Part:BBa_K218021
LuxO D47A under constitutive expression of tetR repressible promoter
This is a circuit designed to produce the LuxO D47A protein, a mutated protein that mimics the dephosphorylated form of the LuxO protein (Vibrio harveyi). In nature, this protein is involved in the AI-2 quorum sensing pathway in Vibrio harveyi. This dephosphorylated form of the LuxO protein cannot bind to the qrr promoter (K131017) and as result, does not transcribe any genes downstream. This circuit, in conjunction with K218017, a circuit that produces LuxO D47E, a mutant protein that mimcs the phosphorylated form of LuxO, can be used to test the functionality of the qrr4 promoter (K131017).
Usage and Biology
Quorum-sensing bacteria produce and release chemical signal molecules termed autoinducers (AIs) whose external concentration increases as a function of increasing cell-population density. Bacteria detect the accumulation of a minimal threshold stimulatory concentration of these autoinducers and alter gene expression, and therefore their behavior. Using these signal-response systems, bacteria synchronize particular behaviors on a population-wide scale and thus function as multicellular organisms. The bioluminescent marine bacterium Vibrio harveyi uses three different AIs—AHL, CAI-1, and AI-2—to control the expression of genes responsible for bioluminescence and numerous other traits. We have designed our System 2 based on V. harveyi AI-2 signaling. V. harveyi AI-2 signal is a furanosyl borate diester, production of which requires the LuxS enzyme. Biosynthesis of AI-2 is dependent on the usage of S-adenosylmethionine (SAM) by the cell in various methylation reactions. For this reason, during periods of exponential growth, there is a very large production of AI-2, thus perhaps signaling to neighbors that a suitable environment for growth (i.e. rich in nutrients) has been found. LuxS catalyzes the formation of the (S)-4,5-dihydroxy-2,3-pentanedione (DPD) intermediate which spontaneously cyclizes and reacts with borate to give AI-2. AI-2 is bound in the periplasm by the protein LuxP, which is constitutively bound to LuxQ, a membrane bound histidine kinase sensor. The binding of AI-2 to LuxP is necessary in regulating the activity of the periplasm-bound LuxQ. At low cell density, in the absence of significant amounts of autoinducers, LuxQ acts as a kinase, autophosphorylates, and subsequently transfers the phosphate to the cytoplasmic protein LuxU. LuxU passes the phosphate to the DNA-binding response regulator protein LuxO. Phospho-LuxO, in conjunction with a transcription factor termed σ54, involved in nitrogen metabolism, activates transcription of the genes encoding five regulatory small RNAs (sRNAs) termed Qrr1–5 (for Quorum Regulatory RNA). The Qrr sRNAs interact with an RNA chaperone termed Hfq, involved in mRNA splicing. The sRNAs, together with Hfq, bind to and destabilize the mRNA encoding the transcriptional activator termed LuxR. LuxR is required to activate transcription of the luciferase operon: luxCDABE. Thus, at low cell density, because the luxR mRNA is degraded, the bacteria do not express the genes necessary for bioluminescence. At high cell density, when the autoinducers accumulate to the level required for detection, the kinase activity of LuxQ is overtaken by its phosphatase activity and thus drains phosphate from LuxO via LuxU. Unphosphorylated LuxO cannot induce expression of the sRNAs. This allows translation of luxR mRNA, production of LuxR, resulting in bioluminescence.
Reference: Reference: Waters C.M. and Bassler B.L. Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol. 2005;21:319-46.
Jeremy A. Freeman and Bonnie L. Bassler. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. 1999a. Molecular Microbiology. 31(2), 665-677.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 904
Illegal SapI.rc site found at 866
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