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Lecture 12

Lecture 12.doc

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Department
Microbiology (Biological Sciences)
Course Code
MICRB316
Professor
Nicolas Vozza

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Lecture 12
AI-1 or AIP are produced and detected in a species specific way.
AI-2:
CELL TO CELL SIGNALLING II
Multiple QS systems per cell
It was previously thought that 2 autoinducers would play
different roles in a cell
V. harveyi (not at all like V. fischeri); has bioluminescence,
but it is marine planktonic, not in squid
LuxLM makes AI-1, binds LuxN (species specific)
LuxS makes AI-2, binds LuxPQ (general)
LuxP is homologous to an E. coli ribose binding protein;
binds AI-2 in the periplasm.
LuxP+AI-2 bind LuxQ which is a hybrid 2C SK with a
periplasmic sensory domain, and cytoplasmic HK & RR
LuxN is also a hybrid 2C SK-RR; binds AI-1. (HAI-1)
1
Lecture 12
AI-1 is detected by LuxN
-Ai-2 is detected by LuxPQ
-Signal is transduced through phosphorelay to LuxU and
then LuxO (RR)
-Activated (phospho) LuxO acts (with sigma 54) as a Tn
activator of genes for small RNAs.
-sRNAs +Hfq destabilize the mRNA of LuxR
-
At low cell densities, LuxN & LuxQ act as kinases and
transfer phosphoryl group to SHARED Hpt protein LuxU
LuxU transmits this to RR protein LuxO activates
expression of the repressor RNA + protein, which
negatively regulates luxCDABE (luciferase) > no light
At high cell density, sensors LuxN & LuxPQ bind AI-1 &
AI-2 convert from kinases to phosphatases, reversing
the flow of phosphate: LuxO > LuxU > LuxN and LuxQ
where the phosphoryl group is hydrolyzed.
Dephosphorylation of LuxO inactivates it and terminates
repressor X expression.
In the absence of the repressor X, Vh. LuxR (unrelated to
LuxIR LuxR) binds at promoter luxCDABE activates Tn
LIGHT PRODUCED.
2
Lecture 12
^Phosphorylation flow. Top is low, phosphate flow to right.
Bottom is high, phosphate flow to the left.
In this case, it appears that AI-1 and AI-2 act synergistically.
Because there are 2 QS systems, the bacteria could
potentially distinguish between 4 states.
No AIs – the cell must be planktonic; no light
Hi AI-1; Lo AI-2 – cell is part of mono-culture of Vh 1%
Lo AI-1; Hi AI-2 – cell is invading another population
0.01%
Both AIs – big Vh subcolony of consortium
3

