Cyclic di-GMP-Responsive Transcriptional Reporter Bioassays in Pseudomonas aeruginosa

Borlee, Bradley R.; Borlee, Grace I.; Martin, Kevin H.; Irie, Yasuhiko

doi:10.1007/978-1-4939-7240-1_9

Cited by 5 publications

(7 citation statements)

References 15 publications

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“…This c-di-GMPresponsive reporter is a transcriptional fusion of the cdrA promoter with the gene encoding luciferase and enables real-time evaluation of c-di-GMP-regulated activities through the quantification of luminescence. c-di-GMP reporters utilizing this transcriptional fusion-based approach were previously published and validated (27)(28)(29)(30). In a swarming assay, the wt harboring P cdrA ::lux showed minimal luminescence 3 h after inoculation on surfaces (i.e., after sufficient time for the transition to a swarming mode) for all media tested, which is expected for motile bacteria (Fig.…”

Section: Figmentioning

confidence: 81%

“…After detailing the divergence in ΔdipA mutant swarm phenotypes on several medium conditions, we were curious whether the mutant was simply producing less c-di-GMP on rich media and more c-di-GMP on minimal media. We utilized a P cdrA ::lux reporter (27) to assess c-di-GMP production levels on LB Lennox broth, lab-mixed LB (tryptone, yeast extract, and NaCl), and M8-glucose-Casamino Acids medium and FAB-glutamate. This c-di-GMPresponsive reporter is a transcriptional fusion of the cdrA promoter with the gene encoding luciferase and enables real-time evaluation of c-di-GMP-regulated activities through the quantification of luminescence.…”

Section: Figmentioning

confidence: 99%

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Multiple Environmental Factors Influence the Importance of the Phosphodiesterase DipA upon Pseudomonas aeruginosa Swarming

Mattingly

Kamatkar

Morales-Soto

et al. 2018

Appl Environ Microbiol

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exhibits flagellum-mediated swimming in liquid and swarming on hydrated surfaces under diverse nutrient conditions. Prior studies have implicated a phosphodiesterase, DipA, in regulating these flagellum-mediated motilities, but collectively, the necessity for DipA was unclear. In this study, we find that the medium composition conditionally constrains the influence of DipA on flagellar motility. We show that DipA exhibits more influence on minimal medium supplemented with glutamate or glucose, where flagellar motility was negated for the mutant. Conversely, a-deficient mutant exhibits flagellar motility when growing with LB Lennox broth and minimal medium supplemented with Casamino Acids. Swarming under these rich medium conditions occurs under elevated levels of c-di-GMP. We also demonstrate that the influence of DipA upon swimming often differs from that upon swarming, and we conclude that a direct comparison of the motility phenotypes is generally valid only when characterizing motility assay results from the same medium composition. Our results are consistent with the explanation that DipA is one of several phosphodiesterases responding to the nutrient environment sensed by On minimal medium with glutamate or glucose, DipA is dominant; however, on rich medium, the necessity of DipA is fully negated. Motile and ubiquitous bacteria such as can quickly colonize surfaces and form biofilms in numerous environments such as water distribution systems, soil, and the human lung. To effectively disrupt bacterial colonization, it is imperative to understand how bacteria regulate motility in these different growth environments. Here, we show that the phosphodiesterase DipA is not required for flagellar motility under all nutrient conditions. Thus, the maintenance of intracellular c-di-GMP levels to promote flagellar motility or biofilm development must be conditionally regulated by differing phosphodiesterases in variation with select nutrient cues.

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Section: Figmentioning

confidence: 81%

Section: Figmentioning

confidence: 99%

Multiple Environmental Factors Influence the Importance of the Phosphodiesterase DipA upon Pseudomonas aeruginosa Swarming

Mattingly

Kamatkar

Morales-Soto

et al. 2018

Appl Environ Microbiol

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“…2b). Additionally, a bioreporter system, which is comprised of a plasmid-borne fusion between the cdrA promoter (P cdrA ) and the gene for green fluorescent protein (gfp) 49 , indicated that cdrA transcription in a PAO1 strain bearing tdcA increased linearly 5.2-fold from 31 to 41°C (Fig. 2c).…”

Section: Resultsmentioning

confidence: 99%

“…This organism was engineered to express tdcA (or the inactive tdcA 162ΔG allele) via site-directed transposon mutagenesis 56 . Subsequently, these strains were transformed with a luminescent c-di-GMP bioreporter, comprised of a fusion between P cdrA and the luxCDABE operon 49 . These transformants were then grown side-by-side in Petri dishes at 25°C, then shifted to 37°C and photographed in the dark via time-lapse imaging.…”

Section: Resultsmentioning

confidence: 99%

Bacterial cyclic diguanylate signaling networks sense temperature

et al. 2021

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Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.

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“…We also evaluated the activity of these genes using a heterologous approach to evaluate the effect on c-di-GMP signaling mediated phenotypes in Pseudomonas aeruginosa PAO1 (20)(21)(22). Inducible expression of cdpA or cdpA-I2285 in either P. Mapping residues important for activity in CdpA, I2285, and II2523.…”

Section: E)mentioning

confidence: 99%

Disruption of c-di-GMP signaling networks unlocks cryptic expression of secondary metabolites during biofilm growth in Burkholderia pseudomallei

Borlee

Mangalea

Martin

et al. 2021

Preprint

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The regulation and production of secondary metabolites during biofilm growth of Burkholderia spp. is not well understood. To learn more about the crucial role and regulatory control of cryptic molecules produced during biofilm growth, we disrupted c-di-GMP signaling in Burkholderia pseudomallei, a soil-borne bacterial saprophyte and the etiologic agent of melioidosis. Our approach to these studies combined transcriptional profiling with genetic deletions that targeted key c-di-GMP regulatory components to characterize responses to changes in temperature. Mutational analyses and conditional expression studies of c-di-GMP genes demonstrates their contribution to phenotypes such as biofilm formation, colony morphology, motility, and expression of secondary metabolite biosynthesis when grown as a biofilm at different temperatures. RNA-seq analysis was performed at varying temperatures in a ΔII2523 mutant background that is responsive to temperature alterations resulting in hypo- and hyper- biofilm forming phenotypes. Differential regulation of genes was observed for polysaccharide biosynthesis, secretion systems, and nonribosomal peptide and polyketide synthase (NRPS/PKS) clusters in response to temperature changes. Deletion mutations of biosynthetic gene clusters (BGCs) clusters 2, 11, 14 (syrbactin), and 15 (malleipeptin) in wild-type and ΔII2523 backgrounds also reveals the contribution of these BGCs to biofilm formation and colony morphology in addition to inhibition of Bacillus subtilis and Rhizoctonia solani. Our findings suggest that II2523 impacts the regulation of genes that contribute to biofilm formation and competition. Characterization of cryptic BGCs under differing environmental conditions will allow for a better understanding of the role of secondary metabolites in the context of biofilm formation and microbe-microbe interactions.

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Cyclic di-GMP-Responsive Transcriptional Reporter Bioassays in Pseudomonas aeruginosa

Cited by 5 publications

References 15 publications

Multiple Environmental Factors Influence the Importance of the Phosphodiesterase DipA upon Pseudomonas aeruginosa Swarming

Multiple Environmental Factors Influence the Importance of the Phosphodiesterase DipA upon Pseudomonas aeruginosa Swarming

Bacterial cyclic diguanylate signaling networks sense temperature

Disruption of c-di-GMP signaling networks unlocks cryptic expression of secondary metabolites during biofilm growth in Burkholderia pseudomallei

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