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Volume 168, Issue 4

Screening transcriptional connections in using high-throughput transduction of bioluminescent reporter plasmids Free

Abstract

Characterization of transcriptional networks is one of the main strategies used to understand how bacteria interact with their environment. To reveal novel regulatory elements in the human pathogen , we adapted a traditional transduction protocol to be used in a high-throughput format in combination with the publicly available Nebraska Transposon Mutant Library. Specifically, plasmid transductions are performed in 96-well format, so that a single plasmid can be simultaneously transferred into numerous recipient strains. When used in conjunction with bioluminescent reporter constructs, this strategy enables parallel and continuous monitoring of downstream transcriptional effects of hundreds of defined mutations. Here, we use this workflow in a proof-of-concept study to identify novel regulators of the staphylococcal metalloprotease aureolysin. Importantly, this strategy can be utilized with any other bacterium where plasmid transduction is possible, making it a versatile and efficient tool to probe transcriptional regulatory connections.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): NTML , reporter plasmid , S. aureus Nebraska Transposon Mutant Library , screening , Staphylococcus aureus and transduction
Funding
This study was supported by the:
  • National Institute of Allergy and Infectious Diseases (Award F32AI157215)
    • Principle Award Recipient: AndyWeiss
  • National Institute of Environmental Health Sciences (Award T32ES007028)
    • Principle Award Recipient: AndyWeiss
  • American Heart Association (Award 18POST33990262)
    • Principle Award Recipient: AndyWeiss
  • National Institute of Allergy and Infectious Diseases (Award AI124458)
    • Principle Award Recipient: LindseyN. Shaw
  • National Institute of Allergy and Infectious Diseases (Award R01AI069233)
    • Principle Award Recipient: EricP. Skaar
  • National Institute of Allergy and Infectious Diseases (Award R01AI073843)
    • Principle Award Recipient: EricP. Skaar
  • National Institute of Allergy and Infectious Diseases (Award AI101171)
    • Principle Award Recipient: EricP. Skaar
© 2022 The Authors
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2022-04-21
2024-04-25
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Screening transcriptional connections in Staphylococcus aureus using high-throughput transduction of bioluminescent reporter plasmids
Microbiology 168, 001174 (2022); https://doi.org/10.1099/mic.0.001174
/content/journal/micro/10.1099/mic.0.001174
/content/journal/micro/10.1099/mic.0.001174
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