Nonheritable Antibiotic Resistance

细菌的染色体多重耐药是一个严重的临床问题。我们的研究表明，大肠杆菌变得耐多种抗生素，有机溶剂和超氧化物的活性时，任何三个旁系同源，但不同的调节，转录激活因子，MarA，SoxS和罗布，增加。这些激活子结合一个叫做marbox的序列，它位于一组大约40个染色体基因的启动子上游，这些基因被称为mar/sox/rob调节子。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. acrAB和tolC基因的转录激活是导致抗生素抗性和有机溶剂耐受的主要机制。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试验，我们研究了这些操纵子的调节由阻遏物AcrR和由MarA，SoxS和罗布。我们发现，tolC有两个以前未确定的强重叠启动子，由MarA激活。它们以独特的方式配置，使得激活剂与单个marbox的结合可以激活来自任一启动子的转录。marbox位于tolC p4启动子处RNA聚合酶的-10信号上游20 bp（I类 * 构型），但位于tolC p3启动子处-10信号上游30 bp（II类构型）。 此外，我们发现，三个顺式作用元件是重要的调节acrAB和acrR：marbox，它允许激活acrAB;一个24 bp的反向重复序列，其中包含acrAB启动子和部分的发散转录acrR启动子，这是可能的网站的acrR结合的acrAB和acrR的镇压;和一个22 bp的反向重复序列，包含部分的acrR启动子。我们还发现，2，2 '-联吡啶（我们以前发现它可以激活Rob）下调AcrR功能。由acrR缺失引起的acrAB的组成型表达在tolC活化不存在的情况下最小程度地影响抗生素抗性，但是当tolC被活化时导致超抗性表型。tolC和acrAB marbox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的程度。我们以前已经确定，虽然一些相关性之间存在的活动和不同marbox的激活剂的结合常数，这种相关性是不足以解释的差异激活。我们现在已经将MarA的表达置于由IPTG激活的lac启动子的控制下，确定了IPTG浓度和MarA的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）的表达作为在不同浓度的IPTG中生长的函数。所得数据用于开发数学模型，以深入了解不同启动子的激活机制。 我们发现，激活所需的MarA的浓度变化至少30倍，不同的启动子，从而确定了以前不受重视的形式的调节子控制，其中一些基因在特定的激活剂浓度被激活，而其他人没有显着激活。野生型mar启动子本身在最低浓度的MarA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启动子的可比较数量为约10，000 MarA/细胞。测试的15个其他启动子中没有一个表现出活性平台，即使在人工最高水平的MarA（约26，000分子/细胞）下也是如此。 为了深入了解调节子激活的多样性，我们开发了一个依赖于MarA的启动子活性的数学模型。在该模型中，MarA增加（吸引）或减少（排斥）RNA聚合酶（RNAP）在启动子处的占有率，并且一旦结合在启动子处，则增加（加速）或减少（延迟）RNAP的正向转录速率。mar启动子激活的最佳模型是结合了联合收割机的吸引力和加速作用。对于其他调节子启动子，结合联合收割机排斥和加速的模型最适合数据。结果表明，转录激活可能涉及排斥（由于激活剂导致RNAP占用率降低），这种效应通常与抑制而不是激活有关。结果还表明，加速（由于激活剂而导致的转录正向速率的增加）是激活的重要组成部分。加速度与排斥力结合在一起，以前并没有被认为在激活中起作用。

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.