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Nicotiana tabacum leaves are covered by trichomes involved in the secretion of large amounts of secondary metabolites, some of which play a major role in plant defense. However, little is known about the metabolic pathways that operate in these structures. We undertook a proteomic analysis of N. tabacum trichomes in order to identify their protein complement. Efficient trichome isolation was obtained by abrading frozen leaves. After homogenization, soluble proteins and a microsomal fraction were prepared by centrifugation. Gel-based and gel-free proteomic analyses were then performed. 2-DE analysis of soluble proteins led to the identification of 1373 protein spots, which were digested and analyzed by MS/MS, leading to 680 unique identifications. Both soluble proteins and microsomal fraction were analyzed by LC MALDI-MS/MS after trypsin digestion, leading to 858 identifications, many of which had not been identified after 2-DE, indicating that the two methods complement each other. Many enzymes putatively involved in secondary metabolism were identified, including enzymes involved in the synthesis of terpenoid precursors and in acyl sugar production. Several transporters were also identified, some of which might be involved in secondary metabolite transport. Various (a)biotic stress response proteins were also detected, supporting the role of trichomes in plant defense.

Aim: To assess the dynamics of plasmid transfer between Bacillus thuringiensis and B. cereus in various food microcosms using the B. thuringiensis pAW63 and Staphylococcus aureus pUB110 plasmids as models. Methods and Results: The conjugative behaviour of pAW63, which resembles the B. anthracis virulence plasmid pXO2, and the mobilization of pUB110 were investigated using kinetics studies performed in reference LB (lysogeny broth) medium, full-cream and skimmed milks, soya milk and rice milk. Transfers of pAW63 and pUB110 were found to occur in the five tested media, with higher frequencies observed in food matrices, most notably in full-cream milk, skimmed milk and soya milk, where the mean transfer frequencies reached 10(-3) transconjugants per recipient cell. The most notable observations were that the higher transfer frequencies obtained in foodstuffs compared to those observed in LB were because of an earlier onset of conjugation in combination with a higher transfer rate and/or a longer mating period. Conclusion: These results indicate that not only the potential for plasmid transfer but also the overall timing of conjugation is affected by each of these food matrices. Significance and Impact of the Study: This new approach to study plasmid transfer provides insights for a better understanding of conjugation in food microcosms from both animal and vegetable origins among members of the B. cereus group.

During the past 15years, atomic force microscopy (AFM) has opened new opportunities for imaging supported lipid bilayers (SLBs) on the nanoscale. AFM offers a means to visualize the nanoscale structure of SLBs in physiological conditions. A unique feature of AFM is its ability to monitor dynamic events, like bilayer alteration, remodelling or digestion, upon incubation with various external agents such as drugs, detergents, proteins, peptides, nanoparticles, and solvents. Here, we survey recent progress made in the area.

X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectroscopy (ToF-SIMS), two surface-sensitive spectroscopic methods, are commonly used to characterize adsorbed protein layers. Principal component analysis (PCA) is a statistical method which aims at reducing the number of variables in complex sets of data while retaining most of the original information. The aim of this paper is to review work carried out in our group regarding the use of PCA with a view to facilitate and deepen the interpretation of ToF-SIMS or XPS spectra acquired on adsorbed protein layers. ToF-SIMS data acquired on polycarbonate membranes after albumin and, or insulin adsorption were treated with PCA. The results reveal the preferential exposure of particular amino acids at the outermost surface depending on the adsorption conditions (nature of the substrate and of the proteins involved, concentration in solution), giving insight into the adsorption mechanisms. PCA was applied on XPS data collected on three different substrates after albumin or fibrinogen adsorption, followed in some cases by a cleaning procedure with oxidizing agents. The results allow samples to be classified according to the nature of the substrate and to the adsorbed amount and, or the level of surface coverage by the protein. Chemical shifts of particular interest are also identified, which may facilitate further peak decomposition. It is useful to recall that the outcome of PCA strongly depends on data selection and normalisation.

Micrococcus luteus (NCTC2665, "Fleming strain") has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content 73%) predicted to encode 2403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 IS elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and fourteen response regulators, indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to beta-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose, and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.

Summary Thanks to their potential to bind virtually all types of molecules; monoclonal antibodies are in increasing demand as therapeutics and diagnostics. To overcome the overloading of current production facilities, alternative expression systems have been developed, of which plants appear the most promising. In this review, we focus on the expression of monoclonal IgG or IgM in plant species. We analyse the data for 32 different antibodies expressed in various ways, differing in DNA construction, transformation method, signal peptide source, presence or absence of an endoplasmic reticulum retention sequence, host species and the organs tested, together resulting in 98 reported combinations. A large heterogeneity is found in the quantity and quality of the antibody produced. We discuss in more detail the strategy used to express both chains, the nature of the transcription promoters, subcellular localization and unintended proteolysis, when encountered.

DNA pol g is the sole polymerase responsible for mtDNA replication. 150 mutations associated with human disorders have been identified. The patients often have several different mutations in cis or in trans, and thus it is difficult to distinguish pathogenic mutations and to establish the impact of each mutation in the disorder. We have introduced 7 mip1 mutations in Mip1p, the yeast pol g, alone or in combination, identical to those found in patients. The aim of this work is to determine the contribution of each mutation to the disorder and the role of these amino acids in the pol g function. R607P, T716P, M920R and T716P-F921I elicit loss of mtDNA. Except for the R607P, Mip1p levels are not detectable suggesting that the protein structure is impaired. Q264H, R607C and F921I allow maintenance of the mtDNA. Whereas the Q264H, R607C and T716P-F921I/Q264H elicit high instability of the mtDNA, the F921I has only a mild effect and shows an increase in the punctual mutation rate compared to wild-type. No effect was observed for the K903C supporting the view that the human equivalent is a SNP. In these mutants, Mip1p levels are not, or weakly, decreased. The gap-filling activity is lower for the Q264H, R607C/P, and similar to wild-type for F921I and K903C. Processive DNA synthesis is decreased in all mutants whereas DNA binding affinity is not (R607C, F921I) or moderately (Q264H, R607P) decreased. The 3’-5’ exonuclease activity is the highest in R607P, similar to wild-type in F921I and R607C, and lower in Q264H. Our data suggest that all mutations, except K903C, play a role in the disease. In conclusion, characterization of human mutations associated with mitochondrial disorders in yeast can generate informations that can be used for understanding the molecular defects in humans., (AGRO 3) -- UCL, 2010

