Identification of a spermidine excretion protein complex (MdtJI) in Escherichia coli
Kyohei Higashi1, Hiroyuki Ishigure, Risa Demizu
1Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan.
Abstract:
A spermidine excretion protein in Escherichia coli was looked for among 33 putative drug exporters thus far identified. Cell toxicity and inhibition of growth due to overaccumulation of spermidine were examined in an E. coli strain deficient in spermidine acetyltransferase, an enzyme that metabolizes spermidine. Toxicity and inhibition of cell growth by spermidine were recovered in cells transformed with pUCmdtJI or pMWmdtJI, encoding MdtJ and MdtI, which belong to the small multidrug resistance family of drug exporters. Both mdtJ and mdtI are necessary for recovery from the toxicity of overaccumulated spermidine. It was also found that the level of mdtJI mRNA was increased by spermidine. The spermidine content in cells cultured in the presence of 2 mM spermidine was decreased, and excretion of spermidine from cells was enhanced by MdtJI, indicating that the MdtJI complex can catalyze excretion of spermidine from cells. It was found that Tyr4, Trp5, Glu15, Tyr45, Tyr61, and Glu82 in MdtJ and Glu5, Glu19, Asp60, Trp68, and Trp81 in MdtI are involved in the excretion activity of MdtJI.
Insights
Researchers identified the MdtJI complex in Escherichia coli as a spermidine excretion protein. This complex, composed of MdtJ and MdtI, alleviates spermidine toxicity and enhances its excretion from cells.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Spermidine is a polyamine essential for cell growth but toxic at high concentrations.
- Escherichia coli possesses mechanisms to regulate intracellular spermidine levels.
- Identifying specific efflux proteins is crucial for understanding polyamine homeostasis.
Purpose of the Study:
- To identify and characterize a protein responsible for spermidine excretion in Escherichia coli.
- To investigate the role of putative drug exporters in managing intracellular spermidine accumulation.
- To elucidate the function of the MdtJI complex in spermidine transport.
Main Methods:
- Screening of 33 putative drug exporters in an E. coli strain with impaired spermidine metabolism.
- Assessing cell toxicity and growth inhibition caused by spermidine overaccumulation.
- Gene expression analysis of mdtJI mRNA levels.
- Measuring intracellular spermidine content and excretion rates.
- Site-directed mutagenesis to identify key amino acid residues in MdtJ and MdtI.
Main Results:
- The MdtJI complex, comprising MdtJ and MdtI from the small multidrug resistance family, was identified as a spermidine excretion protein.
- Transformation with mdtJ and mdtI restored normal growth and alleviated spermidine toxicity in deficient E. coli.
- Spermidine induced increased expression of mdtJI mRNA.
- MdtJI enhanced spermidine excretion and reduced intracellular spermidine levels.
- Specific amino acid residues in MdtJ (Tyr4, Trp5, Glu15, Tyr45, Tyr61, Glu82) and MdtI (Glu5, Glu19, Asp60, Trp68, Trp81) were found to be critical for excretion activity.
Conclusions:
- The MdtJI complex functions as a spermidine exporter in Escherichia coli.
- Regulation of mdtJI expression by spermidine suggests a feedback mechanism for polyamine homeostasis.
- The identified MdtJI exporter plays a significant role in preventing spermidine toxicity by facilitating its efflux.
- Key residues within MdtJ and MdtI are essential for the transporter's function.
