| Abstract [eng] |
Identification of Receptors and Translocators of Klebicins by Transposon Mutagenesis Oportunistic, gram-negative bacteria belonging to the genus Klebsiella causes a wide range of infections, including pneumonias, bacteremias and urinary tract infections. As bacterial strains become increasingly resistant to antibiotics, new approaches to treatment are urgently needed. One of the most promising alternatives to traditional antibiotics are bacteriocins, i.e. antimicrobial ribosomally synthesized proteins. In our laboratory, Klebsiella bacteriocins – klebicins – KpneM, KpneM2, KvarM, KaerA, KpneA, KvarIa and KpneIa were cloned and transiently expressed in plants. Six of these klebicins were purified. The aim of this work was to identify genes encoding receptors and translocators of klebicins. To do so, K. quasipneumoniae subsp. similipneumoniae DSM 28212 strain, sensitive to all seven klebicins, was subjected to several rounds of transposon mutagenesis. Klebicin KpneIa was purified using hydrophobic interaction chromatography. All seven purified klebicins were used for selection of resistant mutants. After performing transposon mutagenesis and selecting mutants with klebicin resistance, PCR analysis was performed to localize transposon insertions which revealed mutations in six genes – fhuA, exbB, ompC, fimB, tonB, yaeT. Complementation assay by ectopic expression of respective wild-type genes confirmed the relation between klebicin sensitivity loss and four genes – fhuA, exbB, tonB and ompC. The results showed that all three colicin M-like klebicins require ferrichrome receptor FhuA. These klebicins are similar to group B colicins and use the TonB-dependent translocation pathway. Mutations in TonB system also lead to resistance to two pore-forming klebicins – KaerA and KpneIa. These klebicins, as well as KvarIa, require in addition functional OmpC. However, the mechanism of import of KpneIa and KvarIa stays unclear, although it seems that BAM system, responsible for protein incorporation into outer membrane, plays a role. |
