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bInfections with the Gram-negative coccobacillus Acinetobacter baumannii are a major threat in hospital settings. The progressing emergence of multidrug-resistant clinical strains significantly reduces the treatment options for clinicians to fight A. baumannii infections. The current lack of robust methods to genetically manipulate drug-resistant A. baumannii isolates impedes research on resistance and virulence mechanisms in clinically relevant strains. In this study, we developed a highly efficient and versatile genome-editing platform enabling the markerless modification of the genome of A. baumannii clinical and laboratory strains, regardless of their resistance profiles. We applied this method for the deletion of AdeR, a transcription factor that regulates the expression of the AdeABC efflux pump in tigecycline-resistant A. baumannii, to evaluate its function as a putative drug target. Loss of adeR reduced the MIC 90 of tigecycline from 25 g/ml in the parental strains to 3.1 g/ml in the ⌬adeR mutants, indicating its importance in the drug resistance phenotype. However, 60% of the clinical isolates remained nonsusceptible to tigecycline after adeR deletion. Evolution of artificial tigecycline resistance in two strains followed by whole-genome sequencing revealed loss-of-function mutations in trm, suggesting its role in an alternative AdeABC-independent tigecycline resistance mechanism. This finding was strengthened by the confirmation of trm disruption in the majority of the tigecycline-resistant clinical isolates. This study highlights the development and application of a powerful genome-editing platform for A. baumannii enabling future research on drug resistance and virulence pathways in clinically relevant strains. O ne of the greatest global health problems results from the limited treatment options to fight bacterial infections caused by multidrug-resistant (MDR) organisms. The group of ESKAPE organisms that is comprised of Enterobacter spp., Staphylococcus aureus/epidermidis, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecalis/faecium is considered to cause the vast majority of, often untreatable, nosocomial infections (1). Among these ESKAPE pathogens A. baumannii is most difficult to treat due to its multiple intrinsic and acquired resistance mechanisms that resulted in the development of MDR, extensively drug resistant (XDR), or even pan-drug-resistant (PDR) phenotypes (2-5).Bacteria have evolved multiple ways to evade antibiotic-mediated cell death, such as (i) enzymatic modification/cleavage of the antibiotic (e.g., beta-lactams), (ii) modification/protection of the antibiotic target (e.g., fluoroquinolones), or (iii) reduction of the intracellular concentration by antibiotic efflux or reduced influx (e.g., tetracyclines) (6). The expression of such defense mechanisms may require an extensive metabolic investment, often leading to a reduced fitness of these resistant bacteria in the absence of the external selection pressure (7). To overcome these ecol...

The P-hydroxyamino structural unit is a key motif in many biologically important molecules. It is difficult to imagine a more efficient means of creating this functionality than by direct addition of the two heteroatom substituents to an olefin, especially if this transformation could also be regio-and/or enantioselective when required. We recently reported the discovery of such a catalytic asymmetric aminohydroxylation (AA) reaction. On treatment with a chiral alkaloid ligand, catalytic osmium, Chloramine-T as the nitrogen source, and water as the oxygen source, olefins undergo enantioselective vicinal addition of a suifonamido and a hydroxyl group."' A limited range of substrates was investigated and enantioselectivities of up to 82 % were achieved.['] These initial results clearly leave much room for improvement. Above all, higher enantioselectivities are needed and, when applicable, higher regioselectivities. Another goal is better chemoselectivity, which principally means suppressing formation of the vicinal diol, sometimes a significant by-product.We report here on progress in all three selectivity categories by use of sulfonamide-derived chloramine salts with smaller organic substituents on the sulfur.L31 The best results to date (Scheme 1, Table 1) were obtained with the methanesulfonamide-derived chloramine salt 2 (Chloramine-ME4]). This reagent can be prepared separately and added as the stable anhydrous salt or it can be generated in sit^.'^.^^ The in situ method is preferred for large-scale applications.As shown in Scheme 1 for methyl trans-cinnamate (l), the procedure is identical to the original AA procedure with the exception that Chloramine-M replaces Chloramine-T. This sub- 1) was the solvent, and an additional 0.5 equiv of lerr-butylhypochlorite was necessary to achieve these results DHQ-H = dihydroquinine, DHQD-H = dihydroquinidine, PHAL = 1.3-phthalazinediyl.

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