分析會影響鼠傷寒沙門氏菌第一型線毛相變化的基因成分

Analysis of the genetic elements that may affect the type 1 fimbrial phase variation in Salmonella enterica serotype Typhimurium

陳怡甄(Chen yi-tseng)

指導教授 ： 葉光勝

臺北醫學大學/醫學院/醫學科學研究所/碩士(2006年)

摘要

沙門氏菌中的第一型線毛 (type 1 fimbriae) 的表現常會有相變化 (phase variation)的情形。也就是說，個別的細菌會在線毛表現相 (fimbriated phase) 及線毛不表現相 (non-fimbriated phase) 之間，會形成的細菌族群包含了兩種不同相的細菌。而在前人的研究中，只知S. Typhimurium生長於靜態液態培養基時，絕大多數的細菌會產生type 1 fimbriae。反之，當培養於固態培養基時，絕大多數的細菌則不產生fimbriae。 目前對於線毛相變化的機制並未有詳盡深入地探討研究。本研究主要以transposon嵌插入S. Typhimurium LB5010菌體DNA中，以建立嵌插突變菌株資料庫。自10,000個菌落中，挑選出2,400個單一菌落，並以酵母菌凝集試驗及甘露糖凝集試驗篩選出37株突變株。而其中有25株突變株，不論那種培養環境均會表現type 1 fimbriae。11株突變株均不會表現type 1 fimbriae。而有1株突變株以agar的培養環境會有type 1 fimbriae的表現情形，但若以broth培養則不會有type 1 fimbriae。南方墨點法 (Southern hybridization) 偵測突變株中是否含有單一kanamycin cassette的DNA片段，結果其中僅有30株突變株為單一DNA片段。接下來定序後分析29株突變株的資料，初步得知S. Typhimurium的第一型線毛調控表現，除了fim gene cluster外，還會受到其他的基因的影響，如與fimbrial biosynthesis、metabolism、membrane-associated protein、enzyme、putative cytoplasmic protein、putative protein、globular regulator等七類有關的基因，進而調控線毛phase on/off的表現情形。回復試驗及後續的研究正持續進行中。

關鍵字

沙門氏菌 ； 酵母菌 ； 第一型線毛

並列摘要

The expression of type 1 fimbriae in Salmonella enterica serotype Typhimurium is subjected to a mode of regulation termed phase variation. The switching of individual cells between a state of expression (fimbriate) and non-expression (nonfimbriate), or vice versa, to produce populations that contain a mixture of both cell types defines fimbrial phase variation. Strongly fimbriate-phase bacteria are isolated following serial passage every 48 hours in unshaken broth culture, while nonfimbriate bacteria are obtained by growth on solid media. The mechanism that control fimbrial phase variation is currently not thoroughly elucidated. Transposon insertion mutagenesis was performed on S. Typhimurium to generate a library to screen for those mutants that are not phenotypically type 1 fimbrial phase variable. Thirty seven mutants were obtained from over 2400 clones screened by yeast agglutination test. Twenty five mutants exhibited type 1 fimbriae at both agar and broth media. Eleven mutants did not produce type 1 fimbriae at any conditions, while one mutant produced type 1 fimbriae at solid agar media and did not exhibit fimbriae in static broth. The flanking sequence of the transposon from each mutant was cloned and sequenced. Our initial analysis reveals that seven major classes of the genes may affect type 1 fimbrial phase variation in S. Typhimurium which are the genes involved in globular regulator and metabolism, the fimbrial-associated and fimbrial subunit genes, the genes in the membrane-associated protein, enzyme , and the genes with putative cytoplasmic protein,putative protein. Complementation tests and detail investigation are being conducted to elucidate the mechanism that may control the type 1 fimbrial phase variation in S. Typhimurium.

並列關鍵字

salmonella ； type 1 fimbriae

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