Transcription factors CysB and SfnR constitute the hierarchical regulatory system for the sulfate starvation response in Pseudomonas putida
Transcription factors CysB and SfnR constitute the hierarchical regulatory system for the sulfate starvation response in Pseudomonas putida
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DOI：
10.1128/jb.00217-08
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发表时间：
2008-07-01
影响因子：
3.2
通讯作者：
Habe, Hiroshi
中科院分区：
文献类型：
--
作者：
Kouzuma, Atsushi;Endoh, Takayuki;Habe, Hiroshi
恶臭假单胞菌DS 1能够利用二甲基砜作为硫源。负责二甲基砜加氧的sfnFG操纵子的表达直接受sigma（54）依赖性转录激活因子SfnR调节，SfnR在sfnECR操纵子内编码。我们研究了硫酸盐饥饿诱导这些sfn操纵子表达的转录机制。使用体内转录测定和体外DNA结合实验，我们发现，SfnR负调控sfnECR的表达通过结合到转录起始点的下游区域。此外，我们证明，LysR型转录调节因子，CysB，直接激活sfnECR的表达结合到其上游区域。CysB是控制sfn操纵子的硫酸盐饥饿反应的主调节因子，如大肠杆菌的磺酸盐利用基因的情况，尽管CysB（DSI）似乎与大肠杆菌的不同。coli CysB中O-乙酰丝氨酸对DNA结合能力的影响。此外，我们通过使用硫酸盐同化基因cysNC和cysI的破坏剂，研究了哪些效应分子在体内抑制sfnFG和sfnECR的表达。这些基因干扰物中sfn操纵子的mRNA水平的测量表明，sfnFG的表达被硫酸盐本身抑制，而sfnECR的表达被硫酸盐同化途径中的下游代谢产物如硫化物和半胱氨酸抑制。这些结果表明，SfnR发挥作用的硫酸盐饥饿诱导的sfn操纵子的表达的CysB独立。
Pseudomonas putida DS1 is able to utilize dimethyl sulfone as a sulfur source. Expression of the sfnFG operon responsible for dimethyl sulfone oxygenation is directly regulated by a sigma(54)-dependent transcriptional activator, SfnR, which is encoded within the sfnECR operon. We investigated the transcription mechanism for the sulfate starvation-induced expression of these sfn operons. Using an in vivo transcription assay and in vitro DNA-binding experiments, we revealed that SfnR negatively regulates the expression of sfnECR by binding to the downstream region of the transcription start point. Additionally, we demonstrated that a LysR-type transcriptional regulator, CysB, directly activates the expression of sfnECR by binding to its upstream region. CysB is a master regulator that controls the sulfate starvation response of the sfn operons, as is the case for the sulfonate utilization genes of Escherichia coli, although CysB(DSI) appeared to differ from that of E. coli CysB in terms of the effect of O-acetylserine on DNA-binding ability. Furthermore, we investigated what effector molecules repress the expression of sfnFG and sfnECR in vivo by using the disruptants of the sulfate assimilatory genes cysNC and cysI. The measurements of mRNA levels of the sfn operons in these gene disruptants suggested that the expression of sfnFG is repressed by sulfate itself while the expression of sfnECR is repressed by the downstream metabolites in the sulfate assimilatory pathway, such as sulfide and cysteine. These results indicate that SfnR plays a role independent of CysB in the sulfate starvation-induced expression of the sfn operons.