Role of the acquisition of a type 3 secretion system in the emergence of novel pathogenic strains of Xanthomonas

Cases of emergence of novel plant-pathogenic strains are regularly reported that reduce the yields of crops and trees. However, the molecular mechanisms underlying such emergence are still poorly understood. The acquisition by environmental non-pathogenic strains of novel virulence genes by horizontal gene transfer has been suggested as a driver for the emergence of novel pathogenic strains. In this study, we tested such an hypothesis by transferring a plasmid encoding the type 3 secretion system (T3SS) and four associated type 3 secreted proteins (T3SPs) to the non-pathogenic strains of Xanthomonas CFBP 7698 and CFBP 7700, which lack genes encoding T3SS and any previously known T3SPs. The resulting strains were phenotyped on Nicotiana benthamiana using chlorophyll fluorescence imaging and image analysis. Wild-type, non-pathogenic strains induced a hypersensitive response (HR)-like necrosis, whereas strains complemented with T3SS and T3SPs suppressed this response. Such suppression depends on a functional T3SS. Amongst the T3SPs encoded on the plasmid, Hpa2, Hpa1 and, to a lesser extent, XopF1 collectively participate in suppression. Monitoring of the population sizes in planta showed that the sole acquisition of a functional T3SS by non-pathogenic strains impairs growth inside leaf tissues. These results provide functional evidence that the acquisition via horizontal gene transfer of a T3SS and four T3SPs by environmental non-pathogenic strains is not sufficient to make strains pathogenic. In the absence of a canonical effector, the sole acquisition of a T3SS seems to be counter-selective, and further acquisition of type 3 effectors is probably needed to allow the emergence of novel pathogenic strains.     

Structural and functional studies of RegB, a new member of a family of sequence-specific ribonucleases involved in mRNA inactivation on the ribosome

The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome.     

Structural Characterization of N-WASP Domain V Using MD Simulations with NMR and SAXS Data

Because of their large conformational heterogeneity, structural characterization of intrinsically disordered proteins (IDPs) is very challenging using classical experimental methods alone. In this study, we use NMR and small-angle x-ray scattering (SAXS) data with multiple molecular dynamics (MD) simulations to describe the conformational ensemble of the fully disordered verprolin homology domain of the neural Aldrich syndrome protein involved in the regulation of actin polymerization. First, we studied several back-calculation software of SAXS scattering intensity and optimized the adjustable parameters to accurately calculate the SAXS intensity from an atomic structure. We also identified the most appropriate force fields for MD simulations of this IDP. Then, we analyzed four conformational ensembles of neural Aldrich syndrome protein verprolin homology domain, two generated with the program flexible-meccano with or without NMR-derived information as input and two others generated by MD simulations with two different force fields. These four conformational ensembles were compared to available NMR and SAXS data for validation. We found that MD simulations with the AMBER-03w force field and the TIP4P/2005s water model are able to correctly describe the conformational ensemble of this 67-residue IDP at both local and global level.     

Sugars en route to the roots. Transport,metabolism and storage within plant roots and towards microorganisms of the rhizosphere

In plants, the root is a typical sink organ that relies exclusively on the import of sugar from the aerial parts. Sucrose is delivered by the phloem to the most distant root tips and, en route to the tip, is used by the different root tissues for metabolism and storage. Besides, a certain portion of this carbon is exuded in the rhizosphere, supplied to beneficial microorganisms and diverted by parasitic microbes. The transport of sugars toward these numerous sinks either occurs symplastically through cell connections (plasmodesmata) or is apoplastically mediated through membrane transporters (MST, mononsaccharide tranporters, SUT/SUC, H+/sucrose transporters and SWEET, Sugar will eventually be exported transporters) that control monosaccharide and sucrose fluxes. Here, we review recent progresses on carbon partitioning within and outside roots, discussing membrane transporters involved in plant responses to biotic and abiotic factors.     

Heat-induced unfolding of neocarzinostatin, a small all-beta protein investigated by small-angle X-ray scattering

Neocarzinostatin is an all-beta protein, 113 amino acid residues long, with an immunoglobulin-like fold. Its thermal unfolding has been studied by small-angle X-ray scattering. Preliminary differential scanning calorimetry and fluorescence measurements suggest that the transition is not a simple, two-state transition. The apparent radius of gyration is determined using three different approaches, the validity of which is critically assessed using our experimental data as well as a simple, two-state model. Similarly, each step of data analysis is evaluated and the underlying assumptions plainly stated. The existence of at least one intermediate state is formally demonstrated by a singular value decomposition of the set of scattering patterns. We assume that the pattern of the solution before the onset of the transition is that of the native protein, and that of the solution at the highest temperature is that of the completely unfolded protein. Given these, actually not very restrictive, boundary constraints, a least-squares procedure yields a scattering pattern of the intermediate state. However, this solution is not unique: a whole class of possible solutions is derived by adding to the previous linear combination of the native and completely unfolded states. Varying the initial conditions of the least-squares calculation leads to very similar solutions. Whatever member of the class is considered, the conformation of this intermediate state appears to be weakly structured, probably less than the transition state should be according to some proposals. Finally, we tried and used the classical model of three thermodynamically well-defined states to account for our data. The failure of the simple thermodynamic model suggests that there is more than the single intermediate structure required by singular value decomposition analysis. Formally, there could be several discrete intermediate species at equilibrium, or an ensemble of conformations differently populated according to the temperature. In the latter case, a third state would be a weighted average of all non native and not completely unfolded states of the protein but, since the weights change with temperature, no meaningful curve is likely to be derived by a global analysis using the simple model of three thermodynamically well-defined states.     

