Genome Sequence of Rickettsia bellii Illuminates the Role of Amoebae in Gene Exchanges between Intracellular Pathogens

The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair–long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic “melting pot” where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1-1 and VvLYK1-2 mediate chitooligosaccharide-triggered immunity

Chitin, a major component of fungal cell walls, is a well-known pathogen-associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM-RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM-RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin-induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1-1, -2, or -3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1-1 and VvLYK1-2 participate in chitin- and chitosan-triggered immunity and that VvLYK1-1 plays an important role in basal resistance against E. necator

Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V

International audienceThe dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (?10(9) g/mol). In this study, we selected a recombinant form, DSR-OK?1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OK?1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10?13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OK?1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis

Biochemical and Structural characterization of a new GH-70 enzyme from Leuconostoc citreum NRRL B-1299

International audienceGlucansucrases (GS) from glycoside hydrolase family 70 (GH70) are a-transglucosylases found in lactic acid bacteria such as Leuconostoc, Lactobacillus, Streptococcus and Weissella sp. They catalyze the polymerization of glucosyl residues from sucrose, an economical and abundant agroresource. Depending on the enzyme specificity, the α-glucans vary in terms of size, types of glucosidic bonds and degree of branching. This structure variety confers to α-glucans diverse properties, making them useful for several applications in food and pharmaceutical industries (Leemhuis et al., 2013). The sequencing of Leuconostoc citreum NRRL B-1299 genome allowed the identification of a new GS called DSR-M (Passerini et al., 2015). Whereas the majority of GS produce glucans of very high molar mass (105 to 108 Da), this enzyme synthesizes only a low molar mass dextran (around 27kDa, from 100 g/L sucrose) composed exclusively of a-(1→6) linkages. Now, by playing with the initial sucrose concentration and/or the reaction temperature, different dextrans of controlled molar mass ranging from 5 to 27 kDa can be produced, directly from sucrose and with excellent yields (Vuillemin et al. 2015). In parallel, we solved the 3D atomic structure of a truncated form of this enzyme comprising domains A, B, C and IV as well as most of the domain V also called Glucan Binding Domain. The crystal structure at 3.8 Å of the inactive mutant DSRM-D2-E715Q in complex with isomaltohexaose (IM6) (Fig. 1.) allowed us to identify new determinants, both near the catalytic site and in the domain V, that play a critical role in the molar masses of the dextran produced (from 1 to 15 kDa)

Biochemical and Structural characterization of a new GH-70 enzyme from Leuconostoc citreum NRRL B-1299

International audienceGlucansucrases (GS) from glycoside hydrolase family 70 (GH70) are a-transglucosylases found in lactic acid bacteria such as Leuconostoc, Lactobacillus, Streptococcus and Weissella sp. They catalyze the polymerization of glucosyl residues from sucrose, an economical and abundant agroresource. Depending on the enzyme specificity, the α-glucans vary in terms of size, types of glucosidic bonds and degree of branching. This structure variety confers to α-glucans diverse properties, making them useful for several applications in food and pharmaceutical industries (Leemhuis et al., 2013). The sequencing of Leuconostoc citreum NRRL B-1299 genome allowed the identification of a new GS called DSR-M (Passerini et al., 2015). Whereas the majority of GS produce glucans of very high molar mass (105 to 108 Da), this enzyme synthesizes only a low molar mass dextran (around 27kDa, from 100 g/L sucrose) composed exclusively of a-(1→6) linkages. Now, by playing with the initial sucrose concentration and/or the reaction temperature, different dextrans of controlled molar mass ranging from 5 to 27 kDa can be produced, directly from sucrose and with excellent yields (Vuillemin et al. 2015). In parallel, we solved the 3D atomic structure of a truncated form of this enzyme comprising domains A, B, C and IV as well as most of the domain V also called Glucan Binding Domain. The crystal structure at 3.8 Å of the inactive mutant DSRM-D2-E715Q in complex with isomaltohexaose (IM6) (Fig. 1.) allowed us to identify new determinants, both near the catalytic site and in the domain V, that play a critical role in the molar masses of the dextran produced (from 1 to 15 kDa)

The xyloglucans : are they new elicitors of <em>Arabidopsis thaliana</em> immunity?

