Related Experiment Videos
Proteolytic activity of YibP protein in Escherichia coli
Toshiharu Ichimura1, Mitsuyoshi Yamazoe, Maki Maeda
1Division of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kuhonji 4-24-1, Kumamoto 862-0976, Japan.
Abstract:
Escherichia coli YibP protein (47.4 kDa) has a membrane-spanning signal at the N-terminal region, two long coiled-coil regions in the middle part, and a C-terminal globular domain, which involves amino acid sequences homologous to the peptidase M23/M37 family. A yibP disrupted mutant grows in rich medium at 37 degrees C but not at 42 degrees C. In the yibP null mutant, cell division and FtsZ ring formation are inhibited at 42 degrees C without SOS induction, resulting in filamentous cells with multiple nucleoids and finally in cell lysis. Five percent betaine suppresses the temperature sensitivity of the yibP disrupted mutation. The mutant has the same sensitivity to drugs, such as nalidixic acid, ethidium bromide, ethylmethane sulfonate, and sodium dodecyl sulfate, as the parental strain. YibP protein is recovered in the inner membrane and cytoplasmic fractions, but not in the outer membrane fraction. Results suggest that the coiled-coil regions and the C-terminal globular domain of YibP are localized in the cytoplasmic space, not in the periplasmic space. Purified YibP has a protease activity that split the substrate beta-casein.
Insights
The Escherichia coli YibP protein is crucial for cell division and FtsZ ring formation, especially at higher temperatures. Its absence leads to cell filamentation and lysis, but betaine can counteract this temperature sensitivity.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Biochemistry
Background:
- Escherichia coli YibP protein possesses a unique structure with a membrane-spanning N-terminus, coiled-coil regions, and a peptidase M23/M37 homologous C-terminal domain.
- A yibP null mutant exhibits temperature-sensitive growth defects, failing to grow at 42°C but not at 37°C.
Purpose of the Study:
- To investigate the function of the Escherichia coli YibP protein in cell division and its localization within the cell.
- To characterize the temperature-sensitive phenotype of the yibP null mutant and identify potential suppressors.
Main Methods:
- Construction and characterization of a yibP disrupted mutant.
- Analysis of cell division, FtsZ ring formation, and filamentation at different temperatures.
- Subcellular localization studies of YibP protein.
- In vitro protease activity assay using purified YibP.
Main Results:
- The yibP null mutant shows inhibited cell division and FtsZ ring formation at 42°C, leading to filamentous cells, multiple nucleoids, and cell lysis without SOS induction.
- Five percent betaine effectively suppresses the temperature sensitivity of the yibP disrupted mutation.
- YibP protein is localized in the inner membrane and cytoplasmic fractions, with its functional domains likely in the cytoplasm.
- Purified YibP exhibits protease activity, capable of cleaving beta-casein.
Conclusions:
- Escherichia coli YibP protein plays a critical role in bacterial cell division and FtsZ ring formation, particularly under temperature stress.
- Betaine can mitigate the detrimental effects of YibP deficiency at elevated temperatures.
- YibP's protease activity and cytoplasmic localization suggest a role in intracellular protein processing or regulation during cell division.