Aspergillus flavus is one of the most common contaminants that produces aflatoxins in foodstuffs. It is also a human allergen and a pathogen of animals and plants. Aspergillus flavus is included in the Aspergillus section Flavi that comprises 11 closely related species producing different profiles of secondary metabolites. A six-step strategy has been developed that allows identification of nine of the 11 species. First, three real-time PCR reactions allowed us to discriminate four groups within the section: (1) A. flavus/Aspergillus oryzae/Aspergillus minisclerotigenes/Aspergillus parvisclerotigenus; (2) Aspergillus parasiticus/Aspergillus sojae/Aspergillus arachidicola; (3) Aspergillus tamarii/Aspergillus bombycis/Aspergillus pseudotamarii; and (4) Aspergillus nomius. Secondly, random amplification of polymorphic DNA (RAPD) amplifications or SmaI digestion allowed us to differentiate (1) A. flavus, A. oryzae and A. minisclerotigenes; (2) A. parasiticus, A. sojae and A. arachidicola; (3) A. tamarii, A. bombycis and A. pseudotamarii. Among the 11 species, only A. parvisclerotigenus cannot be differentiated from A. flavus. Using the results of real-time PCR, RAPD and SmaI digestion, a decision-making tree was drawn up to identify nine of the 11 species of section Flavi. In contrast to conventional morphological methods, which are often time-consuming, the molecular strategy proposed here is based mainly on real-time PCR, which is rapid and requires minimal handling.

Although the chemical composition of many fungal cell walls is known, the spatial organization and interactions of the individual macromolecules remain mysterious. In this context, single-cell and single-molecule atomic force microscopy techniques offer new opportunities for probing the surface of fungal cells down to molecular resolution. Recent breakthroughs include the visualization of the structural dynamics of single cells as they grow or interact with drugs, the quantification and nanoscale imaging of cell surface hydrophobicity and the measurement of the molecular elasticity of cell wall polysaccharides and proteins. Here I highlight the advances made by my team in this area during the past 3 years. Copyright (c) 2010 John Wiley & Sons, Ltd.

Oritavancin, a lipoglycopeptide with marked bactericidal activity against vancomycin-resistant Staphylococcus aureus and enterococci, induces calcein release from CL:POPE and POPG:POPE liposomes, an effect enhanced by an increase in POPG:POPE ratio and decreased when replacing POPG by DPPG (Domenech et al., Biochim Biophys Acta. 2009; 1788:1832-40). Using vesicles prepared from lipids extracted from Staphylococcus aureus, we showed that oritavancin induces holes, erosion of the edges, and decrease of the thickness of the supported lipid bilayers (Atomic Force Microscopy). Oritavancin also induced an increase of membrane permeability (calcein release) on a time- and dose-dependent manner. These effects were probably related to the ability of the drug to bind to lipid bilayers as showed by 8-anilino-1- naphthalene sulfonic acid (ANS) assay. Interaction of oritavancin with phospholipids at the level of their glycerol backbone and hydrophobic domain was studied by monitoring changes of Laurdan excitation generalized polarization (GP(ex)) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy upon temperature increase. Oritavancin increased GP(ex) values and the transition temperature, indicating a more ordered structure at the level of the glycerol backbone. Oritavancin slightly decreased DPH fluorescence depolarization intensities, suggesting an increase in fluidity at the level of acyl chains. Together, our data confirm the interaction of oritavancin with lipids and the potential role of a rigidifying effect at the level of glycerol backbone for membrane permeabilization. This work shows how AFM and biophysical methods may help in characterizing drug-membrane interactions, and sheds further light on the mode of action of oritavancin.

The ability to react to environmental stress is a key feature of microbial cells, which frequently involves the fortification of their cell wall as a primary step. In the model yeast Saccharomyces cerevisiae the biosynthesis of the cell wall is regulated by the so-called cell wall integrity signal transduction pathway, which starts with the detection of cell surface stress by a small family of five membrane-spanning sensors (Wsc1-Wsc3, Mid2, Mtl1). Although genetic evidence indicated that these proteins act as mechanosensors, direct in vivo evidence for their function remained scarce. Here, we review a new approach integrating the tools and concepts of genetics with those of nanotechnology. We show how atomic force microscopy can be combined with advanced protein design by yeast genetics, to study the function and the mechanical properties of yeast sensors in living cells down to the single molecule level. We anticipate that this novel integrated technology will enable a paradigm shift in cell biology, so that pertinent questions can be addressed, such as the nanomechanics of single sensors and receptors, and how they distribute across the cell surface when they respond to extracellular stress.

In Gram-positive bacteria, the functional role of surface polysaccharides (PS) that are not of capsular nature remains poorly understood. Here, we report the presence of a novel cell wall PS pellicle on the surface of Lactococcus lactis. Spontaneous PS-negative mutants were selected using semi-liquid growth conditions, and all mutations were mapped in a single chromosomal locus coding for PS biosynthesis. PS molecules were shown to be composed of hexasaccharide phosphate repeating units that are distinct from other bacterial PS. Using complementary atomic force and transmission electron microscopy techniques, we showed that the PS layer forms an outer pellicle surrounding the cell. Notably, we found that this cell wall layer confers a protective barrier against host phagocytosis by murine macrophages. Altogether, our results suggest that the PS pellicle could represent a new cell envelope structural component of Gram-positive bacteria.

Understanding how cell adhesion proteins form adhesion domains is a key challenge in cell biology. Here, we use single-molecule atomic force microscopy (AFM) to demonstrate the force-induced formation and propagation of adhesion nanodomains in living fungal cells, focusing on the covalently anchored cell-wall protein Als5p from Candida albicans. We show that pulling on single adhesins with AFM tips terminated with specific antibodies triggers the formation of adhesion domains of 100-500 nm and that the force-induced nanodomains propagate over the entire cell surface. Control experiments (with cells lacking Als5p, single-site mutation in the protein, bare tips, and tips modified with irrelevant antibodies) demonstrate that Als5p nanodomains result from protein redistribution triggered by force-induced conformational changes in the initially probed proteins, rather than from nonspecific cell-wall perturbations. Als5p remodeling is independent of cellular metabolic activity because heat-killed cells show the same behavior as live cells. Using AFM and fluorescence microscopy, we also find that nanodomains are formed within ∼30 min and migrate at a speed of ∼20 nm·min(-1), indicating that domain formation and propagation are slow, time-dependent processes. These results demonstrate that mechanical stimuli can trigger adhesion nanodomains in fungal cells and suggest that the force-induced clustering of adhesins may be a mechanism for activating cell adhesion.

The present invention relates to azo dyes, in particular azo dyes of Formula (I) or (II), a tautomer, a quaternary amine, or a salt thereof, wherein R1, R2a, R2b, R3, R4, R5, R3, R7, R8, R9, R10, R11 have the same meaning as in the claims. The present invention also relates to processes for preparing said compounds, compositions containing them and their use as dyes.

