Reconstruction of bacterial transcription-coupled repair at single-molecule resolution

International audienc

Stopped in its tracks: The RNA polymerase molecular motor as a robust sensor of DNA damage.

International audience: DNA repair is often a complex, multi-component, multi-step process; this makes detailed kinetic analysis of the different steps of repair a challenging task using standard biochemical methods. At the same time, single-molecule methods are well-suited for extracting kinetic information despite time-averaging due to diffusion of biochemical components and stochasticity of chemical reaction steps. Here we discuss recent experiments using DNA nanomanipulation in a magnetic trap to study the initiation of transcription-coupled repair in a model bacterial system comprising the canonical Escherichia coli RNA polymerase and the Mfd translocase which specifically binds to it. These experiments provide kinetic insight into the reaction process, helping to explain how Mfd discriminates between transcribing RNAP and stalled RNAP. They also identify a reliably long-lived intermediate containing Mfd translocase and, potentially, RNA polymerase. This intermediate presumably serves as a platform for assembly of downstream repair components UvrAB(C)

Enhanced Antibacterial Activity of Substituted Derivatives of NCR169C Peptide

Medicago truncatula in symbiosis with its rhizobial bacterium partner produces more than 700 nodule-specific cysteine-rich (NCR) peptides with diverse physicochemical properties. Most of the cationic NCR peptides have antimicrobial activity and the potential to tackle antimicrobial resistance with their novel modes of action. This work focuses on the antibacterial activity of the NCR169 peptide derivatives as we previously demonstrated that the C-terminal sequence of NCR169 (NCR169C17–38) has antifungal activity, affecting the viability, morphology, and biofilm formation of various Candida species. Here, we show that NCR169C17–38 and its various substituted derivatives are also able to kill ESKAPE pathogens such as Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. The replacement of the two cysteines with serines enhanced the antimicrobial activity against most of the tested bacteria, indicating that the formation of a disulfide bridge is not required. As tryptophan can play role in the interaction with bacterial membranes and thus in antibacterial activity, we replaced the tryptophans in the NCR169C17–38C12,17/S sequence with various modified tryptophans, namely 5-methyl tryptophan, 5-fluoro tryptophan, 6-fluoro tryptophan, 7-aza tryptophan, and 5-methoxy tryptophan, in the synthesis of NCR169C17–38C12,17/S analogs. The results demonstrate that the presence of modified fluorotryptophans can significantly enhance the antimicrobial activity without notable hemolytic effect, and this finding could be beneficial for the further development of new AMPs from the members of the NCR peptide family

Enantioselective resolution of biologically active dipeptide analogs by high-performance liquid chromatography applying Cinchona alkaloid-based ion-exchanger chiral stationary phases

Sixteen pairs of enantiomeric dipeptides were separated on four chiral ion-exchanger-type stationary phases based on Cinchona alkaloids. Anion-exchangers (QN-AX, QD-AX) and zwitterionic phases [ZWIX(+)(TM) and ZWIX(-)(TM)] were studied in a comparative manner. The effects of the nature and concentrations of the mobile phase solvent components and organic salt additives on analyte retention and enantioseparation were systematically studied in order to get a deeper insight into the enantiorecognition mechanism. Moreover, experiments were performed in the temperature range 10-50 degrees C to calculate thermodynamic parameters like changes in standard enthalpy, Delta(Delta H degrees), entropy, Delta(Delta S degrees), and free energy, Delta(Delta G degrees) on the basis of van't Hoff plots derived from the In alpha vs. 1/T curves. Elution sequences of the dipeptides were determined in all cases and, with a few exceptions, they were found to be opposite on the pseudoenantiomeric stationary phases as of QN-AX/QD-AX and of ZWIX(+) and ZWIX(-). The stere-oselective retention mechanism is based on electrostatically driven intermolecular interactions supported by additional interaction increments mainly determined by the absolute configuration of the chiral C8 and C9 atoms of the quinine and quinidine moieties. (C) 2019 Elsevier B.V. All rights reserved

Interspecific Variation In Juvenile Snapper Otolith Chemical Signatures In the Northern Gulf of Mexico

