This work was supported by a Discovery Grant from the Natural Science and Engineering Council of Canada to AK. The schematics used in the figures are created with BioRender.com.

1. Experimental preparation
<ol>
	<li>Purify the plasmid pUC18T-mini-Tn7T-LAC-Gm9 (insertion plasmid, Figure 2A) with the gene of interest. 
	NOTE: Here, the gene of interest is adeIJK. The final plasmid concentration should be &#8805;100 ng/&#181;L.</li>
	<li>Purify the helper plasmid (pTNS2)9 and the excision plasmid (pFLP2ab)6 (Figure 2B,C, respectively), ideally to a final plasmid DNA concentration of &#8805;100 ng/&#181;L.</li>
	<li>Prepare 50 mL of sterile ultrapure water and 25 mL of sterile LB (Lennox) broth (see Table of Materials).</li>
	<li>Prepare a minimum of 10 LB agar plates, each with the following additives: plain (no additives), gentamicin (Gm) at 50 &#181;g/mL, carbenicillin (Cb) at 200 &#181;g/mL (for selection via the ampicillin resistance gene), and 5% sucrose (see Table of Materials).</li>
	<li>Streak out the strain to be used for insertion on LB agar and incubate at 37 &#176;C for 16-18 h. Here, A. baumannii AB258 is used. 
	NOTE: This protocol must be performed in a sterile environment as much as possible by using a Bunsen burner at the bench or a biological safety cabinet. All consumables (pipette tips, inoculating loops, microfuge tubes, etc.) need to be sterile.</li>
</ol>
2. Culture preparation
<ol>
	<li>Inoculate a single colony of A. baumannii AB258 into 4 mL of LB broth in a sterile 13 mL culture tube using a sterile inoculation loop or sterile wooden inoculating stick. 
	NOTE: A single 4 mL culture is used for one sample and one control. Increase the number of cultures depending on the number of samples needed.</li>
	<li>Incubate overnight at 37 &#176;C with shaking (250 rpm).</li>
	<li>Place a bottle of sterile distilled water (25-50 mL) at 4 &#176;C overnight for use in step 3.</li>
</ol>
3. Preparation of electrocompetent cells
<ol>
	<li>Place all sterile 1.5 mL microfuge tubes (two per culture) and sterile electroporation cuvettes (two per culture) on ice (see Table of Materials). Keep the samples on ice as much as possible throughout the procedure.</li>
	<li>Place the bottle of sterile water that was stored at 4 &#176;C on ice.</li>
	<li>Transfer 1.5 mL of the overnight bacterial culture into one of the 1.5 mL microfuge tubes.</li>
	<li>Centrifuge at 13,000 x g for 2 min to pellet the cells. 
	NOTE: Centrifugation would ideally be performed at 4 &#176;C, but ambient temperature centrifugation is acceptable and not detrimental.</li>
	<li>Using a 1 mL pipette, remove all of the supernatant without disturbing the cell pellet.</li>
	<li>Add another 1.5 mL of bacterial culture into the same microfuge tube. Centrifuge at 13,000 x g for 2 min, then remove all of the supernatant.</li>
	<li>Repeat step 3.6 one final time with the remaining 1 mL of culture.</li>
	<li>Add 1 mL of ice-cold sterile water to the cell pellet and resuspend with gentle pipetting until the pellet no longer sits at the bottom of the microfuge tube.</li>
	<li>Centrifuge the resuspended cells at 13,000 x g for 2 min.</li>
	<li>Carefully remove the supernatant using a 1 mL pipette. Do not pour off the supernatant, especially in the subsequent wash steps when the cells tend to form less compact pellets.</li>
	<li>Repeat this wash step with ice-cold sterile water, steps 3.8 to step 3.10, twice more.</li>
	<li>Gently resuspend the final cell pellet in 200 &#181;L of ice-cold sterile water.</li>
	<li>Transfer 100 &#181;L of the final cell suspension into the second ice-cold 1.5 mL microfuge tube. This second aliquot will become the negative control for electroporation. Keep the cell samples on ice.</li>
</ol>
4. Electroporation
<ol>
	<li>Pre-warm 1 mL of LB broth and one LB + Gm50 agar plate (prepared in step 1.4) for each sample and control in a static incubator set to 37 &#176;C.</li>
	<li>In a combined volume of 5 &#181;L or less, add 100-200 ng each of the pTNS2 helper plasmid and the pUC18T-mini-Tn7T-LAC-Gm-adeIJK insertion plasmid to an aliquot of electrocompetent cells.</li>
	<li>Mix with gentle fingertip tapping to ensure complete mixing of the plasmids with the electrocompetent cells without introducing bubbles.</li>
	<li>Add an equivalent volume of sterile distilled water into the negative control cell aliquot and mix gently as above.</li>
	<li>Incubate the samples on ice for 20 min.</li>
	<li>Transfer the entire cell sample into an ice-cold electroporation cuvette, then place the cuvette back on ice. Repeat for the negative control cell sample.</li>
	<li>Electroporate the cell sample.
