通过抗生素诱导的 SOS 反应、质粒测序、MALDI-TOF-TOF 质谱分析和自上而下的蛋白质组分析检测病原菌的 Colicin 免疫蛋白。

IF 1.8 3区 化学 Q4 BIOCHEMICAL RESEARCH METHODS
Clifton K.  Fagerquist, Yanlin  Shi, Jihyun  Park
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引用次数: 0

摘要

理由:质粒在致病菌的存活中起着重要作用。质粒是通过水平基因转移获得的,导致它们在不同菌株、物种和属的细菌中传播。粘菌素是一种由质粒基因表达的细菌蛋白毒素,可以释放出来对抗同处位置的细菌竞争者。方法:采用抗生素诱导、MALDI-TOF-TOF质谱、自上而下蛋白质组学分析和小质粒测序相结合的方法,对3株产志贺毒素大肠杆菌(STEC)进行基因组测序。蛋白质生物标志物鉴定使用内部软件,匹配蛋白质质量和片段离子骨干裂解的天冬氨酸效应。预测的硅蛋白质结构有助于解释蛋白质离子断裂。结果:除了噬菌体编码志贺毒素的蛋白质组学鉴定外,我们还能够鉴定质粒编码的大肠杆菌素D和E3免疫蛋白。这些质粒编码蛋白的基因在之前的基因组测序中未被发现。然而,对这些菌株的小质粒重测序显示,这些基因存在于7-8 kb大小的质粒上。大肠杆菌素/免疫基因的上游是SOS/LexA盒子的反向重复,抑制基因表达直到抗生素攻击。结论:我们自上而下的蛋白质组学方法表明,在SOS控制下,可以筛选假定的致病菌(其基因组已全部、部分或根本没有测序),以确定噬菌体和质粒编码的毒素和粘菌素基因的存在。小质粒测序证实了诱导和自上而下的蛋白质组学分析提示的大肠杆菌素/免疫基因(及其调节控制)的存在。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Colicin Immunity Proteins of Pathogenic Bacteria Detected by Antibiotic-Induced SOS Response, Plasmid Sequencing, MALDI-TOF-TOF Mass Spectrometry, and Top-Down Proteomic Analysis

Rationale:

Plasmids can play a major role in the survival of pathogenic bacteria. Plasmids are acquired through horizontal gene transfer resulting in their spread across various strains, species and genera of bacteria. Colicins are bacterial protein toxins expressed by plasmid genes and released against co-located bacterial competitors.

Methods:

Three Shiga toxin-producing E. coli (STEC), whose genomes were sequenced previously, were analyzed using a combination of antibiotic induction, MALDI-TOF-TOF mass spectrometry, top-down proteomic analysis, and small plasmid sequencing. Protein biomarkers were identified using in-house software that matches protein mass and fragment ions of backbone cleavage by the aspartic acid effect. Predicted in silico protein structures assisted in the interpretation of protein ion fragmentation.

Results:

In addition to proteomic identification of phage-encoded Shiga toxin, we were able to identify plasmid-encoded immunity proteins for colicin D and E3. The genes for these plasmid-encoded proteins were not found in the previous genomic sequencing. However, resequencing of these strains for small plasmids revealed the genes to be present on 7–8 kb sized plasmids. Upstream of the colicin/immunity genes was an inverted repeat of the SOS/LexA box that represses gene expression until antibiotic challenge.

Conclusions:

Our top-down proteomic method demonstrates that it is possible to screen putative pathogenic bacteria (whose genomes have been sequenced in full, in part or not at all) for the presence of phage- and plasmid-encoded toxin and colicin genes under SOS control. Small plasmid sequencing confirmed the presence of colicin/immunity genes (and their regulatory control) suggested from induction and top-down proteomic analysis.

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来源期刊
CiteScore
4.10
自引率
5.00%
发文量
219
审稿时长
2.6 months
期刊介绍: Rapid Communications in Mass Spectrometry is a journal whose aim is the rapid publication of original research results and ideas on all aspects of the science of gas-phase ions; it covers all the associated scientific disciplines. There is no formal limit on paper length ("rapid" is not synonymous with "brief"), but papers should be of a length that is commensurate with the importance and complexity of the results being reported. Contributions may be theoretical or practical in nature; they may deal with methods, techniques and applications, or with the interpretation of results; they may cover any area in science that depends directly on measurements made upon gaseous ions or that is associated with such measurements.
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