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Editor's Pick Research Article

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

Paul D. Fey, Jennifer L. Endres, Vijaya Kumar Yajjala, Todd J. Widhelm, Robert J. Boissy, Jeffrey L. Bose, Kenneth W. Bayles
Karen Bush, Editor
Paul D. Fey
Department of Pathology and Microbiology, Center for Staphylococcal Research, University of Nebraska Medical Center, Omaha, Nebraska, USA
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Jennifer L. Endres
Department of Pathology and Microbiology, Center for Staphylococcal Research, University of Nebraska Medical Center, Omaha, Nebraska, USA
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Vijaya Kumar Yajjala
Department of Pathology and Microbiology, Center for Staphylococcal Research, University of Nebraska Medical Center, Omaha, Nebraska, USA
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Todd J. Widhelm
Department of Pathology and Microbiology, Center for Staphylococcal Research, University of Nebraska Medical Center, Omaha, Nebraska, USA
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Robert J. Boissy
Department of Internal Medicine, Center for Bioinformatics and Systems Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA
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Jeffrey L. Bose
Department of Pathology and Microbiology, Center for Staphylococcal Research, University of Nebraska Medical Center, Omaha, Nebraska, USA
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Kenneth W. Bayles
Department of Pathology and Microbiology, Center for Staphylococcal Research, University of Nebraska Medical Center, Omaha, Nebraska, USA
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Karen Bush
Indiana University, Bloomington
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DOI: 10.1128/mBio.00537-12
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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.

  • 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.

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A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes
Paul D. Fey, Jennifer L. Endres, Vijaya Kumar Yajjala, Todd J. Widhelm, Robert J. Boissy, Jeffrey L. Bose, Kenneth W. Bayles
mBio Feb 2013, 4 (1) e00537-12; DOI: 10.1128/mBio.00537-12

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A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes
Paul D. Fey, Jennifer L. Endres, Vijaya Kumar Yajjala, Todd J. Widhelm, Robert J. Boissy, Jeffrey L. Bose, Kenneth W. Bayles
mBio Feb 2013, 4 (1) e00537-12; DOI: 10.1128/mBio.00537-12
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