Around 40 P-type ATPases have been identified in Arabidopsis and rice, for which the genomes are known. None seems to exchange sodium and potassium, as does the animal Na(+)/K(+)-ATPase. Instead, plants, together with fungi, possess a proton pumping ATPase (H(+)-ATPase), which couples ATP hydrolysis to proton transport out of the cell, and so establishes an electrochemical gradient across the plasma membrane, which is dissipated by secondary transporters using protons in symport or antiport, as sodium is used in animal cells. Additional functions, such as stomata opening, cell growth, and intracellular pH homeostasis, have been proposed. Crystallographic data and homology modeling suggest that the H(+)-ATPase has a broadly similar structure to the other P-type ATPases but has an extended C-terminal region, which is involved in enzyme regulation. Phosphorylation of the penultimate residue, a Thr, and the subsequent binding of regulatory 14-3-3 proteins result in the formation of a dodecamer (six H(+)-ATPase and six 14-3-3 molecules) and enzyme activation. This type of regulation is unique to the P-type ATPase family. However, the recent identification of additional phosphorylated residues suggests further regulatory features.

The combination of atomic force microscopy (AFM) and the Langmuir trough technique was used in this work to investigate the molecular interactions of fengycin with lipid monolayers constituted of the major lipid classes found in human stratum corneum (SC). AFM imaging of spread SC lipids/fengycin monolayers showed that fengycin preferentially partitions into cholesterol-rich phases surrounding 2D domains mainly constituted of ceramide and fatty acid molecules. Penetration experiments of fengycin from the subphase into SC-mimicking monolayers clearly indicated that the lipopeptide insertion at the lipid interface is enhanced in the presence of cholesterol. AFM analysis of mixed SC lipids/fengycin monolayers obtained after lipopeptide penetration revealed that cholesterol strongly interacts with fengycin and undergoes specific molecular interactions with more disordered, loosely packed ceramide molecules. These results highlight the capacity of fengycin to interact with the lipid constituents of the extracellular matrix of SC and, in particular, with cholesterol.

Conjugation, mobilisation and retromobilisation are three related mechanisms of horizontal gene transfer in bacteria. They have been extensively studied in Gram-negative species, where retromobilisation, the capture of DNA from a recipient by a donor cell, was shown to result from two successive steps: the transfer of the conjugative plasmid from the donor to the recipient, followed by the retrotransfer of the mobilisable plasmid to the donor. This successive model was established for Gram-negative bacteria but was lacking experimental data from the Gram-positive counterparts. In the present work, mobilisation and retromobilisation abilities of the conjugative plasmid pXO16 from Bacillus thuringiensis sv israelensis were studied using the mobilisable plasmids pUB110 and pE194 and the "non-mobilisable" element pC194 lacking the mob and oriT features (all from Staphylococcus aureus). Experimental data suggested a successive model since different retromobilisation frequencies were observed between the small plasmids. More importantly, retromobilisation was shown to be delayed by 50 and 150 min for pUB110 and pE194, respectively, compared to pXO16 conjugation. Natural liquid foods (cow milk, soy milk and rice milk) were used to evaluate the putative ecological impact of these transfers. In cow and soy milk, conjugation, mobilisation and retromobilisation were shown to occur at frequencies of 8.0 x 10(-1), 1.0 x 10(-2) and 1.2 x 10(-4) transconjugants per recipient, respectively. These data are comparable to those obtained in LB medium and about 10-fold lower than in the case of rice milk. Taken together, these results emphasize the potential role of plasmid capture played by B. thuringiensis in natural environments.

The effects of abscisic acid (ABA) on aquaporin content, root hydraulic conductivity (Lpr), whole-plant hydraulic conductance and leaf growth are controversial. We addressed these effects via a combination of experiments at different scales of plant organisation, and tested their consistency via a model. We analysed under moderate water deficit a series of transformed maize (Zea mays L.) lines, one sense and three antisense, affected on the NCED gene expression and which differed in the concentration of ABA in the xylem sap. In roots, the mRNA expression of most aquaporin PIP genes was increased in S plants and decreased in AS plants. The same pattern was observed for the protein contents of 4 PIPs. This resulted in more than 6-fold differences between lines in Lpr under both hydrostatic and osmotic gradients of water potential. This effect was probably due to differences in aquaporin activity because it was nearly abolished by an H2O2 treatment, which blocks the water channel activity of aquaporins. The hydraulic conductance of intact whole plants was affected in the same way when measured either in steady state conditions or via the rate of recovery of leaf water potential after rewatering. The recoveries of leaf water potential and elongation upon rehydration differed between lines and were accounted for by the experimentally measured Lpr in a model of water transfer. Overall, these results suggest that ABA has long-lasting effects on plant hydraulic properties via aquaporin activity, which contributes to maintenance of a favourable plant water status.

The proton pump ATPase (H+-ATPase) of the plant plasma membrane is regulated by an auto-inhibitory C-terminal domain, which can be displaced by phosphorylation of the penultimate Thr residue and the subsequent binding of 14-3-3 proteins. We performed a mass spectrometric analysis of plasma membrane H+-ATPase isoform 2 (PMA2) isolated from Nicotiana tabacum suspension cells and identified two new phosphorylated residues in the enzyme 14-3-3 protein binding site: Thr931 and Ser938. When PMA2 was expressed in Saccharomyces cerevisiae, mutagenesis of each of these two residues into Asp prevented growth of a yeast strain devoid of its own H+-ATPases. When the Asp mutations were individually introduced in a constitutively activated mutant of PMA2 (Glu14Asp), they still allowed yeast growth, but at a reduced rate. Purification of His-tagged PMA2 showed that the Thr931Asp or Ser938Asp mutation prevented 14-3-3 protein binding, although the penultimate Thr955 was still phosphorylated, indicating that Thr955 phosphorylation is not sufficient for full enzyme activation. Expression of PMA2 in an N. tabacum cell line also showed absence of 14-3-3 protein binding resulting from the Thr931Asp or Ser938Asp mutation. Together, the data show that activation of H+-ATPase by the binding of 14-3-3 proteins is negatively controlled by phosphorylation of two residues in the H+-ATPase 14-3-3 protein binding site. The data also show that phosphorylation of the penultimate Thr and 14-3-3 binding each contribute in part to H+-ATPase activation.

Actively growing extraradical hyphae extending from mycorrhizal plants are an important source of inoculum in soils which has seldom been considered in vitro to inoculate young plantlets. Seedlings of Medicago truncatula were grown in vitro in the extraradical mycelium network extending from mycorrhizal plants. After 3, 6, 9, 12, and 15 days of contact with the mycelium, half of the seedlings were harvested and analyzed for root colonization. The other half was carefully transplanted in vitro on a suitable growth medium and mycelium growth and spore production were evaluated for 4 weeks. Seedlings were readily colonized after 3 days of contact with the mycelium. Starting from 6 days of contact, the newly colonized seedlings were able to reproduce the fungal life cycle, with the production of thousands of spores within 4 weeks. The fast mycorrhization process developed here opens the door to a broad range of in vitro studies for which either homogenous highly colonized seedlings or mass-produced in vitro inoculum is necessary.

