Characterization and Modeling of Transcriptional Cross-Regulation in Acinetobacter baylyi ADP1 Dayi Zhang, †,+ Yun Zhao, ‡,§,+ Yi He, ∥ Yun Wang, † Yiyu Zhao, †,‡ Yi Zheng, ‡ Xia Wei, ‡ Litong Zhang, ‡ Yuzhen Li, ‡ Tao Jin, ‡ Lin Wu, ∥ Hui Wang, ⊥ Paul A. Davison, † Junguang Xu, ‡,§, * and Wei E. Huang* ,† † Kroto Research Institute, University of Sheffield, Broad Lane, Sheffield S3 7HQ, U.K. ‡ BGI-Shenzhen, Shenzhen 518083, P.R. China § Shenzhen Key Laboratory of Environmental Microbial Genomics and Application, Shenzhen 518083, P.R. China ∥ Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100029, China ⊥ Centre for Ecology and Hydrology, Wallingford, Banson Road, Wallingford OX10 8BB, U.K. * S Supporting Information ABSTRACT: Synthetic biology involves reprogramming and engineering of regulatory genes in innovative ways for the implementation of novel tasks. Transcriptional gene regulation systems induced by small molecules in prokaryotes provide a rich source for logic gates. Cross-regulation, whereby a promoter is activated by different molecules or different promoters are activated by one molecule, can be used to design an OR-gate and achieve cross-talk between gene networks in cells. Acinetobacter baylyi ADP1 is naturally transformable, readily editing its chromosomal DNA, which makes it a convenient chassis for synthetic biology. The catabolic genes for salicylate, benzoate, and catechol metabolism are located within a supraoperonic cluster (-sal- are-ben-cat-) in the chromosome of A. baylyi ADP1, which are separately regulated by LysR-type transcriptional regulators (LTTRs). ADP1-based biosensors were constructed in which salA, benA, and catB were fused with a reporter gene cassette luxCDABE under the separate control of SalR, BenM, and CatM regulators. Salicylate, benzoate, catechol, and associated metabolites were found to mediate cross-regulation among sal, ben, and cat operons. A new mathematical model was developed by considering regulator-inducer binding and promoter activation as two separate steps. This model fits the experimental data well and is shown to predict cross-regulation performance. KEYWORDS: cross-regulation, Acinetobacter baylyi ADP1, catechol, salicylate, benzoate, LysR-type gene regulation, mathematic model, repressor O ne of the important goals of synthetic biology is to reprogram and rewire regulatory genes in innovative ways for the implementation of novel tasks. To help better design a controllable gene network, it is crucial to understand naturally occurring gene regulatory systems and develop mathematic models to predict gene regulation performance. The regulated gene transcription is an essential strategy in prokaryotes for the economic use of energy and enables a rapid response to the changing environment. The highly naturally transformable bacterium Acinetobacter baylyi ADP1 is a convenient chassis for synthetic biology, because its chromosome is readily editable by cutting, deleting, duplicating, and inserting DNA. 1−7 One quarter of the A. baylyi ADP1 genome is composed of five major “islands of catabolic pathways”. 1 LysR-type transcriptional regulators (LTTRs) control the largest family of transcriptional gene regulation system in prokaryotes. 8 The salicylate, benzoate, and catechol degradation pathways are located in the supercluster sal-are-ben- cat in the chromosome of A. baylyi ADP1, which are controlled by LysR-type transcriptional regulators SalR, BenM, and CatM separately. 9−12 The salAR is controlled by SalR, which can be activated by salicylate. 10 The benABCDE operon is regulated by BenM, responding to both benzoate and its metabolite cis,cis- muconate. 13 CatM controls transcription of catA and the catBCIJFD operon and is specifically activated by cis,cis- muconate. 14 BenM and CatM are 59% identical in DNA sequence, and both respond to cis,cis-muconate to activate transcription. 12 It was previously found that CatM was not involved in benA expression. 15 The upstream metabolic pathway of catechol is shown in Figure 1, together with the Special Issue: Synthetic Biology: Research Perspectives from China Received: April 1, 2012 Research Article pubs.acs.org/synthbio © XXXX American Chemical Society A dx.doi.org/10.1021/sb3000244 | ACS Synth. Biol. XXXX, XXX, XXX−XXX