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標題: Klebsiella pneumoniae 及 Photobacterium phosphoreum ampicillin 抗藥基因的選殖及其特性分析
Characterization of the Ampicillin Resistance Genes from Klebsiella pneumoniae and Photobacterium phosphoreum
作者: 莊宜婷
Chuang, Yi-Ting
關鍵字: 抗藥基因;ampicillin Resistance Genes;Photobacterium phosphoreum;Klebsiella pneumoniae
出版社: 生物化學研究所
自 Klebsiella pneumoniae 及 Photobacterium phosphoreum 中分別篩選含 ampC DNA 片段 pYTB-1 (~4.5 kb) 及 pYTS-2 (~6.4 kb)。pYTB-1所含基因及可能調節區域依序為 ←recF-R&R←ufoI-R&R-ampC→←deoR-。pYTS-2上所含基因及可能調節區域依序為 ←ampC-R&R-ufoII→。K. pneumoniae 與 E. coli, A. sp. SUN-72 及 S. dysenteriae 之 AmpC 胺基酸序列分別有 100%, 100%, 99.7% 相同,具有 66S*XXK69, 126SDN128, 162EXXN166, 230KTG232, Cys119 及 Asp159 保留區,其中 66S 為 AmpC active site serine 。P. phosphoreum 與 P. damselae subsp., V. cholerae non-01/non- 0139 及 V. parahaemolyticus RIMD 2210633 之 AmpC 胺基酸序列分別有 48.6%, 47%, 48.4% 相同,含 65S*XXK68, 125SDN127, 161EXXN165, Asp158 及 Cys118 保留區,雖缺乏 KTG conserved motif ,但 229RSG331 與 KTG 具有相似性;因此K. pneumoniae 及 P. phosphoreum AmpC 皆屬A 型 b-lactamases 。演化分析顯示陸生菌 K. pneumoniae 與 E. coli 具同源性 (homologous) ,與海螢光菌屬於不同的族群 (groups)。P. phosphoreum AmpC 與其他相近菌株,如 V. fischeri, V. harveyi 演化差異亦大。推測陸生菌與海生菌 ampC 基因於演化上早已分為不同的族群,海生菌中的 ampC 並非來自陸生菌。蛋白比活性結果顯示 K. pneumoniae AmpC 於 E. coli 中的功能與原始菌相似。K. pneumoniae 與 E. coli JM103y(pYT4133) AmpC之 pI 分別為 ~7.6 與 ~7.35。K. pneumoniae 及 P. phosphoreum 中 AmpC 分子量為 ~28 kDa。K. pneumoniae 及 P. phosphoreum ampC 皆具有兩組啟動子 P[I]-promoter 及 P[II]-promote,轉錄起始點分析確認 K. pneumoniae及 P. phosphoreum ampC 主要以 P[I]-promoter 來帶動基因的表現。P. phosphoreum ampC 啟動子於 E. coli 之表現屬於持續型,ampicillin 加入不影響 ampC 調節區域的表現。K. pneumoniae ampC 於 E. coli JM103y 中啟動子表現量偏低,於 E. coli DH5a 的表現屬於持續型,於 E. coli LE392 中啟動子的表現可低量持續表現,ampicillin 加入可誘導其表現,其表現亦屬於劑量依賴型 (dose-dependent)。

Genome library was constructed to isolate the ampC genes form K. pneumoniae and P. phosphorenum. pYTB-1 (about ~4.5 kb) and pYTS-2 (about ~6.4 kb) were selected. Nucleotide sequence of the ampC and the neighboring genes have been determined; the gene order is ←recF-R&R←ufo-R&R-ampC→←deoR- in K. pneumoniae, and ←ampC-R&R-ufoII→ in P. phosphorenum. Alignment and comparison of the AmpC from K. pneumoniae and various species, among E. coli, Acinetobacter sp. SUN-72, and S. dysenteriae, there are 100%, 100%, and 99.7% identity. K. pneumoniae AmpC contains four conserved motifs, 66S*XXK69 (66S is the active site serine), 126SDN128, 162EXXN166, 230KTG232, and two conserved residues, C119 and D159. Alignment and comparison of the AmpC from P. phosphoreum and various species, among P. damselae subsp., V. cholerae non-01/non-0139, and V. parahaemolyticus RIMD 2210633, there are 48.6%, 47%, and 48.4%. P. phosphoreum AmpC contains three conserved motifs, 65S*XXK68, 125SDN127, 161EXXN165, and two conserved residues, C118 and D158. K. pneumoniae and P. phosphoreum AmpCs are classified as class A b-lactamases. The evolu- tionary dendrogram of b-lactamases elicits that K. pneumoniae and E. coli are closer in a group; P. phosphoreum is alone in a group. Primer extension assays confirm K. pneumoniae and P. phosphoreum ampC genes transcription initia- tion +1. Protein activity assays indicate AmpC in E. coli or K. pneumoniae are similar. K. pneumoniae and E. coli JM103y(pYT4133) produce AmpCs with an isoelectric point pI ~7.6 and 7.35. The AmpC Mr in wild type and E. coli were all ~28 kDa. P. phosphoreum ampC in E. coli is expressed consitiutively, and ampicillin is not induced the ampC gene. K. pneumoniae ampC gene in E. coli JM103y(pYT4133) and DH5a(pYT4133) are expressed constitutively, but in LE392(pYT4133) is expressed inducible.
Appears in Collections:生物化學研究所

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