Our objective of this project is to construct a broadly applicable transcription-based biosensor for gauging c-di-GMP levels in real time in different bacteria. In this design part, we will have a full understanding of the connection between biofilm formation and c-di-GMP. We will also show you how we design our biosensor to achieve the sensing by linking the presence of c-di-GMP to the reporter gene expression through a special FleQ response promoter.

Bacteria switch their lifestyles between planktonic and sessile modes in response to various environmental cues. This switch is often regulated by sophisticated intracellular signaling networks that modulate the levels of small molecules. Among them,c-di-GMP networks is the most-studied chemical signaling systems.







Fig 1. Principles of cyclic di-GMP (c-di-GMP) signalling^[1].

The c-di-GMP monomer exhibits two-fold symmetry, with two GMP moieties that are fused by a 5'-3' macrocyclic ring. The synthesis of c-di-GMP is catalysed by diguanylate cyclases(DGCs) through the cooperative action of their two catalytic GGDEF domains. Specific phosphodiesterases(PDEs) that contain EAL or HD-GYP domains hydrolyse c-di-GMP into GMP. Through binding to effector molecules, c-di-GMP regulates diverse cellular processes, including motility, adherence, biofilm formation, virulence, development and cell cycle progression^[1].



Fig 2. Role of c-di-GMP in biofilm formation and dispersal^[1]

Earlier studies have shown that c-di-GMP plays an important role in mediating several cellular functions and various aspects of bacterial physiology ^{[2, 3]}. High levels of c-di-GMP promote biofilm formation by increasing cell aggregation and exopolysaccharide production, whereas low levels of c-di-GMP reduce biofilm formation by decreasing bacterial motility and extracellular DNA production ^[4].

In response to c-di-GMP, the enhancer binding protein FleQ from Pseudomonas aeruginosa derepresses the expression of Pel exopolysaccharide genes required for biofilm formation when a second protein, FleN is present^[5]. The c-di-GMP- responsive transcriptional regulator FleQ in P. aeruginosa has been demonstrated to repress pelA transcription by binding to the pelA promoter, whereas a high concentration of c-di-GMP relieved the repression and induced Pel synthesis^[6].

FleQ binds to two sites called box 1 and 2. FleN binds to FleQ bound at these sites causing the intervening DNA to bend. Binding of c-di-GMP to FleQ relieves the DNA distortion but FleQ remains bound to the two sites. Previous research showed that FleQ represses gene expression from box 2 and activates gene expression in response to c-di-GMP from box 1. The role of c-di-GMP is thus to convert FleQ from a repressor to an activator. The mechanism of action of FleQ is distinct from that of other bacterial transcription factors that both activate and repress gene expression from a single promote^[5].



Fig. 3 Model of pel regulation. FleQ binds to two FleQ boxes on the pel promoter a| FleQ interacts with FleN in the absence of ATP. b| as well as in the presence of ATP, but in this case it induces absorption of pel DNA . c|We propose that FleN forms a bridge between two FleQ bound to their binding sites. The binding of FleQ to FleQ box 2 is essential for repression. The binding of c-di-GMP to FleQ induces a conformational change of FleQ, probably propagated through FleN, which induces the relief of pel distortion and leads to pel expression. d|The binding of FleQ to FleQ box 1 is essential for activation.

In our project, we describe the design of a transcription-based fluorescent reporter that is readily adaptable for gauging c-di-GMP levels in different bacteria. The components of the reporter comprise a transcription factor FleQ that is originated from P. aeruginosa, a tandem promoter PcI/pel that is composed of a constitutive promoter PcI and a c-di-GMP responsive promoter Ppel, as well as a fluorescent protein GFP as a reporter. In P. aeruginosa, the transcriptional factor FleQ represses expression of the pel operon for Ppel exopolysaccharide biosynthesis by a simple roadblock mechanism in which FleQ sits at the pel promoter and prevents RNA polymerase from binding, while FleQ derepresses gene expression due to the conformational change of FleQ upon binding c-di-GMP. The detail of how we test the working performance and improve our design based on the result will be in the engineering success page.

https://2022.igem.wiki/cug-china/engineering



Fig 4. Schematic illustration of the transcription-based fluorescent c-di-GMP reporter. A| The tandem promoter PcI/pel is constructed by fusing the cI promoter to the c-di-GMP responsive pel promoter. PcI constitutively drives gfp transcription in the absence of FleQ. B| In the presence of FleQ, it binds to two FleQ boxes in the pel promoter to repress gfp transcription. C| The binding of c-di-GMP to FleQ induces the conformational change of FleQ, which relieves FleQ from pel promoter to activate gfp transcription promoted by cI promoter.

1. Choose of chassis

We have no P2 lab, therefore, we cannot test our biosensor in pathogen and choose a common model organism E. coli BL21. Why choose Shewanella oneidensis MR-1? To test weather our biosensor can used in different hosts, we choose S. oneidensis MR-1 as another tested organisms because it is an attractive model microbe for elucidating the biofilm-metal interactions that contribute to the billions of dollars in corrosion damage to industrial applications each year. Our biosensor is potentially applied for the screen of chemicals that decrease c-di-GMP of MR-1 to control its biofilm formation on engineering settings.

2. Performance test

To test biosensor performance, we changed intracullelar c-di-GMP of BL21 and MR-1 by overexpressing c-di-GMP hydrolyses gene yjhH originated from E.coli. By adding different level of IPTG, we created a c-di-GMP gradient, and detect the florescence intensity under these conditions.



Fig. 5 Testing biosensor function by creating different level through yhjH^[5]

3. Using biosensor to screen c-di-GMP-decreasing chemical

After verifying the response to different c-di-GMP levels of our biosensor, we decided to stimulate the practical applications of the biosensor to screen the biofilm dissociating drugs. Our engineered strain was cultured by adding the literature reported dispersal agent^[7]. Then we will detect the florescence intensity to see whether it is reduced. We further measured c-di-GMP level by using HPLC and found that these chemical indeed reduced c-di-GMP level. It evidences the possibility of biosensor to be used for screening biofilm-dispersing agents.

[1] Jenal U, Reinders A, Lori C. Cyclic di-GMP: second messenger extraordinaire[J]. Nat Rev Microbiol, 2017,15(5):271-284.

[2] Cotter P A, Stibitz S. c-di-GMP-mediated regulation of virulence and biofilm formation[J]. Curr Opin Microbiol, 2007,10(1):17-23.

[3] Hengge R. Principles of c-di-GMP signalling in bacteria[J]. Nat Rev Microbiol, 2009,7(4):263-273.

[4] Hu Y, Wu Y, Mukherjee M, et al. A near-infrared light responsive c-di-GMP module-based AND logic gate in Shewanella oneidensis[J]. Chem Commun (Camb), 2017,53(10):1646-1648.

[5] Baraquet C, Murakami K, Parsek M R, et al. The FleQ protein from Pseudomonas aeruginosa functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c-di-GMP[J]. Nucleic Acids Res, 2012,40(15):7207-7218.

[6] Hickman J W, Harwood C S. Identification of FleQ from Pseudomonas aeruginosa as ac-di-GMP-responsive transcription factor[J]. Molecular microbiology, 2008,69(2):376-389.

[7] Ho C L, Chong K S J, Oppong J A, et al. Visualizing the perturbation of cellular cyclic di-GMP levels in bacterial cells[J]. Journal of the American Chemical Society, 2013,135(2):566-569.