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Kinetic Screening of Nuclease Activity using Nucleic Acid Probes
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Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.

Megan R Kiedrowski1, Heidi A Crosby1, Frank J Hernandez2

  • 1Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, United States of America.

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Staphylococcus aureus possesses a second surface-bound nuclease, Nuc2, which is active during infections. This bacterial enzyme, similar to Nuc, degrades DNA and impacts biofilm formation.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • Staphylococcus aureus is a significant bacterial pathogen.
  • The secreted nuclease (Nuc) is a known virulence factor.
  • A second nuclease, Nuc2, in S. aureus is less understood.

Purpose of the Study:

  • To characterize the localization, expression, and biochemical properties of Staphylococcus aureus Nuc2.
  • To determine Nuc2's role in infection and biofilm formation.

Main Methods:

  • Utilized fluorescence microscopy and alkaline phosphatase fusions for Nuc2 localization.
  • Employed fluorescence resonance energy transfer (FRET) assays to detect cell surface enzyme activity.
  • Assessed Nuc2 activity in vivo using a mouse pyomyositis model and activatable probes.
  • Purified Nuc2 for biochemical characterization and in vitro biofilm assays.

Main Results:

  • Nuc2 is a signal-anchored Type II membrane protein with its C-terminus exposed extracellularly.
  • Nuc2 enzyme activity and transcription are highest during early logarithmic growth.
  • Nuc2 activity was detected in vivo during S. aureus infections.
  • Purified Nuc2 exhibits DNA cleavage activity, thermostability, and calcium dependence, similar to Nuc.
  • Nuc2 inhibited biofilm formation in vitro.

Conclusions:

  • Staphylococcus aureus encodes a second, functional, surface-attached DNase (Nuc2).
  • Nuc2 is expressed during infection and shares biochemical properties with the secreted Nuc enzyme.
  • Nuc2 plays a role in modulating biofilm formation.