Meier Petra, Wackernagel Wilfried
Genetics, Department of Biology and Environmental Sciences, Carl von Ossietzky University of Oldenburg, Germany.
J Bacteriol. 2005 Jan;187(1):143-54. doi: 10.1128/JB.187.1.143-154.2005.
In prokaryotic mismatch repair the MutS protein and its homologs recognize the mismatches. The mutS gene of naturally transformable Pseudomonas stutzeri ATCC 17587 (genomovar 2) was identified and characterized. The deduced amino acid sequence (859 amino acids; 95.6 kDa) displayed protein domains I to IV and a mismatch-binding motif similar to those in MutS of Escherichia coli. A mutS::aac mutant showed 20- to 163-fold-greater spontaneous mutability. Transformation experiments with DNA fragments of rpoB containing single nucleotide changes (providing rifampin resistance) indicated that mismatches resulting from both transitions and transversions were eliminated with about 90% efficiency in mutS+. The mutS+ gene of strain ATCC 17587 did not complement an E. coli mutant but partially complemented a P. stutzeri JM300 mutant (genomovar 4). The declining heterogamic transformation by DNA with 0.1 to 14.6% sequence divergence was partially alleviated by mutS::aac, indicating that there was a 14 to 16% contribution of mismatch repair to sexual isolation. Expression of mutS+ from a multicopy plasmid eliminated autogamic transformation and greatly decreased heterogamic transformation, suggesting that there is strong limitation of MutS in the wild type for marker rejection. Remarkably, mutS::aac altered foreign DNA acquisition by homology-facilitated illegitimate recombination (HFIR) during transformation, as follows: (i) the mean length of acquired DNA was increased in transformants having a net gain of DNA, (ii) the HFIR events became clustered (hot spots) and less dependent on microhomologies, which may have been due to topoisomerase action, and (iii) a novel type of transformants (14%) had integrated foreign DNA with no loss of resident DNA. We concluded that in P. stutzeri upregulation of MutS could enforce sexual isolation and downregulation could increase foreign DNA acquisition and that MutS affects mechanisms of HFIR.
在原核生物错配修复中，MutS蛋白及其同源物识别错配。对可自然转化的斯氏假单胞菌ATCC 17587（基因组变种2）的mutS基因进行了鉴定和表征。推导的氨基酸序列（859个氨基酸；95.6 kDa）显示出与大肠杆菌MutS中相似的I至IV蛋白结构域和错配结合基序。一个mutS::aac突变体显示出自发性突变率提高了20至163倍。用含有单核苷酸变化（赋予利福平抗性）的rpoB DNA片段进行的转化实验表明，在mutS+菌株中，由转换和颠换产生的错配约90%的效率被消除。ATCC 17587菌株的mutS+基因不能互补大肠杆菌突变体，但能部分互补斯氏假单胞菌JM300突变体（基因组变种4）。mutS::aac部分缓解了序列差异为0.1%至14.6%的DNA导致的异配转化下降，表明错配修复对性隔离的贡献为14%至16%。多拷贝质粒中mutS+的表达消除了同配转化并大大降低了异配转化，这表明野生型中MutS对标记排斥有很强的限制。值得注意的是，mutS::aac在转化过程中通过同源性促进的非法重组（HFIR）改变了外源DNA的获取，具体如下：（i）在DNA净增加的转化体中，获取的DNA平均长度增加；（ii）HFIR事件变得聚集（热点）且对微同源性的依赖性降低，这可能是由于拓扑异构酶的作用；（iii）一种新型转化体（14%）整合了外源DNA而没有损失宿主DNA。我们得出结论，在斯氏假单胞菌中，MutS的上调可加强性隔离，下调可增加外源DNA的获取，并且MutS影响HFIR机制。
