Development of a Pseudomonas aeruginosa Agmatine Biosensor
Abstract
:1. Introduction
2. Experimental Section
2.1. Bacterial Strains and Plasmids
Parent strain | Phenotype | Genotype | Source or reference |
---|---|---|---|
PA14 | Wild-type | Wild-type | [18] |
WT Agmatine Reporter | aguRB-CTX-Lux | This work | |
WT Reporter Vector Control | CTX-Lux | This work | |
AgDi Knockout | aguA:gm, ∆agu2ABCA’ | [12] | |
AgDi Knockout, Agmatine Reporter | aguA:gm, ∆agu2ABCA’, AguRB-CTX-Lux | This work | |
AgDi Knockout, Reporter Vector Control | aguA:gm, ∆agu2ABCA’, CTX-Lux | This work | |
Arginine Decarboxylase Knockout | ∆speA | This work | |
Arginine Decarboxylase Knockout, Agmatine Reporter | ∆speA, AguRB-CTX-Lux | This work | |
Arginine Decarboxylase Knockout, Reporter Vector Control | ∆speA, CTX-Lux | This work | |
AgDi Knockout, Arginine Decarboxylase Knockout | aguA:gm, ∆agu2ABCA’, ∆speA | This work | |
Agmatine Biosensor-AgDi Knockout, Arginine Decarboxylase Knockout, Agmatine Reporter | aguA:gm, ∆agu2ABCA’, ∆speA, AguR-CTX-Lux | This work | |
AgDi Knockout, Arginine Decarboxylase Knockout, Reporter Vector Control | aguA:gm, ∆agu2ABCA’, ∆speA, CTX-Lux | This work | |
E. coli Top10 | Competent cells for cloning purposes | F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ- | Invitrogen |
E. coli SM10 | Vehicle for conjugative mating to P. aeruginosa | KmR, thi-1, thr, leu, tonA, lacY, supE, recA::RP4-2-Tc::Mu, pir. | [19] |
Plasmid | Description | Features | Source or reference |
---|---|---|---|
Mini-ctx-lux | luxCDABE based reporter vector with site specific integration at attB site in P. aeruginosa chromosome | See Reference | [20] |
pEX18-Ap | Gene replacement vector | See Reference | [17] |
CTXnoT7 | Contains Lux operon, T7 promoter removed to reduce background expression | No T7 | This work |
AguRB-lux | Contains Lux operon induced by agmatine via the inserted promotion system of the primary agmatine deiminase, T7 promoter removed to reduce background expression | No T7, AguRB promoter fusion to luxCDABE | This work |
pBAD202 | Arabinose induced expression vector | See manufacturer | Invitrogen |
SpeBAD | speA from PA14 cloned into MCS of pBAD202 | Arabinose induced expression vector of SpeA | This work |
speA KO | pEX18-Ap based cloning vector with speA knockout construct | Designed for disruption of chromosomal speA in P. aeruginosa | This work |
2.2. Development of the Agmatine Biosensor
2.3. Mass Spectrometry Measurement of Agmatine
2.4. Bioassay Technique
2.5. Spinal Cord Infection
3. Results and Discussion
3.1. Construction of a Bioluminescent Agmatine Reporter Construct for Use in P. aeruginosa
3.2. Behavior of Biosensor Construct in Mutants of the Arginine Decarboxylase and Agmatine Deiminase Pathways
3.3. Validation of the ΔspeA, aguA:gm, Δagu2ABCA’, aguRB:lux Mutant as an Agmatine Biosensor
3.4. Use of the Agmatine Biosensor in Biologic Assays
3.4.1. Rapid Detection of Agmatine Secretion by Clinical P. aeruginosa Isolates
3.4.2. Detection of Arginine Decarboxylase Enzyme Activity
3.4.3. In Vivo Detection of Agmatine during Infection in Mouse Spinal Cords
4. Conclusions
Acknowledgments
Ethics Statement
Author Contributions
Conflicts of Interest
References
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Gilbertsen, A.; Williams, B. Development of a Pseudomonas aeruginosa Agmatine Biosensor. Biosensors 2014, 4, 387-402. https://doi.org/10.3390/bios4040387
Gilbertsen A, Williams B. Development of a Pseudomonas aeruginosa Agmatine Biosensor. Biosensors. 2014; 4(4):387-402. https://doi.org/10.3390/bios4040387
Chicago/Turabian StyleGilbertsen, Adam, and Bryan Williams. 2014. "Development of a Pseudomonas aeruginosa Agmatine Biosensor" Biosensors 4, no. 4: 387-402. https://doi.org/10.3390/bios4040387