Sna4p, a vacuolar membrane protein, belongs to a small family of proteins conserved in plant and fungi. It is transported to the vacuolar membrane via the alkaline phosphatase (ALP) pathway, which bypasses the multivesicular bodies (MVB). Here, we show that transfer of Sna4p by the ALP route involves the AP-3 adaptor protein complex, which binds to an acidic di-leucine sorting signal in the cytoplasmic region of Sna4p. In addition, Sna4p can use the MVB pathway using a PPPY motif, which is involved in the interaction with ubiquitin ligase Rsp5p. Deletion or mutation of the Sna4p PPPY motif or a low level of Rsp5p inhibits the entrance of Sna4p into MVBs. Sna4p is polyubiquitylated on its only lysine and Sna4p lacking this lysine shows defective MVB sorting. These data indicate that Sna4p has two functional motifs, one for interaction with the AP-3 complex, followed by entry into the ALP pathway, and one for binding Rsp5p, which directs the protein to the MVB pathway. The presence of these two motifs allows Sna4p to localise to both the vacuolar membrane and lumen.

Although the chemical composition of mycobacterial cell walls is well known, the 3D organization of the various constituents is not fully understood. In particular, it is unclear whether the major wall component lipoarabinomannan (LAM) is exposed on the outermost surface or hindered by other constituents such as mycolic acids. To address this pertinent question, we used atomic force microscopy (AFM) with tips bearing anti-LAM antibodies to detect single LAM molecules on Mycobacterium bovis BCG cells. First, we showed the ability of anti-LAM tips to detect isolated, purified LAM molecules. We then mapped the distribution of LAM on mycobacteria, prior to and after treatment with the drug isoniazid. We found that LAM was not exposed on the surface of native cells, pointing to the presence of a homogeneous layer of mycolic acids, whereas it was greatly exposed on isoniazid-treated cells, in agreement with the action mode of the drug. This single-molecule study provides novel insight into the architecture of mycobacterial cell walls and offers promising perspectives for understanding the action modes of antimycobacterial drugs.

Chicory (Cichorium intybus) roots contain high amounts of inulin, a fructose polymer used as a storage carbohydrate by the plant and as a human dietary and prebiotic compound. We performed 2-D electrophoretic analysis of proteins from root material before the first freezing period. The proteins were digested with trypsin and the peptides analyzed by MS (MALDI-TOF/TOF). From the 881 protein spots analyzed, 714 proteins corresponded to a database accession, 619 of which were classified into functional categories. Besides expected proteins (e.g. related to metabolism, energy, protein synthesis, or cell structure), other well-represented categories were proteins related to folding and stability (49 spots), proteolysis (49 spots), and the stress response (67 spots). The importance of abiotic stress response was confirmed by the observation that 7 of the 21 most intense protein spots are known to be involved in cold acclimation. These results suggest a major effect of the low temperature period that preceded root harvesting.

Cell surface polysaccharides have an established role as virulence factors in human bacterial pathogens. Less documented are the biosynthesis and biological functions of surface polysaccharides in beneficial bacteria. We identified a gene cluster that encodes the enzymes, regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the well-documented probiotic strain Lactobacillus rhamnosus GG. Subsequent mutation of the welE gene encoding the priming glycosyltransferase within this cluster, and comparative phenotypic analyses of wild-type versus mutant confirmed the specific function of this gene cluster in the biosynthesis of high molecular weight galactose-rich heteropolymeric EPS molecules. The phenotypic analyses included monomer composition determination, polymer length estimation of the isolated EPS molecules and single-molecule force spectroscopy of the surface polysaccharides. Further characterization of the welE mutant also showed that deprivation of these long galactose-rich EPS molecules results in an increased adherence and biofilm formation capacity of L. rhamnosus GG, possibly by less shielding of adhesins such as fimbriae-like structures.

The presence of pXO1- and/or pXO2-like plasmid(s) in clinical isolates of B. cereus (sensu stricto) and in strains of the biopesticide Bacillus thuringiensis have been reported recently, and the pXO2-like plasmid pBT9727 was found conjugative as well as another pXO2-like plasmid pAW63. In this study, a total of 1,000 B. cereus group isolates were analyzed for the presence of pXO1- and pXO2-like replicons and for pXO2-related conjugative modules. The occurrence frequency of pXO1- and pXO2-like replicons in random environmental samples was ca. 6.6% and 7.7%, respectively, and ca. 1.54% of the strains were positive for pXO2-like transfer module genes. Only the strains harboring a pXO2-like replicon also contained the corresponding transfer genes. For the strains which contained pXO1- and/or pXO2-like replicon(s), large plasmid(s) with a size similar to that of pXO1-like and/or pXO2-like plasmids were also observed, but none of these isolates were found to carry the B. anthracis toxin or capsule virulence genes. Furthermore, 17 out of 22 pXO2-like plasmids containing the transfer modules displayed the capability of self-transfer and of mobilizing small plasmids. No pXO1-, or pXO2-like plasmid lacking the cognate transfer modules has been found to have transfer potential. Among the strains possessing the putative pXO2-like conjugative apparatus, variations on the presence of the group II introns B.th.I.1 and B.th.I.2 were observed, suggesting an important flexibility of the conjugation modules and their regulation. There was no consistent correlation between pXO2-like repA dendrogram and presence/absence of tra region, or between virB4 dendrogram and transfer capabilities. Discrepancies between pXO2-like repA and virB4 dendrograms were also observed, indicating that the evolution of pXO2 is an active process.

BACKGROUND: Early radiological tumor shrinkage may be associated with better long-term outcome in chemorefractory metastatic colorectal cancer (cmCRC) treated with cetuximab. We aimed at validating this in a large and independent series. PATIENTS AND METHODS: Of the 329 patients, 289 had a measurement both at baseline and week 6. Tumor shrinkage was expressed as a relative decrease compared with baseline and categorized according to a previously reported cut-off value ( approximately 10%) or used as a continuous variable. RESULTS: Median time to progression (TTP) was 6.1 [95% confidence interval (CI) 5.1-7.2] versus 1.5 months (95% CI 1.4-1.7) in patients with [99 patients (34.3%)] or without [190 patients (65.7%)] tumor shrinkage, respectively, at week 6 [hazard ratio (HR) 0.23 (95% CI 0.17-0.32)]. The median overall survival (OS) was 13.7 (CI NA) versus 6.9 months (95% CI 6.1-7.7) [HR 0.21 (95% CI 0.14-0.32)], respectively. In a multivariate model, early tumor decrease outperformed skin toxicity as a predictor of long-term outcome. CONCLUSIONS: Tumor shrinkage at 6 weeks is a strong predictor of TTP and OS in cmCRC patients treated with cetuximab with or without irinotecan. This suggests early tumor shrinkage is the hallmark of efficacy of cetuximab and reliably identifies the subpopulation that is sensitive to the drug. Early tumor shrinkage can be used as a marker of efficacy in clinical practice, as such or in combination.

