Marcas O Muineachan

 Quorum sensing (QS): Cell – density gene expression.

 Release, detect and respond to a signalling molecule.
 Signalling molecule in G- bacteria is an N-acylated
homoserine lactone (AHL).
 QS systems are key regulators for the expression of virulence
factors in certain pathogens – Pseudomonas aeruginosa.
 Marine sponges: source of bioactive compounds
 Haliclona simulans
 QS inhibitors to be found in marine sponges – Lactonases
(aiiA gene)

 We have a metagenomic library from Haliclona simulans–
E.coli/pCC1phos.

 We have 30 sporeformers which have been characterised in detail.

Of these 30, PCR screening has shown that two have genes (aiiA)
for lactonases: CH8A and #11.

1. Devolop an assay to screen for QSIs using Chromobacterium

violaceum DSM30191 as an indicator.

2. Apply the assay to the sporeformers and the metagenomic

library.

3. Evaluate the effect of lactonases from sponge associated

sporeformers CH8a and #11 on QS regulated virulence
determinants of P. aeruginosa.
 Chromobacterium violaceum (DSM30191)

 Regulates pigment production (purple) by different AHLs (3-

hydroxy-C10-HSL is dominant) which is inhibited by AHL
analogues and other antagonists.

 Positive result indicated by a lack of pigmentation surrounding

the QSI.

 PAO1 is a “positive control”: its long chain AHL competitively

binds and inhibits the receptor for 3-hydroxy-C10-HSL.
 Carried out 3 more assays
 Test microorganisms: CH8a, #11, #12, #1 grown in nutrient broth
(NB) and marine broth (MB). PAO1 in NB.

 For assays 1 and 2: Spot the test m/os on MA and NA (assay 1)

and streak the test m/os on MA and NA (assay 2). Have control
NA and MA plates also. Let dry and incubate overnight (o/n) at
30◦C.

 o/n of C. violaceum DSM30191 (CVDSM) prepared (NB).

 Next day, inoculated nutrient soft agar with Chromobacterium

violaceum and overlayed. (Done individually to all MA and NA
plates). Control plates were also overlayed.

 The plates were then incubated for 96 hours at 30◦C and at room
temperature for 48 hours. Photographs below were taken then.
 MA
PAO1 NA CH8a NA

NB spot MB
streak

NB
streak
NB
streak

MA #11 NA
#11, NA, NB spot
MB
spot

NB
spot
 MA #1 NA
MA #12 NA

MB
streak

NB
streak
PAO1 in NB #11, MB

MB #12 NB
MB CH8a NB
 Q plates, 384 well microtitre plates, 6 per Q plate, screened
13,824 clones.
 260ml LB / chloramphenicol / arabinose, machine spots the
clones, next day o/l with C. violaceum (o/n culture) in NSA.
Results monitored over a number of days.
 Also PA01 (on base and o/l), #11, #12, CH8a (o/l only)
spotted on top part of plate. 72h
24h
7
7
24h
72h
 PBBR1MCS5 as cloning vector. Purified, restriction digest
using Hind3 and BamHI at MCS of the plasmid. After excision
from the band (post- restriction digest) and further
purification plasmid “disappeared” somewhere in the process
– Repeat once more, if no band again move ahead without
further purtification.
 Also, tested conditions for

PCR amplification of aiiA gene