Construction of a tetracycline inducible expression vector and characterization of its use in Vibrio cholerae

Bina, Xiaowen R.; Wong, Eileen A.; Bina, Thomas F.; Bina, James E.

doi:10.1016/j.plasmid.2014.10.004

Cited by 13 publications

(11 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…was constructed in two steps: first, the plasmid pMK47 containing an inducible mKate2 construct was constructed by digesting the pUC18-mini-Tn7T-LAC vector with NsiI and Eco53kI restriction enzymes (NEB) 43 . A tet regulation cassette was PCR amplified from plasmid pXB300 44 using primers pMK47_F1.For and pMK47_F1.Rev and the gene coding for the fluorescent protein mKate2 was amplified from plasmid pPaQa 5 using primers pMK47_F2.For and pMK47_F2.Rev. All three fragments were joined using Gibson assembly.…”

Section: Methodsmentioning

confidence: 99%

Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili

Koch

Fei

Wingreen

et al. 2020

Preprint

View full text Add to dashboard Cite

10The functions of type IV pili (TFP) are mediated by cycles of extension and retraction. The 11 coordination of these cycles remains mysterious due to poor quantification of TFP dynamics. Here we 12 fluorescently label the TFP in the opportunistic pathogen Pseudomonas aeruginosa and track the full 13 extension and retraction cycles of individual TFP to quantify their dynamics. We test several models for 14 the switch between extension and retraction using quantitative experiments, biophysical modeling and 15 genetics. We invalidate the prominent hypothesis that this switch is triggered by surface contact. Instead, 16 we show that the entire repetitive cycle of extension and retraction of individual TFP is governed by the 17 stochastic binding of antagonistic extension and retraction motors and explain how this mechanism 18 quantitatively defines physiologically-important features like TFP length and their production rate. 19Interestingly, our results suggest that the major throttle of TFP production is the unbinding of the 20 retraction motor. 21 22 microns 22 . Despite the limitations of these approaches, they have led to several competing models for 42 how the switch between TFP extension and retraction is controlled. A cryo-EM study did not observe 43 motors at the base of unpiliated structures, suggesting that the motors do not remain bound after TFP 44 retraction 12 . Meanwhile, an interferometry study focusing on the longest subpopulation of TFP suggested 45 that TFP retraction is triggered by surface association 22 . However, the inability to directly visualize the 46 dynamics of the entire TFP population previously limited the ability to directly test these models. 47Here we addressed the above limitations by directly fluorescently labeling the TFP of P. 48 aeruginosa. Fluorescent labeling of TFP was first achieved with non-specific labeling of extracellular 49 proteins 23 . However, similar to the interferometry approach, this surface labeling approach led to a strong 50 halo from staining of the cell body that prevented analysis of short pili. More recently, TFP from 51Caulobacter crescentus and Vibrio cholerae were directly labeled by introducing a reactive cysteine 52 residue into the pilin sequence 24-26 . Here we apply this approach to P. aeruginosa and use it to perform 53 the first direct quantitative analysis of full TFP extension and retraction cycles of individual pili. We go 54 on to develop and test quantitative models for the behaviors we observe. We show that TFP production 55 rate, length, and dynamics can be fully explained by the mutually exclusive stochastic binding of the 56 extension and retraction motors, and that this stochasticity persists in the presence or absence of surface 57 association. 58 59 Results 60Quantifying TFP dynamics reveals that P. aeruginosa makes mostly short pili that are highly dynamic 61We fluorescently labeled the major protein of the P. aeruginosa pilus fiber (PilA) by introducing 62 a cysteine point mutation, A86C, that we then labeled with the thiol-r...

show abstract

Section: Methodsmentioning

confidence: 99%

Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili

Koch

Fei

Wingreen

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…qRT-PCR. V. cholerae strains harboring either pBAD33-ompR or pBAD33 empty vector were grown under AKI conditions with 0.05% arabinose for 5 h, at which time total RNA was isolated from the cultures using TRIzol (Invitrogen), according to the manufacturer's instructions (80)(81)(82)(83)(84). cDNA was generated from the purified RNA using the Maxima first-strand cDNA synthesis kit (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

Vibrio cholerae OmpR Contributes to Virulence Repression and Fitness at Alkaline pH

Kunkle

Bina

2020

Infect Immun

View full text Add to dashboard Cite

Vibrio cholerae is a Gram-negative human pathogen and the causative agent of the life-threatening disease cholera. V. cholerae is a natural inhabitant of marine environments and enters humans through the consumption of contaminated food or water. The ability to transition between aquatic ecosystems and the human host is paramount to the pathogenic success of V. cholerae. The transition between these two disparate environments requires the expression of adaptive responses, and such responses are most often regulated by two-component regulatory systems such as the EnvZ/OmpR system, which responds to osmolarity and acidic pH in many Gram-negative bacteria. Previous work in our laboratory indicated that V. cholerae OmpR functioned as a virulence regulator through repression of the LysR-family transcriptional regulator aphB; however, the role of OmpR in V. cholerae biology outside virulence regulation remained unknown. In this work, we sought to further investigate the function of OmpR in V. cholerae biology by defining the OmpR regulon through RNA sequencing. This led to the discovery that V. cholerae ompR was induced at alkaline pH to repress genes involved in acid tolerance and virulence factor production. In addition, OmpR was required for V. cholerae fitness during growth under alkaline conditions. These findings indicate that V. cholerae OmpR has evolved the ability to respond to novel signals during pathogenesis, which may play a role in the regulation of adaptive responses to aid in the transition between the human gastrointestinal tract and the marine ecosystem.

show abstract

“…Due to its location far upstream of the PLOS GENETICS gene, the putative Qrr1 base pairing region for VIBHAR_RS16980 could not be excluded [30]. Translational reporters employing mVenus were designed using a previously described method and transcribed from the aTc inducible tetA promoter [30,68]. Plasmids were introduced into E. coli by electroporation using a Bio-Rad Micro Pulser.…”

Section: Dna Manipulation and Strain Constructionmentioning

confidence: 99%

LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA

et al. 2021

View full text Add to dashboard Cite

Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacterium Vibrio harveyi monitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs largely function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor of qrr1. LuxT does not regulate qrr2-5, demonstrating that qrr genes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression of qrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved among Vibrionaceae and sequence comparisons predict that LuxT represses qrr1 in additional species. The present findings reveal that the QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors.

show abstract

Construction of a tetracycline inducible expression vector and characterization of its use in Vibrio cholerae

Cited by 13 publications

References 33 publications

Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili

Competitive binding of independent extension and retraction motors explains the quantitative dynamics of type IV pili

Vibrio cholerae OmpR Contributes to Virulence Repression and Fitness at Alkaline pH

LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA

Contact Info

Product

Resources

About