SYNTHESIS OF THE ESCHERICHIA-COLI K-12 NUCLEOID-ASSOCIATED DNA-BINDING PROTEIN H-NS IS SUBJECTED TO GROWTH-PHASE CONTROL AND AUTOREGULATION
SYNTHESIS OF THE ESCHERICHIA-COLI K-12 NUCLEOID-ASSOCIATED DNA-BINDING PROTEIN H-NS IS SUBJECTED TO GROWTH-PHASE CONTROL AND AUTOREGULATION
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DOI：
10.1111/j.1365-2958.1993.tb01634.x
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发表时间：
1993-05-01
影响因子：
3.6
通讯作者：
BREMER, E
中科院分区：
文献类型：
--
作者：
DERSCH, P;SCHMIDT, K;BREMER, E
大肠杆菌类核相关DNA结合蛋白H-NS的结构基因(HNS)突变对基因表达、定点重组、噬菌体Mu转座、遗传物质的稳定性和DNA的拓扑状态具有高度的多效性影响。我们研究了HNS的表达调控，发现HNS的转录受到稳定期的诱导和负的自我调节。在体外构建了一组HNS-LacZ蛋白和操纵子的融合基因，并以单拷贝形式整合到细菌基因组的attB位点。沿着细菌生长曲线对β-半乳糖苷酶活性的量化表明，HNS在稳定期的表达比指数级生长的细胞增加了大约10倍。对生长期和稳定期细胞中H-NS蛋白的免疫检测支持了遗传学数据，并表明H-NS的合成随着生长阶段的不同而不同。此外，引物区延伸实验表明，HNS的mRNA在静止相培养中的量增加，并且HNS的转录是由一个独特的启动子控制的，该启动子在对数和静止相都起作用。插入突变破坏HNS基因，导致整合在attB位点的HNS-LacZ操纵子融合表达去抑制(约四倍)，表明HNS转录受到其自身基因产物的负调控。在对数生长期，HNS表达的自动调节尤为明显。HNS转录的稳定期控制和自身调节都与包含HNS启动子的130bp片段相关。为了研究H-NS与其自身调控区的相互作用，我们开发了一种高效的过量生产程序和一个简单的H-NS纯化方案。DNA凝胶滞留分析表明，H-NS蛋白可以优先与携带HNS启动子的限制性片段相互作用。在4℃和60℃下进行的双向聚丙烯酰胺凝胶电泳法判断，该限制性片段呈现弯曲的DNA特征。
Mutations in the structural gene (hns) for the Escherichia coli nucleoid-associated DNA-binding protein H-NS cause highly pleiotropic effects on gene expression, site-specific recombination, transposition of phage Mu, the stability of the genetic material and the topological state of the DNA. We have investigated the regulation of hns expression and found that hns transcription is subjected to stationary phase induction and negative autoregulation. A set of hns-lacZ protein and operon fusions was constructed in vitro and integrated in single copy into the attB site of the bacterial genome. Quantification of beta-galactosidase activity along the bacterial growth curve showed that hns expression increases approximately 10-fold in stationary phase compared with exponentially growing cells. Immunological detection of the H-NS protein in growing and stationary phase cells supported the genetic data and showed that H-NS synthesis varies with growth phase. In addition, primer extension experiments demonstrated that the amount of hns mRNA is elevated in stationary phase cultures and that hns transcription is directed by a unique promoter functioning in both log and stationary phase. Disruption of the hns gene by an insertion mutation led to the derepression (approximately fourfold) of the expression of an hns-lacZ operon fusion integrated at the attB site, showing that hns transcription is subjected to negative regulation by its own gene product. Autoregulation of hns expression is particularly pronounced in log phase. Both stationary phase control and autoregulation of hns transcription are associated with a 130 bp fragment that contains the hns promoter. In order to study the interaction of H-NS with its own regulatory region, we developed an efficient overproduction procedure and a simple purification scheme for H-NS. DNA gel retardation assays showed that the H-NS protein can preferentially interact with a restriction fragment carrying the hns promoter. This restriction fragment showed features of curved DNA as judged by two-dimensional polyacrylamide gel electrophoresis performed at 4-degrees-C and 60-degrees-C.