Use and Evaluation of a pES213-Derived Plasmid for the Constitutive Expression of
            <i>gfp</i>
            Protein in Pathogenic Vibrios: a Tagging Tool for
            <i>In Vitro</i>
            Studies

Norfolk, William A.; Lipp, Erin K.

doi:10.1128/spectrum.02490-22

Cited by 6 publications

(5 citation statements)

References 58 publications

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“…We created 7 plasmids (Fig. 1), all of them being able to replicate in E. coli , among which four can replicate in Vibrionaceae and Pseudoalteromonaceae families ( 18, 19 ) (pFD115, pFD116, pFD145 and pFD150, the latter being promoterless and served as a negative control), two being able to replicate in Rhizobiaceae (pFD141 and pFD148), and one containing the broad host-range origin of replication pBBR1 ( 20 ) (pFD149).…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, these circuits were implemented into various plasmidic backbones, varying in their origin of replications for E. coli (colE1 for pFD141 and 148, R6K for pFD115, pFD116 and pFD145 and pBBR1 for pFD149). These plasmids were also chosen, because they are shuttle vectors and can replicate in bacteria outside E. coli : Vibrionaceae ( 18, 19 ) and Pseudoalteromonaceae ( 18 ) for pFD115, pFD116 and pFD145, Rhizobiaceae for pFD141 and pFD148, while pFD149 can maintain in various Gram negative bacteria.…”

Section: Discussionmentioning

confidence: 99%

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Development and utilization of new O₂-independent bioreporters

Agranier,

Crétin,

Joublin-Delavat

et al. 2023

Preprint

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Fluorescent proteins have revolutionized science since their discovery in 1962. They have enabled imaging experiments to decipher the function of proteins, cells and organisms, as well as gene regulation. GFP and all its derivatives are now standard tools in cell biology, immunology, molecular biology and microbiology laboratories around the world. A common feature of these proteins is their O2-dependent maturation allowing fluorescence, which precludes their use in anoxic contexts. In this work, we report the development andin cellulocharacterization of genetic circuits encoding the O2-independent KOFP-7 protein, a flavin-binding fluorescent protein. We have optimized the genetic circuit for high bacterial fluorescence at population and single-cell level, implemented this circuit in various plasmids differing in host range, and quantified their fluorescence under both aerobic and anaerobic conditions. Finally, we showed that KOFP-7 based constructions can be used to produce fluorescing cells ofV. diazotrophicus, a facultative anaerobe, demonstrating the usefulness of the genetic circuits for various anaerobic bacteria. These genetic circuits can thus be modified at will, both to solve basic and applied research questions, opening a highway to shed light on the obscure anaerobic world.ImportanceFluorescent proteins are used since decades, and have allowed major discoveries in biology in a wide variety of fields, and are used in environmental as well as clinical contexts. GFP and all its derivatives share a common feature: they rely on the presence of O2for protein maturation and fluorescence. This dependency precludes their use in anoxic environments. Here, we constructed a series of genetic circuits allowing production of KOFP-7, an O2-independant Flavin-Binding Fluorescent Protein. We demonstrated thatEscherichia colicells producing KOFP-7 are fluorescent, both at the population and single-cell levels. Importantly, we showed that, unlike cells producing GFP, cells producing KOFP-7 are fluorescent in anoxia. Finally, we demonstrated thatVibrio diazotrophicusNS1, a facultative anaerobe, is fluorescent in the absence of O2when KOFP-7 is produced.Altogether, the development of new genetic circuits allowing O2-independent fluorescence will open new perspective to study anaerobic processes.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Development and utilization of new O₂-independent bioreporters

Agranier,

Crétin,

Joublin-Delavat

et al. 2023

Preprint

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“…used in this experiment were tagged with GFP to enable localization and quantification of the bacterium. Tagging was accomplished using the methods outlined in Norfolk & Lipp, (2022). In short, a tri-parental mating assay was used to transfer a gfp -containing plasmid to the target Vibrio sp.…”