Rhizosecretion is an attractive technology for the production of recombinant proteins from transgenic plants. However, to date, yields of plant-derived recombinant pharmaceuticals by this method have been too low for commercial viability. Studies conducted focused on three transgenic plant lines grown in hydroponic culture medium, two expressing monoclonal antibodies Guy's 13 and 4E10 and one expressing a small microbicide polypeptide cyanovirin-N. Rhizosecretion rates increased significantly by the addition of the plant growth regulator alpha-naphthalene acetic acid. The maximum rhizosecretion rates achieved were 58 microg/g root dry weight/24 h for Guy's 13, 10.43 microg/g root dry weight/24 h for 4E10, and 766 microg/g root dry weight/24 h for cyanovirin-N, the highest figures so far reported for a full-length antibody and a recombinant protein, respectively. The plant growth regulators indole-butyric acid, 6-benzylaminopurine, and kinetin were also demonstrated to increase rhizosecretion of Guy's 13. The effect of the growth regulators differed, as alpha-naphthalene acetic acid and indole-butyric acid increased the root dry weight of hydroponic plants, whereas the cytokinins benzylaminopurine and kinetin increased rhizosecretion without affecting root mass. A comparative glycosylation analysis between MAb Guy's 13 purified from either hydroponic culture medium or from leaf extracts demonstrated a similar pattern of glycosylation comprising high mannose to complex glycoforms. Analysis of the hydroponic culture medium at harvest revealed significantly lower and less complex levels of proteolytic enzymes, in comparison with leaf extracts, which translated to a higher proportion of intact Guy's 13 IgG in relation to other IgG products. Hydroponic medium could be added directly to a chromatography column for affinity purification, allowing simple and rapid production of high purity Guy's 13 antibody. In addition to the attractiveness of controlled cultivation within

Understanding the molecular interactions between bacterial adhesion proteins (adhesins) and their receptors is essential for elucidating the molecular mechanisms of bacterial pathogenesis. Here, atomic force microscopy (AFM) is used to explore the specific interactions between the heparin-binding hemagglutinin (HBHA) from Mycobacterium tuberculosis, and heparan sulphate proteoglycan (HSPG) receptors on live A549 pneumocytes. First, we show that the specific binding forces between single HBHA-HSPG pairs, 57+/-16 pN, are similar to the forces measured earlier between HBHA and heparin molecules. Second, we mapped the distribution of single HSPG receptors on the surface of A549 cells, revealing that the proteins are widely and homogeneously exposed. Third, we observed force curves with constant force plateaus at large pulling velocities, reflecting the extraction of membrane tethers or nanotubes. These single-molecule measurements provide new avenues in pathogenesis research, particularly for elucidating the molecular basis of pathogen-host interactions.

The fungal lipid-translocating exporter family consists of conserved membrane proteins, with six or seven transmembrane spans. Phylogenetic trees and conserved gene order relationships show that the common ancestor of five closely related hemiascomycetous yeast species contained the RSB1 and PUG1 paralogous genes. In Saccharomyces cerevisiae, Rsb1 functions as a transporter or translocase of sphingoid bases, whereas Pug1 facilitates the inducible transport of protoporphyrin IX and hemin. The budding yeast contains two other paralogs, Ylr046p, of unknown function, and Rta1p, overexpression of which confers resistance to an ergosterol biosynthesis inhibitor. Large-scale mRNA expression profiling has shown that transcription of PUG1, RTA1 and YLR046 is induced under hypoxic conditions. Ergosterol biosynthesis is impaired under low-oxygen conditions as a consequence of the decreased synthesis of heme and heme-containing proteins. These genes may encode transporters or sensors that facilitate the excretion of excessive or aberrant biosynthetic intermediates, either directly or indirectly. The expression of RSB1 and RTA1 is under the control of pleiotropic drug resistance transcription factors, suggesting that the encoded proteins may have additional roles in cell resistance to xenobiotics. This review summarizes current knowledge concerning the lipid-translocating exporter family and its potential functions, focusing on multidrug resistance and membrane phospholipid homeostasis.

The denitration of 2,4,6-trinitrotoluene (TNT) can produce mono- or dinitro aromatic compounds susceptible to microbial mineralization. In the present study, denitration of TNT and other nitro aromatic compounds was investigated with a solid-phase extract obtained from the culture supernatant of Pseudomonas aeruginosa ESA-5 grown on a chemically defined aerobic medium. When the C18 solid-phase extract containing extracellular catalysts (EC) was incubated with TNT and NAD(P)H, we observed a significant release of nitrite. The concentration of nitrite released in the reaction medium was strongly dependent on the concentration of NAD(P)H and EC. Denitration also occurred with two TNT-related molecules, 2,4,6-trinitrobenzaldehyde, and 2,4,6-trinitrobenzyl alcohol. The release of nitrite was coupled with the formation of two polar metabolites, and mass spectrometry analyses indicated that each of these compounds had lost two nitro groups from the trinitro aromatic parent molecule. During this process, the production of toxic reduced TNT metabolites was minimal. The incubation of EC with TNT, NAD(P)H, and specific scavengers of reactive oxygen species suggested the involvement of superoxide radicals (O2*-) and hydrogen peroxide in the denitration process. Results obtained in this study reveal for the first time that extracellular small-molecular-weight substance(s) of bacterial origin can serve as green catalyst(s) to initiate TNT denitration. In addition, this study gives clear evidence for the production of a TNT metabolite bearing a single nitro groupfollowing a denitration reaction with catalyst(s) of biotic origin.

Despite their widespread occurrence in soils, the ecology of Acidobacteria and their response to environmental perturbations due to human activities remain very poorly documented. This study was aimed at assessing changes in the diversity and abundance of Acidobacteria in soils contaminated with 2,4,6-trinitrotoluene (TNT) compared with nonpolluted soils. The analysis of Acidobacteria communities at two sites with long-term and short-term contamination revealed that TNT has a drastic impact on the relative abundance of Acidobacteria in soil bacterial 16S rRNA gene libraries. The disappearance of most Acidobacteria from these soils was concomitant with a shift in Acidobacteria community composition and a loss of diversity, although the extent of diversity erosion depended on the sampling site.

