PURIFICATION AND MUTANT ANALYSIS OF CITROBACTER-FREUNDII AMPR, THE REGULATOR FOR CHROMOSOMAL AMPC BETA-LACTAMASE
PURIFICATION AND MUTANT ANALYSIS OF CITROBACTER-FREUNDII AMPR, THE REGULATOR FOR CHROMOSOMAL AMPC BETA-LACTAMASE
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DOI：
10.1111/j.1365-2958.1991.tb01920.x
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发表时间：
1991-07-01
影响因子：
3.6
通讯作者：
NORMARK, S
中科院分区：
文献类型：
--
作者：
BARTOWSKY, E;NORMARK, S
对弗氏柠檬酸杆菌AmpC-β-内酰胺酶基因的转录调控因子AMPR进行了纯化。纯化的AMPR具有与DNA结合的活性，在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳法上的分子量为32 kDa，前15个氨基酸的N末端测序与根据核苷酸序列预测的一致。分离到两个突变株，它们分别映射在AMPR的氨基末端和羧基末端，从而取消了DNA结合和β-内酰胺酶的诱导。突变的氨基末端(S35F)位于螺旋-转角-螺旋区域，与LysR调节家族的其他成员有很高的同源性。因此，该突变可能直接取消AMPR与其操纵子序列之间的接触。提示AMPR的C端突变(Y264N)影响亚单位间的相互作用。分离到一个位于ampr基因中心的构件性突变。这个G102E突变体在没有β-内酰胺诱导剂和ampg的情况下导致结构性β-内酰胺酶的表达，ampg是在野生型肠杆菌中诱导必不可少的基因。另一种突变蛋白D135Y在AMPG+和AMPG：：Kan背景下显示出野生型特性，但与野生型AMPR不同，它可以在ampG1突变背景下激活ampC基因。目前认为ampG1是一种错义突变。这两种类型的AMPR突变体表明，AMPC转录的激活依赖于AMPR向转录激活剂的转化，这种激活通常可能涉及与AMPG的相互作用。
AmpR, the transcriptional regulator for the Citrobacter freundii ampC-beta-lactamase gene, was purified. The purified AmpR had DNA-binding activity, the same molecular mass (32 kDa) on sodium dodecyl sulphate/polyacrylamide gel electrophoresis as previously described, and N-terminal sequencing of the first 15 amino acids was in agreement with that predicted from the nucleotide sequence. Two mutants were isolated that abolish DNA-binding and beta-lactamase induction and which map in the amino- and carboxyl-terminal ends of AmpR, respectively. The mutation in the amino terminus (S35F) was located in a helix-turn-helix region showing high homology to other members of the LysR regulator family. Therefore this mutation may directly abolish the contact between AmpR and its operator sequence. It is suggested that the C-terminal mutation (Y264N) affects subunit interactions in AmpR. One constitutive mutant was isolated which mapped in the centre of the ampR gene. This G102E mutant leads to constitutive beta-lactamase expression in the absence of both beta-lactam inducer and ampG, a gene essential for induction in wild-type enterobacteria. Another mutant protein, D135Y, showed wild-type properties in an ampG+ and an ampG::kan background, but could, unlike wild-type AmpR, activate the ampC gene in an ampG1 mutant background. It is thought that ampG1 is a missense mutant. These two types of ampR mutants suggest that activation of ampC transcription is dependent on the conversion of AmpR into a transcriptional activator and that this activation may normally involve interactions with AmpG.