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Tools, Strains, and Strategies To Effectively Conduct Anaerobic and Aerobic Transcriptional Reporter Screens and Assays in Staphylococcus aureus

Price, Erin E ; Rudra, Paulami ; Norambuena, Javiera ; Román-Rodríguez, Franklin ; Boyd, Jeffrey M Semrau, Jeremy D ; Semrau, Jeremy D.

Applied and environmental microbiology, 2021-10, Vol.87 (21), p.e0110821-e0110821 [Periódico revisado por pares]

United States: American Society for Microbiology

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  • Título:
    Tools, Strains, and Strategies To Effectively Conduct Anaerobic and Aerobic Transcriptional Reporter Screens and Assays in Staphylococcus aureus
  • Autor: Price, Erin E ; Rudra, Paulami ; Norambuena, Javiera ; Román-Rodríguez, Franklin ; Boyd, Jeffrey M
  • Semrau, Jeremy D ; Semrau, Jeremy D.
  • Assuntos: Anaerobiosis ; Applied and Industrial Microbiology ; Binding sites ; Expression vectors ; Ferricyanide ; Ferrocyanide ; Fluorescence ; Galactosidase ; Gene Expression Regulation, Bacterial ; Gene regulation ; Genes, Reporter ; Genetic screening ; Genetics and Molecular Biology ; Histocompatibility antigen HLA ; Insertion ; Iron cyanides ; Mutants ; Screening ; Spotlight ; Spotlight Selection ; Staphylococcus aureus ; Staphylococcus aureus - genetics ; Transcription ; Transcription, Genetic ; β-Galactosidase
  • É parte de: Applied and environmental microbiology, 2021-10, Vol.87 (21), p.e0110821-e0110821
  • Notas: ObjectType-Article-1
    SourceType-Scholarly Journals-1
    ObjectType-Feature-2
    content type line 23
    Citation Price EE, Rudra P, Norambuena J, Román-Rodríguez F, Boyd JM. 2021. Tools, strains, and strategies to effectively conduct anaerobic and aerobic transcriptional reporter screens and assays in Staphylococcus aureus. Appl Environ Microbiol 87:e01108-21. https://doi.org/10.1128/AEM.01108-21.
    Erin E. Price and Paulami Rudra contributed equally to the manuscript. The order of first authors was determined alphabetically.
  • Descrição: Transcriptional reporters are reliable and time-tested tools to study gene regulation. In Staphylococcus aureus, β-galactosidase ( )-based genetic screens are not widely used because of the necessity of selectable markers for strain construction and the production of staphyloxanthin pigment, which obfuscates results. We describe a series of vectors that allow for markerless insertion of codon-optimized -based transcriptional reporters. The vectors code for different ribosomal binding sites, allowing for tailored expression. A Δ :: deletion insertion mutant was constructed that prevents the synthesis of staphyloxanthin, thereby permitting blue-white screening without the interference of carotenoid production. We demonstrate the utility of these vectors to monitor aerobic and anaerobic transcriptional activities. For the latter, we describe the use of a ferrocyanide-ferricyanide redox system [Fe(CN) ] permitting blue-white screening in the absence of oxygen. We also describe additional reporter systems and methods for monitoring transcriptional activity during anaerobic culture, including an FAD-binding fluorescent protein ( ), alpha-hemolysin ( ), or lipase ( ). The systems and methods described are compatible with vectors utilized to create and screen high-density transposon mutant libraries. Staphylococcus aureus is a human pathogen and a leading cause of infectious disease-related illness and death worldwide. For S. aureus to successfully colonize and invade host tissues, it must tightly control the expression of genes encoding virulence factors. Oxygen tension varies greatly at infection sites, and many abscesses are devoid of oxygen. In this study, we have developed novel tools and methods to study how and when S. aureus alters transcription of genes. A key advantage of these methods and tools is that they can be utilized in the presence and absence of oxygen. A better understanding of anaerobic gene expression in S. aureus will provide important insights into the regulation of genes in low-oxygen environments.
  • Editor: United States: American Society for Microbiology
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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