A laccase from the aquatic ascomycete Phoma sp. UHH 5-1-03 (DSM 22425) was purified upon hydrophobic interaction and size exclusion chromatography (SEC). Mass spectrometric analysis of the laccase monomer yielded a molecular mass of 75.6 kDa. The enzyme possesses an unusual alkaline isoelectric point above 8.3. The Phoma sp. laccase undergoes pH-dependent dimerisation, with the dimer ( approximately 150 kDa, as assessed by SEC) predominating in a pH range of 5.0 to 8.0. The enzyme oxidises common laccase substrates still at pH 7.0 and 8.0 and is remarkably stable at these pH values. The laccase is active at high concentrations of various organic solvents, all together indicating a considerable biotechnological potential. One laccase gene (lac1) identified at the genomic DNA level and transcribed in laccase-producing cultures was completely sequenced. The deduced molecular mass of the hypothetical protein and the predicted isoelectric point of 8.1 well agree with experimentally determined data. Tryptic peptides of electrophoretically separated laccase bands were analysed by nano-liquid chromatography-tandem mass spectrometry. By using the nucleotide sequence of lac1 as a template, eight different peptides were identified and yielded an overall sequence coverage of about 18%, thus confirming the link between lac1 and the expressed laccase protein.

Sna3p and Sna4p are small proteins of unknown function possessing two transmembrane domains and belong to a small family of conserved proteins present in plant and fungi. The budding yeast has four SNA proteins (Sna1–4) that have different localizations in the cell. Sna3p is targeted to the vacuolar lumen by the multivesicular body pathway. Two observations marked Sna3p as a multivesicular body cargo that is sorted in an ubiquitin-independent manner. First, Sna3p-GFP is still correctly transported to internal multivesicular body vesicles under conditions of ubiquitin depletion, which impairs multivesicular body sorting of certain other cargoes. Second, a mutant form of Sna3p-GFP lacking the only potential positions for ubiquitylation is still correctly targeted to the vacuolar lumen. It has thus been postulated that ubiquitylation marks, but not all, membrane proteins for sorting into the interior of the vacuole. In this study we present a further characterization of the Golgi to vacuole trafficking of Sna3p together with its ubiquitylation status. We observed that Sna3p physically interacts with the E3 ligase Rsp5p and that this interaction is essential for sorting of Sna3p to the endosomal pathway. Sna3p is ubiquitylated on its Lys125 residue by Rsp5p and modified by Lys 63-linked ubiquitin chains. In contrast to the conclusions from prior reports, we demonstrated that, as noticed for most other multivesicular body cargoes, Sna3p ubiquitylation is required for its multivesicular body sorting. Sna4p is localized to the vacuolar membrane and interior. Sna4p contains an acidic di-leucine motif, that could be a sorting signal specific for AP-3 dependent pathway directing Sna4p to the vacuolar membrane. In apm3∆ cells, where µ subunit of the AP-3 complex is deleted, Sna4p is missorted to the vacuolar interior. Strikingly, this localization is different from localization of markers of AP-3 dependent pathway. This dissimilarity indicates that Sna4p possesses an additiona, (AGRO 3) -- UCL, 2009

Summary The Arabidopsis gene At2g47770 encodes a membrane-bound protein designated AtTSPO (Arabidopsis thaliana TSPO-related). AtTSPO is related to the bacterial outer membrane tryptophan-rich sensory protein (TspO) and the mammalian mitochondrial 18 kDa Translocator Protein (18 kDa TSPO), members of the TspO/MBR domain-containing membrane proteins. In this study we showed that AtTSPO is mainly detected in dry seeds, but can be induced in vegetative tissues by osmotic or salt stress or abscisic acid treatment, corroborating available transcriptome data. Using subcellular fractionation, immunocytochemistry, and fluorescent protein tagging approaches we present evidence that AtTSPO is targeted to the secretory pathway in plant. Induced or constitutively expressed AtTSPO can be detected in the endoplasmic reticulum and the Golgi stacks of plant cell. AtTSPO tagged with fluorescent protein in transgenic plant (Arabidopsis and tobacco) was mainly detected in Golgi stacks of leaf epidermal cells. Constitutive expression of AtTSPO resulted in increased sensitivity to NaCl, but not to osmotic stress, and in reduced greening of cultured Arabidopsis cells under light growing conditions. Transgenic Arabidopsis plants overexpressing AtTSPO were more sensitive to ABA-induced growth inhibition, indicating that constitutive expression of AtTSPO may enhance ABA sensitivity. AtTSPO is rapidly downregulated during seeds imbibition, and the ABA-dependent induction in plant is transient. Downregulation of AtTSPO seems to be boosted by aminolevulenic acid treatment. Taken together, these results suggest that AtTSPO is a highly regulated protein, induced by abiotic stress to modulate, at least in part, transient intracellular ABA-dependent stress perception and/or signalling.

PURPOSE: Our aim was to compare perfusion magnetic resonance imaging (MRI) and positron emission tomography (PET) using carbon-11 labelled methionine (MET) in gliomas and their value in differentiating tumour recurrence from necrosis. MATERIALS AND METHODS: We retrospectively reviewed 28 patients with a high-grade glioma. A total of 33MR perfusions and MET-PET were ultimately analysable for comparison between the relative cerebral blood volume (rCBV) and MET-PET examinations. Intra- and interobserver reproducibility was assessed and diagnostic value of rCBV compared to MET-PET and histology was assessed by the area under the receiver operating characteristic (ROC) curve. RESULTS: ROC curve analysis showed that rCBV had at least equal performances in differentiating tumour recurrence and necrosis than MET-PET. Cut-off value of rCBV for differentiating tumour from necrosis was 182% with a sensitivity of 81.5% and a specificity of 100%. CONCLUSION: In clinical practice, perfusion MRI could replace MET-PET for differentiating necrosis from tumour recurrence.

Crystalline bacterial cell surface layers (S-layers) are monomolecular arrays of (glyco)proteins that have recently produced a wealth of new opportunities in nanotechnology. Whereas the in vitro imaging of isolated S-layers is well established, their direct imaging on live cells remains very challenging. Here we use atomic force microscopy (AFM) to visualize S-layer nanoarrays on living Corynebacterium glutamicum bacteria. We demonstrate the presence of two highly ordered surface layers. The most external layer represents the hexagonal S-layer, and the inner layer displays regular patterns of nanogrooves that could act as a biomolecular template promoting the 2-D assembly of S-layer monomers. These nanoscale analyses open new avenues for understanding the structure of protein monomolecular arrays, which is a crucial challenge in current nanoscience and life science research.

Genes encoding the heavy and light chains of LO-BM2, a therapeutic IgG antibody, were assembled in the tandem or inverted convergent orientation and expressed in Nicotiana tabacum plants and BY-2 suspension cells. The tandem construct allowed higher expression in both expression systems. A similar degradation pattern was observed for the secreted antibody recovered from the leaf intercellular fluid and BY-2 culture medium. Degradation increased with leaf age or culture time. Antibodies purified from leaf tissues and BY-2 cells were both functional. However, MS analysis of the N-glycosylation showed complex plant-type glycans to be the major type in the antibody purified from plants, whereas, oligomannosidic was the major glycosylation type in that purified from BY-2 cells. LO-BM2 was observed mainly in the endoplasmic reticulum of BY-2 cells while, in leaf cells, it was localized mostly to vesicles resembling prevacuolar compartments. These results and those from endoglycosidase H studies suggest that LO-BM2 is secreted from BY-2 cells more readily than from leaf cells where it accumulates in a post-Golgi compartment.

