Toxin-antitoxin (TA) systems are ubiquitous modules existed in bacterial genomes. The transcription of a toxin gene is often coupled with its cognate antitoxin. Toxins are small polypeptides that negatively regulate cellular growth by inhibiting important cellular processes while antitoxins can neutralize them and bring cells back to normal.
TA systems are classified into six groups based on the type of antitoxin and mechanism of how antitoxin blocks toxin activity. We choose the CbeA-CbtA TA pair. CbeA -CbtA is a novel type IV TA system for indirect interaction between toxin and antitoxin. Toxin CbtA can bind and inhibit the polymerization of MerB and FtsZ, leading to effect on cellular morphology and division. Antitoxin CbeA suppresses CbtA toxicity by stabilizing the CbtA-targeted proteins rather than by directly interacting with CbtA to suppress its toxicity. Specifically, CbeA directly binds to both MreB and FtsZ, enhancing the bundling of their filaments in vitro. Notably, this is a unique feature of the CbeA–CbtA system, distinguishing it from all the other TA systems, that toxin CbtA and antitoxin CbeA do not form a complex. Thus, toxicity of CbtA can be considered as reversible, which means even if cellular division has been repressed by CbtA, CbeA can still neutralize CbtA toxicity and enable cellular division.
Figure 1. The mechanism of CbeA and CbtA(type IV TA system). CbeA blocks the CbtA toxicity by promoting the assembly of FtsZ and MreB filaments, which is inhibited by the CbtA.
DmpR, the product of the Pseudomonas sp Strain CF600 dmpR gene[3,4], mediates expression of the dmp operon to allow growth on simple phenols. Pr is a constitutive promoter allowing expression of DmpR. Transcription from P0, the promoter of the dmp operon, is activated when DmpR detects the presence of an inducing phenol. It depends on a direct physical interaction between the sensor domain of DmpR and the inducing phenol. A productive association between the sensor domain and a phenolic molecule causes DmpR to undergo a conformational change that results in a polymerase-activating form of the protein[5,6], promoting the expression of gene downstream P0.
Figure 2. The mechanism of DmpR sensor.
Domain-swapping experiments to form XylR-DmpR hybrids demonstrated that the sensor activity of these regulatory proteins is localized in the amino-terminal region. Modification of the sensor domain should allow the creation of novel proteins that respond to xenobiotics which remain undetected by the wild-type protein. Such altered proteins have the potential to extend the chemical target range of biosensors beyond that based on natural systems. Phenol and substituted phenols are common starting materials and waste by-products in the manufacture of chemical, industrial, and agricultural products. The natural interaction of DmpR with a subset of phenols suggested that modification of its sensor domain might create protein derivatives with the capacity to detect phenolic molecules commonly used in industry.
It’s reported that high expression of DmpR would delay cell growth with longer incubation time required to culture the bacterial cells to early stationary phase but did not help to increase the sensitivity towards pollutants. As such, promoters have different promoting strength belongs to Anderson Promoter Collection are in front of dmpR to certify it.
Naturally, the first two steps in phenolics degradation evolve two enzymes, ortho hydroxyl addition by monooxygenase and cleavage of aromatic ring by dioxygenase.
Figure 3. Intial two steps in phenolics degradation.
TfdB-JLU is a novel 2,4-dichlorophenol hydroxylase whose amino acid sequence exhibits less than 48% homology with other known TfdBs. The rate-limited step of phenolic degradation is the ortho hydroxylation. Compared to wild-type TfdB, TfdB-JLU has a wilder substrate range and higher catalysis activity. Thus, the enzyme has advantages in efficient disposing of phenolic effluents.
CphA-I and CaO19_12036 are dioxygenase which from A. chlorophenolicus A6 and Candida albicans TL3 respectively[9,10]. These two enzymes have a similar optimum temperature with TfdB-JLU and have a high catalytic activity towards pyrocatechol.
DmpR and toxin are downstream of a constitutive promoter, antitoxin and enzyme are downstream of pdmp operon (Fig 4). When there are no phenolic components in environment, DmpR protein can be ready to activate dmp operon and toxin can be expressed to repress growth of our engineered bacteria. When aromatic substrates appear, DmpR protein can combine pdmp and trigger antitoxin and enzyme expression, leading to toxin neutralization and phenolic degradation, respectively.
Figure 4. The design of whole circuit.
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