The recombination initiation functions DprA and RecFOR suppress microindel mutations in Acinetobacter baylyi ADP1.

Short-Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single-strands anneal with genomic DNA at microhomologies and get integrated during DNA replication, presumably acting as primers for Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR is tightly controlled by genome maintenance functions including RecA. It is thought that RecA scavenges DNA single-strands and renders them unable to anneal. To further elucidate the role of RecA in this process, we investigate the roles of the upstream functions DprA, RecFOR, and RecBCD, all of which load DNA single-strands with RecA. Here we show that all three functions suppress SPDIR mutations in the wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA, they are strongly increased in the absence of both DprA and RecA. This SPDIR-avoiding function of DprA is not related to its role in natural transformation. These results suggest a function for DprA in combination with RecA to avoid potentially harmful microindel mutations, and offer an explanation for the ubiquity of dprA in the genomes of naturally non-transformable bacteria.

Testing for the fitness benefits of natural transformation during community-embedded evolution.

Natural transformation is a process where bacteria actively take up DNA from the environment and recombine it into their genome or reconvert it into extra-chromosomal genetic elements. The evolutionary benefits of transformation are still under debate. One main explanation is that foreign allele and gene uptake facilitates natural selection by increasing genetic variation, analogous to meiotic sex. However, previous experimental evolution studies comparing fitness gains of evolved transforming- and isogenic non-transforming strains have yielded mixed support for the 'sex hypothesis.' Previous studies testing the sex hypothesis for natural transformation have largely ignored species interactions, which theory predicts provide conditions favourable to sex. To test for the adaptive benefits of bacterial transformation, the naturally transformable wild-type Acinetobacter baylyi and a transformation-deficient ∆comA mutant were evolved for 5 weeks. To provide strong and potentially fluctuating selection, A. baylyi was embedded in a community of five other bacterial species. DNA from a pool of different Acinetobacter strains was provided as a substrate for transformation. No effect of transformation ability on the fitness of evolved populations was found, with fitness increasing non-significantly in most treatments. Populations showed fitness improvement in their respective environments, with no apparent costs of adaptation to competing species. Despite the absence of fitness effects of transformation, wild-type populations evolved variable transformation frequencies that were slightly greater than their ancestor which potentially could be caused by genetic drift.

Collection of Annotated Acinetobacter Genome Sequences.

The genus Acinetobacter contains environmental species as well as opportunistic pathogens of humans. Several species are competent for natural transformation, an important mechanism of horizontal gene transfer. Here, we present the genome sequences of 19 Acinetobacter strains used in past and upcoming studies of natural transformation.

Evolutionary Instability of Collateral Susceptibility Networks in Ciprofloxacin-Resistant Clinical Escherichia coli Strains.

Collateral sensitivity and resistance occur when resistance development toward one antimicrobial either potentiates or deteriorates the effect of others. Previous reports on collateral effects on susceptibility focus on newly acquired resistance determinants and propose that novel treatment guidelines informed by collateral networks may reduce the evolution, selection, and spread of antimicrobial resistance. In this study, we investigate the evolutionary stability of collateral networks in five ciprofloxacin-resistant, clinical Escherichia coli strains. After 300 generations of experimental evolution without antimicrobials, we show complete fitness restoration in four of five genetic backgrounds and demonstrate evolutionary instability in collateral networks of newly acquired resistance determinants. We show that compensatory mutations reducing efflux expression are the main drivers destabilizing initial collateral networks and identify rpoS as a putative target for compensatory evolution. Our results add another layer of complexity to future predictions and clinical application of collateral networks. IMPORTANCE Antimicrobial resistance occurs due to genetic alterations that affect different processes in bacteria. Thus, developing resistance toward one antimicrobial drug may also alter the response toward others (collateral effects). Understanding the mechanisms of such collateral effects may provide clinicians with a framework for informed antimicrobial treatment strategies, limiting the emergence of antimicrobial resistance. However, for clinical implementation, it is important that the collateral effects of resistance development are repeatable and temporarily stable. Here, we show that collateral effects caused by resistance development toward ciprofloxacin in clinical Escherichia coli strains are not temporarily stable because of compensatory mutations restoring the fitness burden of the initial resistance mutations. Consequently, this instability is complicating the general applicability and clinical implementation of collateral effects into treatment strategies.

