Dissection of Bacterial Wilt on Medicago truncatula Revealed Two Type III Secretion System Effectors Acting on Root Infection Process and Disease Development

Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease, which colonizes susceptible Medicago truncatula via the intact root tip. Infection involves four steps: appearance of root tip symptoms, root tip cortical cell invasion, vessel colonization, and foliar wilting. We examined this pathosystem by in vitro inoculation of intact roots of susceptible or resistant M. truncatula with the pathogenic strain GMI1000. The infection process was type III secretion system dependent and required two type III effectors, Gala7 and AvrA, which were shown to be involved at different stages of infection. Both effectors were involved in development of root tip symptoms, and Gala7 was the main determinant for bacterial invasion of cortical cells. Vessel invasion depended on the host genetic background and was never observed in the resistant line. The invasion of the root tip vasculature in the susceptible line caused foliar wilting. The avrA mutant showed reduced aggressiveness in all steps of the infection process, suggesting a global role in R. solanacearum pathogenicity. The roles of these two effectors in subsequent stages were studied using an assay that bypassed the penetration step; with this assay, the avrA mutant showed no effect compared with the GMI1000 strain, indicating that AvrA is important in early stages of infection. However, later disease symptoms were reduced in the gala7 mutant, indicating a key role in later stages of infection.     

Does dietary DHA improve neural function in children? Observations in phenylketonuria

Children with phenylketonuria (PKU) have a restricted protein intake and thus low dietary intakes of long-chain polyunsaturated fatty acids (LC-PUFA), which may cause subtle neurological deficits. We measured plasma phospholipid fatty acids and visual evoked potential (VEP) in 36 children with well-controlled PKU (6.3±0.6 years, 19 girls), before and after 3 months of supplementing fish oil capsules providing 15 mg docosahexaenoic acid (DHA)/kg daily. The motometric Rostock-Oseretzky Scale (ROS) was performed before and after supplementation in the 24 PKU children aged >4 years. VEP latencies and ROS were also assessed in omnivorous, age-matched controls without fish oil supply at baseline and after 3 months. Fish oil supply increased plasma phospholipid eicosapentaenoic acid (EPA) (0.40±0.03 vs 3.31±0.19%, p<0.001) and DHA (2.37±0.10 vs 7.05±0.24%, p<0.001), but decreased arachidonic acid (AA) (9.26±0.23 vs 6.76±0.16%, p<0.001). Plasma phenylalanine was unchanged. VEP latencies and ROS results significantly improved after fish oil in PKU children, but remained unchanged in controls. The improvements of VEP latencies, fine motor and coordination skills indicate that preformed n-3 LC-PUFA are needed for neural normalcy in PKU children. The optimal type and dose of supply still needs to be determined. Since PKU children are generally healthy and have normal energy and fatty acid metabolism, these data lead us to conclude that childhood populations in general require preformed n-3 LC-PUFA to achieve optimal neurological function.     

The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis

The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACP_Pks13). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACP_Pks13) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce α-alkyl β-ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening.Mycolic acids, α-branched and β-hydroxylated fatty acids of unusual chain length (C30-C90), are the hallmark of the Corynebacterineae suborder that includes the causative agents of tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). Members of each genus biosynthesize mycolic acids of specific chain lengths, a feature used in taxonomy. For example, Corynebacterium holds the simplest prototypes (C32-C36), called “corynomycolic acids,” which result from an enzymatic condensation between two regular size fatty acids (C16–C18). In contrast, the longest mycolates (C60-C90) are the products of condensation between a very long meromycolic chain (C40-C60) and a shorter α-chain (C22-C26) (1). These so-called “eumycolic acids” are found in mycobacteria and display various structural features present on the meromycolic chain. Eumycolic acids are major and essential components of the mycobacterial envelope where they contribute to the formation of the outer membrane (2, 3) that plays a crucial role in the permeability of the envelope. They also impact on the pathogenicity of some mycobacterial species (4).The first in vitro mycolate biosynthesis assays have been developed using Corynebacterium cell-wall extracts in the presence of a radioactive precursor (5, 6) and have brought key information about this pathway. Yet, any attempt to fractionate these extracts to identify the proteins involved has ended in failure. Later, enzymes catalyzing the formation of the meromycolic chain and the introduction of functions have been discovered with the help of novel molecular biology tools (for review, see Ref. 1), culminating with the identification of the putative operon fadD32-pks13-accD4 that encodes enzymes implicated in the mycolic condensation step in both corynebacteria and mycobacteria (see Fig. 1) (7–9). AccD4, a putative carboxyltransferase, associates at least with the AccA3 subunit to form an acyl-CoA carboxylase (ACC)³ complex that most likely activates, through a C₂-carboxylation step, the extender unit to be condensed with the meromycolic chain (see Fig. 1). In Corynebacterium glutamicum, the carboxylase would metabolize a C16 substrate (8, 10), whereas in M. tuberculosis the purified complex AccA3-AccD4 was shown to carboxylate C24-C26 acyl-CoAs (11). Furthermore, FadD32, predicted to belong to a new class of long-chain acyl-AMP ligases (FAAL) (12), is most likely required for the activation of the meromycolic chain prior to the condensation reaction. At last, the cmrA gene controls the reduction of the β-keto function to yield the final mycolic motif (13) (see Fig. 1).Open in a separate windowFIGURE 1.Proposed scheme for the biosynthesis of mycolic acids. The asymmetrical carbons of the mycolic motif have a R,R configuration. R₁-CO, meromycolic chain; R₂, branch chain. In mycobacteria, R₁-CO = C40-C60 and R₂ = C20-C24; in corynebacteria, R₁-CO = C16-C18 and R₂ = C14-C16; X₁, unknown acceptor of the mycolic α-alkyl β-ketoacyl chains; X₂, unknown acceptor of the mycolic acyl chains.Although the enzymatic properties of the ACC complex have been well characterized (9, 11), those of Pks13 and FadD32 are poorly or not described. Pks13 is a type I polyketide synthase (PKS) made of a minimal module holding ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domains, and additional N-terminal ACP and C-terminal thioesterase domains (Fig. 1). Its ACP domains are naturally activated by the 4′-phosphopantetheinyl (P-pant) transferase PptT (14). The P-pant arm has the general function of carrying the substrate acyl chain via a thioester bond involving its terminal thiol group. In the present article we report the purification of a soluble activated form of the large Pks13 protein. For the first time, the loading mechanisms of both types of substrates on specific domains of the PKS were investigated. We describe a unique catalytic mechanism of the Pks13-FadD32 enzymatic couple and the development of an in vitro condensation assay that generates the formation of α-alkyl β-ketoacids, the precursors of mycolic acids.     

Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin‐binding entities

Post‐translational modification with ubiquitin is one of the most important mechanisms in the regulation of protein stability and function. However, the high reversibility of this modification is the main obstacle for the isolation and characterization of ubiquitylated proteins. To overcome this problem, we have developed tandem‐repeated ubiquitin‐binding entities (TUBEs) based on ubiquitin‐associated (UBA) domains. TUBEs recognize tetra‐ubiquitin with a markedly higher affinity than single UBA domains, allowing poly‐ubiquitylated proteins to be efficiently purified from cell extracts in native conditions. More significant is the fact that TUBEs protect poly‐ubiquitin‐conjugated proteins, such as p53 and IκBα, both from proteasomal degradation and de‐ubiquitylating activity present in cell extracts, as well as from existing proteasome and cysteine protease inhibitors. Therefore, these new ‘molecular traps’ should become valuable tools for purifying endogenous poly‐ubiquitylated proteins, thus contributing to a better characterization of many essential functions regulated by these post‐translational modifications.     

Carbon and arsenic metabolism in Thiomonas strains: differences revealed diverse adaptation processes

Background  

Thiomonas strains are ubiquitous in arsenic-contaminated environments. Differences between Thiomonas strains in the way they have adapted and respond to arsenic have never been studied in detail. For this purpose, five Thiomonas strains, that are interesting in terms of arsenic metabolism were selected: T. arsenivorans, Thiomonas spp. WJ68 and 3As are able to oxidise As(III), while Thiomonas sp. Ynys1 and T. perometabolis are not. Moreover, T. arsenivorans and 3As present interesting physiological traits, in particular that these strains are able to use As(III) as an electron donor.     

Desulfosporosinus acidiphilus sp. nov.: a moderately acidophilic sulfate-reducing bacterium isolated from acid mining drainage sediments

An obligately anaerobic, spore-forming, acidophilic sulfate-reducing bacterium, strain SJ4^T, was isolated from an acid mining effluent decantation pond sediment sample (pH around 3.0). Cells were Gram negative, non-motile, curved rods occurring singly. Strain SJ4^T grew at pH 3.6–5.5 with an optimum at pH 5.2. Strain SJ4^T utilized H₂, lactate, pyruvate, glycerol, glucose, and fructose as electron donors. Lactate and glucose were weakly used. Sulfate was used as electron acceptors, but not sulfite, elemental sulfur, arsenate (V), and fumarate. The G + C content of genomic DNA was 42.3 mol% (HPLC). 16S rRNA gene sequence analysis indicated that strain SJ4^T belonged to the genus Desulfosporosinus within the family Peptococcaceae in the phylum Firmicutes. The level of 16S rRNA gene sequence similarity with other Desulfosporosinus species was 94.7–96.2%, D. orientis DSM 765^T (similarity of 96.2%) and D. auripigmenti DSM 13351^T (similarity of 95%) being its closest relatives. DNA–DNA relatedness values with D. orientis and D. auripigmenti were 16.5 and 31.8%, respectively. On the basis of phenotypic, phylogenetic, and genetic characteristics, strain SJ4^T represents a novel species within the genus Desulfosporosinus, for which the name Desulfosporosinus acidiphilus sp. nov. is proposed. The type strain is SJ4^T (=DSM 22704^T = JCM 16185^T).     

Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines

The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium-dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis.