Pyocyanin Extraction and Quantitative Analysis in Swarming Pseudomonas aeruginosa

King, Michelle M.; Guragain, Manita; Sarkisova, S. A.; Patrauchan, Marianna A.

doi:10.21769/bioprotoc.2042

Cited by 11 publications

(8 citation statements)

References 7 publications

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“…For this assay 2 ml of fresh overnight culture of the plasmid-expressed mutants or PA14 DlasR and PA14 DpqsA strains, were used to inoculate wells in a 96-well plate filled with 200 ml of Pseudomonas Isolation Agar (PIA) containing gentamicin and 0.1% L-arabinose. The production of pyocyanin was observed after 24 hours incubation at 37 C. Pyocyanin quantification from plates was performed as previously described (King et al, 2016); 2ml of overnight cultures were spotted onto PIA plates and the plates were incubated at 37 C for 24 hours. Samples of agar of 2 cm 2 were excised from the plate.…”

Section: Pyocyanin Production Assaymentioning

confidence: 99%

A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa

et al. 2021

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Section: Pyocyanin Production Assaymentioning

confidence: 99%

A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa

et al. 2021

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“…The clear color of this agar allowed pigmentation to be easily seen. Pyocyanin production of mutants that appeared bluer or less blue than the wild-type were then quantified by first scraping cells off of the agar surface, and then extracting pyocyanin from the agar in the plates, as described previously (82). Preliminary assays were done in ABTGC agar solidified in 24 well plates(1 mL agar per well) for rapid visualization.…”

Section: Measurement Of Secreted Virulence Factorsmentioning

confidence: 99%

Two-Component Signaling Systems Regulate Diverse Virulence-Associated Traits in Pseudomonas aeruginosa

Wang

Cady

Oyarce

et al. 2021

Appl Environ Microbiol

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Pseudomonas aeruginosa is an opportunistic pathogen that can cause problematic infections at different sites throughout the human body. P. aeruginosa encodes a large suite of over 60 two-component signaling systems that enable cells to rapidly sense and respond to external signals. Previous work has shown that some of these sensory systems contribute to P. aeruginosa pathogenesis, but the virulence-associated processes and phenotypic traits that each of these systems controls is still largely unclear. To aid investigations of these sensory systems, we have generated deletion strains for each of 64 genes encoding histidine kinases and one histidine phosphotransferase in P. aeruginosa PA14. We provide initial phenotypic characterizations of this collection by assaying these mutants for over a dozen virulence-associated traits, and find that each of these phenotypes is regulated by multiple sensory systems. Our work highlights the usefulness of this collection for further studies of P. aeruginosa two-component signaling systems and provides insight into how these systems may contribute to P. aeruginosa infection. Importance Pseudomonas aeruginosa can grow and survive in a wide range of conditions, including as a human pathogen. As such, P. aeruginosa must be able to sense and respond to diverse signals and cues in its environment. This sensory capability is endowed in part by the hundreds of two-component signaling proteins encoded in the P. aeruginosa genome, but the precise roles of each remain poorly defined. To facilitate systematic study of the signaling repertoire of P. aeruginosa PA14, we generated a library of deletion strains, each lacking one of the 65 histidine kinases. By subjecting these strains to a battery of phenotypic assays, we confirm the functions of many and unveil roles for dozens of previously uncharacterized histidine kinases in controlling various traits, many of which are associated with P. aeruginosa virulence. Thus, this work provides new insight into the functions of two-component signaling proteins and provides a resource for future investigations.

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“…To assess the role of EfhP and its ability to bind Ca 2+ in the production of pyocyanin, we quantified the production of the pigment in P. aeruginosa, PAO1, Δ efhP, Δ efhP :: efhP and Δ efhP::efhP_q strains by using chloroform-HCl method as described in 92 with modifications. Cultures were grown in BMM for 18 h and normalized to an OD 600 of 0.3, following which 100 µl of each culture was plated on BMM plates with or without 5 mM Ca 2+ and incubated at 37 °C for 24 h. Following incubation, the cells were removed from the agar surface by using a glass spreader and 4 ml of saline and collected in 15 ml falcon tubes.…”

Section: Methodsmentioning

confidence: 99%

EF-hand protein, EfhP, specifically binds Ca2+ and mediates Ca2+ regulation of virulence in a human pathogen Pseudomonas aeruginosa

Kayastha

Kubo

Burch-Konda

et al. 2022

Sci Rep

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Calcium (Ca2+) is well known as a second messenger in eukaryotes, where Ca2+ signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca2+ signaling, little is known about the mechanisms of bacterial Ca2+ signaling. Previously, we have identified a putative Ca2+-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca2+ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca2+ with 13.7 µM affinity, and that mutations at the +X and −Z positions within each or both EF-hand motifs abolished Ca2+ binding. We also show that the hydrophobicity of EfhP increased in a Ca2+-dependent manner, however no such response was detected in the mutated proteins. 15 N-NMR showed Ca2+-dependent chemical shifts in EfhP confirming Ca2+-binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca2+ binding abolished Ca2+ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca2+, which triggers its structural changes required for the Ca2+ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca2+ sensor.

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Pyocyanin Extraction and Quantitative Analysis in Swarming Pseudomonas aeruginosa

Cited by 11 publications

References 7 publications

A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa

A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa

Two-Component Signaling Systems Regulate Diverse Virulence-Associated Traits in Pseudomonas aeruginosa

EF-hand protein, EfhP, specifically binds Ca2+ and mediates Ca2+ regulation of virulence in a human pathogen Pseudomonas aeruginosa

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