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      A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes

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          ABSTRACT

          To enhance the research capabilities of investigators interested in Staphylococcus aureus, the Nebraska Center for Staphylococcal Research (CSR) has generated a sequence-defined transposon mutant library consisting of 1,952 strains, each containing a single mutation within a nonessential gene of the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) isolate USA300. To demonstrate the utility of this library for large-scale screening of phenotypic alterations, we spotted the library on indicator plates to assess hemolytic potential, protease production, pigmentation, and mannitol utilization. As expected, we identified many genes known to function in these processes, thus validating the utility of this approach. Importantly, we also identified genes not previously associated with these phenotypes. In total, 71 mutants displayed differential hemolysis activities, the majority of which were not previously known to influence hemolysin production. Furthermore, 62 mutants were defective in protease activity, with only 14 previously demonstrated to be involved in the production of extracellular proteases. In addition, 38 mutations affected pigment formation, while only 7 influenced mannitol fermentation, underscoring the sensitivity of this approach to identify rare phenotypes. Finally, 579 open reading frames were not interrupted by a transposon, thus providing potentially new essential gene targets for subsequent antibacterial discovery. Overall, the Nebraska Transposon Mutant Library represents a valuable new resource for the research community that should greatly enhance investigations of this important human pathogen.

          IMPORTANCE

          Infections caused by Staphylococcus aureus cause significant morbidity and mortality in both community and hospital environments. Specific-allelic-replacement mutants are required to study the biology of this organism; however, this process is costly and time-consuming. We describe the construction and validation of a sequence-defined transposon mutant library available for use by the scientific community through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) strain repository. In addition, complementary resources, including a website ( http://app1.unmc.edu/fgx/) and genetic tools that expedite the allelic replacement of the transposon in the mutants with useful selectable markers and fluorescent reporter fusions, have been generated. Overall, this library and associated tools will have a significant impact on studies investigating S. aureus pathogenesis and biology and serve as a useful paradigm for the study of other bacterial systems.

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          Most cited references38

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          Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database.

          Oxacillin-resistant Staphylococcus aureus (ORSA) is a virulent pathogen responsible for both health care-associated and community onset disease. We used SmaI-digested genomic DNA separated by pulsed-field gel electrophoresis (PFGE) to characterize 957 S. aureus isolates and establish a database of PFGE patterns. In addition to PFGE patterns of U.S. strains, the database contains patterns of representative epidemic-type strains from the United Kingdom, Canada, and Australia; previously described ORSA clonal-type isolates; 13 vancomycin-intermediate S. aureus (VISA) isolates, and two high-level vancomycin-resistant, vanA-positive strains (VRSA). Among the isolates from the United States, we identified eight lineages, designated as pulsed-field types (PFTs) USA100 through USA800, seven of which included both ORSA and oxacillin-susceptible S. aureus isolates. With the exception of the PFT pairs USA100 and USA800, and USA300 and USA500, each of the PFTs had a unique multilocus sequence type and spa type motif. The USA100 PFT, previously designated as the New York/Tokyo clone, was the most common PFT in the database, representing 44% of the ORSA isolates. USA100 isolates were typically multiresistant and included all but one of the U.S. VISA strains and both VRSA isolates. Multiresistant ORSA isolates from the USA200, -500, and -600 PFTs have PFGE patterns similar to those of previously described epidemic strains from Europe and Australia. The USA300 and -400 PFTs contained community isolates resistant only to beta-lactam drugs and erythromycin. Noticeably absent from the U.S. database were isolates with the previously described Brazilian and EMRSA15 PFGE patterns. These data suggest that there are a limited number of ORSA genotypes present in the United States.
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            Interactive microbial genome visualization with GView

            Summary: GView is a Java application for viewing and examining prokaryotic genomes in a circular or linear context. It accepts standard sequence file formats and an optional style specification file to generate customizable, publication quality genome maps in bitmap and scalable vector graphics formats. GView features an interactive pan-and-zoom interface, a command-line interface for incorporation in genome analysis pipelines, and a public Application Programming Interface for incorporation in other Java applications. Availability: GView is a freely available application licensed under the GNU Public License. The application, source code, documentation, file specifications, tutorials and image galleries are available at http://gview.ca Contact: gary.van.domselaar@phac-aspc.gc.ca
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              Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity

              Golden color imparted by carotenoid pigments is the eponymous feature of the human pathogen Staphylococcus aureus. Here we demonstrate a role of this hallmark phenotype in virulence. Compared with the wild-type (WT) bacterium, a S. aureus mutant with disrupted carotenoid biosynthesis is more susceptible to oxidant killing, has impaired neutrophil survival, and is less pathogenic in a mouse subcutaneous abscess model. The survival advantage of WT S. aureus over the carotenoid-deficient mutant is lost upon inhibition of neutrophil oxidative burst or in human or murine nicotinamide adenine dinucleotide phosphate oxidase–deficient hosts. Conversely, heterologous expression of the S. aureus carotenoid in the nonpigmented Streptococcus pyogenes confers enhanced oxidant and neutrophil resistance and increased animal virulence. Blocking S. aureus carotenogenesis increases oxidant sensitivity and decreases whole-blood survival, suggesting a novel target for antibiotic therapy.
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                Author and article information

                Journal
                mBio
                MBio
                mbio
                mbio
                mBio
                mBio
                American Society of Microbiology (1752 N St., N.W., Washington, DC )
                2150-7511
                12 February 2013
                Jan-Feb 2013
                : 4
                : 1
                : e00537-12
                Affiliations
                Department of Pathology and Microbiology, Center for Staphylococcal Research, University of Nebraska Medical Center, Omaha, Nebraska, USA [ a ];
                Department of Internal Medicine, Center for Bioinformatics and Systems Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA [ b ]
                Author notes
                Address correspondence to Kenneth W. Bayles, kbayles@ 123456unmc.edu .

                Editor Karen Bush, Indiana University, Bloomington

                Article
                mBio00537-12
                10.1128/mBio.00537-12
                3573662
                23404398
                74949b73-bfd4-46cc-b538-5dd0888f8ea1
                Copyright © 2013 Fey et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 20 November 2012
                : 14 January 2013
                Page count
                Pages: 8
                Categories
                Research Article
                Custom metadata
                January/February 2013

                Life sciences
                Life sciences

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