	<ol>
		<li>Turn on the electroporator and set it to 2.0 kV (25 &#181;F, 200 &#937;) (see Table of Materials).</li>
		<li>Wipe the surface of the cuvette with a soft tissue to remove any adhering ice or moisture.</li>
		<li>Insert the cuvette into the electroporator and deliver the electric shock.</li>
		<li>Immediately add 0.9 mL of pre-warmed LB broth to the cells in the cuvette and gently pipette up and down to mix the cells with the media.</li>
		<li>Transfer the entire cell suspension into a new 1.5 mL microfuge tube (room temperature).</li>
		<li>Check the time constant value on the electroporator; for best results, this value should be between 4 and 6.</li>
	</ol>
	</li>
	<li>Repeat the electroporation procedure (steps 4.7.1 to step 4.7.6) for the negative control cell sample.</li>
	<li>Incubate the electroporated samples at 37 &#176;C for 1 h at 250 rpm to allow for cell recovery.</li>
	<li>Using an inoculation spreader, spread 100 &#181;L of each electroporated cell sample onto a pre-warmed LB + Gm50 agar plate.
	<ol>
		<li>Centrifuge the remaining samples at 13,000 x g for 2 min to pellet the cells.</li>
		<li>After removing all of the supernatant using a pipette, resuspend the cells in 100 &#181;L of LB broth.</li>
		<li>Spread each entire sample onto individual pre-warmed LB + Gm50 agar plates. 
		NOTE: Electroporation efficiency can be strain-dependent. When electroporating a strain for the first time, it can be informative to plate out different volumes, or even dilutions, of the electroporation sample to ensure the resulting plates have isolated colonies.</li>
	</ol>
	</li>
	<li>Incubate the plates at 37 &#176;C for 16-18 h.</li>
</ol>
5. Selecting transformed colonies for PCR-based screening
<ol>
	<li>Check the electroporation plates. The negative control should have no colonies; the sample should have distinct colonies. 
	NOTE: Plates with colonies, at this step and at all subsequent steps where agar plates with colonies or patches are produced, can be stored at 4 &#176;C for up to 3 days before proceeding.</li>
	<li>Using sterile toothpicks, pick up to 10 single colonies from the LB + Gm50 agar plates and patch them onto a fresh LB + Gm50 agar plate.</li>
	<li>Incubate the plates at 37 &#176;C for 16-18 h.</li>
</ol>
6. Verifying chromosomal insertion by colony PCR
<ol>
	<li>Remove the LB + Gm50 agar plates containing the patched colonies from the incubator.</li>
	<li>Using a sterile toothpick or sterile pipette tip, remove a small portion (about the size of a large colony) from a patch into 20 &#181;L of sterile distilled water in a 0.2 mL PCR tube; mix well. The water sample should become visibly cloudy as the cells are released from the toothpick. Prepare any number of samples that need to be screened, but 6 should be sufficient.</li>
	<li>Incubate the PCR tube containing the bacterial suspension at 100 &#176;C for 5-10 min.</li>
	<li>Using a mini-centrifuge (see Table of Materials), spin the sample at the fixed maximum speed for 2 min to pellet cellular debris.</li>
	<li>Transfer the supernatant to a new 0.2 mL PCR tube and place it on ice. This sample contains the template DNA for the PCR reaction. 