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Description
Lecture 12 1 AI-1 or AIP are produced and detected in a species specific way. AI-2: CELL TO CELL SIGNALLING II Multiple QS systems per cell It was previously thought that 2 autoinducers would play different roles in a cell V. harveyi (not at all like V. fischeri); has bioluminescence, but it is marine planktonic, not in squid LuxLM makes AI-1, binds LuxN (species specific) LuxS makes AI-2, binds LuxPQ (general) LuxP is homologous to an E. coli ribose binding protein; binds AI-2 in the periplasm. LuxP+AI-2 bind LuxQ which is a hybrid 2C SK with a periplasmic sensory domain, and cytoplasmic HK & RR LuxN is also a hybrid 2C SK-RR; binds AI-1. (HAI-1) Lecture 12 2 AI-1 is detected by LuxN -Ai-2 is detected by LuxPQ -Signal is transduced through phosphorelay to LuxU and then LuxO (RR) -Activated (phospho) LuxO acts (with sigma 54) as a Tn activator of genes for small RNAs. -sRNAs +Hfq destabilize the mRNA of LuxR - At low cell densities, LuxN & LuxQ act as kinases and transfer phosphoryl group to SHARED Hpt protein LuxU LuxU transmits this to RR protein LuxO  activates expression of the repressor RNA + protein, which negatively regulates luxCDABE (luciferase) > no light At high cell density, sensors LuxN & LuxPQ bind AI-1 & AI-2  convert from kinases to phosphatases, reversing the flow of phosphate: LuxO > LuxU > LuxN and LuxQ where the phosphoryl group is hydrolyzed. Dephosphorylation of LuxO inactivates it and terminates repressor X expression. In the absence of the repressor X, Vh. LuxR (unrelated to LuxIR LuxR) binds at promoter luxCDABE  activates Tn  LIGHT PRODUCED. Lecture 12 3 ^Phosphorylation flow. Top is low, phosphate flow to right. Bottom is high, phosphate flow to the left. In this case, it appears that AI-1 and AI-2 act synergistically. Because there are 2 QS systems, the bacteria could potentially distinguish between 4 states. No AIs – the cell must be planktonic; no light Hi AI-1; Lo AI-2 – cell is part of mono-culture of Vh 1% Lo AI-1; Hi AI-2 – cell is invading another population 0.01% Both AIs – big Vh subcolony of consortium Lecture 12 4 OR old monoculture of Vh 100% light output This produces a graded response: coincident detector. Other bacteria have two systems in parallel or series. Two systems in parallel: B. subtillis. Unlike V. haveyi, system has antagonistic effect. ComX AIP activates receptor ComP -Phosphorelay activates ComA. -Phospjho-ComA activates transcription of competence genes. -CSF AIP is internalized and inhibits ComA activation. Repressing sporu;ation. Two systems control competance and sporulation. Signaling in Pathogenic Bacteria P. aeruginosa LasIR & RhlIR work in tandem to control genes involved in timing of gene expression for biofilms & virulence factors. LasI (synthase that makes inducer)  3-oxododeconoyl-L- HSL (3oxoC12 HSL)  Lecture 12 5 LasR (receptor) + 3oxoC12 (inducer) regulates lasA, lasB, aprA, toxA, lasI and rhII The las system also produces a negative regulator RslA that can down-regulate lasI although its upreg’d by LasR+3oxo ?? Feedback control The second QS system suggests that Pa. regulation even more complex than previously thought. C12-AHL inhibits RhIR (timing control) RhlI  N-butyryl-L-HSL (C4 HSL) RhlR + C4 HSL regulates rhlAB rhamnolipid, a surfactant, as well as rpoS (σS), and pyocyanin & cyanide production. Do the 2 systems interact? There is little interchangeability in spite of high AA homology between LasR & RhlR, therefore R proteins have high specificity Genes activated by one system, minimally activated by other. EXCEPT, LasR activates not only lasIR, but also rhlIR. And 3oxoC12 HSL competes with C4 HSL for RhIR binding, therefor, 3oxoC12 HSL acts as an antagonist for the the rhI system. Lecture 12 6 Signal for first system inhibits the second system. Second system only activated when system one makes its activator (therefor, systems are in series). AND 3oxoC12 HSL competes with C4 HSL for RhlR binding, therefore, 3oxoC12 HSL acts as an antagonist for the rhl system. Therefore, it appears that the 2 systems are arranged hierarchically; las is dominant over the rhl system. Minor HSLs are also produced when fatty acid availability is low  C6, C8, C10 HSLs. B. cepacia Also has at least 2 QS systems: cepIR / cciIR / bviIR In CF infections, Bc. can take up AIs from Pa. Pa. spent cultures fed to Bc. Lecture 12 7 • Increases protease prod. 2-fold • Increases siderophore prod. 7-fold Why does Bc. let Pa. control its gene expression? Pa produces 1000 times the amount of AIs that Bc does. Maybe Bc uses other bacteria’s HSLs to initiate infection; it only infects where a good growth environment is proven to exist Agrobacterium tumefaciens – crown gall tumors in plants QS controls the conjugation of a plasmid called Ti
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