Difference between revisions of "Part:BBa K1666001"

Latest revision as of 03:33, 19 September 2015

LsrB 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 LsrB is a solute-binding protein which could transport the AI-2 into the cytoplasm. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus for the final purpose of constructing an integrated AI-2 response pathway of Salmonella in the engineered bacteria.

Usage and Biology

AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In a group of bacteria exemplified by Salmonella, AI-2 response involves lsr genes that encode ATP binding cassette-type transporter. LsrB is a solute-binding protein that can bind AI-2 specifically and help to transport the AI-2 into the cytoplasm.

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.

In our project, we set this protein-coding part under the regulation of a nisA promoter which can be activated by food-grade inducer, nisin. We linearized the related expression vectors and stably integrated them into the genome of the hosts. And together with other parts, we will construct a response pathway for AI-2 generated by pathogens in the engineered bacteria. LsrB can bind AI-2 specifically and help to transport the AI-2 into the cytoplasm

Fig2. Part of the vector containing lsrB We use nisA promoter to initiate the transcription of lsrB gene

Characterization

We transformed the plasmid pHY300PLK containing Plsr with a blue pigment gene at its downstream into E. coli Trans T1. As we can see, after overnight incubation, we observed blue colonies on the plate. That means without LsrR-mediated repression, the Plsr promoter will be constitutively active and promote the production of visible blue pigment.

Fig3. Engineered E. coli Trans T1 contains pHY300PLK-Plsr-amilCP

After we have successfully integrated pNZ9530 and the expression vectors for lsrB, R and K into the Lactobacillus genome, we sequentially transformed the Plsr-amilCP vector. We cultured our engineered Lactobacillus in the medium containing AI-2 secreted by E. coli CD-2. We used DH5alpha bacteria as a control because they do not produce any AI-2. Our engineered Lactobacillus showed clear blue color compared to the control. Without AI-2, the engineered bacteria did not generate blue pigment due to the repression of LsrR. It is phosphorylated AI-2 that caused LsrR to relieve its repression, and LsrB is necessary when AI-2 entered the bacteria. This indicated that LsrB functions as expected.

Fig4. Engineered Lactobacillus incubated with culture supernatant of different bacteria

Sequence and Features