Lewis acid-base adducts of Al(N(C6F5)2)3 and Ga(N(C6F5)2)3 - structural features and dissociation enthalpies.

Herein we present the molecular structures of six neutral Lewis acid-base adducts of the Lewis superacid Al(N(C6F5)2)3 and its higher homolog Ga(N(C6F5)2)3 with the electron pair donors MeCN, CNtBu, THF and PMe3. The crystal structures reveal crucial structural changes compared to the free Lewis acids as a consequence of the adduct formation. Furthermore, we calculated the corresponding dissociation enthalpies of the adducts which lie between 69 and 141 kJ mol-1 and are therefore considerably lower compared to the values for the formation of the anionic fluoride or chloride metallates.

Antimicrobial resistance acquisition via natural transformation: context is everything.

Natural transformation is a process where bacterial cells actively take up free DNA from the environment and recombine it into their genome or reconvert it into extra-chromosomal genetic elements. Although this mechanism is known to mediate the uptake of antibiotic resistance determinants in a range of human pathogens, its importance in the spread of antimicrobial resistance is not always appreciated. This review highlights the context in which transformation takes place: in diverse microbiomes, in interaction with other forms of horizontal gene transfer and in increasingly polluted environments. This examination of the abiotic and biotic drivers of transformation reveals that it could be more important in the dissemination of resistance genes than is often recognised.

2,9-Diazadibenzoperylene and 2,9-Dimethyldibenzoperylene-1,3,8,10-tetratriflates: Key to Functionalized 2,9-Diazaperopyrenes.

The synthesis of 2,9-diaza-1,3,8,10-tetratriflato-dibenzoperylene (DDP 3 a) and corresponding 2,9-dimethyl-1,3,8,10-tetratriflato-dibenzoperylene (DBP 3 b) has been developed at multigram scale via reduction of one of the industrially most important high-performance dyes, perylene-3,4,9,10-tetracarboxylic diimide (PTCDI), and of the corresponding dihydroxy peropyrenequinone precursor. The focus of this paper is on the reactivity pattern of 3 a as key intermediate towards highly functionalized 2,9-diazadibenzopyrelenes (DDPs) obtained via catalytic substitution of four triflate by aryl, heteroaryl, alkynyl, aminyl, and O-phosphanyl substituents. The influence of electron-donating substituents (OSiMe3 , OPt-Bu2 , N-piperidinyl), electron-withdrawing (OTf, 3,5-bis-trifluoromethyl-phenyl), and of electron-rich π-conjugated (2-thienyl, 4-tert-butylphenyl, trimethylsilyl-ethynyl) substituents on optoelectronic and structural properties of these functionalized DDPs has been investigated via XRD analyses, UV/Vis, PL spectroscopy, and by electroanalytical CV. These results were correlated to results of DFT and TD-DFT calculations. Thus, functionalized DPPs with easily tunable HOMO and LUMO energies and gap became available via a new and reliable synthetic strategy starting from readily available PTCDI.

Bis-Cyclometalated Indazole and Benzimidazole Chiral-at-Iridium Complexes: Synthesis and Asymmetric Catalysis.

A new class of bis-cyclometalated iridium(III) catalysts containing two inert cyclometalated 6-tert-butyl-2-phenyl-2H-indazole bidentate ligands or two inert cyclometalated 5-tert-butyl-1-methyl-2-phenylbenzimidazoles is introduced. The coordination sphere is complemented by two labile acetonitriles, and a hexafluorophosphate ion serves as a counterion for the monocationic complexes. Single enantiomers of the chiral-at-iridium complexes (>99% er) are obtained through a chiral-auxiliary-mediated approach using a monofluorinated salicyloxazoline and are investigated as catalysts in the enantioselective conjugate addition of indole to an α,ß-unsaturated 2-acyl imidazole and an asymmetric Nazarov cyclization.

Bacterial evolution on demand.

