| It's proved that many antimicrobial peptides kill bacteria by acting on the cell membrane. Besides, some peptides can penetrate into cells and kill bacteria quickly by binding with large molecules inner to affect the metabolism. But the function of antimicrobial peptides on bacteria's nucleic acid and followed effect on the cell cycle is researched little. So we study the cell membrane and intracellular specific function of analogues buforinâ…¡-A (BF2-A) and buforinâ…¡-B (BF2-B) of the antimicrobial peptide buforinâ…¡on bacteria.the bond of genomic DNA with BF2-A/BF2-B was investigated with gel retardation assay and The change of DNA structure after the binding was observed by ultraviolet spectra, Circular Dichroism (CD) and fluorescence spectra respectively, and the competitive intercalation of BF2-A/BF2-B and ethidium bromide (EB) into genomic DNA were also analyzed by fluorescence spectra. Results showed that after peptides acted on DNA, the intensity of the specific peaks in the spectras changed but the peak sites almost had no change. It's proved that both BF2-A and BF2-B bond to gennemic DNA of both Staphylococcus aureus and Escherich coli ,and the double-helix structure was not broken. Besides, at the same condition the action of both BF2-A and BF2-B on gennemic DNA of Staphylococcus aureus was stronger than on Escherich coli. Maybe it's because that the AT % of Staphylococcus aureus is higher than Escherich coli. Besides, the action of BF2-B on genomic DNA was stronger than BF2-A.flow cytometry analyzed the change of the bacterial cell membrane of bacteria after treated by BF2-A/BF2-B. The results showed that, at the minimal inhibitory concentration of BF2-A/BF2-B, the cell membrane was almost intact, especially that treated by BF2-A. as the concentration of BF2-A/BF2-B improved, ,both BF2-A and BF2-B only caused a little breakage, indicating that BF2-A/BF2-B pecentrates the cell membrane and gets into the cell. And the gel retardation assay observed the gennemic DNA change after BF2-A/BF2-B acted on bacteria. The result showed that BF2-A/BF2-B didn't break the gennemic DNA. Then flow cytometry analyzed the cell cycle change of bacteria after treated by BF2-A/BF2-B. The result was that the cells were blocked at the DNA synthesis phase of cell cycle,which must be one of the most important reasons that BF2-A/BF2-B has a very good inhibitory function. Besides, both BF2-A and BF2-B had a better antimicrobial action on Staphylococcus aureus than Escherich coli. The results maybe also be owe to the AT % of Staphylococcus aureus higher than Escherich coli. Also the action of BF2-B on both the cell membrane and Intracellular molecules was better than BF2-A.Finally, binding action between peptide and genes related to DNA synthesis that were harvested by PCR was researched by gel retardation assay. It's discovered that both BF2-A and BF2-B bond to genes related to DNA synthesis (DnaA, DNA polymerase III subunit beta, gyrA, gyrB), and also both BF2-A and BF2-B were easier to bind to genes related to DNA synthesis that had more number of AT. That's why S. aureus(Staphylococcus aureus)/E.coli (Escherichia coli) was blocked at DNA synthesis phase of cell cycle. And the bond of BF2-B to genes related to DNA synthesis was stronger than BF2-A.All the experiment results prove that BF2-A/BF2-B can penetrate into bacteria while hardly affecting the cell membrane, and block the DNA synthesis phase of cell cycle. The reason is that both BF2-A and BF2-B were easier to bind to genes related to DNA synthesis that had more number of AT specifically without breaking DNA. Besides, all the results show that the action effect of BF2-B are better than BF2-A, which is why BF2-B can inhibit bacteria better than BF2-A. |
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