Summary: | 碩士 === 國立中興大學 === 化學系所 === 98 === Pectobacterium carotovorum subsp. carotovorum (Pcc) is a phytopathogenic enterobacterium for the soft-rot disease of many plants species. This has led to important economic losses without efficient method to control, particularly over the past few decades as agricultural production has intensified. To face these drawbacks, biological control using avirulent bacteriocin-producing mutants of Pcc is under investigation.
Carocin S3, is a chromosome-encoded toxic protein with DNase activity, produced from Pcc strain, H-rif-8-6. This low-molecular-weight bacteriocin is active against indicator strain Ea1068 with 2 μg/mL. The DNA fragment containing carocin S3 was isolated, analyzed and expressed by the method mentioned before. Two structural genes, one responsible for antibiotic activity (killing protein) which we named it as CaroS3K (95.6 kDa), and the other conferring to inhibit antibiotic activity of the killing protein (immunity protein) referred as CaroS3I (10.2 kDa).
Here, multiple sequence alignment against several sequence and structure databases showed that caroS3K can be divided into three functional domains, translocation, receptor-binding and toxicity. The toxicity of Carocin S3 is associated with the 40-45 kDa C-terminal part of the carocin molecule, where an H-N-H motif is located. The H-N-H motif has been identified in many DNA hydrolysis standing with the most conserved residues, His and Asn. To find out the role of H-N-H motif in Carocin S3, alanine mutagenesis was used as a means of scanning for functional positions in proteins, including H778A, H815A and H845A. The cell killing and gel shift assay showed those three mutants have abolished activity. These results suggest that H-N-H motif plays an important role in DNA hydrolysis mechanism.
To determine the contents of metal elements in the protein complex, Carocin S3 was measured by ICP-Mass. The results showed that the major elements including Mg2+, Ca2+ and Zn2+. Metal-dependent cleavage assay were incubated with chromosome DNA from Ea1068 as the substrate. Accordingly, the metal ions exist in Carocin S3 all contained nuclease activity, in the order of Mg2+ > Ca2+ > Mn2+ > Ni2+ > Zn2+. Additionally, DNA hydrolysis of Carocin S3 with Mg2+ was initially activated with increasing concentration, followed by a gradual decrease in activity.
From this study, Carocin S3 was used to confirm and clarify the binding modes of transition and alkaline metal ions. Based on the results of gel shift assay, we suggest that 10mM Mg2+ is the optimal relevant metal ion used here. We also decipher the critical residues in the toxicity domain, especially within H-N-H motif. In particular, the biological significance of CaroS3I in regulating expression of the carocin operon is also under progressing. Finally, structure pattern determination has carried out for providing the molecular mechanism that is involved in specific recognition processes and protein-protein interactions.
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