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Open AccessArticle

Modification and Assembly of a Versatile Lactonase for Bacterial Quorum Quenching

by Melissa K. Rhoads, Pricila Hauk, Valerie Gupta, Michelle L. Bookstaver, Kristina Stephens, Gregory F. Payne and William E. Bentley

Molecules 2018, 23(2), 341; https://doi.org/10.3390/molecules23020341 - 6 Feb 2018

Cited by 7 | Viewed by 4253

Abstract

This work sets out to provide a self-assembled biopolymer capsule activated with a multi-functional enzyme for localized delivery. This enzyme, SsoPox, which is a lactonase and phosphotriesterase, provides a means of interrupting bacterial communication pathways that have been shown to mediate pathogenicity. Here we demonstrate the capability to express, purify and attach SsoPox to the natural biopolymer chitosan, preserving its activity to “neutralize” long-chain autoinducer-1 (AI-1) communication molecules. Attachment is shown via non-specific binding and by engineering tyrosine and glutamine affinity ‘tags’ at the C-terminus for covalent linkage. Subsequent degradation of AI-1, in this case N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL), serves to “quench” bacterial quorum sensing (QS), silencing intraspecies communication. By attaching enzymes to pH-responsive chitosan that, in turn, can be assembled into various forms, we demonstrate device-based flexibility for enzyme delivery. Specifically, we have assembled quorum-quenching capsules consisting of an alginate inner core and an enzyme “decorated” chitosan shell that are shown to preclude bacterial QS crosstalk, minimizing QS mediated behaviors. Full article

(This article belongs to the Section Organic Chemistry)

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Open AccessReview

Microbial Biofilm as a Smart Material

by Christian Garde, Martin Welch, Jesper Ferkinghoff-Borg and Thomas Sams

Sensors 2015, 15(2), 4229-4241; https://doi.org/10.3390/s150204229 - 12 Feb 2015

Cited by 5 | Viewed by 5782

Abstract

Microbial biofilm colonies will in many cases form a smart material capable of responding to external threats dependent on their size and internal state. The microbial community accordingly switches between passive, protective, or attack modes of action. In order to decide which strategy to employ, it is essential for the biofilm community to be able to sense its own size. The sensor designed to perform this task is termed a quorum sensor, since it only permits collective behaviour once a sufficiently large assembly of microbes have been established. The generic quorum sensor construct involves two genes, one coding for the production of a diffusible signal molecule and one coding for a regulator protein dedicated to sensing the signal molecules. A positive feedback in the signal molecule production sets a well-defined condition for switching into the collective mode. The activation of the regulator involves a slow dimerization, which allows low-pass filtering of the activation of the collective mode. Here, we review and combine the model components that form the basic quorum sensor in a number of Gram-negative bacteria, e.g., Pseudomonas aeruginosa. Full article

(This article belongs to the Special Issue Smart Materials for Switchable Sensors)

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442 KiB

Open AccessArticle

Kinetic Model for Signal Binding to the Quorum Sensing Regulator LasR

by Anetta Claussen, Tim Holm Jakobsen, Thomas Bjarnsholt, Michael Givskov, Martin Welch, Jesper Ferkinghoff-Borg and Thomas Sams

Int. J. Mol. Sci. 2013, 14(7), 13360-13376; https://doi.org/10.3390/ijms140713360 - 27 Jun 2013

Cited by 8 | Viewed by 6710

Abstract

We propose a kinetic model for the activation of the las regulon in the opportunistic pathogen Pseudomonas aeruginosa. The model is based on in vitro data and accounts for the LasR dimerization and consecutive activation by binding of two OdDHL signal molecules. Experimentally, the production of the active LasR quorum-sensing regulator was studied in an Escherichia coli background as a function of signal molecule concentration. The functional activity of the regulator was monitored via a GFP reporter fusion to lasB expressed from the native lasB promoter. The new data shows that the active form of the LasR dimer binds two signal molecules cooperatively and that the timescale for reaching saturation is independent of the signal molecule concentration. This favors a picture where the dimerized regulator is protected against proteases and remains protected as it is activated through binding of two successive signal molecules. In absence of signal molecules, the dimerized regulator can dissociate and degrade through proteolytic turnover of the monomer. This resolves the apparent contradiction between our data and recent reports that the fully protected dimer is able to “degrade” when the induction of LasR ceases. Full article

(This article belongs to the Special Issue Quorum Sensing Research in Microbial Systems)

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1464 KiB

Open AccessArticle

Intra-Species Bacterial Quorum Sensing Studied at Single Cell Level in a Double Droplet Trapping System

by Yunpeng Bai, Santoshkumar N. Patil, Steven D. Bowden, Simon Poulter, Jie Pan, George P. C. Salmond, Martin Welch, Wilhelm T. S. Huck and Chris Abell

Int. J. Mol. Sci. 2013, 14(5), 10570-10581; https://doi.org/10.3390/ijms140510570 - 21 May 2013

Cited by 22 | Viewed by 9707

Abstract

In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP) from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets. Full article

(This article belongs to the Special Issue Quorum Sensing Research in Microbial Systems)

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