Difference between revisions of "Part:BBa K218017"

Revision as of 02:06, 22 October 2009

LuxO D47E under constitutive expression of TetR repressible promoter

LuxOD47E with constitutive promoter, RBS and terminator.

LuxOD47E from Vibrio harveyi with constitutive promoter and RBS. This circuit contains luxO D47E with constitutive promoter and RBS (J13002) and terminator (B0015). This circuit allows for the expression of the LuxO D47E protein, a mutant variant of the Vibrio harveyi LuxO protein. The LuxO D47E protein mimics the active (i.e.phosphorylated) form of the LuxO protein and is a transcriptional activator. Together with sigma factor 54, LuxO D47E binds to Vibrio harveyi's quorum regulatory small RNA promoters (Pqrr1, Pqrr2, Pqrr3, Pqrr4 and Pqrr5). We have also added the qrr4 promoter to the Registry as part of last year's project (BBa_K131017) and have created a reporter circuit containing the Pqrr4-gfp fusion (BBa_K218011) for the functional verification of this circuit. Furthermore, the KT1144 E. coli strain containing the Pqrr4-gfp fusion (obtained and used with permission from Dr. Bonnie Bassler of Princeton University) was used as an additional standard to test the functionality of the LuxOD47E protein. For the protocol and the results of this test, please refer to the main page of Part BBa_K131011.

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: 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