Subtractive hybridization and optical mapping of the enterotoxigenic Escherichia coli H10407 chromosome:: isolation of unique sequences and demonstration of significant similarity to the chromosome of E coli K-12
Subtractive hybridization and optical mapping of the enterotoxigenic Escherichia coli H10407 chromosome:: isolation of unique sequences and demonstration of significant similarity to the chromosome of E coli K-12
复制标题
DOI：
10.1099/mic.0.28648-0
复制
发表时间：
2006-04-01
期刊：
影响因子：
2.8
通讯作者：
Venkatesan, MM
中科院分区：
文献类型：
--
作者：
Chen, Q;Savarino, SJ;Venkatesan, MM
产肠毒素大肠杆菌（ETEC）是发展中国家婴儿和前往流行地区的旅行者腹泻的主要原因。虽然已经在ETEC质粒上发现了几个毒力基因，但对ETEC染色体知之甚少，尽管预计它与大肠杆菌K-12在骨干序列上具有显著的同源性。在缺乏基因组序列信息的情况下，采用减法杂交方法和最近描述的光学作图技术来确定毒力强的ETEC菌株H10407和非致病性大肠杆菌K-12菌株MG1655之间的基因组变异程度。在一轮基于pcr的抑制减法杂交中，鉴定出153个片段，代表菌株H10407特有的序列。BLAST搜索显示，很少有独特的序列与相关大肠杆菌病原体中已知的致病性岛基因具有同源性。总共有65个片段包含的序列要么与假设的蛋白质相连，要么与数据库中的任何已知序列没有同源性。其余的序列要么与噬菌体或前噬菌体相关，要么与在细菌代谢的各个方面起作用的可分类基因同源。153个独特的序列在不同的ETEC菌株中表现出不同的分布，包括ETEC菌株137A，该菌株毒力较弱，缺乏多个H 10407特异性序列。将菌株h10407的限制性内切酶光学图与菌株MG1655及相关大肠杆菌病原体的硅酶切图进行比较。5.1 Mb的ETEC染色体长度比E coliK-12染色体长约500 kb，与E coliK-12共线，与菌株MG1655的染色体相比，ETEC染色体有几个插入和/或缺失的离散区域。在ETEC染色体上未观察到明显的倒位、转位或重排。基于与已知基因组序列的比较和相关的光学图谱限制性内切位点相似性，预计h10407染色体序列与大肠杆菌K-12的同源性接近96%。
Enterotoxigenic Escherichia coli (ETEC) is a primary cause of diarrhoea in infants in developing countries and in travellers to endemic regions. While several virulence genes have been identified on ETEC plasmids, little is known about the ETEC chromosome, although it is expected to share significant homology in backbone sequences with E coli K-12. In the absence of genomic sequence information, the subtractive hybridization method and the more recently described optical mapping technique were carried out to determine the degree of genomic variation between virulent ETEC strain H10407 and the non-pathogenic E coli K-12 strain MG1655. In one round of PCR-based suppression subtractive hybridization, 153 fragments representing sequences unique to strain H10407 were identified. BLAST searches indicated that few unique sequences showed homology to known pathogenicity island genes identified in related E coli pathogens. A total of 65 fragments contained sequences that were either linked to hypothetical proteins or showed no homology to any known sequence in the database. The remaining sequences were either phage or prophage related or displayed homology to classifiable genes that function in various aspects of bacterial metabolism. The 153 unique sequences showed variable distribution across different ETEC strains including ETEC strain 137A, which is attenuated in virulence and lacked several H 10407-specific sequences. Restriction-enzyme-based optical maps of strain H 10407 were compared to in silico restriction maps of strain MG1655 and related E coli pathogens. The 5.1 Mb ETEC chromosome was similar to 500 kb greater in length than the chromosome of E coliK-12, collinear with it and indicated several discrete regions where insertions and/or deletions had occurred relative to the chromosome of strain MG1655. No major inversions, transpositions or gross rearrangements were observed on the ETEC chromosome. Based on comparisons with known genomic sequences and related optical-map-based restriction site similarity, the sequence of the H 10407 chromosome is expected to demonstrate similar to 96 % identity with that of E coli K-12.