Toward a role of dendritic cells in the germinal center reaction: triggering of B cell proliferation and isotype switching

We have reported previously that in vitro generated dendritic cells (DC) can directly regulate B cell responses. Recently, germinal center DC (GCDC) were identified within B cell follicles. Due to their particular localization, we have tested in the present study whether GCDC could contribute to key events characteristic of the GC reaction. Our present results demonstrate that 1) ex vivo GCDC induce a dramatic GC B cell expansion upon CD40 and IL-2 activation and drive plasma cell differentiation, 2) this property is shared by GCDC and blood DC, but not by Langerhans cells, 3) IL-12 production by GCDC is critical in GC B cell expansion and differentiation, and 4) importantly, GCDC also induce IL-10-independent isotype switching toward IgG1. These observations support the novel concept that GCDC directly contribute to the germinal center reaction.     

Three steroidogenic factor-1 binding elements are required for constitutive and cAMP-regulated expression of the human adrenocorticotropin receptor gene

In the present study, we characterized two new SF-1 binding sites, SF-209 and SF-98, in the promoter of the human ACTH receptor (hACTH-R) gene. Both sites, together with the previously described SF-35 site, are required for full constitutive activity of this gene. This was demonstrated by the use of constructs containing part of the promoter upstream of the luciferase gene and carrying mutation in one of these sites, to transiently transfect H295R cells. Mutations of either SF-35, SF-98, or SF-209 induced a decrease of luciferase activity. This effect was amplified when two or three elements were mutated together in the same construct. Only SF-35 and SF-98 seem to play a major role in the cAMP-induced regulation of the hACTH-R gene, since mutation of either one of these sites reduced the forskolin induction of luciferase activity by 50%. When both elements were mutated, no stimulation was obtained over the control. This indicates that SF-1 protein must bind to both sites for the cAMP response.     

Influence of the ionic strength on the heat-induced aggregation of the globular protein beta-lactoglobulin at pH 7

The influence of the ionic strength on the structure of beta-lactoglobulin aggregates formed after heating at pH 7 has been studied using static and dynamic light scattering. The native protein depletion has been monitored using size exclusion chromatography. Above a critical association concentration (CAC) well-defined clusters are formed containing about 100 monomers. The CAC increases with decreasing ionic strength. The so-called primary aggregates associate to form self similar semi-flexible aggregates with a large scale structure that is only weakly dependent on the ionic strength. The local density of the aggregates increases with increasing ionic strength. At a critical gel concentration, Cg, the size of the aggregates diverges. Cg decreases from 100 g/l without added salt to 1 g/l at 0.4M NaCl. For C > Cg the system gels except at high ionic strength close to Cg where the gels collapse under gravity and a precipitate is formed.     

Phase diagram of nucleosome core particles

We present a phase diagram of the nucleosome core particle (NCP) as a function of the monovalent salt concentration and applied osmotic pressure. Above a critical pressure, NCPs stack on top of each other to form columns that further organize into multiple columnar phases. An isotropic (and in some cases a nematic) phase of columns is observed in the moderate pressure range. Under higher pressure conditions, a lamello-columnar phase and an inverse hexagonal phase form under low salt conditions, whereas a 2D hexagonal phase or a 3D orthorhombic phase is found at higher salt concentration. For intermediate salt concentrations, microphase separation occurs. The richness of the phase diagram originates from the heterogeneous distribution of charges at the surface of the NCP, which makes the particles extremely sensitive to small ionic variations of their environment, with consequences on their interactions and supramolecular organization. We discuss how the polymorphism of NCP supramolecular organization may be involved in chromatin changes in the cellular context.     

The naphthoquinol oxidizing cytochrome bc1 complex of the hyperthermophilic knallgasbacterium Aquifex aeolicus: properties and phylogenetic relationships

Phylogenetic analysis of constituent proteins of Rieske/cytochrome b complexes [Schütz et al. (2000) J. Mol. Biol. 300, 663-675] indicated that the respective enzyme from the hyperthermophile Aquifex (A.) aeolicus is closely related to proteobacterial counterparts, in disagreement with positioning of its parent species on small subunit rRNA trees. An assessment of the details and possible reasons for this discrepancy necessitates a thorough understanding of the biochemical and biophysical properties of the enzyme in addition to the bioinformatic data. The cytochrome bc(1) complex from A. aeolicus, which is part of the "Knallgasreaction" pathway, was therefore studied in membranes and in detergent-solubilized, isolated complex. Hemes b(L) (E(m,7) = -190 mV; g(z)= 3.7), b(H) (E(m,7) = -60 mV; g(z )= 3.45), and c(1) (E(m,7) = +160 mV; g(z )= 3.55) were identified by EPR and optical spectroscopy in combination with electrochemical methods. Two electrochemically distinct (E(m,7) = +95 mV; E(m,7) = +210 mV) Rieske centers were detected in membranes, and the +210 mV species was shown to correspond to the Rieske center of the cyt bc(1) complex. The gene coding for this latter Rieske protein was heterologously expressed in Escherichia coli, and the resulting protein was characterized in detail. The pool quinone of A. aeolicus was determined to be naphthoquinone. The redox poises of the individual electron-transfer steps are compared to those of other Rieske/cyt b complexes. The Aquifex enzyme was found to represent the only extant naphthoquinol oxidizing true cyt bc(1) complex described so far. An improved scenario for the phylogenetic positioning of the Aquifex cyt bc(1) complex is proposed.