	NOTE: This template DNA can be stored at -20 &#176;C before proceeding with PCR.</li>
	<li>Prepare a PCR reaction mixture containing 1x polymerase buffer, 200 &#181;M dNTPs, 0.18 &#181;M ABglmS2_F_New forward primer, 0.18 &#181;M Tn7R reverse primer (Table 1), 1 U of Taq DNA polymerase, and 1 &#181;L of the prepared template DNA in a total volume of 25 &#181;L. Prepare a no-template control (NTC), including all but the template DNA (see Table of Materials).</li>
	<li>Enter the reaction conditions into a thermal cycler: 95 &#176;C for 2 min; 95 &#176;C for 30 s, 49 &#176;C for 30 s, and 72 &#176;C for 30 s for a total of 35 cycles; 72 &#176;C for 10 min; 12 &#176;C hold. Run the samples.</li>
	<li>Following the addition of DNA loading dye to a final concentration of 1x, load 10 &#181;L of each PCR reaction on a 2% agarose gel and run at 80 V for 40 min. The expected amplicon size for this primer pair is 382 bp. The NTC should have no bands.</li>
	<li>Identify the samples that produced the expected PCR product. Prepare a streak plate on LB + Gm50 agar plates from any of the PCR-positive patches; incubate at 37 &#176;C for 16-18 h. The goal is to generate a plate with single colonies for the preparation of a glycerol stock to preserve this marked strain and as a starting point for creating an unmarked strain (described below).</li>
</ol>
7. Removal of the GmR marker using pFLP2ab
<ol>
	<li>Follow the procedure mentioned in step 2: Culture preparation. The sample used to prepare the overnight culture is a single colony from the LB + Gm50 agar plates prepared to generate discrete colonies in step 6.9.</li>
	<li>Follow the procedure mentioned in step 3: Preparation of&#160;electrocompetent cells. Prepare the cells from the overnight culture for electroporation.</li>
	<li>Follow the procedure mentioned in step 4: Electroporation. Here, the plasmid to introduce to the cells is pFLP2ab (100-200 ng), which will remove the gentamicin resistance gene, flanking the chromosomally inserted gene of interest.</li>
	<li>After the cells have recovered for 1 h (step 4.9), spread 100 &#181;L of the electroporated cell sample onto a pre-warmed LB + Cb200 agar plate (prepared in step 1.4). This selective pressure ensures that only cells harboring the pFLP2ab excision plasmid will grow.</li>
	<li>Incubate the plates at 37 &#176;C for 16-18 h.</li>
	<li>Check the plate for colonies. The negative control plate should have no colonies; the LB + Cb200 agar sample plate should have distinct colonies.</li>
	<li>Using sterile toothpicks, cross-patch up to 20 isolated colonies onto an LB + Cb200 agar plate and an LB + Gm50 agar plate.</li>
	<li>Incubate the plates for 16-18 h at 37 &#176;C.</li>
	<li>Check the plates for patches. Clones that grow on the LB + Cb200 agar plate but not the LB + Gm50 agar plate have had the gentamicin resistance gene removed from the original insert.</li>
	<li>Select the patches that are carbenicillin resistant and gentamicin susceptible for streaking onto individual LB agar plates supplemented with 5% (w/v) sucrose, which forces the expulsion of the pFLP2ab plasmid from the bacteria. Choose up to 10 colonies.</li>
	<li>Incubate the plates for 16-18 h at 37 &#176;C.</li>
	<li>Check the plates for colonies.</li>
	<li>As a final confirmation of pFLP2ab plasmid loss, cross-patch 4-6 isolated colonies from the 5% sucrose plates onto an LB + Cb200 agar plate and an LB agar plate.</li>
	<li>Incubate the plates for 16-18 h at 37 &#176;C.</li>
	<li>Choose a clone that is growing on the LB agar plate, and that is carbenicillin sensitive for the preparation of a glycerol stock to preserve this unmarked strain.
