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，当三种旁系同源但受不同调控的转录激活因子 MarA、SoxS 和 Rob 的活性增加时，大肠杆菌会对多种抗生素、有机溶剂和超氧化物产生耐药性。这些激活剂结合一个称为 marbox 的序列，该序列位于一组约 40 个称为 mar/sox/rob 调节子的染色体基因的启动子上游。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重抗生素耐药性的机制。 A. 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延伸测定，我们研究了阻遏物 AcrR 以及 MarA、SoxS 和 Rob 对这些操纵子的调节。我们发现 tolC 有两个先前未识别的强重叠启动子，它们被 MarA 激活。它们以独特的方式配置，因此激活子与单个 marbox 的结合可以激活任一启动子的转录。 marbox 位于 tolC p4 启动子处 RNA 聚合酶（I* 类配置）-10 信号上游 20 bp，但位于 tolC p3 启动子处 -10 信号（II 类配置）上游 30 bp。 此外，我们发现三个顺式作用元件在acrAB和acrR的调节中很重要：允许激活acrAB的marbox； 24 bp 反向重复序列，包含 acrAB 启动子和部分转录的 acrR 启动子，并且是 AcrR 结合抑制 acrAB 和 acrR 的可能位点；以及包含 acrR 启动子部分的 22 bp 反向重复序列。我们还发现 2,2'-联吡啶（我们之前发现它会在翻译后激活 Rob）下调 AcrR 功能。在没有 tolC 激活的情况下，acrR 缺失导致的 acrAB 组成型表达对抗生素耐药性影响最小，但当 tolC 激活时，会导致超耐药表型。 tolC 和 acrAB marboxes 协调三方外排泵的激活。 B. 不同的调节子基因被激活剂激活到不同程度。我们之前已经确定，虽然不同 marbox 的活性和激活剂的结合常数之间存在某种相关性，但这种相关性不足以解释差异激活。我们现在将 MarA 的表达置于由 IPTG 激活的 lac 启动子的控制下，确定了 IPTG 浓度与 MarA 细胞内浓度之间的关系，并检查了许多调节子基因（使用 lacZ 作为转录报告基因）的表达作为不同浓度 IPTG 中生长的函数。由此产生的数据用于开发数学模型，从而深入了解不同启动子的激活机制。 我们发现，对于不同的启动子，激活所需的 MarA 浓度至少有 30 倍的差异，从而识别出一种以前未被重视的调节子控制形式，其中一些基因在特定的激活剂浓度下被激活，而其他基因则没有显着激活。野生型mar启动子本身在最低MarA浓度下被激活，在约900个MarA分子/细胞的浓度下达到半最大刺激。 micF 启动子的可比数量约为 10,000 MarA/细胞。即使在人为最高水平的 MarA（约 26,000 个分子/细胞）下，测试的 15 个其他启动子中没有一个表现出活性平台期。 为了深入了解调节子激活的多样性，我们开发了 MarA 依赖性启动子活性的数学模型。在该模型中，MarA 会增加（吸引）或减少（排斥）启动子处 RNA 聚合酶 (RNAP) 的占用，并且一旦与启动子结合，就会增加（加速）或减少（延迟）RNAP 的转录正向速率。 mar 启动子激活的最佳模型将吸引力与加速结合起来。对于其他调节子启动子，将排斥力与加速度相结合的模型最适合数据。结果表明，转录激活可能涉及排斥（由于激活剂导致 RNAP 占用减少），这种效应通常与抑制而不是激活相关。结果还表明加速（由于激活剂导致转录的正向速率增加）是激活的重要部分。加速度与排斥力的结合此前并未被认为在激活中发挥着作用。