Nonheritable Antibiotic Resistance

非遗传性抗生素耐药性

• 批准号: 7593539
• 负责人: MARTIN F. GELLERT
• 金额: $ 33.27万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593539
• 关键词: Acceleration; Affect; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biological Assay; Cells; Class; Clinical; DNA-Directed RNA Polymerase; Data; Escherichia coli; Exhibits; Genes; Genetic Transcription; Goals; Growth; Isopropyl Thiogalactoside; LacZ Genes; Modeling; Numbers; Operon; Organic solvent product; Phenotype; Planet Mars; Play; Primer Extension; Rate; Regulation; Regulon; Reporter Genes; Repression; Resistance; Role; Signal Transduction; Site; Superoxides; Testing; Transcription Coactivator; Transcriptional Activation; antibiotic efflux; cis acting element; efflux pump; insight; mathematical model; promoter

Chromosomal multiple antibiotic resistance in bacteria is a serious clinical problem. Our studies have shown that Escherichia coli becomes resistant to a variety of antibiotics, organic solvents and superoxides when the activities of any of three paralogous, but differently regulated, transcriptional activators, MarA, SoxS and Rob, are increased. These activators bind a sequence called the marbox which lies upstream of the promoters of a set of about 40 chromosomal genes called the mar/sox/rob regulon. The major goals of this project are to understand the regulation of these activators, the mechanisms whereby they activate the regulon promoters, and the mechanisms whereby the multiple antibiotic resistance is generated. A. The primary mechanism for the antibiotic resistance and organic solvent tolerance is the transcriptional activation of acrAB and tolC. These genes encode the major antibiotic efflux pump of Escherichia coli. Using transcriptional fusions and primer extension assays, we investigated the regulation of these operons by the repressor AcrR and by MarA, SoxS and Rob. We showed that tolC has two previously unidentified strong overlapping promoters which are activated by MarA. They are configured in a unique way so that the binding of an activator to a single marbox can activate transcription from either promoter. The marbox is 20 bp upstream of the -10 signal for RNA polymerase (Class I* configuration) at the tolC p4 promoter, but 30 bp upstream of the -10 signal (Class II configuration) at the tolC p3 promoter. Furthermore, we showed that three cis-acting elements are important in the regulation of acrAB and acrR: the marbox which allows activation of acrAB; a 24 bp inverted repeat which contains the acrAB promoter and part of the divergently transcribed acrR promoter, and which is the likely site of AcrR binding for repression of both acrAB and acrR; and a 22 bp inverted repeat that contains part of the acrR promoter. We found also that 2,2'-dipyridyl (which we previously found post-translationally activates Rob) down-regulates AcrR function. Constitutive expression of acrAB resulting from an acrR deletion minimally affects antibiotic resistance in the absence of tolC activation but leads to a hyper-resistant phenotype when tolC is activated. The tolC and acrAB marboxes coordinate the activation of the tripartite efflux pump. B. The different regulon genes are activated to different extents by the activators. We previously had determined that while some correlation exists between activity and the binding constant of the activator for the different marboxes, this correlation is insufficient to explain the differential activation. We have now placed the expression of MarA under the control of a lac promoter which is activated by IPTG, determined the relationship between IPTG concentration and the intracellular concentration of MarA, and examined the expression of a number of the regulon genes (using lacZ as a transcriptional reporter gene) as a function of growth in different concentrations of IPTG. The resulting data were used to develop mathematical models that yield insight into the mechanisms of activation at the different promoters. We found that the concentration of MarA required for activation varies by at least 30-fold for different promoters, thus identifying a previously unappreciated form of regulon control in which some genes are activated at a particular activator concentration whereas others are not significantly activated. The wild-type mar promoter itself is activated at the lowest concentration of MarA, reaching half-maximal stimulation at a concentration of about 900 MarA molecules/cell. The comparable number for the micF promoter is about 10,000 MarA/cell. None of the 15 other promoters tested exhibits a plateau in activity, even at the artificially highest levels of MarA (about 26,000 molecules/cell). To gain insight innto the diversity in activation of the regulon, we developed a mathematical model of MarA-dependent promoter activity. In the model, MarA either increases (attraction) or decreases (repulsion) the occupancy of RNA polymerase (RNAP) at the promoter, and either increases (acceleration) or decreases (retardation) the forward rate of transcription by RNAP once bound at the promoter. The best models of mar promoter activation combine attraction with acceleration. For other regulon promoters, models that combine repulsion with acceleration fit the data best. The results suggest that transcriptional activation can involve repulsion (a decrease in the occupancy of RNAP due to activator), an effect commonly associated with repression rather than activation. The results also suggest that acceleration (an increase in the forward rate of transcription due to activator) is an important part of activation. Acceleration combined with repulsion has not been previously appreciated as playing a role in activation.

