The development of a panel of bacterial reporter strains for the detection of antimicrobial compounds at sub-inhibitory levels
T.K.S Janssens1, M. Naderman1, B. van der Burg1 and B. Brouwer1,2
1BioDetection Systems BV, Science Park 406, 1098XH Amsterdam, the Netherlands 2VU University Amsterdam, Department of Animal Ecology, de Boelelaan 1085, 1081HV Amsterdam, the Netherlands
Introduction Mining (meta)genomic libraries or isolates for useful small antimicrobial molecules requires screening of biological activities Problem: false negatives in inhibition assays due to sub-inhibitory concentrations in inhibitory assays Detection of antimicrobial compounds based on structural and mechanistic properties by transcriptional regulatory mechanisms of • antibiotic resistance systems • intra- and inter-specific signalling • general stress pathways
Aim Development of a panel of microbial reporters for biomining purposes based on biological activities based on mechanisms reported in the literature. To be applied in: •De-replication of downstream screens •Promising clone selection •Mode of action assessment of new compounds Proof of principle in host E. coli DH5α Low copy number luxCDABE plasmid (pCS26-Pac) constructs Future panel
Class specific
Receptor mediated -macrolides
-tetracyclines -phloroglucinoles -streptogramins
-β-lactams
Sensor kinase signaling -cationic polypeptides
-glycopeptides
Mode of action
Inhibition of Quorum Sensing
Replication Transcription Translation
Cell wall synthesis Fatty acid synthesis
Stress responses
Cell cycle arrest Cell envelope Genotoxicity
Oxidative
Whole cell biosensor
Results pPHZlux-1: reporter for redox-active compounds (P. aeruginosa SoxR and mono-oxygenase promoter) BPlux: whole cell bioreporter for cytotoxicity (strong synthetic basal promoter) pPHZlux-1 responds to redox-cycling small molecules (e.g. the phenazine pyocyanine) directly but not to ROS pPHZlux-1 is induced by pure redox-active model compounds, but not by the negative control PQS pPHZlux-1 detects redox activity in microbial cultures at sub-inhibitory concentrations
0 0,5 5 500.0
0.2
0.4
0.6
0.8
0
50
100
150
pPHZlux-1pCS26-PacBPlux
mg/l Na-arsenite
RLU
BP
lux
0 20 200 20000.0
0.2
0.4
0.6
0.8
0
50
100
150
nM pyocyanine
RLU
Activities in pellets of biocontrol strainsPseudomonas chlororaphis Phz24 and
P. fluorescens Pf-5
Blank
1.8
ng P
hz24
18 n
g Phz
24
180
ng P
hz24
g Ph
z24
µ
1.8 1.
8 ng
Pf-5
18 n
g Pf-5
180
ng P
f-5
g Pf
-5
µ
1.8
0
1000
2000
3000
4000
0
200000
400000
600000
800000
pPHZlux-1BPlux
WW pellet in filter sterile extract
RLU
BP
lux R
LU
10 - 1 5 10- 1 0 10- 50
2000
4000
6000
8000
10000
1.05 E-7 Mpyocyanine eq./µgPhz24 pellet
M pyocyanine
RLU
Conclusion/Outlook The combined application of a specific and a whole cell bioreporter on redox active producing biocontrol strains, confirms the feasibility of the approach. The application of a panel of reporters for antimicrobial activities can assist in the directed mining of unknown microbial metabolic diversity.
P. fluorescens Pf-5: control P. chlororaphis Phz24:
phenazine-1-carboxylate producer
10- 9 10- 8 10- 7 10- 6 10- 50
5000
10000
15000
20000 Pyocyanine
4-nitroquinoline-N-oxide
ParaquatdiCl
2-heptyl-3-hydroxy-4(1H)-quinolone
M
RLU
(PQS)
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