Part:BBa_K1666000

LsrA of LuxS/AI-2 signaling pathway in Salmonalla

Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And LsrA is the ATP-binding protein which provides energy for AI-2 transport. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus or Lactococcus for the final purpose of constructing an integrated AI-2 response pathway of Salmonella in the engineered bacteria.

Usage and Biology

In quorum sensing process, bacteria communicate with one another using secreted chemical signaling molecules termed autoinducers. As the density of the bacterial population increases, so does the concentration of secreted autoinducer molecules. When the concentration of the autoinducer reaches a critical threshold, it diffuses back into the cell and activates or represses certain target genes.

An autoinducer called AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. Depending on the bacteria, response to AI-2 can follow one of the two identified routes. In one group of bacteria exemplified by Salmonella, AI-2 response involves genes named Lsr (LuxS regulated) that encode ATP binding cassette-type transporter. And LsrA is ATP-binding protein of the ABC transporter.

Fig1. Schematic overview of the AI-2 response pathway in Salmonella TyphimuriumThe precursor of AI-2, 4,5-Dihydroxy-2,3-Pentanedione (DPD) , is a byproduct generated when LuxS converts S-Ribosylhomocysteine (SRH) to Homocysteine (HCY). DPD then undergoes spontaneously cyclization, forming AI-2, and exports to the culture supernatant. After that, extracellular AI-2 bounds to LsrB, following by passing the membrane channel and importing the cytoplasm. LsrK phosphorylates AI-2 afterwards. The lsr operon is repressed until phosphorylated AI-2 causes LsrR to relieve its repression on the promoter. And this allows further AI-2 import.

However, in many other bacteria, such as our chassis, Lactobacillus and Lactococcus, AI-2 response follows a different route from the one mentioned above.

In our project, we set this part encoding LsrA of Salmonella under the regulation of a nisA promoter. The promoter can be activated by food-grade inducer, nisin. We try to integrate this part in the genome of our engineered bacteria. And Together with other parts, we will construct a membrane channel for AI-2 generated by pathogens. And LsrA is an essential part due to its function of providing energy for AI-2 transport.

Fig2. Schematic overview of the pathogen detection process in Lactobacillus bulgaricusWe set LsrABCDRK genes under the regulation of nisA promoter respectively and integrated them to the genome of our engineered bacteria (Lactobacillus bulgaricus was taken as an example). The nisA promoter will be activated by food-grade inducer, nisin, leading to the formation of membrane channels. Besides, we use the promoter of lsr operon to regulate the transcription of the report gene which encodes blue pigment. Eventually, our engineered bacteria will be capable of receiving AI-2 molecules from pathogens and lead to the expression of the report gene.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 939
  
• 1000
  COMPATIBLE WITH RFC[1000]