以跳躍因子系統性突變發現幽門桿菌一個全新的限制-修飾系統

利用1500株幽門桿菌突變株，篩選其對胃癌上皮細胞株黏著能力，以尋找幽門桿菌之黏著因子，發現6株突變株黏著能力降低，但光學及電子顯微鏡下觀察發現菌體型態明顯延長。定序後發現這6株突變株破壞的基因是相同的，但幽門桿菌26695及J99菌株全基因體中並無該核酸及胺基酸的相似序列。這個開放讀架由1617個鹼基對組成，胺基酸序列和Bacillus halodurans的一個推斷的單股剪切酶有24%的相似性，和二個第IIS型限制內切酶，PleI和MlyI，分別有23%及20%相似度。分析這個純化的未知限制酶，HpyC1I，其辨識及剪切位置為5’-CCATC(4/5)-3’。此基因上游二個開放讀架，M1.HpyC1I和M2.HpyC1I，都含有甲基轉移酶的弁鈰礡A因此這三個基因可能構成一個調控組，且負責限制-修飾弁遄C這段序列和全基因體比較，G+C含量明顯較低，推測可能經由水平基因轉移獲得。測試15株幽門桿菌菌株，有9株菌株具有這個HpyC1I限制-修飾基因。質體DNA轉型和染色體裁切的實驗證明擁有HpyC1I限制-修飾系統可以使DNA受到保護不被HpyC1I剪切。在本研究中我們發現一個新的限制-修飾系統-HpyC1I限制-修飾系統，此系統存在於~60%的幽門桿菌菌株中，且破壞會導致細菌型態延長和HpyC1I限制酶的裁切。

關鍵字

限制-修飾系統；跳躍因子；幽門桿菌；黏著因子

並列摘要

To identify adhesion-associated loci, we constructed a mutant library from a clinical isolate of Helicobacter pylori. A total of 1500 mutant strains were screened by cell adhesion assay. We identified six mutants from NTUH-C1 strain that exhibited decreased adherence and cell elongation. Inverse polymerase chain reaction and DNA sequencing revealed that the same locus was interrupted in these six mutants. Nucleotide and amino acid sequences showed no homologies with H. pylori 26695 and J99 strains. This novel open reading frame (ORF) contained 1617 base pairs. The amino acid sequence shared 24% identity with a putative nicking enzyme in Bacillus halodurans, and 23% and 20% identity with Type-IIS restriction endonucleases PleI and MlyI, respectively. The purified protein, HpyC1I, showed endonuclease activity with the recognition and cleavage site 5’-CCATC(4/5)-3’. Two ORFs were located upstream of the gene encoding HpyC1I. Together, HpyC1I and these two putative methyltransferases (M1.HpyC1I and M2.HpyC1I) function as a restriction-modification (R-M) system. The HpyC1I R-M genes were found in nine of the fifteen H. pylori strains tested. When compared to the full genome, significantly lower G+C content of HpyC1I R-M genes implied that these genes might have been acquired by horizontal gene transfer. Plasmid DNA transformation efficiencies and chromosomal DNA digestion assays demonstrated protection from HpyC1I digestion by the R-M system. In conclusion, we have identified a novel R-M system present in ~60% of H. pylori strains. Disruption of this R-M system results in cell elongation and susceptibility to HpyC1I digestion.

並列關鍵字

restriction-modification system ； Helicobacter pylori ； adhesin ； transposon

參考文獻

1. Akopyants NS, Fradkov A, Diatchenko L, Hill JE, Siebert PD, Lukyanov SA, Sverdlov ED, Berg DE. PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc Natl Acad Sci U S A. 1998;95(22):13108-13.

3. Ando T, Xu Q, Torres M, Kusugami K, Israel DA, Blaser MJ. Restriction-modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer. Mol Microbiol. 2000;37(5):1052-65.

4. Ang S, Lee CZ, Peck K, Sindici M, Matrubutham U, Gleeson MA, Wang JT. Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray. Infect Immun. 2001;69(3):1679-86.

5. Aras RA, Small AJ, Ando T, Blaser MJ. Helicobacter pylori interstrain restriction-modification diversity prevents genome subversion by chromosomal DNA from competing strains. Nucleic Acids Res. 2002;30(24):5391-7.

6. Asahi M, Azuma T, Ito S, Ito Y, Suto H, Nagai Y, Tsubokawa M, Tohyama Y, Maeda S, Omata M, Suzuki T, Sasakawa C. Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells. J Exp Med. 2000;191(4):593-602.

國際替代計量

以跳躍因子系統性突變發現幽門桿菌一個全新的限制-修飾系統

全文下載

主題瀏覽