Diamant Eran, Palti Yniv, Gur-Arie Riva, Cohen Helit, Hallerman Eric M, Kashi Yechezkel
Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Appl Environ Microbiol. 2004 Apr;70(4):2464-73. doi: 10.1128/AEM.70.4.2464-2473.2004.
Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.
看家基因的多位点测序先前已用于细菌菌株分型及推断大肠杆菌菌株间的进化关系。在本研究中，我们使用了包含重复单核苷酸基序（单核苷酸重复序列[MNRs]）的简单序列重复（SSRs）的较短基因间序列来推断致病性和共生性大肠杆菌菌株的系统发育。对27株菌株（包括肠出血性大肠杆菌[6株O157:H7分离株]、肠致病性大肠杆菌、产肠毒素性大肠杆菌、B群和K-12菌株）的7个非编码位点（4个MNRs和3个非SSRs）进行了测序。还对大肠杆菌参考（ECOR）菌株库中的20株代表性菌株的4个MNRs进行了测序。MNR位点（包括侧翼序列）的序列多态性显著更高，表明MNR区域侧翼序列的突变率更高。在标准的ECOR A、B1和D组中，4个MNR位点可通过PCR扩增，但B2组中只有1个位点（yaiN）能被扩增，这与之前认为B2是最古老菌群的研究结果一致。4个MNR位点之间具有高度的序列兼容性，表明它们处于同一克隆框架内。根据序列数据构建的系统发育树与之前使用多位点酶电泳的研究结果高度一致。结果表明，MNR位点可用于推断系统发育关系，并且比看家基因提供更高的序列变异。因此，使用MNR位点进行多位点序列分型在细菌的临床诊断、流行病学及进化研究中应是有效的。
