Role of the 'cre/blr-tag' DNA sequence in regulation of gene expression by the Aeromonas hydrophila β-lactamase regulator, BlrA
Role of the 'cre/blr-tag' DNA sequence in regulation of gene expression by the Aeromonas hydrophila β-lactamase regulator, BlrA
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DOI：
10.1093/jac/dkh077
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发表时间：
2004-02-01
影响因子：
5.2
通讯作者：
Bennett, PM
中科院分区：
文献类型：
--
作者：
Avison, MB;Niumsup, P;Bennett, PM
目的：进一步了解嗜水气单胞菌T429125 blr调控的调控机制，包括三个非连锁β -内酰胺酶基因ampH、cepH和imiH，并研究“cre/blr-tag”DNA序列（TTCAC）在双组分系统BlrAB的转录调控中的作用。方法：采用标准方法克隆blab - amph连锁基因；采用RT-PCR或β -内酰胺酶法检测基因表达；通过反转录分析确定转录起始位点；通过PCR构建cepH启动子探针报告基因，包括cre/blr标签缺失；利用pBAD质粒在大肠杆菌中过表达BlrA。结果：blrD基因编码一种推测的内膜蛋白，位于blab - amph的下游。RT-PCR分析显示blrD是嗜水单胞菌blr调控子的一部分，转录起点测定显示blr调控子启动子（包括blrD的启动子）之前至少有一个cre/blr标签。靶向删除16bp的cepH cre/blr-tag二聚体可阻断blra诱导的大肠杆菌中cepH的过量产生。结论：这是关于非β -内酰胺酶基因与通常共同存在的β -内酰胺酶基因协同调节的首次报道，也是cre/blr-tag序列在BlrA调控转录中的作用的第一个直接证据。
Objectives: To further understand the mechanisms used to regulate expression of the blr regulon of Aeromonas hydrophila T429125, including three unlinked beta-lactamase genes, ampH, cepH and imiH, and to examine the role of the 'cre/blr-tag' DNA sequence (TTCAC) in transcriptional control exerted by the two-component system, BlrAB.Methods: Genes linked to blrAB-ampH were cloned using standard methods; gene expression was measured by RT-PCR or beta-lactamase assays; transcription start sites were determined by reversed-transcript analysis; cepH promoter probe reporter constructs including cre/blr-tag deletions were generated by PCR; and BlrA was overexpressed in Escherichia coli using the pBAD plasmid.Results: The blrD gene, encoding a putative inner membrane protein, was found to be located downstream of blrAB-ampH. RT-PCR analysis showed that blrD is part of the A. hydrophila blr regulon, and transcript start-point determinations revealed that blr-regulon promoters (including that of blrD) are preceded by at least one cre/blr-tag. Targeted deletion of the 16 bp cepH cre/blr-tag dimer blocked BlrA-induced overproduction of cepH in E. coli.Conclusions: This is the first report of non-beta-lactamase genes being co-ordinately regulated with a normally co-resident beta-lactamase gene, and the first direct evidence for a role of the cre/blr-tag sequence in the regulation of transcription by BlrA.