	<ol>
		<li>Genetic verification of the unmarked strain can be performed with colony PCR (step 6: Verifying chromosomal insertion by colony PCR) using the primers that target the gentamicin resistance gene.</li>
		<li>Prepare a PCR reaction mixture containing 1x polymerase buffer, 200 &#181;M dNTPs, 0.18 &#181;M Gm_F forward primer, 0.18 &#181;M Gm_R reverse primer (Table 1), 1 U of Taq DNA polymerase, and 1 &#181;L of the prepared template DNA in a total volume of 25 &#181;L. Prepare a no-template control (NTC) including all but the template DNA, and a positive control using DNA from the marked strain.</li>
		<li>Enter the reaction conditions into a thermal cycler: 95 &#176;C for 2 min; 95 &#176;C for 30 s, 50 &#176;C for 30 s, and 72 &#176;C for 40 s for a total of 35 cycles; 72 &#176;C for 10 min; 12 &#176;C hold. Run the samples.</li>
		<li>Following the addition of DNA loading dye to a final concentration of 1x, load 10 &#181;L of each PCR reaction on a 1% agarose gel and run at 80 V for 40 min. The expected amplicon size for this primer pair is 525 bp. The positive control should have a single band; the samples and the NTC should have no bands.</li>
	</ol>
	</li>
</ol>

Single&#45;Copy Gene Expression for Characterizing Multidrug Efflux Systems

<ol>
	<li>Prioritization of pathogens to guide research, discovery, research and development of new antibiotics for drug-resistant bacterial infections, including tuberculosis. World Health Organization Available from: <a target="_blank" href="https://www.who.int/publications/i/item/WHO-EMP-IAU-2017.12">https://www.who.int/publications/i/item/WHO-EMP-IAU-2017.12</a> (2017)</li><li>Kornelsen, V., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+multidrug+resistance+efflux+pumps+in+Acinetobacter+spp.">Update on multidrug resistance efflux pumps in Acinetobacter spp.</a> Antimicrob Agents Chemother. 65 (7), e00514-00521 (2021).</li><li>Sykes, E. M., Deo, S., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+genetic+tools+for+Acinetobacter+baumannii.">Recent advances in genetic tools for Acinetobacter baumannii.</a> Front Genet. 11, 601380 (2020).</li><li>Kumar, A., Chua, K. L., Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+regulated+expression+of+single-copy+efflux+pump+genes+in+a+surrogate+Pseudomonas+aeruginosa+strain:+identification+of+the+BpeEF-OprC+chloramphenicol+and+trimethoprim+efflux+pump+of+Burkholderia+pseudomallei+1026b.">Method for regulated expression of single-copy efflux pump genes in a surrogate Pseudomonas aeruginosa strain: identification of the BpeEF-OprC chloramphenicol and trimethoprim efflux pump of Burkholderia pseudomallei 1026b.</a> Antimicrob Agents Chemother. 50 (10), 3460-3463 (2006).</li><li>Kumar, A., Dalton, C., Cortez-Cordova, J., Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-Tn7+vectors+as+genetic+tools+for+single+copy+gene+cloning+in+Acinetobacter+baumannii.">Mini-Tn7 vectors as genetic tools for single copy gene cloning in Acinetobacter baumannii.</a> J Microbiol Methods. 82 (3), 296-300 (2010).</li><li>Ducas-Mowchun, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+of+Tn7-based+single-copy+insertion+elements+for+use+in+multi-+and+pan-drug+resistant+strains+of+Acinetobacter+baumannii.">Next generation of Tn7-based single-copy insertion elements for use in multi- and pan-drug resistant strains of Acinetobacter baumannii.</a> Appl Environ Microbiol. 85 (11), e00066-00119 (2019).</li><li>Ducas-Mowchun, K., De Silva, P. M., Patidar, R., Schweizer, H. P., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tn7-based+single-copy+insertion+vectors+for+Acinetobacter+baumannii.">Tn7-based single-copy insertion vectors for Acinetobacter baumannii.</a> Methods Mol Biol. 1946, 135-150 (2019).</li><li>Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+Saccharomyces+cerevisiae+Flp-FRT+system+in+bacterial+genetics.">Applications of the Saccharomyces cerevisiae Flp-FRT system in bacterial genetics.