Characterization of the Ampicillin Resistance Genes from Klebsiella pneumoniae and Photobacterium phosphoreum

• Main Authors: Yi-Ting Chuang, 莊宜婷
• Other Authors: Juey-Wen Lin
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/50982048030243394020

碩士 === 國立中興大學 === 生物化學研究所 === 91 === 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.