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

Enkeltkopi genekspression til karakterisering af multidrug efflux-systemer

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 Spotlight: Fremme af antibiotikaresistensforskning ved hjælp af en bakteriestamme med udstrømningsmangel og et enkeltkopi af genekspressionssystem

Karakterisering af multidrug efflux systemer i Acinetobacter baumannii ved hjælp af en efflux-mangelfuld bakteriestamme og et enkelt-kopi genekspressionssystem

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.

Elektroporationsprocedure for enkeltkopigenekspression i en bakteriestamme med effluxmangel

Forvarm en milliliter LB bouillon og en LB plus gentamicin agarplade for hver prøve og kontrol i en statisk inkubator indstillet til 37 grader Celsius. I et kombineret volumen på fem mikroliter eller mindre tilsættes 100 til 200 nanogram hver af pTNS2-hjælperplasmidet og pUC18-Tmini-Tn7-Tlac gentamicin adeIJK-indsættelsesplasmidet til en alikvot af elektrokompetente celler. Bland med let fingerspidstryk for at sikre fuldstændig blanding af plasmiderne med de elektrokompetente celler uden at indføre bobler.Der tilsættes en tilsvarende mængde sterilt destilleret vand i den negative kontrolcelle, der er en delprøve, og blandes forsigtigt som vist før. Prøverne inkuberes på is i 20 minutter. Overfør hele celleprøven til en iskold elektroporationskuvette, og læg derefter kuvetten tilbage på is.For at elektroporate celleprøven skal du tænde elektroporatoren og indstille den til to kilovolt. Tør kuvettens overflade af med et blødt væv for at fjerne klæbende is eller fugt. Sæt kuvetten i elektroporatoren, og giv det elektriske stød.Tilsæt straks 0,9 ml forvarmet LB-bouillon til cellerne i kuvetten, og pipette forsigtigt op og ned for at blande cellerne med mediet. Overfør hele cellesuspensionen til et nyt 1,5 ml mikrofugerør ved stuetemperatur. Kontroller derefter tidskonstantværdien på elektroporatoren.For de bedste resultater skal denne værdi være mellem fire og seks. De elektroporerede prøver inkuberes ved 37 grader Celsius i en time ved 250 RPM for at muliggøre cellegendannelse. Efter en time spredes 100 mikroliter af hver prøve på en forvarmet LB plus gentamicin agarplade ved hjælp af en podningsspreder.Inkuber pladerne ved 37 grader Celsius i 16 til 18 timer. Kontroller elektroporationspladerne. Brug sterile tandstikkere til at hente op til 10 enkeltkolonier fra LB plus gentamicin agarpladerne og lappe dem på en frisk LB plus gentamicin agarplade.Inkuber pladerne ved 37 grader Celsius i 16 til 18 timer. Patching af transformationspladekolonier på selektive medier bevarer den transformerede stamme og giver udgangsmateriale til koloni-PCR-screening. PCR-produktet til screeningsprimerne, ABglmS2 fremad nyt og Tn7 omvendt er 382 basepar.Negative kontroller for reaktionen omfatter vildtype A.baumannii ATCC 17978, AB 258 og ingen skabelon.

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 af elektrokompetente celler for at opnå enkeltkopi-genekspression i en efflux-mangelfuld bakteriestamme 