Organocatalytic, Michael-Stetter reaction and rhodium(I)-catalyzed hydroheteroarylation of acrylates with benzoxazoles: reaction development and investigations into origins of enantioselectivity

2015 Spring.Includes bibliographical references.The chapters that follow describe two independent investigations. Both relay the development of experimental methods for the catalytic, asymmetric addition of carbon-hydrogen bonds to alkenes. In the first chapter, nucleophilic amine and N-heterocyclic carbene cocatalysts cooperate in the organocatalytic, cascade synthesis of benzofuranone products in good yields and high enantioselectivities. Importantly, the cascade protocol is found to outperform a two-pot procedure in which reaction intermediates are isolated and purified before the second step. Mechanistic studies reveal that additives and geometry of an olefin intermediate crucially influence reaction enantioselectivity. In the second method, a bulky Rh(I)-bisphosphine complex catalyzes the asymmetric, intermolecular addition of benzoxazoles to methacrylate derivatives in fair to excellent yields and good to excellent enantioselectivities. Detailed deuterium labeling and epimerization studies provide considerable insight into the reaction mechanism: C-H activation is reversible; migratory insertion is likely enantiodetermining; and the bulky- bisphosphine ligand likely boosts reactivity and selectivity by discouraging deleterious ligation of benzoxazole starting materials to on- or off-cycle rhodium complexes and by impeding coordination-induced product epimerization

Causalities of war: The connection between type VI secretion system and microbiota

Cellular Microbiology published by John Wiley & Sons Ltd Microbiota niches have space and/or nutrient restrictions, which has led to the coevolution of cooperation, specialisation, and competition within the population. Different animal and environmental niches contain defined resident microbiota that tend to be stable over time and offer protection against undesired intruders. Yet fluxes can occur, which alter the composition of a bacterial population. In humans, the microbiota are now considered a key contributor to maintenance of health and homeostasis, and its alteration leads to dysbiosis. The bacterial type VI secretion system (T6SS) transports proteins into the environment, directly into host cells or can function as an antibacterial weapon by killing surrounding competitors. Upon contact with neighbouring cells, the T6SS fires, delivering a payload of effector proteins. In the absence of an immunity protein, this results in growth inhibition or death of prey leading to a competitive advantage for the attacker. It is becoming apparent that the T6SS has a role in modulating and shaping the microbiota at multiple levels, which is the focus of this review. Discussed here is the T6SS, its role in competition, key examples of its effect upon the microbiota, and future avenues of researc

Biofilms and cyclic di-GMP (c-di-GMP) signaling: lessons from Pseudomonas aeruginosa and other bacteria

The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities, and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa. This bacterium is frequently adopted as a model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation, and transmission

Agrobacterium tumefaciens Deploys a Superfamily of Type VI Secretion DNase Effectors as Weapons for Interbacterial Competition In Planta

The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many Proteobacteria to target effectors/toxins into both eukaryotic and prokaryotic cells. We report that Agrobacterium tumefaciens, a soil bacterium that triggers tumorigenesis in plants, produces a family of type VI DNase effectors (Tde) that are distinct from previously known polymorphic toxins and nucleases. Tde exhibits an antibacterial DNase activity that relies on a conserved HxxD motif and can be counteracted by a cognate immunity protein, Tdi. In vitro, A. tumefaciens T6SS could kill Escherichia coli but triggered a lethal counterattack by Pseudomonas aeruginosa upon injection of the Tde toxins. However, in an in planta coinfection assay, A. tumefaciens used Tde effectors to attack both siblings cells and P. aeruginosa to ultimately gain a competitive advantage. Such acquired T6SS-dependent fitness in vivo and conservation of Tde-Tdi couples in bacteria highlights a widespread antibacterial weapon beneficial for niche colonization

TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha

The bacterial type VI secretion system (T6SS) delivers effectors into eukaryotic host cells or toxins into bacterial competitor for survival and fitness. The T6SS is positively regulated by the threonine phosphorylation pathway (TPP) and negatively by the T6SS-accessory protein TagF. Here, we studied the mechanisms underlying TagF-mediated T6SS repression in two distinct bacterial pathogens, Agrobacterium tumefaciens and Pseudomonas aeruginosa. We found that in A. tumefaciens, T6SS toxin secretion and T6SS-dependent antibacterial activity are suppressed by a two-domain chimeric protein consisting of TagF and PppA, a putative phosphatase. Remarkably, this TagF domain is sufficient to post-translationally repress the T6SS, and this inhibition is independent of TPP. This repression requires interaction with a cytoplasmic protein, Fha, critical for activating T6SS assembly. In P. aeruginosa, PppA and TagF are two distinct proteins that repress T6SS in a TPP-dependent and -independent pathways, respectively. P. aeruginosa TagF interacts with Fha1, suggesting that formation of this complex represents a conserved TagF-mediated regulatory mechanism. Using TagF variants with substitutions of conserved amino acid residues at predicted protein-protein interaction interfaces, we uncovered evidence that the TagF-Fha interaction is critical for TagF-mediated T6SS repression in both bacteria. TagF inhibits T6SS without affecting T6SS protein abundance in A. tumefaciens, but TagF overexpression reduces the protein levels of all analyzed T6SS components in P. aeruginosa. Our results indicate that TagF interacts with Fha, which in turn could impact different stages of T6SS assembly in different bacteria, possibly reflecting an evolutionary divergence in T6SS control

