preparation of electrocompetent cells to achieve single&#45;copy gene expression in an efflux&#45;deficient bacterial strain

Espressione genica a copia singola per la caratterizzazione di sistemi di efflusso multifarmaco

Acinetobacter baumannii is a World Health Organization top priority pathogen due to its encompassing resistance to all classes of antibiotics1. It is an opportunistic pathogen mostly affecting hospitalized, injured, or immunocompromised people. A. baumannii largely evades antibiotics via efflux pumps, the most relevant being the Resistance-Nodulation-Division (RND) family of exporters2. Understanding how these efflux pumps work mechanistically will allow one to develop targeted therapeutic options.
One common way that cellular processes can be specifically distinguished is through genetic manipulation. However, the tools available for A. baumannii genetic studies are limited, and to further confound experimental design, clinical isolates often are resistant to the antibiotics routinely used for selection in genetic manipulations3. A second hurdle encountered when studying efflux pumps specifically is that they are strictly regulated-often by unknown factors-making it difficult to accurately isolate and attribute function to a single pump4. Seeing this need to expand the research toolbox, we developed a mini-Tn7-based, single-copy-insertion, inducible expression system that incorporates a Flp recombinase target (FRT) cassette, which allows for the removal of the selection marker5,6,7 (Figure 1). First created for Pseudomonas8,9,10, this elegant cloning and expression system was used to generate single-copy efflux pump complements into an RND efflux pump-deficient strain of A. baumannii (ATCC 17978::&#916;adeIJK,&#916;adeFGH,&#916;adeAB: hereafter referred to as A. baumannii AB258) that we generated11. Being able to study one efflux pump at a time and not overwhelm the bacterial cells with high-copy expression (as generally seen with plasmid-based expression systems), one can better learn about the critical, physiological aspects of each efflux pump with minimal interference and reduced complications.
This article describes how to use the mini-Tn7 system to complement a deleted gene of interest, RND efflux pump adeIJK, into the chromosome of A. baumannii AB258 through a series of uncomplicated steps performed over the course of 9 days7. The first set of steps re-introduces the deleted efflux pump genes cloned into the mini-Tn7-based insertion plasmid (Figure 2A) at the single attTn7 insertion site downstream of the well-conserved glmS gene (Figure 3A). This process is facilitated by a non-replicative helper plasmid (Figure 2B) that encodes for the transposase genes needed for Tn7-driven insertion. The second set of steps uses an excision plasmid (Figure 2C) for Flp recombinase-mediated removal of the gentamicin gene flanked by FRT sites (Figure 3B) to create an unmarked strain. Though this system is used to elucidate the essential roles and possible inhibitors of RND efflux pumps with respect to antibiotic resistance, it can be used to investigate any gene of interest.

Autore in primo piano: Avanzamento della ricerca sulla resistenza agli antibiotici utilizzando un ceppo batterico carente di efflusso e un sistema di espressione genica a copia singola

Caratterizzazione di sistemi di efflusso multifarmaco in Acinetobacter baumannii utilizzando un ceppo batterico carente di efflusso e un sistema di espressione genica a singola copia

We study multi-drug resistance efflux systems in gram-negative bacteria. We are interested in learning about the natural functions of these pumps and how these pumps can be used as a potential drug target. Expression of multi-drug resistance efflux systems is tightly regulated, making it difficult to study their functions.Thus, an expression system that bypasses the regulatory system can be quite useful in the characterization of these pumps. This protocol allows for the expression of genes at levels usually seen in natural settings. Thus, the activity of the gene products mimics what is observed in nature.

Preparazione di cellule elettrocompetenti per ottenere l'espressione genica a singola copia in un ceppo batterico carente di efflusso 

Per iniziare, posizionare tutte le provette sterili da 1,5 millilitri di microfuge e le cuvette sterili per elettroporazione sul ghiaccio. Quindi metti la bottiglia di acqua sterile, che è stata conservata a quattro gradi Celsius, sul ghiaccio. Trasferire 1,5 millilitri di coltura batterica durante la notte in una delle provette da 1,5 millilitri e centrifugare a 13.000 G per due minuti per pellettare le cellule.Utilizzando una pipetta da un millilitro, rimuovere tutto il surnatante senza disturbare il pellet cellulare. Aggiungere altri 1,5 millilitri di coltura batterica nella stessa provetta per microfuge, centrifugare a 13.000 G per due minuti e rimuovere il surnatante. Aggiungere un millilitro di acqua sterile ghiacciata al pellet cellulare e risospendere con un pipettaggio delicato fino a quando il pellet non si trova più sul fondo del tubo del microfugo.Centrifugare le cellule risospese a 13.000 g per due minuti e rimuovere con cautela il surnatante utilizzando una pipetta da un millilitro. Risospendere delicatamente il pellet cellulare finale in 200 microlitri di acqua sterile ghiacciata e trasferire 100 microlitri della sospensione cellulare finale nella seconda provetta microfuge ghiacciata da 1,5 millilitri.

Research

JoVE Journal

Immunology and Infection

Preparation of Electrocompetent Cells to Achieve Single&#45;Copy Gene Expression in an Efflux&#45;Deficient Bacterial Strain

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend ...

Ricerca

Immunologia e infezioni

Electroporation Procedure for the Single&#45;Copy Gene Expression in an Efflux&#45;Deficient Bacterial Strain

Procedura di elettroporazione per l'espressione genica a copia singola in un ceppo batterico carente di efflusso