Molecular Cloning of Cecropin B Responsive Endonucleases in Yersinia ruckeri
Molecular Cloning of Cecropin B Responsive Endonucleases in Yersinia ruckeri
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DOI：
10.1007/s10126-010-9269-z
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发表时间：
2011-02
影响因子：
3
通讯作者：
Ulysses W. Sallum;Thomas T. Chen
中科院分区：
文献类型：
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作者：
Ulysses W. Sallum;Thomas T. Chen
我们先前已经证明鲁氏耶尔森菌以可诱导的方式抵抗天蚕素B。在这项研究中，我们试图确定负责诱导天蚕素B抗性的Y的分子变化。ruckeri。研究了与诱导抗性相关的基因表达差异。Y.的文化。将ruckeri暴露于亚致死浓度的天蚕素B，并使用差异显示逆转录聚合酶链反应（DD-RT-PCR）分析细菌信使RNA群体的变化。在实验的所有重复中，单个条带的强度一致增加。切下条带，克隆，测序，并用于筛选aY。ruckeri基因组DNA文库。该片段与Y染色体上克服溶原性缺陷（OLD）家族的ATP依赖性内切酶的cDNA序列同源性为100%。ruckeri29473.回收的基因组克隆与DD-RT-PCR克隆不相同，但含有分泌型内切核酸酶1（nucM）同源物的基因。通过RT-PCR确定了在暴露于天蚕素B后该基因的转录上调。此外，Y.证实了暴露于天蚕素B后的ruckeri。这些发现表明，天蚕素B暴露增加了至少两种核酸内切酶在Y。ruckeri。Y.产生和分泌一种内切核酸酶。鲁氏菌素对抗微生物肽的反应表明细胞内和细胞外DNA都参与了天蚕素B的毒性作用。
We have previously demonstrated thatYersinia ruckeriresists cecropin B in an inducible manner. In this study, we sought to identify the molecular changes responsible for the inducible cecropin B resistance ofY. ruckeri. Differences in gene expression associated with the inducible resistance were investigated. Cultures ofY. ruckeriwere exposed to a sublethal concentration of cecropin B and resultant changes in the messenger RNA population of the bacteria were assayed using the differential display reverse transcription polymerase chain reaction (DD-RT-PCR). A single band was consistently increased in intensity in all repeats of the experiment. The band was excised, cloned, sequenced, and used to screen aY. ruckerigenomic DNA library. The DD-RT-PCR fragment shared 100% identity to the cDNA sequence of an ATP-dependent endonuclease of the overcome lysogenization defect (OLD) family ofY. ruckeri29473. The genomic clone that was recovered was not identical to the DD-RT-PCR clone, but harbored a gene for a secreted endonuclease 1 (nucM) homologue. It was determined that transcription of the gene was upregulated following exposure to cecropin B via RT-PCR. Furthermore, an increase in the nuclease activity of culture supernatants ofY. ruckerifollowing exposure to cecropin B was demonstrated. These findings demonstrate that cecropin B exposure increases the expression of at least two endonucleases inY. ruckeri. The production and secretion of an endonuclease byY. ruckeriin response to an antimicrobial peptide indicates the involvement of both intracellular and extracellular DNA in the toxic effects of cecropin B.