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An Assay for Quantifying Protein-RNA Binding in Bacteria
Published on: June 12, 2019
Identification of neisserial DNA binding components.
Emma Lång1,2, Kristine Haugen1, Burkhard Fleckenstein3
1Centre for Molecular Biology and Neuroscience, Institute of Microbiology, Rikshospitalet, Oslo, Norway.
Microbiology (Reading, England)
|February 28, 2009
Summary
Neisseria meningitidis natural transformation involves inner membrane proteins like PilG and ComL in DNA binding. These proteins, along with pilus machinery, are crucial for DNA uptake and transformation competence.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Neisseria meningitidis causes meningitis and septicaemia.
- Type IV pili are involved in DNA uptake via natural transformation.
- The outer membrane protein PilQ binds DNA in its pore region.
Purpose of the Study:
- To identify DNA-binding proteins in the inner membrane of Neisseria meningitidis.
- To elucidate the mechanism of DNA transport across bacterial membranes during natural transformation.
Main Methods:
- Solid-phase overlay assay using DNA substrates on inner membrane fractions.
- Mass spectrometry (MS) for protein identification.
- Construction and phenotypic analysis of null mutants for identified DNA-binding proteins.
Main Results:
- Identified several DNA-binding proteins including PilG, ComL, FtsE, and two hypothetical proteins.
- DNA-binding activity was independent of the neisserial DNA uptake sequence.
- Only mutants defective in pilus biogenesis showed defects in competence and pili formation.
Conclusions:
- Pilus biogenesis components PilQ and PilG, along with other identified proteins, are directly involved in natural transformation.
- These proteins play active roles in DNA uptake, not just indirectly through pilus assembly.