细菌中染色体多重抗生素耐药性是一个严重的临床问题。我们的研究表明，当三种寄生虫的活性中，大肠杆菌对多种抗生素，有机溶剂和超氧化物具有抵抗力，当时三种寄生虫的活性（但有不同的调节），转录激活剂，Mara，Soxs和Rob增加了。这些激活剂结合了一个称为marbox的序列，该序列位于一个约40个称为MAR/SOX/ROB REGULON的启动子上游的序列。该项目的主要目标是了解这些激活剂的调节，它们激活调节子启动子的机制以及产生多种抗生素耐药性的机制。 答：抗生素耐药性和有机溶剂耐受性的主要机制是ACRAB和TOLC的转录激活。这些基因编码大肠杆菌的主要抗生素外排泵。使用转录融合和引物扩展测定法，我们研究了阻遏物ACRR和Mara，Soxs和Rob对这些操纵子的调节。我们表明，TOLC具有两个先前未识别的强重叠启动子，这些启动子被MARA激活。它们以独特的方式配置，以便激活剂与单个marbox的结合可以激活两个启动子的转录。 Marbox是在TOLC P4启动子处的RNA聚合酶（I类配置）的-10信号上游的20 bp，但在TOLC P3启动子处-10信号（II类配置）上游30 bp。 此外，我们表明，三个顺式作用元件在调节ACRAB和ACRR：允许激活ACRAB的Marbox的调节中很重要。一个24 bp的倒置重复，其中包含ACRAB启动子和一部分分发的ACRR启动子，并且可能是ACRR结合的位点，以抑制ACRAB和ACRR。 22 bp的倒置重复包含ACRR启动子的一部分。我们还发现，2,2'-二吡啶基（以前我们发现翻译后激活ROB）下调了ACRR功能。 ACRR缺失引起的ACRAB的组成型表达在没有TOLC激活的情况下极大地影响抗生素耐药性，但在激活TOLC时会导致过度耐药表型。 TOLC和ACRAB MARBOX协调三方外排泵的激活。 B.不同的调节基因被激活剂激活到不同的范围。我们以前已经确定，尽管活性与激活剂的结合常数在不同的marbox中存在一些相关性，但这种相关性不足以解释差异激活。现在，我们将MARA的表达置于LAC启动子的控制下，LAC启动子被IPTG激活，确定了IPTG浓度与MARA的细胞内浓度之间的关系，并检查了许多调节基因（将LACZ作为转录报告基因）的表达表达为IPTG浓度不同的生长功能。所得数据用于开发数学模型，以深入了解不同启动子处激活机制。 我们发现，激活所需的MARA浓度的浓度与不同启动子的至少30倍不同，因此确定了先前未批准的调节量对照形式，其中某些基因在特定的激活因子浓度下被激活，而其他基因并未显着激活。野生型MAR启动子本身以最低的MARA浓度激活，在约900个MARA分子/细胞的浓度下达到一半最大的刺激。 MICF启动子的可比数值约为10,000 Mara/Cell。其他15个测试的启动子中没有一个表现出活性的平稳，即使在人为的MARA水平（约26,000个分子/细胞）中也没有表现出来。 为了获得洞察力的激活中的多样性，我们开发了一种依赖Mara依赖性启动子活性的数学模型。在模型中，MARA要么增加（吸引力）或降低（排斥）RNA聚合酶（RNAP）在启动子处的占用率，要么增加（加速度）或降低（加速度）（延迟）RNAP一旦结合在启动子处的RNAP的转录率。 MAR启动子激活的最佳模型将吸引力与加速度相结合。对于其他法规启动子，将排斥力与加速度结合的模型最适合数据。结果表明，转录激活可能涉及排斥（由于激活剂引起的RNAP占用率降低），这通常与抑制而不是激活有关。结果还表明，加速度（因激活剂引起的转录率提高）是激活的重要组成部分。加速与排斥相结合，以前尚未被认为是在激活中发挥作用。