Part:BBa_K5317013

PknB

Usage and Biology

PknB is a eukaryote-like serine/threonine kinase from Staphylococcus aureus that plays an important role in the bacterial response to antibiotics, particularly beta-lactams, via its PASTA domain (Stehle et al.2012). PknB is a membrane-localized protein consisting of an N-terminal cytosolic kinase domain, a central transmembrane segment, and three C-terminal extracellular PASTA domains. The PASTA (penicillin-binding protein and serine/threonine kinase-associated) domain plays a critical role in the recognition and binding of beta-lactam antibiotics (Stehle et al.2012). Upon binding these compounds, the PASTA domain initiates a signaling cascade by inducing the N-terminal kinase domain autophosphorylation. This activation leads to the initiation of downstream signaling pathways (Cheung et al.2010). In S. aureus, this mechanism is critical for the early detection of antibiotics and helps the bacteria adapt to antibiotic stress (Sauer et al. 2018).

Cloning

Theoretical Part Design

The sequence of pknB was obtained from S. aureus. The construct was codon optimized for human cell lines and synthesized by Eurofins.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1213
  Illegal SpeI site found at 1876
  Illegal PstI site found at 638
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 1876
  Illegal PstI site found at 638
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 888
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1213
  Illegal SpeI site found at 1876
  Illegal PstI site found at 638
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1213
  Illegal SpeI site found at 1876
  Illegal PstI site found at 638
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The kinase was analyzed by composing a gene cassette where its placed upstream of the reporter gene EGFP to assess the ß-lactam dependent effect based on the fluorescent signal. Please visit the K5317018 registry entry to view the results.

References

Pensinger, D. A., Schaenzer, A. J., & Sauer, J. D. (2018). Do Shoot the Messenger: PASTA Kinases as Virulence Determinants and Antibiotic Targets. Trends in microbiology, 26(1), 56–69. https://doi.org/10.1016/j.tim.2017.06.010

Rakette S, Donat S, Ohlsen K, Stehle T (2012) Structural Analysis of Staphylococcus aureus Serine/Threonine Kinase PknB. PLOS ONE 7(6): e39136. https://doi.org/10.1371/journal.pone.0039136

Tamber, S., Schwartzman, J., & Cheung, A. L. (2010). Role of PknB kinase in antibiotic resistance and virulence in community-acquired methicillin-resistant Staphylococcus aureus strain USA300. Infection and immunity, 78(8), 3637–3646. https://doi.org/10.1128/IAI.00296-10