Synthesis and inclusion ability of anthracene appended β-cyclodextrins: unexpected effect of triazole linker

A new fluorescent β-cyclodextrin has been synthesized by coupling an anthracene moiety to the cyclic oligosaccharide via click chemistry. The influence of the triazole spacer was compared to the simple amino and amido linkers. While a sensing ability toward adamantan-1-ol was observed with the latter two spacers, the absence of inclusion capacity prevents the triazole modified cyclodextrin from showing any fluorescence variations. The difference in the binding behaviors studied by Isothermal Titration Calorimetry, UV–vis and fluorescence spectroscopies, was highlighted by the NOESY NMR spectra of the modified cyclodextrins: whereas a free cavity was observed for the amino and amido linkers, an important obstruction was obtained in the case of the triazole.     

Greenhouse gas fluxes respond to different N fertilizer types due to altered plant-soil-microbe interactions

The application of inorganic nitrogen (N) fertilizers strongly influences the contribution of agriculture to the greenhouse effect, especially by potentially increasing emissions of nitrous oxide (N₂O), carbon dioxide (CO₂) and methane (CH₄) from soils. The present microcosm-study investigates the effect of different forms of inorganic N fertilizers on greenhouse gas (GHG) emissions from two different agricultural soils. The relationship between greenhouse gas emissions and soil microbial communities, N transformation rates and plant (Hordeum vulgare L. cv. Morex) growth were investigated. Repeated N fertilization led to increased N₂O emissions. In a parallel survey of functional microbial population dynamics we observed a stimulation of bacterial and archaeal ammonia oxidisers accompanied with these N₂O emissions. The ratio of archaeal to bacterial ammonium monooxygenase subunit A (amoA) gene copies (data obtained from Inselsbacher et al., 2010) correlated positively with N₂O fluxes, which suggests a direct or indirect involvement of archaea in N₂O fluxes. Repeated N fertilization also stimulated methane oxidation, which may also be related to a stimulation of ammonia oxidizers. The fertilizer effects differed between soil types: In the more organic Niederschleinz soil N-turnover rates increased more strongly after fertilization, while in the sandy Purkersdorf soil plant growth and soil respiration were accelerated depending on fertilizer N type. Compared to addition of NH ₄ ⁺ and NO ₃ ^? , addition of NH₄NO₃ fertilizer resulted in the largest increase in global warming potential as a summary indicator of all GHG related effects. This effect resulted from the strongest increase of both N₂O and CO₂ emission while plant growth was not equally stimulated, compared to e.g. KNO₃ fertilization. In order to decrease N losses from agricultural ecosystems and in order to minimize soil derived global warming potential, this study points to the need for interdisciplinary investigations of the highly complex interactions within plant-soil-microbe-atmosphere systems. By understanding the microbial processes underlying fertilizer effects on GHG emissions the N use efficiency of crops could be refined.     

Genetic diversity, structure, gene flow and evolutionary relationships within the Sorghum bicolor wild�Cweedy�Ccrop complex in a western African region

Gene flow between domesticated plants and their wild relatives is one of the major evolutionary processes acting to shape their structure of genetic diversity. Earlier literature, in the 1970s, reported on the interfertility and the sympatry of wild, weedy and cultivated sorghum belonging to the species Sorghum bicolor in most regions of sub-Saharan Africa. However, only a few recent surveys have addressed the geographical and ecological distribution of sorghum wild relatives and their genetic structure. These features are poorly documented, especially in western Africa, a centre of diversity for this crop. We report here on an exhaustive in situ collection of wild, weedy and cultivated sorghum assembled in Mali and in Guinea. The extent and pattern of genetic diversity were assessed with 15 SSRs within the cultivated pool (455 accessions), the wild pool (91 wild and weedy forms) and between them. F (ST) and R (ST) statistics, distance-based trees, Bayesian clustering methods, as well as isolation by distance models, were used to infer evolutionary relationships within the wild-weedy-crop complex. Firstly, our analyses highlighted a strong racial structure of genetic diversity within cultivated sorghum (F (ST) = 0.40). Secondly, clustering analyses highlighted the introgressed nature of most of the wild and weedy sorghum and grouped them into two eco-geographical groups. Such closeness between wild and crop sorghum could be the result of both sorghum's domestication history and preferential post-domestication crop-to-wild gene flow enhanced by farmers' practices. Finally, isolation by distance analyses showed strong spatial genetic structure within each pool, due to spatially limited dispersal, and suggested consequent gene flow between the wild and the crop pools, also supported by R (ST) analyses. Our findings thus revealed important features for the collection, conservation and biosafety of domesticated and wild sorghum in their centre of diversity.     

Multicopy suppression screen in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain lacking the Rab GTPase-activating protein Msb3p

The yeast proteins, Msb3p and Msb4p, are two Ypt/Rab-specific GTPase-activating proteins sharing redundant functions in exocytosis, organization of the actin cytoskeleton, and budding site selection. To see if Msb3p might play an additional, specific role, we first tested the sensitivities of msb3 and msb4 mutant strains to different drugs and then screened a genomic library for multicopy suppressors of msb3 sensitivity to CdCl₂ or to the calcium channel blocker diltiazem hydrochloride. Three genes (ADH1, RNT1, and SUI1) were found to suppress the CdCl₂ sensitivity of the msb3 strain and three others (YAP6, ZEO1, and SLM1) its diltiazem-HCl sensitivity. The results suggest a possible involvement of Msb3p in calcineurin-mediated signalling.     

