| Research Abstract |
1. The conjugative plasmid pYI14 (61kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E.faecalis. Genetic analysis showed that the determinant was located in a 6.6-kbp region Six open reading frames (ORFs) were identified in this region and designated ORF7 (bacL_1) ORF8 (bacL_2), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL_1, bacL_2, bacA, and bacI were essential for expression of the bacteriocin in E.faecalis. Extracellular complementation of bacteriocin expression was possible for bacL_1 and -L_2 and bacA mutants. bacL_1 and -L_2 and bacA encoded bacteriocin component L and activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity,
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providing the host with resistance to its own bacteriocin activity.
2. The bacteriocin 51 (Bac51) was encoded on the mobilizable plasmid pHY (6,037bp) isolated from vancomycin resistant E.faecium V38. Bacteriocin 51 was active against E.faecium, E.hirae, and E.duraus. The sequence analysis of pHY showed that plasmid pHY encoded nine ORFs from ORF1 to ORF9 in this order. ORF3, ORF4 and ORF5 showed homology to mobC, mobA, and mobB, respectively. ORF7 and ORF8 showed homology to repA and repB, respectively. ORF1, ORF2, ORF6 and ORF9 had no homology to the reported genes. The Tn5 insertion mutants in ORF1 did not show both bacteriocin and immunity activities, implying that ORF1 and ORF2 were the bacteriocin determinant, and were designated as bacA and bacB, respectively. Detailed DNA sequence analysis of bacA and bacB was performed. bacA encoded a 144-amino-acid protein. The ATG start codon was preceded by a potential ribosome binding site 8 bp upstream. The deduced bacA protein had a span of hydrophobic residues typical of the signal sequence at its amino terminus and a potential signal peptidase processing site corresponding to the VEA sequence was located at position 37 to 39 amino acid. The predicted BacA mature protein consisted of 105 amino acids. There was a potential promoter sequence upstream of the start codon. bacB encoded a 55 amino acid protein. The ATG start codon was preceded by a potential ribosome binding site 12 bp upstream. There was no obvious promoter sequence upstream of the ribosome binding site. A putative transcription terminator signal was identified downstream of bacB. There was no obvious promoter or terminator sequence between bacA and bacB. The transcript of bacA and bacB were analyzed by Northern hybridization. The results of Northern analysis of bacA and bacB with bacA-RNA probe showed about 700 nucleotides which corresponded to the nucleotide size of both bacA and bacB, indicating that a transcription initiated from the promoter upstream bacA, continued through bacB, and terminated at the terminator downstream of bacB. The transcription start site was determined at the nucleotide T located at six nucleotides downstream from -10 promoter sequence by Rapid Amplification of cDNA Ends (RACE) method. These results indicated that the bacA and bacB composed of operon structure, and bacA was bacteriocin structure gene and bacB was the immunity gene. Less
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