The objective of this study was to evaluate whether age-0 lane snapper Lutjanus synagris otolith chemical signatures could serve as accurate proxies for those of its congener, red snapper L. campechanus, among northern Gulf of Mexico (GOM) nursery regions. Red (n = 90) and lane (n = 53) snappers were sampled from 3 regions of the northern GOM in fall 2005, and their otolith chemistry was analyzed with sector field-inductively coupled plasma-mass spectrometry (Ba:Ca, Mg:Ca, Mn:Ca, Sr:Ca, Li:Ca) or stable isotope ratio-mass spectrometry (δ13C and δ18O). Chemical signatures were significantly different among regions (MANOVA, p \u3c 0.001) and between species (MANOVA, p = 0.029), with the species effect being driven by significant differences in 4 of the 7 constituents analyzed (ANOVA, p \u3c 0.036). The significant region effect persisted (MANOVA, p \u3c 0.001), but the species effect was non-significant (MANOVA, p = 0.964) when constituent values were normalized to species-specific means. Mean regional classification accuracies from linear discriminant functions computed with otolith constituent data were 84% for lane snapper and 80% for red snapper whether data were normalized or not. Maximum likelihood models parameterized with normalized lane snapper otolith chemistry data estimated red snapper regional composition reasonably well among mixed-region samples (mean error = 9.7% among models). Therefore, it appears age-0 lane snapper otolith chemical signatures can serve as accurate proxies for those of red snapper in the northern GOM. These results have broader implications for deriving natural tags based on otolith chemistry for fishes that may have low abundance in parts of their range

Legume Plant Peptides as Sources of Novel Antimicrobial Molecules Against Human Pathogens

Antimicrobial peptides are prominent components of the plant immune system acting against a wide variety of pathogens. Legume plants from the inverted repeat lacking clade (IRLC) have evolved a unique gene family encoding nodule-specific cysteine-rich NCR peptides acting in the symbiotic cells of root nodules, where they convert their bacterial endosymbionts into non-cultivable, polyploid nitrogen-fixing cells. NCRs are usually 30–50 amino acids long peptides having a characteristic pattern of 4 or 6 cysteines and highly divergent amino acid composition. While the function of NCRs is largely unknown, antimicrobial activity has been demonstrated for a few cationic Medicago truncatula NCR peptides against bacterial and fungal pathogens. The advantages of these plant peptides are their broad antimicrobial spectrum, fast killing modes of actions, multiple bacterial targets, and low propensity to develop resistance to them and no or low cytotoxicity to human cells. In the IRLC legumes, the number of NCR genes varies from a few to several hundred and it is possible that altogether hundreds of thousands of different NCR peptides exist. Due to the need for new antimicrobial agents, we investigated the antimicrobial potential of 104 synthetic NCR peptides from M. truncatula, M. sativa, Pisum sativum, Galega orientalis and Cicer arietinum against eight human pathogens, including ESKAPE bacteria. 50 NCRs showed antimicrobial activity with differences in the antimicrobial spectrum and effectivity. The most active peptides eliminated bacteria at concentrations from 0.8 to 3.1 μM. High isoelectric point and positive net charge were important but not the only determinants of their antimicrobial activity. Testing the activity of shorter peptide derivatives against Acinetobacter baumannii and Candida albicans led to identification of regions responsible for the antimicrobial activity and provided insight into their potential modes of action. This work provides highly potent lead molecules without hemolytic activity on human blood cells for novel antimicrobial drugs to fight against pathogens

Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization

International audienceLittle is known about the molecular mechanisms that induce gametefusion during mammalian fertilization. After initial contact, adhesionbetween gametes only leads to fusion in the presence of threemembrane proteins that are necessary, but insufficient, for fusion:Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. Whathappens during this adhesion phase is a crucial issue. Here, wedemonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conservedacross species, as the adhesion partners, Izumo1 and its receptor,are interchangeable between mouse and human. Our findingssuggest that Cd9 is a partner of Juno, and these discoveries allowus to propose a new model of the molecular mechanisms leading togamete fusion, in which the adhesion-induced membraneorganization assembles all key players of the fusion machinery