Transcription Factors CysB and SfnR Constitute the Hierarchical Regulatory System for the Sulfate Starvation Response in Pseudomonas putida

Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; Department of Industrial Chemistry, Faculty of Engineering, Shibaura Institute of Technology, 3-9-14 Shibaura, Minato-ku, Tokyo 108-8548, Japan; National Institute of Advanced Industrial Science and Technology (AIST), Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan

^* To whom correspondence should be addressed. Email: hiroshi.habe{at}aist.go.jp.

	Abstract

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 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_DS1 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 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 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.