Cyclic di-GMP inactivates T6SS and T4SS activity in Agrobacterium tumefaciens

© 2019 The Authors. The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged as a key player in regulating the microbial diversity in a population. In the plant pathogen Agrobacterium tumefaciens, the signalling cascades regulating the activity of this secretion system are poorly understood. Here, we outline how the universal eubacterial second messenger cyclic di‐GMP impacts the production of T6SS toxins and T6SS structural components. We demonstrate that this has a significant impact on the ability of the phytopathogen to compete with other bacterial species in vitro and in planta. Our results suggest that, as opposed to other bacteria, c‐di‐GMP turns down the T6SS in A. tumefaciens thus impacting its ability to compete with other bacterial species within the rhizosphere. We also demonstrate that elevated levels of c‐di‐GMP within the cell decrease the activity of the Type IV secretion system (T4SS) and subsequently the capacity of A. tumefaciens to transform plant cells. We propose that such peculiar control reflects on c‐di‐GMP being a key second messenger that silences energy‐costing systems during early colonization phase and biofilm formation, while low c‐di‐GMP levels unleash T6SS and T4SS to advance plant colonization.Biotechnology and Biological Sciences Research Council. Grant Numbers: BB/L007959/1, BB/M02735X/1 Ministry of Science and Technology, Taiwan. Grant Number: 104-2311-B-001-025-MY

The Diguanylate Cyclase SadC Is a Central Player in Gac/Rsm-Mediated Biofilm Formation in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immune-compromised and Cystic Fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the c-di-GMP signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population towards a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than forty c-di-GMP-related enzymes it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understand this insulation mechanism

Valorization of surface-water RO brines via Assisted-Reverse Electrodialysis for minerals recovery: Performance analysis and scale-up perspectives

Reverse osmosis (RO) processes have been recently identified as mostly capable of quantitative removal of salts and contaminants from saline and surface waters, though posing the problem of a concentrated brine to be disposed of and a produced permeate too low in minerals, thus requiring a sometimes expensive remineralization step. In the present paper, Assisted-Reverse Electrodialysis (A-RED) has been proposed for the remineralization of surface-water RO permeate by recovering minerals from its brine. A purposely developed and validated model has been adopted to carry out a parametric analysis for design and optimization of an industrial-scale plant. The techno-economic analysis underlined that full permeate remineralization can be achieved with minimum specific energy consumption of 0.08 kWh m(-3), while a minimum remineralization cost of 2.2 c(sic) m(-3) was found applying a permeate by-pass and feed & bleed scheme to (i) increase the plant remineralization capacity and (ii) maintain a stack inlet conductivity above 100-160 mu S cm(-1) (starting from a permeate similar to 10 mu S cm(-1)). Compared to current post-treatment techniques, results appear very promising thanks to the reduction of chemicals and total costs as well as environmental concerns related to brine disposal

Transcriptional organization and regulation of the Pseudomonas putida K1 type VI secretion system gene cluster

The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive micro-organisms. Three T6SSs have recently been described in Pseudomonas putida KT2440 and it has been shown that one, K1-T6SS, is used to outcompete a wide range of phytopathogens, protecting plants from pathogen infections. Given the relevance of this system as a powerful and innovative mechanism of biological control, it is critical to understand the processes that govern its expression. Here, we experimentally defined two transcriptional units in the K1-T6SS cluster. One encodes the structural components of the system and is transcribed from two adjacent promoters. The other encodes two hypothetical proteins, the tip of the system and the associated adapters, and effectors and cognate immunity proteins, and it is also transcribed from two adjacent promoters. The four identified promoters contain the typical features of σ70-dependent promoters. We have studied the expression of the system under different conditions and in a number of mutants lacking global regulators. P. putida K1-T6SS expression is induced in the stationary phase, but its transcription does not depend on the stationary σ factor RpoS. In fact, the expression of the system is indirectly repressed by RpoS. Furthermore, it is also repressed by RpoN and the transcriptional regulator FleQ, an enhancer-binding protein typically acting in conjunction with RpoN. Importantly, expression of the K1-T6SS gene cluster is positively regulated by the GacS–GacA two-component regulatory system (TCS) and repressed by the RetS sensor kinase, which inhibits this TCS. Our findings identified a complex regulatory network that governs T6SS expression in general and P. putida K1-T6SS in particular, with implications for controlling and manipulating a bacterial agent that is highly relevant in biological control