electroporation procedure for the single&#45;copy gene expression in an efflux&#45;deficient bacterial strain

Enkeltkopi genuttrykk for karakterisering av multidrug efflukssystemer

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.

Forfatter i søkelyset: Fremme antibiotikaresistensforskning ved bruk av en effluks-mangelfull bakteriestamme og et enkeltkopi av genuttrykkssystem

Karakterisering av multidrug efflux systemer i Acinetobacter baumannii ved bruk av en effluks-mangelfull bakteriestamme og en single-copy genet ekspresjonssystem

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.

Elektroporeringsprosedyre for enkeltkopigenuttrykket i en effluksmangel bakteriestamme

Forvarm en milliliter LB-buljong og en LB pluss gentamicin-agarplate for hver prøve og kontroll i en statisk inkubator satt til 37 grader Celsius. I et kombinert volum på fem mikroliter eller mindre, tilsett 100 til 200 nanogram hver av pTNS2-hjelperplasmidet og pUC18-Tmini-Tn7-Tlac-gentamicin adeIJK-innføringsplasmidet til et alikot av elektrokompetente celler. Bland med forsiktig fingertupptapping for å sikre fullstendig blanding av plasmidene med de elektrokompetente cellene uten å introdusere bobler.Tilsett et tilsvarende volum sterilt destillert vann i den negative kontrollcellealikoten og bland forsiktig som vist før. Inkuber prøvene på is i 20 minutter. Overfør hele celleprøven til en iskald elektroporasjonskuvett, og legg deretter kyvetten tilbake på is.For å elektroporere celleprøven, slå på elektroporatoren og sett den på to kilovolt. Tørk overflaten av kyvetten med et mykt vev for å fjerne is eller fuktighet som kleser seg. Sett kyvetten inn i elektroporatoren og lever det elektriske støtet.Tilsett umiddelbart 0,9 ml forvarmet LB-buljong til cellene i kyvetten, og pipetter forsiktig opp og ned for å blande cellene med mediet. Overfør hele cellesuspensjonen til et nytt 1,5 milliliters mikrofugerør ved romtemperatur. Kontroller deretter tidskonstantverdien på elektroporatoren.For best resultat bør denne verdien være mellom fire og seks. Inkuber de elektroporerte prøvene ved 37 grader Celsius i en time ved 250 RPM for å tillate cellegjenoppretting. Etter en time, spred 100 mikroliter av hver prøve på en forvarmet LB pluss gentamicin agarplate, ved hjelp av en inokulasjonsspreder.Inkuber platene ved 37 grader Celsius i 16 til 18 timer. Kontroller elektroporasjonsplatene. Bruk sterile tannpirkere, plukk opp til 10 enkeltkolonier fra LB pluss gentamicin agarplater og dem på en fersk LB pluss gentamicin agar plate.Inkuber platene ved 37 grader Celsius i 16 til 18 timer. Patching transformasjonsplatekolonier på selektive medier bevarer den transformerte stammen og gir utgangsmateriale for koloni-PCR-screening. PCR-produktet for screeningprimerne, ABglmS2 fremover nye, og Tn7 revers er 382 basepar.Negative kontroller for reaksjonen inkluderer villtype A.baumannii ATCC 17978, AB 258, og ingen mal.

Research

JoVE Journal

Immunology and Infection

Electroporation Procedure for the 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 ...

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

Fremstilling av elektrokompetente celler for å oppnå enkeltkopigenuttrykk i en effluksmangel bakteriestamme 