Over-expression of a zeatin O-glucosylation gene in maize leads to growth retardation and tasselseed formation

To study the effects of cytokinin O-glucosylation in monocots, maize (Zea mays L.) transformants harbouring the ZOG1 gene (encoding a zeatin O-glucosyltransferase from Phaseolus lunatus L.) under the control of the constitutive ubiquitin (Ubi) promoter were generated. The roots and leaves of the transformants had greatly increased levels of zeatin-O-glucoside. The vegetative characteristics of hemizygous and homozygous Ubi:ZOG1 plants resembled those of cytokinin-deficient plants, including shorter stature, thinner stems, narrower leaves, smaller meristems, and increased root mass and branching. Transformant leaves had a higher chlorophyll content and increased levels of active cytokinins compared with those of non-transformed sibs. The Ubi:ZOG1 plants exhibited delayed senescence when grown in the spring/summer. While hemizygous transformants had reduced tassels with fewer spikelets and normal viable pollen, homozygotes had very small tassels and feminized tassel florets, resembling tasselseed phenotypes. Such modifications of the reproductive phase were unexpected and demonstrate a link between cytokinins and sex-specific floral development in monocots

Characteristics of Positive Deviants in Western Chimpanzee Populations

With continued expansion of anthropogenically modified landscapes, the proximity between humans and wildlife is continuing to increase, frequently resulting in species decline. Occasionally however, species are able to persist and there is an increased interest in understanding such positive outliers and underlying mechanisms. Eventually, such insights can inform the design of effective conservation interventions by mimicking aspects of the social-ecological conditions found in areas of species persistence. Recently, frameworks have been developed to study the heterogeneity of species persistence across populations with a focus on positive outliers. Applications are still rare, and to our knowledge this is one of the first studies using this approach for terrestrial species conservation. We applied the positive deviance concept to the western chimpanzee, which occurs in a variety of social-ecological landscapes. It is now categorized as Critically Endangered due to hunting and habitat loss and resulting excessive decline of most of its populations. Here we are interested in understanding why some of the populations did not decline. We compiled a dataset of 17,109 chimpanzee survey transects (10,929 km) across nine countries and linked them to a range of social and ecological variables. We found that chimpanzees seemed to persist within three social-ecological configurations: first, rainforest habitats with a low degree of human impact, second, steep areas, and third, areas with high prevalence of hunting taboos and low degree of human impact. The largest chimpanzee populations are nowadays found under the third social-ecological configuration, even though most of these areas are not officially protected. Most commonly chimpanzee conservation has been based on exclusion of threats by creation of protected areas and law enforcement. Our findings suggest, however, that this approach should be complemented by an additional focus on threat reduction, i.e., interventions that directly target individual human behavior that is most threatening to chimpanzees, which is hunting. Although changing human behavior is difficult, stakeholder co-designed behavioral change approaches developed in the social sciences have been used successfully to promote pro-environmental behavior. With only a fraction of chimpanzees and primates living inside protected areas, such new approaches might be a way forward to improve primate conservation

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein

Pseudomonas aeruginosa MipA-MipB envelope proteins act as new sensors of polymyxins

International audienceABSTRACT Due to the rising incidence of antibiotic-resistant infections, the last-line antibiotics, polymyxins, have resurged in the clinics in parallel with new bacterial strategies of escape. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa develops resistance to colistin/polymyxin B by distinct molecular mechanisms, mostly through modification of the lipid A component of the LPS by proteins encoded within the arnBCDATEF-ugd ( arn ) operon. In this work, we characterized a polymyxin-induced operon named mipBA , present in P. aeruginosa strains devoid of the arn operon. We showed that mipBA is activated by the ParR/ParS two-component regulatory system in response to polymyxins. Structural modeling revealed that MipA folds as an outer-membrane β-barrel, harboring an internal negatively charged channel, able to host a polymyxin molecule, while the lipoprotein MipB adopts a β-lactamase fold with two additional C-terminal domains. Experimental work confirmed that MipA and MipB localize to the bacterial envelope, and they co-purify in vitro . Nano differential scanning fluorimetry showed that polymyxins stabilized MipA in a specific and dose-dependent manner. Mass spectrometry-based quantitative proteomics on P. aeruginosa membranes demonstrated that ∆ mipBA synthesized fourfold less MexXY-OprA proteins in response to polymyxin B compared to the wild-type strain. The decrease was a direct consequence of impaired transcriptional activation of the mex operon operated by ParR/ParS. We propose MipA/MipB to act as membrane (co)sensors working in concert to activate ParS histidine kinase and help the bacterium to cope with polymyxin-mediated envelope stress through synthesis of the efflux pump, MexXY-OprA. IMPORTANCE Due to the emergence of multidrug-resistant isolates, antibiotic options may be limited to polymyxins to eradicate Gram-negative infections. Pseudomonas aeruginosa , a leading opportunistic pathogen, has the ability to develop resistance to these cationic lipopeptides by modifying its lipopolysaccharide through proteins encoded within the arn operon. Herein, we describe a sub-group of P. aeruginosa strains lacking the arn operon yet exhibiting adaptability to polymyxins. Exposition to sub-lethal polymyxin concentrations induced the expression and production of two envelope-associated proteins. Among those, MipA, an outer-membrane barrel, is able to specifically bind polymyxins with an affinity in the 10-µM range. Using membrane proteomics and phenotypic assays, we showed that MipA and MipB participate in the adaptive response to polymyxins via ParR/ParS regulatory signaling. We propose a new model wherein the MipA-MipB module functions as a novel polymyxin sensing mechanism

A Soluble Metabolon Synthesizes the Isoprenoid Lipid Ubiquinone

International audienceUbiquinone (UQ) is a polyprenylated lipid that is conserved from bacteria to humans and is crucial to cellular respiration. How the cell orchestrates the efficient synthesis of UQ, which involves the modification of extremely hydrophobic substrates by multiple sequential enzymes, remains an unresolved issue. Here, we demonstrate that seven Ubi proteins form the Ubi complex, a stable metabolon that catalyzes the last six reactions of the UQ biosynthetic pathway in Escherichia coli. The SCP2 domain of UbiJ forms an extended hydrophobic cavity that binds UQ intermediates inside the 1 MDa Ubi complex. We purify the Ubi complex from cytoplasmic extracts and demonstrate that UQ biosynthesis occurs in this fraction, challenging the current thinking of a membrane-associated biosynthetic process. Collectively, our results document a rare case of stable metabolon and highlight how the supramolecular organization of soluble enzymes allows the modification of hydrophobic substrates in a hydrophilic environment

An algal photoenzyme converts fatty acids to hydrocarbons

International audienceAlthough many organisms capture or respond to sunlight, few enzymes are known to be driven by light. Among these are DNA photolyases and the photosynthetic reaction centers. Here, we show that the microalga $Chlorella\ variabilis$ NC64A harbors a photoenzyme that acts in lipid metabolism. This enzyme belongs to an algae-specific clade of the glucose-methanol-choline oxidoreductase family and catalyzes the decarboxylation of free fatty acids to n-alkanes or-alkenes in response to blue light. Crystal structure of the protein reveals a fatty acid–binding site in a hydrophobic tunnel leading to the light-capturing flavin adenine dinucleotide (FAD) cofactor. The decarboxylation is initiated through electron abstraction from the fatty acid by the photoexcited FAD with a quantum yield >80%. This photoenzyme, which we name fatty acid photodecarboxylase, may be useful in light-driven, bio-based production of hydrocarbons