scholarly journals A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae

2020 ◽  
Author(s):  
Triana N. Dalia ◽  
Jennifer L. Chlebek ◽  
Ankur B. Dalia
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Industrial Applications ◽  
Model System ◽  
Promoter Element ◽  
Reporter System ◽  
Dynamic Activity ◽  
Type Iv ◽  
Ectopic Gene Expression ◽  
Integrated Platform

AbstractThe ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements Ptac and PBAD to versions that have an additional theophylline-responsive riboswitch (Ptac-riboswitch and PBAD-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with Ptac > PBAD > PBAD-riboswitch > Ptac-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for Ptac > Ptac-riboswitch > PBAD; while the newly developed PBAD-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible PBAD-riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of Vibrio cholerae and could be adapted for use in other species.

scholarly journals A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Triana N. Dalia ◽  
Jennifer L. Chlebek ◽  
Ankur B. Dalia
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Industrial Applications ◽  
Model System ◽  
Promoter Element ◽  
Reporter System ◽  
Dynamic Activity ◽  
Type Iv ◽  
Ectopic Gene Expression ◽  
Integrated Platform

Abstract The ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements Ptac and PBAD to versions that have an additional theophylline-responsive riboswitch (Ptac-riboswitch and PBAD-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with Ptac > PBAD > PBAD-riboswitch > Ptac-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for Ptac > Ptac-riboswitch > PBAD; while the newly developed PBAD-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible PBAD-riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of V. cholerae and could be adapted for use in other species.

scholarly journals Fresh extension of Vibrio cholerae competence type IV pili predisposes them for motor-independent retraction

2021 ◽  
Author(s):  
Jennifer L. Chlebek ◽  
Triana N. Dalia ◽  
Nicolas Biais ◽  
Ankur B. Dalia
Keyword(s):  
Cell Surface ◽  
Vibrio Cholerae ◽  
Conformational Changes ◽  
Pathogenic Bacteria ◽  
Ectopic Expression ◽  
Type Iv Pili ◽  
Therapeutic Interventions ◽  
Dynamic Activity ◽  
Type Iv ◽  
Additional Support

ABSTRACTBacteria utilize dynamic appendages called type IV pili (T4P) to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can either occur with the aid of an ATPase motor, or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remains poorly characterized. We have previously demonstrated that Vibrio cholerae competence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken, but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization.SIGNIFICANCEExtracellular pilus fibers are critical to the virulence and persistence of many pathogenic bacteria. A crucial function for most pili is the dynamic ability to extend and retract from the cell surface. Inhibiting this dynamic pilus activity represents an attractive approach for therapeutic interventions, however, a detailed mechanistic understanding of this process is currently lacking. Here, we use the competence pilus of Vibrio cholerae to study how pili retract in the absence of dedicated retraction motors. Our results reveal a novel regulatory mechanism of pilus retraction that is an inherent property of the external pilus filament. Thus, understanding the conformational changes that pili adopt under different conditions may be critical for the development of novel therapeutics that aim to target the dynamic activity of these structures.

scholarly journals Fresh extension of Vibrio cholerae competence type IV pili predisposes them for motor-independent retraction

2021 ◽  
Author(s):  
Jennifer L. Chlebek ◽  
Triana N. Dalia ◽  
Nicolas Biais ◽  
Ankur B. Dalia
Keyword(s):  
Cell Surface ◽  
Vibrio Cholerae ◽  
Conformational Changes ◽  
Pathogenic Bacteria ◽  
Ectopic Expression ◽  
Type Iv Pili ◽  
Therapeutic Interventions ◽  
Dynamic Activity ◽  
Type Iv ◽  
Additional Support

Bacteria utilize dynamic appendages called type IV pili (T4P) to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can either occur with the aid of an ATPase motor, or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remains poorly characterized. We have previously demonstrated that Vibrio cholerae competence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken, but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization. IMPORTANCE Extracellular pilus fibers are critical to the virulence and persistence of many pathogenic bacteria. A crucial function for most pili is the dynamic ability to extend and retract from the cell surface. Inhibiting this dynamic pilus activity represents an attractive approach for therapeutic interventions, however, a detailed mechanistic understanding of this process is currently lacking. Here, we use the competence pilus of Vibrio cholerae to study how pili retract in the absence of dedicated retraction motors. Our results reveal a novel regulatory mechanism of pilus retraction that is an inherent property of the pilus filament. Thus, understanding the conformational changes that pili adopt under different conditions may be critical for the development of novel therapeutics that aim to target the dynamic activity of these structures.

scholarly journals Oxygen depletion and nitric oxide stimulate type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA

2021 ◽  
Author(s):  
Hannah Q Hughes ◽  
Kyle A Floyd ◽  
Sajjad Hossain ◽  
Sweta Anantharaman ◽  
David T Kysela ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Bacterial Pathogen ◽  
Dna Uptake ◽  
Dynamic Activity ◽  
Regulatory Pathways ◽  
Type Iv ◽  
Microaerobic Conditions

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. Dynamic extension and retraction of pili is often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To study this question, we use the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we find that V. cholerae cells retract MSHA pili and detach from a surface in microaerobic conditions. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP (c-di-GMP) under microaerobic conditions to induce MSHA pilus retraction. CdpA contains a putative NO-sensing NosP domain, and we demonstrate that nitric oxide (NO) is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that microaerobic conditions result in endogenous production of NO (or an NO-like molecule) in V. cholerae. Together, these results describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. More broadly, these results show how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

scholarly journals Comparative approaches to the study of physiology: Drosophila as a physiological tool

2013 ◽  
Vol 304 (3) ◽  
pp. R177-R188 ◽  
Author(s):  
Wendi S. Neckameyer ◽  
Kathryn J. Argue
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Pattern Formation ◽  
Signaling Pathways ◽  
Human Disease ◽  
Cognitive Disorders ◽  
Model System ◽  
Critical Questions ◽  
Developmental Signaling ◽  
Shed Light

Numerous studies have detailed the extensive conservation of developmental signaling pathways between the model system, Drosophila melanogaster, and mammalian models, but researchers have also profited from the unique and highly tractable genetic tools available in this system to address critical questions in physiology. In this review, we have described contributions that Drosophila researchers have made to mathematical dynamics of pattern formation, cardiac pathologies, the way in which pain circuits are integrated to elicit responses from sensation, as well as the ways in which gene expression can modulate diverse behaviors and shed light on human cognitive disorders. The broad and diverse array of contributions from Drosophila underscore its translational relevance to modeling human disease.

A model system for T-lymphocyte differentiation: Regulation of CD4 and CD8 gene expression in SL12.4 T-lymphoma cell clones

1991 ◽  
Vol 28 (1-2) ◽  
pp. 57-68 ◽  
Author(s):  
Miles F. Wilkinson ◽  
Alan M. Fong ◽  
Hong Huynh ◽  
Esther F. Hays ◽  
Carol L. MacLeod
Keyword(s):  
Gene Expression ◽  
T Lymphocyte ◽  
Model System ◽  
Lymphoma Cell ◽  
Lymphocyte Differentiation ◽  
Cell Clones ◽  
T Lymphoma
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