Section: Methodsmentioning

confidence: 99%

Coral disease and ingestion: investigating the role of heterotrophy in the transmission of pathogenicVibriospp. using a sea anemone (Exaiptasia pallida) model system

Norfolk

Melendez-Declet

Lipp

2023

Preprint

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Understanding disease transmission in corals can be complicated given the intracity of the holobiont and difficulties associated withex situcoral cultivation. As a result, most of the established transmission pathways for coral disease are associated with perturbance (i.e., damage) rather than evasion of immune defenses. Here we investigate ingestion as a potential pathway for the transmission of coral pathogens that evades the mucus membrane. Using sea anemones (Exaiptasia pallida) and brine shrimp (Artemiasp.) to model coral feeding, we tracked the acquisition of the putative pathogens,Vibrio alginolyticus,V. harveyi, andV. mediterraneiusing GFP-tagged strains.Vibriosp. were provided to anemones using three experimental exposures 1) direct water exposure alone, 2) water exposure in the presence of a food source (cleanArtemia), and 3) through a "spiked" food source (Vibrio-colonizedArtemia) created by exposingArtemiacultures to GFP-Vibriovia the ambient water overnight. Following a 3 h feeding/exposure duration, the level of acquired GFP-Vibriowas quantified from anemone tissue homogenate. Ingestion of spikedArtemiaresulted in a significantly greater burden of GFP-Vibrioequating to an 829.7-fold, 3,108.2-fold, and 435.0-fold increase in CFU mL-1when compared to water exposed trials and a 206.8-fold, 62.2-fold, and 27.3-fold increase in CFU mL-1compared to water exposed with food trials forV. alginolyticus,V. harveyi, andV. mediterranei, respectively. These data suggest that ingestion can facilitate delivery of an elevated dose of pathogenic bacteria in cnidarians and may describe an important portal of entry for pathogens in the absence of perturbing conditions.

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“…used in this experiment were tagged with GFP to enable localization and quantification of the bacterium. Tagging was accomplished using the methods outlined in Norfolk & Lipp, (2022) ( 70 ). In short, a tri-parental mating assay was used to transfer a gfp -containing plasmid to the target Vibrio sp.…”

Section: Methodsmentioning

confidence: 99%

Coral Disease and Ingestion: Investigating the Role of Heterotrophy in the Transmission of Pathogenic Vibrio spp. using a Sea Anemone ( Exaiptasia pallida ) Model System

Norfolk

Melendez-Declet

Lipp

2023

Appl Environ Microbiol

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Use and Evaluation of a pES213-Derived Plasmid for the Constitutive Expression of gfp Protein in Pathogenic Vibrios: a Tagging Tool for In Vitro Studies

Cited by 6 publications

References 58 publications

Development and utilization of new O₂-independent bioreporters

Development and utilization of new O₂-independent bioreporters

Coral disease and ingestion: investigating the role of heterotrophy in the transmission of pathogenicVibriospp. using a sea anemone (Exaiptasia pallida) model system

Coral Disease and Ingestion: Investigating the Role of Heterotrophy in the Transmission of Pathogenic Vibrio spp. using a Sea Anemone ( Exaiptasia pallida ) Model System

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Use and Evaluation of a pES213-Derived Plasmid for the Constitutive Expression of gfp Protein in Pathogenic Vibrios: a Tagging Tool for In Vitro Studies

Cited by 6 publications

References 58 publications

Development and utilization of new O2-independent bioreporters

Development and utilization of new O2-independent bioreporters

Coral disease and ingestion: investigating the role of heterotrophy in the transmission of pathogenicVibriospp. using a sea anemone (Exaiptasia pallida) model system

Coral Disease and Ingestion: Investigating the Role of Heterotrophy in the Transmission of Pathogenic Vibrio spp. using a Sea Anemone ( Exaiptasia pallida ) Model System

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Development and utilization of new O₂-independent bioreporters

Development and utilization of new O₂-independent bioreporters