Bacteria carry antibiotic resistant genes on movable sections of DNA that allow them to select the relevant genes on demand.

Cryptic ß-Lactamase Evolution Is Driven by Low ß-Lactam Concentrations.

Our current understanding of how low antibiotic concentrations shape the evolution of contemporary ß-lactamases is limited. Using the widespread carbapenemase OXA-48, we tested the long-standing hypothesis that selective compartments with low antibiotic concentrations cause standing genetic diversity that could act as a gateway to developing clinical resistance. Here, we subjected Escherichia coli expressing blaOXA-48, on a clinical plasmid, to experimental evolution at sub-MICs of ceftazidime. We identified and characterized seven single variants of OXA-48. Susceptibility profiles and dose-response curves showed that they increased resistance only marginally. However, in competition experiments at sub-MICs of ceftazidime, they demonstrated strong selectable fitness benefits. Increased resistance was also reflected in elevated catalytic efficiencies toward ceftazidime. These changes are likely caused by enhanced flexibility of the Ω- and ß5-ß6 loops and fine-tuning of preexisting active site residues. In conclusion, low-level concentrations of ß-lactams can drive the evolution of ß-lactamases through cryptic phenotypes which may act as stepping-stones toward clinical resistance.IMPORTANCE Very low antibiotic concentrations have been shown to drive the evolution of antimicrobial resistance. While substantial progress has been made to understand the driving role of low concentrations during resistance development for different antimicrobial classes, the importance of ß-lactams, the most commonly used antibiotics, is still poorly studied. Here, we shed light on the evolutionary impact of low ß-lactam concentrations on the widespread ß-lactamase OXA-48. Our data indicate that the exposure to ß-lactams at very low concentrations enhances ß-lactamase diversity and drives the evolution of ß-lactamases by significantly influencing their substrate specificity. Thus, in contrast to high concentrations, low levels of these drugs may substantially contribute to the diversification and divergent evolution of these enzymes, providing a standing genetic diversity that can be selected and mobilized when antibiotic pressure increases.

Chalcogenido-Dimethylgallates and -Indates DMPyr2 [Me2 M(µ2 -E)]2 (M=Ga, In; E=S, Se): Building Blocks for Higher and Lower Order Chalcogenidoindates.

Metalation of the anions in the ionic liquids DMPyr[SH] and DMPyr[SeH] (DMPyr=1,1-dimethylpyrrolidinium) by trimethylgallium and trimethylindium is investigated. The reaction proceeds via pre-coordination of [EH]- , methane elimination and formation of an unprecedented series of chalcogenido metalates DMPyr2 [Me2 M(µ2 -E)]2 (M=Ga, In; E=S, Se). These show the presences of dinuclear dianions with four-membered ring structures displaying highly nucleophilic bridging chalcogenide ligands in their crystallographically determined molecular structures. Some representative reactions of these building blocks with amphoteric electrophiles were studied: Addition of two equivalents of E(SiMe3 )2 (E=S, Se) to the indates DMPyr2 [Me2 In(µ2 -S)]2 and DMPyr2 [Me2 In(µ2 -Se)]2 leads to a cleavage of the ring, E silylation and formation of mononuclear, monoanionic indates DMPyr[Me2 In(SSiMe3 )2 ], DMPyr[Me2 In(SeSiMe3 )2 ], and even a mixed sulfido-selenido dimethylindate DMPyr[Me2 In(SSiMe3 )(SeSiMe3 )]. Reaction of DMPyr2 [Me2 In(µ2 -S)]2 with two equivalents of Lewis acid Me3 In leads to charge delocalization, ring expansion and formation of six-membered ring DMPyr3 [Me2 In(µ2 -S-InMe3 )]3 . The latter is a key intermediate in the formation of dianionic sulfidoindate DMPyr2 [(Me2 In)6 (µ3 -S)4 ] displaying an unusual inverse heteroadamantane cage structure with four capping sulfido ligands.

Tn1 transposition in the course of natural transformation enables horizontal antibiotic resistance spread in Acinetobacter baylyi.

