Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Jeremy Kubik
Date created: 2009-10-15 11:00:00
Date modified: 2015-05-08 01:11:30
LuxPQ from Vibrio harveyi
| Types | DnaRegion |
| Roles | Translational_Unit engineered_region |
| Sequences | BBa_K218000_sequence (Version 1) |
Description
LuxP is a ABC-type sugar transport system periplasmic protein (Protein ID = YP_001447483.1). LuxQ is a histidine kinase (Protein ID = YP_001447484.1).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.
Notes
This part was submitted to the Registry in 2008 (K131015); however, this part was cloned again and sequenced and is being re-submitted.LuxP and LuxQ are found in the operon, thus we decided to keep them together. We kept natural RBS in front of LuxP, as well as the intrinsic terminator. Silent mutations were performed on a EcoRI site located within LuxP, EcorI site within LuxQ, and XbaI site within LuxQ.
Source
GenBank Accession: NC_009784 Vibrio harveyi ATCC BAA-1116 chromosome II, REGION: 579654..580751.Neiditch MB, Federle MJ, Pompeani AJ, Kelly RC, Swem DL, Jeffrey PD, Bassler BL, Hughson FM. Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing. Cell. 2006 Sep 22;126(6):1095-108.
| Sequence Annotation | Location | Component / Role(s) |
| start of LuxP start of LuxQ stop for LuxP LuxP stop for LuxQ LuxQ Silent mutation to remove EcoRI site Silent mutation to remove EcoRI site Silent mutation to remove XbaI site | 30,32 1127,1129 1125,1127 30,1127 3704,3706 1127,3706 599,599 1465,1465 1618,1618 | start_codon feature/start feature/start start_codon stop_codon feature/stop feature/cds CDS feature/stop stop_codon feature/cds CDS feature/s_mutation point_mutation point_mutation feature/s_mutation point_mutation feature/s_mutation |