A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential <named-content content-type="genus-species">Staphylococcus aureus</named-content> Genes

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 ge...

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Autores principales:	Paul D. Fey, Jennifer L. Endres, Vijaya Kumar Yajjala, Todd J. Widhelm, Robert J. Boissy, Jeffrey L. Bose, Kenneth W. Bayles
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2013
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/9ab586a47c944699b6d2c8865c047ec5
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spelling	oai:doaj.org-article:9ab586a47c944699b6d2c8865c047ec52021-11-15T15:40:23ZA Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential <named-content content-type="genus-species">Staphylococcus aureus</named-content> Genes10.1128/mBio.00537-122150-7511https://doaj.org/article/9ab586a47c944699b6d2c8865c047ec52013-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00537-12https://doaj.org/toc/2150-7511ABSTRACT 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.Paul D. FeyJennifer L. EndresVijaya Kumar YajjalaTodd J. WidhelmRobert J. BoissyJeffrey L. BoseKenneth W. BaylesAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 4, Iss 1 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Paul D. Fey Jennifer L. Endres Vijaya Kumar Yajjala Todd J. Widhelm Robert J. Boissy Jeffrey L. Bose Kenneth W. Bayles A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential <named-content content-type="genus-species">Staphylococcus aureus</named-content> Genes
description	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.
format	article
author	Paul D. Fey Jennifer L. Endres Vijaya Kumar Yajjala Todd J. Widhelm Robert J. Boissy Jeffrey L. Bose Kenneth W. Bayles
author_facet	Paul D. Fey Jennifer L. Endres Vijaya Kumar Yajjala Todd J. Widhelm Robert J. Boissy Jeffrey L. Bose Kenneth W. Bayles
author_sort	Paul D. Fey
title	A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential <named-content content-type="genus-species">Staphylococcus aureus</named-content> Genes
title_short	A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential <named-content content-type="genus-species">Staphylococcus aureus</named-content> Genes
title_full	A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential <named-content content-type="genus-species">Staphylococcus aureus</named-content> Genes
title_fullStr	A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential <named-content content-type="genus-species">Staphylococcus aureus</named-content> Genes
title_full_unstemmed	A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential <named-content content-type="genus-species">Staphylococcus aureus</named-content> Genes
title_sort	genetic resource for rapid and comprehensive phenotype screening of nonessential <named-content content-type="genus-species">staphylococcus aureus</named-content> genes
publisher	American Society for Microbiology
publishDate	2013
url	https://doaj.org/article/9ab586a47c944699b6d2c8865c047ec5
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A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential <named-content content-type="genus-species">Staphylococcus aureus</named-content> Genes

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