Transposons are genetic elements that change their intracellular genomic position by transposition and are spread horizontally between bacteria when located on plasmids. It was recently discovered that transposition from fully heterologous DNA also occurs in the course of natural transformation. Here, we characterize the molecular details and constraints of this process using the replicative transposon Tn1 and the naturally competent bacterium Acinetobacter baylyi. We find that chromosomal insertion of Tn1 by transposition occurs at low but detectable frequencies and preferably around the A. baylyi terminus of replication. We show that Tn1 transposition is facilitated by transient expression of the transposase and resolvase encoded by the donor DNA. RecA protein is essential for the formation of a circular, double-stranded cytoplasmic intermediate from incoming donor DNA, and RecO is beneficial but not essential in this process. Absence of the recipient RecBCD nuclease stabilizes the double-stranded intermediate. Based on these results, we suggest a mechanistic model for transposition during natural transformation.

Asymmetric Ring-Closing Aminooxygenation of Alkenes en Route to 2-Amino-1,3-Diols with Vicinal Stereocenters.

A ring-closing aminooxygenation of alkenes with N-benzoyloxycarbamates occurs with very high diastereoselectivity (typically >20:1 d.r.) and very high enantioselectivity (up to 99% ee). The reaction is catalyzed by a recently developed chiral-at-metal ruthenium complex at catalyst loadings of 0.5-1.0 mol %. The reaction is proposed to proceed through a ruthenium nitrenoid intermediate that depending on the nature of the substrate undergoes either an aminooxygenation (1,2-disubstituted alkenes) or stops at the stage of the aziridination (trisubstituted alkenes), which can then be ring opened with benzoic acid. The resulting chiral cyclic carbamates can be hydrolyzed under basic conditions to provide versatile chiral 2-amino-1,3-diols with vicinal stereocenters.

Total Synthesis of (-)-Preussochromone A.

An enantioselective total synthesis of the natural product (-)-preussochromone A is reported. The tricyclic thiopyrane skeleton could be assembled via Lewis acid-mediated cycloisomerization of a precursor with a 2-thiochromenone substructure and an α-ketoester moiety. The chromenone core was synthesized by cyclization of a dithioketene acetal and oxidation to a 2-sulfonylchromenone to set up the subsequent thia-Michael-retro-Michael addition of an aliphatic thiol producing the highly oxidized side chain.

Unexpected formation of a co-crystal containing the chalcone (E)-1-(5-chloro-thio-phen-2-yl)-3-(3-methyl-thio-phen-2-yl)prop-2-en-1-one and the keto-enol tautomer (Z)-1-(5-chloro-thio-phen-2-yl)-3-(3-methyl-thio-phen-2-yl)prop-1-en-1-ol.

The title crystal structure is assembled from the superposition of two mol-ecular structures, (E)-1-(5-chloro-thio-phen-2-yl)-3-(3-methyl-thio-phen-2-yl)prop-2-en-1-one, C12H9ClOS2 (93%), and (Z)-1-(5-chloro-thio-phen-2-yl)-3-(3-methyl-thio-phen-2-yl)prop-1-en-1-ol, C12H11ClOS2 (7%), 0.93C12H9ClOS2·0.07C12H11ClOS2. Both were obtained from the reaction of 3-methyl-thio-phene-2-carbaldehyde and 1-(5-chloro-thio-phen-2-yl)ethanone. In the extended structure of the major chalcone component, mol-ecules are linked by a combination of C-H⋯O/S, Cl⋯Cl, Cl⋯π and π-π inter-actions, leading to a compact three-dimensional supra-molecular assembly.

Directed C(sp3)-H arylation of tryptophan: transformation of the directing group into an activated amide.

The 8-aminoquinoline (8AQ) directed C(sp3)-H functionalization was applied in the synthesis of ß-arylated tryptophan derivatives. The laborious protecting group reorganization towards α-amino acids compatible for solid phase peptide synthesis (SPPS) was cut short by the transformation of the directing group into an activated amide, which was either used directly in peptide coupling or in the gram scale synthesis of storable Fmoc-protected amino acids for SPPS. In this work, directed C-H activation and nonplanar amide chemistry complement each other for the synthesis of hybrids between phenylalanine and tryptophan with restricted side chain mobility.

