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)