sensory mother cells (SMCs) in the PNS. The SMCs that express poxn produce the poly-innervated external sense organs of the larva. In poxn- embryos, poly-innervated sense organs are transformed into mono-innervated. Conversely, ectopic expression of poxn in embryos transformed with a heat-inducible poxn gene can switch mono-innervated to poly-innervated sense organs. Expression of poxn in the wing disc is restricted to the SMCs of the poly-innervated sense organs, suggesting that poxn also determines the lineage of poly-innervated adult sense organs.     

High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.     

Brachypodium distachyon as a model plant toward improved biofuel crops: Search for secreted proteins involved in biogenesis and disassembly of cell wall polymers

Polysaccharides make up about 75% of plant cell walls and can be broken down to produce sugar substrates (saccharification) from which a whole range of products can be obtained, including bioethanol. Cell walls also contain 5–10% of proteins, which could be used to tailor them for agroindustrial uses. Here we present cell wall proteomics data of Brachypodium distachyon, a model plant for temperate grasses. Leaves and culms were analyzed during active growth and at mature stage. Altogether, 559 proteins were identified by LC‐MS/MS and bioinformatics, among which 314 have predicted signal peptides. Sixty‐three proteins were shared by two organs at two developmental stages where they could play housekeeping functions. Differences were observed between organs and stages of development, especially at the level of glycoside hydrolases and oxidoreductases. Differences were also found between the known cell wall proteomes of B. distachyon, Oryza sativa, and the Arabidopsis thaliana dicot. Three glycoside hydrolases could be immunolocalized in cell walls using polyclonal antibodies against proteotypic peptides. Organ‐specific expression consistent with proteomics results could be observed as well as cell‐specific localization. Moreover, the high number of proteins of unknown function in B. distachyon cell wall proteomes opens new fields of research for monocot cell walls.     

Pioneer marine biofilms on artificial surfaces including antifouling coatings immersed in two contrasting French Mediterranean coast sites

Marine biofilm communities that developed on artificial substrata were investigated using molecular and microscopic approaches. Polystyrene, Teflon® and four antifouling (AF) paints were immersed for 2 weeks at two contrasting sites near Toulon on the French Mediterranean coast (Toulon military harbour and the natural protected area of Porquerolles Island). Biofilms comprising bacteria and diatoms were detected on all the coatings. The population structure as well as the densities of the microorganisms differed in terms of both sites and coatings. Lower fouling densities were observed at Porquerolles Island compared to Toulon harbour. All bacterial communities (analysed by PCR-DGGE) showed related structure, controlled both by the sites and the type of substrata. Pioneer microalgal communities were dominated by the same two diatom species, viz. Licmophora gracilis and Cylindrotheca closterium, at both sites, irrespective of the substrata involved. However, the density of diatoms followed the same trend at both sites with a significant effect of all the AF coatings compared to Teflon and polystyrene.     

Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory

Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates.     

Control of expression of a gene encoding an extensin by phytochrome and a blue light receptor in spores of Adiantum capillus-veneris L.

In the present study, using a newly developed fluorescent differential display technique, we have carried out large-scale screening for genes whose expression was regulated by phytochrome and antagonistically by a blue light receptor in the spores of the fern Adiantum capillus-veneris L. Spores after imbibition were briefly irradiated with red, red/blue or blue light and collected 8 h after the irradiation. Total RNA was isolated from each sample and used to make cDNA with an oligo-dT primer. The cDNA was then used as a template for PCR with the oligo-dT primer and 80 arbitrary primers. The resulting PCR products were analyzed by an automated fluorescent DNA sequencer. Among 8000 displayed bands, we identified 15 upregulated and four down-regulated bands by red light, and this red light effect was irreversibly reversed by blue light. We cloned one of the up-regulated cDNA fragments and used it to screen a cDNA library prepared from the spores. The isolated insert is predicted to encode Ser-(Pro) _n repeats and showed homology with cell wall-associated extensins. The expression of this cDNA was induced 8 h after a red light treatment and the red light induction was photoreversibly prevented by far-red light and photo-irreversibly by blue light. The mRNA of this gene was detectable 4 h after red light irradiation and gradually increased in germinating spores.     

Expression of the Tobacco Ext 1.4 Extensin Gene Upon Mechanical Constraint and Localization of Regulatory Regions

Abstract: The regulation of the Ext 1.4 gene encoding a tobacco ( Nicotiana tabacum L.) extensin was studied in response to mechanical constraints. Transgenic plants carrying chimeric Ext 1.4 promoter/GUS (β-glucuronidase)/ nos terminator or Ext 1.4 3'-end constructs were obtained. Expression of gene fusions was found in tissues where mechanical stresses occur, e.g., during germination, as well as in root and stem tissues. Chimeric genes were successively and transiently expressed in different tissues during germination, i.e., at the tip of the root and then in the hypocotyl, during their growth through the seed coat. Moreover, they were expressed in cortical cells surrounding the emergence of adventitious and lateral roots and developmentally-regulated in nodes. The expression of Ext 1.4 could be induced by imposing mechanical constraints due to curving of either the stems or roots. Expression then occurred in cells where it does not normally occur, i.e., in cortical cells of internodes and in the distal piliferous zone of roots. Accumulation of RNAs occurs several days after the start of the constraint. Promoter regions involved in regulation of expression of Ext 1.4 in stems, roots, and in seedlings upon mechanical constraint could be localized. Moreover, the 3' non-coding region was shown to modulate expression in roots. These results suggest that the regulation of Ext 1.4 following mechanical stress is dependent on both tissue-specific and mechanical-responsive elements.