Non-C2-Symmetric Chiral-at-Ruthenium Catalyst for Highly Efficient Enantioselective Intramolecular C(sp3)-H Amidation.

A new class of chiral ruthenium catalysts is introduced in which ruthenium is cyclometalated by two 7-methyl-1,7-phenanthrolinium heterocycles, resulting in chelating pyridylidene remote N-heterocyclic carbene ligands (rNHCs). The overall chirality results from a stereogenic metal center featuring either a Λ or Δ absolute configuration. This work features the importance of the relative metal-centered stereochemistry. Only the non-C2-symmetric chiral-at-ruthenium complexes display unprecedented catalytic activity for the intramolecular C(sp3)-H amidation of 1,4,2-dioxazol-5-ones to provide chiral γ-lactams with up to 99:1 er and catalyst loadings down to 0.005 mol % (up to 11 200 TON), while the C2-symmetric diastereomer favors an undesired Curtius-type rearrangement. DFT calculations elucidate the origins of the superior C-H amidation reactivity displayed by the non-C2-symmetric catalysts compared to related C2-symmetric counterparts.

Bis-Cyclometalated Indazole Chiral-at-Rhodium Catalyst for Asymmetric Photoredox Cyanoalkylations.

A new class of bis-cyclometalated rhodium(III) catalysts containing two inert cyclometalated 6-tert-butyl-2-phenyl-2H-indazole ligands and two labile acetonitriles is introduced. Single enantiomers (>99 % ee) were obtained through a chiral-auxiliary-mediated approach using a monofluorinated salicyloxazoline. The new chiral-at-metal complex is capable of catalyzing the visible-light-induced enantioselective α-cyanoalkylation of 2-acyl imidazoles in which it serves a dual function as the chiral Lewis acid catalyst for the asymmetric radical chemistry and at the same time as the photoredox catalyst for the visible-light-induced redox chemistry (up to 80 % yield, 4:1 d.r., and 95 % ee, 12 examples).

Synthesis, characterization, crystal structure and supra-molecularity of ethyl (E)-2-cyano-3-(3-methyl-thio-phen-2-yl)acrylate and a new polymorph of ethyl (E)-2-cyano-3-(thio-phen-2-yl)acrylate.

The synthesis, crystal structure and structural motif of two thio-phene-based cyano-acrylate derivatives, namely, ethyl (E)-2-cyano-3-(3-methyl-thio-phen-2-yl)acrylate (1), C11H11NO2S, and ethyl (E)-2-cyano-3-(thio-phen-2-yl)acrylate (2), C10H9NO2S, are reported. Derivative 1 crystallized with two independent molecules in the asymmetric unit, and derivative 2 represents a new monoclinic (C2/m) polymorph. The mol-ecular conformations of 1 and the two polymorphs of 2 are very similar, as all non-H atoms are planar except for the methyl of the ethyl groups. The inter-molecular inter-actions and crystal packing of 1 and 2 are described and compared with that of the reported monoclinic (C2/m) polymorph of derivative 2 [Castro Agudelo et al. (2017 ▸). Acta Cryst. E73, 1287-1289].

Asymmetric Photocatalysis by Intramolecular Hydrogen-Atom Transfer in Photoexcited Catalyst-Substrate Complex.

3-(2-Formylphenyl)-1-pyrazol-1-yl-propenones undergo an asymmetric photorearrangement to benzo[d]cyclopropa[b]pyranones with up to >99 % ee, which is catalyzed by a bis-cyclometalated rhodium catalyst in the presence of visible light. Mechanistic experiments and DFT calculations support a mechanism in which a photoexcited catalyst/substrate complex triggers an intramolecular hydrogen-atom transfer followed by a highly stereocontrolled hetero-Diels-Alder reaction. In this reaction scheme, the rhodium catalyst fulfills multiple functions by 1) enabling visible-light π→π* excitation of the catalyst-bound enone substrate, 2) facilitating the hydrogen-atom transfer, and 3) providing the asymmetric induction for the hetero-Diels-Alder reaction.