International audienceHighlights • Fragments derived from plant cell wall xyloglucans induce Arabidopsis thaliana defence responses and protection against Botrytis cinerea • Xyloglucan-triggered immunity against B. cinerea requires the phytoalexin, ethylene and jasmonic acid-dependent pathways Results and discussion Xh treatment induced a dose-dependent MAPK phosphorylation in Arabidopsis cell suspensions. From 5 to 60 min, Xh treatment induced a rapid phosphorylation of two MAPKs with relative molecular masses of 43 and 47 kDa. Treatment with Xh did not induce any free [Ca2+]cyt variations whereas OG treatment induced a rapid and transient increase in free [Ca2+]cyt that peaked after 30 sec. Xh did not trigger any H2O2 production, as observed in control cells but OG treatment induced an oxidative burst with maximal H2O2 production detected at 10 min. To investigate late defence responses, we analysed callose deposition at the site of infection by B. cinerea after elicitor treatments. Xh and OG-treatment resulted in a significant increase of callose production 3 days post infection with the pathogen. The expression of different defence genes was analysed by qPCR. Xh triggered the accumulation of PR-1, PAD3, LOX3 and ICS1 transcripts. To further investigate the efficacy of xyloglucans to induce resistance, we performed protection assays against the necrotrophic fungi B. cinerea and the biotrophic oomycete H. arabidopsidis. Xh treatment applied 48 h before pathogen infection significantly reduced both the B. cinerea lesion diameter and the H. arabidopsidis sporulation on Arabidopsis leaves. Together, these results suggest that Xh are new elicitors of Arabidopsis immunity. Interestingly, some defence responses triggered by Xh are different from those induced by OG. As Arabidopsis responds to Xh treatment, we aimed to identify some signalling components. By using a genetic approach with T-DNA mutants in different defence responses, our data indicated that the Xh-triggered immunity against B. cinerea requires the phytoalexin (cyp71A13, pad3, pad2), ethylene (etr1, ein2) and jasmonic acid-dependent pathways (dde2, lox3, coi1).These results show that Xh are recognised by Arabidopsis. In order to identify a receptor involved in Xh perception or signalling, knock-out mutants of previously known A. thaliana receptors or candidate receptors up-regulated in microarray analysis have been

An eco-design approach for an innovative production process of low molar mass dextran

An approach for early-stage eco-design of an enzyme-based process has been developed by coupling process modeling, Life Cycle Assessment (LCA) and flowsheet design, in order to evaluate the real advantages of the direct synthesis of low molar mass dextrans (5-25 kg mol(-1)) from a sucrose substrate. This approach identifies the most promising development pathways and crucial unit operations that require, as a matter of priority, further investigation and experimentation. Process modeling is based on a comprehensive and multi-fidelity building of Life Cycle Inventories (LCI) to establish all materials and energy inputs and outputs of the processes involved with a flexible and satisfactory level of accuracy. This essentially binds (i) a high-fidelity polymerization model, namely PolyEnz, for the description of the synthesis of dextrans from sucrose using the DSR-M enzyme following a non-processive mechanism, (ii) flexible-fidelity models for description of subsequent purification, separation and drying steps, (iii) upstream processes in the value chain and life cycle system using real-world data from ecoinvent datasets. Three process benchmarks were constructed and compared to determine the most appropriate purification processes and operation conditions at a larger scale. In addition, various process triggers, including the initial concentration and type of substrate, the type of process water, the use of size exclusion chromatography for separation, and the use of freeze drying for the last production stage were subjected to a sensitivity analysis with the criteria being the overall energy demand, the potential environmental damage evaluated by the ReCiPe endpoint and the global warming potential