Laccase from the white rot fungus strain Coriolopsis polyzona was immobilized covalently on the diatomaceous earth support Celite((R)) R-633 using different strategies. A first methodology involved the sequential activation of the support surface with gamma-aminopropyltriethoxysilane followed by the reaction of the functionalized surface with glutaraldehyde (GLU) or glyoxal (GLY) and the immobilization of laccase on the activated surface. Another strategy tested the simultaneous internal cross-linking of the protein with GLU or GLY and the immobilization of the laccase on the silanized surface. Finally, these two strategies were modified to test the impact of the concomitant addition of bovine serum albumin (BSA) as a stabilizing agent during the immobilization steps. The highest laccase activity and the greatest degree of activity recovery (tested using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as the substrate) were achieved by the sequential immobilization procedure using GLU as the cross-linking agent. The solid catalysts featuring internal cross-linking of the protein showed significantly higher stability against several denaturants. The Michaelis-Menten kinetic parameters with respect to ABTS revealed a higher affinity for this substrate in the case of the sequential procedure compared to the simultaneous approach. The biocatalyst formed using GLU in the sequential procedure was applied in a packed bed reactor for the continuous treatment of 5mgl(-1) solutions of the endocrine disrupting chemicals (EDCs) nonylphenol (NP), bisphenol A (BPA) and triclosan (TCS) through repeated batch treatments. All of these EDCs could be eliminated at a contact time of less than 200min by using, respectively, 3.75 units (U) of laccase activity for BPA and TCS and 1.88U for NP. These performances of elimination were maintained over five consecutive treatment cycles using the same biocatalyst. This system could also remove these EDCs from 100mgl(-1) solutions.

The knowledge of molecular mechanisms underlying the adhesive and mechanical properties of cell surface-associated molecules is a key to understanding their functions. In this context, single-molecule force spectroscopy (SMFS) has recently offered new opportunities for probing the adhesion and mechanics of polysaccharides and proteins on live cells. Here we present a protocol that we have used to analyze polysaccharide chains of different nature on the bacterium Lactobacillus rhamnosus GG. We describe procedures (i) for functionalizing atomic force microscopy (AFM) tips with Pseudomonas aeruginosa-I or concanavalin A lectins, (ii) for stretching specific polysaccharide molecules on live bacteria using SMFS with lectin tips and (iii) for mapping the localization, adhesion and extension of individual polysaccharide chains. We also discuss data treatment, emphasizing how to gain insight into the elasticity of the stretched macromolecules using the extended freely jointed chain model. Even though the presented protocol is for L. rhamnosus, it can be easily modified for other cell types. For users having expertise in the field, the entire protocol can be completed in about 5 d.

Mitochondria are metabolically highly active cell organelles that are also implicated in reactive oxygen species production and in cell death regulation. Cyclophilin D, the only human mitochondrial isoform of cyclophilins, plays an essential role in the formation of the mitochondrial permeability transition pore leading to cell necrosis. Recently, it has been shown that redox environment modifies structural and functional properties of some plant cyclophilins. Here, it is shown that oxidation of human cyclophilin D influences the conformation of the enzyme but also its activity. Site-directed mutagenized variants of cyclophilin D allowed the identification of cysteine 203 as an important redox-sensitive residue. Moreover, the redox modulation of cyclophilin D was confirmed in human neuroblastoma SH-SY5Y cells exposed to oxidative stress. Altogether, our results suggest that cyclophilin D may play a role as a redox sensor in mitochondria of mammalian cells transmitting information on the redox environment to target proteins.

Root colonization by arbuscular mycorrhizal (AM) fungi is a dynamic process involving major changes in plant gene expression. Here, the expression of a phosphate transporter gene (PT3) and several defense genes, already known to be involved in the various stages of AM establishment, were monitored in the mycelium donor plant (MDP) in vitro culture system associating potato plantlets with an AM fungus. This system allows fast and homogenous mycorrhization of seedlings at their early stage of development by growing the plantlets in active mycelial networks, but has never been validated for gene expression analysis. Here, QRT-PCR analyses were conducted in parallel to pre- (1 day), early (2 and 3 days), and late (6, 9, and 15 days) stages of root colonization. We observed the induction of a plant gene marker of AM root colonization (PT3) at the late stage and the induction of MAPK and PAL genes at the early and late stages of root colonization. We also demonstrated the induction of PR1 and PR2 genes at pre- and late stages and of GST1 and Lox genes at a late stage of root colonization. These results validated the MDP in vitro culture system as an optimal tool to study gene expression analysis during the AM fungi establishment. This system further opened the door to investigate gene networks associated with the plants-AM fungi symbiosis.

Recent work has revealed the importance of polyfunctional thiols in young Sauternes wines, but very little is yet known about the fate of such compounds during aging in the bottle. In this study, two Sauternes wines were investigated by gas chromatography-olfactometry (GC-O) aroma extract dilution analysis (AEDA), gas chromatography-mass spectrometry (GC-MS), and gas chromatography-pulsed flame photometric detector (GC-PFPD) after XAD 2 and thiol-specific extractions. Most polyfunctional thiols (3-sulfanylpropyl acetate, 2-sulfanylethyl acetate, 3-methyl-3-sulfanylbutanal, etc.) proved to be completely degraded after 2 years of bottle aging in a cellar. Only 3-sulfanylhexan-1-ol was still found in aged samples at concentrations above its threshold value. Most other key odorants found in the young noble rot wine were still detected 5-6 years after harvest: varietal aroma (alpha-terpineol), sotolon, fermentation alcohols (3-methylbutan-1-ol and 2-phenylethanol) and esters (ethyl butyrate, isobutyrate, hexanoate, and isovalerate), and oak maturation-related compounds (guaiacol, vanillin, eugenol, beta-damascenone, trans-non-2-enal, beta-methyl-gamma-octalactone, gamma-nonalactone, and furaneol), as well as three newly identified aromas exhibiting interesting cake, honey-like, and dried apricot odors: homofuraneol, theaspirane, and gamma-decalactone. Interestingly, abhexon, never mentioned in sweet wines before, was found to be synthesized during bottle aging. An optimized extraction method allowed us to quantify this honey/spicy compound at levels close to its threshold value (up to 7 mug/L after 5-6 years), thus suggesting a key role of this strong odorant in old Sauternes wines.

Here we report on in vivo measurement of the mechanical behavior of a cell surface sensor using single-molecule atomic force microscopy. We focus on the yeast wall stress component sensor Wsc1, a plasma membrane protein that is thought to function as a rigid probe of the cell wall status. We first map the distribution of individual histidine-tagged sensors on living yeast cells by scanning the cell surface with atomic force microscopy tips carrying nitrilotriacetate groups. We then show that Wsc1 behaves like a linear nanospring that is capable of resisting high mechanical force and of responding to cell surface stress. Both a genomic pmt4 deletion and the insertion of a stretch of glycines in Wsc1 result in substantial alterations in protein spring properties, supporting the important role of glycosylation at the extracellular serine/threonine-rich region.

A method is described for the site-directed manipulation of single filamentous bacteriophages, by using phage display technology and atomic force microscopy. f1 filamentous bacteriophages were genetically engineered to display His-tags on their pIX tail. Following adsorption on nitrilotriacetate-terminated surfaces, force spectroscopy with tips bearing monoclonal anti-pIII antibodies was used to pull on individual phages via their pIII head. Analysis of the force-extension profiles revealed that upon pulling, the phages are progressively straightened into an extended orientation until rupture of the anti-pIII/pIII complex. The single-virus manipulation technique presented here provides new opportunities for understanding the forces driving cell-virus and material-virus interactions, and for characterizing the binding properties of polypeptide sequences or proteins selected by the phage display technology.

Analyzing microbial cell surface proteins is a challenging task in current microbial proteomic research, which has major implications for drug design, vaccine development, and microbial monitoring. In this context, atomic force microscopy (AFM) has recently emerged has a powerful characterization platform, providing valuable insights into the surface proteome of microbial cells. The aim of this article is to show how advanced AFM techniques, that all have in common functionalization of the AFM tip with specific molecules, can be used to answer pertinent questions related to surface-associated proteins, such as what is their spatial arrangement on the cell surface, and what are the forces driving their interaction with the environment?.

Banana (Musa spp.) is an important crop in the East African Highlands (EAH). Although root health and interaction with soil microorganisms are amongst its major yield determinants, they remain poorly investigated in this region. Understanding these relationships may help to improve yield. In this PhD thesis, we investigated the effect of plant parasitic nematodes, in particular, Praty-lenchus goodeyi, and arbuscular mycorrhizal (AM) fungi on the EAH banana. We demonstrated that P. goodeyi was the main species contributing to root necrosis. However, its abundance was positively correlated with high plant density and mulch. Pratylenchus goodeyi had limited impact on yields in flatter fields (crest and valley bottom) where soil conditions were more optimal for root growth, whereas on steep slopes root death was increased and yield was reduced even under moderate pressure from P. goodeyi. A field experiment showed that P. goodeyi had low impact on yield of highland banana (AAA-EA). Hence, the studies conducted at country, regional and field level suggested that root necrosis caused by P. goodeyi has very little impact on banana plant vigour. These results challenge the general perceptions and previous findings on the negative impact of root lesion nematodes on banana performance. Our studies showed that AM fungi were present in all the sites investigated, but their population density, diversity and root colonization highly varied as a function of rainfall, soil texture, P content, and tillage. The AM fungi colonized banana roots up to 120 cm depth, and plants with poor vigour were more dependent on this symbiotic association than the plants with good vigour. Root colonization decreased with soil depth and reduction in soil water content, and higher colonization lead to more vigorous root systems. Inoculation with AM fungi resulted in greatest plant growth in poor soils and varieties with least developed root system. Our findings suggest that AM fungi play an important r, (AGRO 3) -- UCL, 2009

Spaceflight experiments have suggested a possible effect of microgravity on the plasmid transfer among strains of the Gram-positive Bacillus thuringiensis, as opposed to no effect recorded for Gram-negative conjugation. To investigate these potential effects in a more affordable experimental setup, three ground-based microgravity simulators were tested: the Rotating Wall Vessel (RWV), the Random Positioning Machine (RPM), and a superconducting magnet. The bacterial conjugative system consisted in biparental matings between two B. thuringiensis strains, where the transfer frequencies of the conjugative plasmid pAW63 and its ability to mobilize the nonconjugative plasmid pUB110 were assessed. Specifically, potential plasmid transfers in a 0 g position (simulated microgravity) were compared to those obtained under 1 g (normal gravity) condition in each device. Statistical analyses revealed no significant difference in the conjugative and mobilizable transfer frequencies between the three different simulated microgravitational conditions and our standard laboratory condition. These important ground-based observations emphasize the fact that, though no stimulation of plasmid transfer was observed, no inhibition was observed either. In the case of Gram-positive bacteria, this ability to exchange plasmids in weightlessness, as occurs under Earth's conditions, should be seen as particularly relevant in the scope of spread of antibiotic resistances and bacterial virulence. Key Words: Bacillus thuringiensis-Conjugation-Microgravity-pAW63. Astrobiology 9, 797-805.

Aquaporins (AQPs) are channel proteins that facilitate and regulate the permeation of water across biological membranes. Black mMexican sweet suspension cultured cells are a convenient model for studying the regulation of maize AQP expression and activity. Among other advantages, a single cell system allows the contribution of plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) to the membrane water permeability coefficient (P(f)) to be determined using biophysical measurement methods, such as the cell pressure probe or protoplast swelling assay. We generated a transgenic cell culture line expressing a tagged version of ZmPIP2;6 and used this material to demonstrate that the ZmPIP2;6 and ZmPIP2;1 isoforms physically interact. This kind of interaction could be an additional mechanism for regulating membrane water permeability by acting on the activity and/or trafficking of PIP hetero-oligomers.

Friedreich's ataxia is a neurodegenerative disease caused by the low expression of frataxin, a mitochondrial iron-binding protein which plays an important, but non essential, role in the formation of iron-sulfur (Fe/S) clusters. It has been shown that Yfh1, the yeast frataxin homologue, interacts functionally and physically with Isu1, the scaffold protein on which the Fe/S clusters are assembled. The large beta-sheet platform of frataxin is a good ligand candidate for this interaction. We have generated 12 yeast mutants in conserved residues of the beta-sheet protruding at the surface or buried in the protein core. The Q129A, I130A, W131A(F) and R141A mutations, which reside in surface exposed residues of the 4(th) and 5(th) beta-strands, result in severe cell growth inhibition on high iron media and low aconitase activity, indicating that Fe/S cluster biosynthesis is impaired. The null phenotype of the I130A mutant results from the high instability of the protein, pointing that this buried residue is essential for folding. In contrast, Gln-129, Trp-131 and Arg-141 residues which are spatially closely clustered define a patch important for protein function. Co-immunoprecipitation experiments using cell extracts show that W131A, unlike W131F, is the sole mutation that strongly decreases the interaction with Isu1. Therefore, Trp-131, which is the only strictly conserved frataxin residue in all sequenced species, appears as a major contributor to the interaction with Isu1 through its surface-exposed aromatic side chain.