Methodologies for Salmonella enterica subsp. enterica subtyping: gold standards and alternatives

For more than 80 years, subtyping of Salmonella enterica has been routinely performed by serotyping, a method in which surface antigens are identified based on agglutination reactions with specific antibodies. The serotyping scheme, which is continuously updated as new serovars are discovered, has generated over time a data set of the utmost significance, allowing long-term epidemiological surveillance of Salmonella in the food chain and in public health control. Conceptually, serotyping provides no information regarding the phyletic relationships inside the different Salmonella enterica subspecies. In epidemiological investigations, identification and tracking of salmonellosis outbreaks require the use of methods that can fingerprint the causative strains at a taxonomic level far more specific than the one achieved by serotyping. During the last 2 decades, alternative methods that could successfully identify the serovar of a given strain by probing its DNA have emerged, and molecular biology-based methods have been made available to address phylogeny and fingerprinting issues. At the same time, accredited diagnostics have become increasingly generalized, imposing stringent methodological requirements in terms of traceability and measurability. In these new contexts, the hand-crafted character of classical serotyping is being challenged, although it is widely accepted that classification into serovars should be maintained. This review summarizes and discusses modern typing methods, with a particular focus on those having potential as alternatives for classical serotyping or for subtyping Salmonella strains at a deeper level.     

Entianin, a novel subtilin-like lantibiotic from Bacillus subtilis subsp. spizizenii DSM 15029T with high antimicrobial activity

Lantibiotics, such as nisin and subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for each lantibiotic, and reporter systems for nisin and subtilin autoinduction are available. In this report, we used the previously reported subtilin autoinduction bioassay in combination with mass spectrometric analyses to identify the novel subtilin-like lantibiotic entianin from Bacillus subtilis subsp. spizizenii DSM 15029(T). Linearization of entianin using Raney nickel-catalyzed reductive cleavage enabled, for the first time, the use of tandem mass spectrometry for the fast and efficient determination of an entire lantibiotic primary structure, including posttranslational modifications. The amino acid sequence determined was verified by DNA sequencing of the etnS structural gene, which confirmed that entianin differs from subtilin at 3 amino acid positions. In contrast to B. subtilis ATCC 6633, which produces only small amounts of unsuccinylated subtilin, B. subtilis DSM 15029(T) secretes considerable amounts of unsuccinylated entianin. Entianin was very active against several Gram-positive pathogens, such as Staphylococcus aureus and Enterococcus faecalis. The growth-inhibiting activity of succinylated entianin (S-entianin) was much lower than that of unsuccinylated entianin: a 40-fold higher concentration was required for inhibition. For succinylated subtilin (S-subtilin), a concentration 100-fold higher than that of unsuccinylated entianin was required to inhibit the growth of a B. subtilis test strain. This finding was in accordance with a strongly reduced sensing of cellular envelope stress provided by S-entianin relative to that of entianin. Remarkably, S-entianin and S-subtilin showed considerable autoinduction activity, clearly demonstrating that autoinduction and antibiotic activity underlie different molecular mechanisms.     

Flow cytometry confirms reticulate evolution and reveals triploidy in Central European Diphasiastrum taxa (Lycopodiaceae, Lycophyta)

Background and Aims

Interspecific Diphasiastrum hybrids have been assumed to be homoploid and to produce well-formed spores serving sexual reproduction. If this were the case, forms intermediate between hybrids and parents or hybrid swarms should be expected. The purpose of this study was: (1) to check whether homoploidy consistently applies to the three hybrids throughout their Central European range; (2) to examine whether their genome sizes confirm their parentage as assumed by morphology; and (3) to perform a screening for detection of ploidy levels other than diploid and variation in DNA content due to backcrossing.

Methods

Flow cytometry was used first to measure the relative DNA values [with 4′,6-diamidino-2-phenylindole (DAPI) staining] and ploidy level as a general screening, and secondly to determine the absolute DNA 2C values [with propidium iodide (PI) staining] in a number of selected samples with the main focus on the hybrids.

Key Results

A considerable variation of DNA 2C values (5·26–7·52 pg) was detected between the three European Diphasiastrum species. The values of the diploid hybrids are highly constant without significant variation between regions. They are also intermediate between their assumed parents and agree closely with those calculated from their putative parents. This confirms their hybrid origin, assumed parentage and homoploid status. Considerably higher DNA amounts (9·48–10·30 pg) were obtained for three populations, suggesting that these represent triploid hybrids, an interpretation that is strongly supported by their morphology.

Conclusions

Diploid hybrids have retained their genetic and morphological identites throughout their Central European range, and thus no indications for diploid backcrossing were found. The triploid hybrids have probably originated from backcrossing between a diploid gametophyte of a hybrid (derived from a diplospore) and a haploid gametophyte of a diploid parental species. By repeated crossing events, reticulate evolution patterns arise that are similar to those known for a number of ferns.     

A general strategy to characterize calmodulin-calcium complexes involved in CaM-target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin-calcium complexes

Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four “EF hand” type Ca²⁺ binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca²⁺ ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca_n (n = 0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.