K Akiba1, T Ando, E Isogai, T Nakae, H Yoneyama. 1. Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
Abstract
BACKGROUND: Pseudomonas aeruginosa produces the Sec and Tat protein secretion machineries. The latter appears to be involved in the secretion of virulence factors, including phospholipase C (PlcH), and hence is a potential target of chemotherapeutic agents. METHODS: The signal sequence of OprM, the outer membrane subunit of the xenobiotic extrusion pumps, was substituted with that of PlcH. The antibiotic susceptibility of oprM-deficient cells expressing the hybrid protein PlcH-OprM was evaluated using the agar dilution method. RESULTS: The PlcH-OprM-expressing cells showed resistance to various MexAB-OprM substrate antibiotics. To evaluate the translocation route of PlcH-OprM, tatC encoding an indispensable component of the Tat machinery was knocked out in oprM-deficient cells. The tatC-oprM double mutant expressing PlcH-OprM exhibited antibiotic hypersusceptibility like the oprM-deficient cells, indicating that PlcH-OprM was translocated across the inner membrane exclusively through the Tat system. CONCLUSIONS: This system can be used for the screening of Tat system inhibitors and will be an excellent model for the study of secretion and biogenesis of the β-barrel outer membrane proteins.
BACKGROUND:Pseudomonas aeruginosa produces the Sec and Tat protein secretion machineries. The latter appears to be involved in the secretion of virulence factors, including phospholipase C (PlcH), and hence is a potential target of chemotherapeutic agents. METHODS: The signal sequence of OprM, the outer membrane subunit of the xenobiotic extrusion pumps, was substituted with that of PlcH. The antibiotic susceptibility of oprM-deficient cells expressing the hybrid protein PlcH-OprM was evaluated using the agar dilution method. RESULTS: The PlcH-OprM-expressing cells showed resistance to various MexAB-OprM substrate antibiotics. To evaluate the translocation route of PlcH-OprM, tatC encoding an indispensable component of the Tat machinery was knocked out in oprM-deficient cells. The tatC-oprM double mutant expressing PlcH-OprM exhibited antibiotic hypersusceptibility like the oprM-deficient cells, indicating that PlcH-OprM was translocated across the inner membrane exclusively through the Tat system. CONCLUSIONS: This system can be used for the screening of Tat system inhibitors and will be an excellent model for the study of secretion and biogenesis of the β-barrel outer membrane proteins.