</a> J Mol Microbiol Biotechnol. 5 (2), 67-77 (2003).</li><li>Choi, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tn7-based+broad-range+bacterial+cloning+and+expression+system.">A Tn7-based broad-range bacterial cloning and expression system.</a> Nat Methods. 2 (6), 443-448 (2005).</li><li>Choi, K. H., Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-Tn7+insertion+in+bacteria+with+single+attTn7+sites:+example+Pseudomonas+aeruginosa.">Mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa.</a> Nat Protoc. 1 (1), 153-161 (2006).</li><li>Kornelsen, V., Unger, M., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atorvastatin+does+not+display+an+antimicrobial+activity+on+its+own+nor+potentiates+the+activity+of+other+antibiotics+against+Acinetobacter+baumannii+ATCC+17978+or+A.+baumannii+AB030.">Atorvastatin does not display an antimicrobial activity on its own nor potentiates the activity of other antibiotics against Acinetobacter baumannii ATCC 17978 or A. baumannii AB030.</a> Access Microbiol. 3, 000288 (2021).</li><li>Reyrat, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counterselectable+markers:+untapped+tools+for+bacterial+genetics+and+pathogenesis.">Counterselectable markers: untapped tools for bacterial genetics and pathogenesis.</a> Infect Immun. 66 (9), 4011-4017 (1998).</li><li>Gay, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+procedure+for+entrapment+of+insertion+sequence+elements+in+gram-negative+bacteria.">Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.</a> J Bacteriol. 164, 918-921 (1985).</li><li>Kyriakidis, I., Vasileiou, E., Pana, Z. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tragiannidis,+Acinetobacter+baumannii+antibiotic+resistance+mechanisms.">Tragiannidis, Acinetobacter baumannii antibiotic resistance mechanisms.</a> Pathogens. 10 (3), 373 (2021).</li><li>Clinical and Laboratory Standards Institute. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+standards+for+antimicrobial+susceptibility+testing.">Performance standards for antimicrobial susceptibility testing.</a> CLSI Supplement M100., 31st edn. , (2021).</li><li>Dijkshoorn, L., Nemec, A., Seifert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+increasing+threat+in+hospitals:+multidrug-resistant+Acinetobacter+baumannii.">An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii.</a> Nature Reviews Microbiology. 5, 939-951 (2007).</li><li>Yildirim, S., Thompson, M. G., Jacobs, A. C., Zurawski, D. V., Kirkup, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+parameters+for+high+efficiency+transformation+of+Acinetobacter+baumannii.">Evaluation of parameters for high efficiency transformation of Acinetobacter baumannii.</a> Sci Rep. 25 (6), 22110 (2016).</li><li>Thompson, M. G., Yildirim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Acinetobacter+baumannii:+electroporation.">Transformation of Acinetobacter baumannii: electroporation.</a> Methods Mol Biol. 1946, 69-74 (2019).</li><li>Choi, K. H., DeShazer, D., Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-Tn7+insertion+in+bacteria+with+multiple+glmS-linked+attTn7+sites:+example+Burkolderia+mallei+ATCC+2334.">Mini-Tn7 insertion in bacteria with multiple glmS-linked attTn7 sites: example Burkolderia mallei ATCC 2334.</a> Nat Protoc. 1 (1), 162-169 (2006).</li><li>Jittawuttipoka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-Tn7+vectors+as+genetic+tools+for+gene+cloning+at+a+single+copy+number+in+an+industrially+important+and+phytopathogenic+bacteria,+Xanthomonas+spp.">Mini-Tn7 vectors as genetic tools for gene cloning at a single copy number in an industrially important and phytopathogenic bacteria, Xanthomonas spp.</a> FEMS Microbiol Lett. 298 (1), 111-117 (2009).</li><li>P&#233;rez-Varela, M., Tierney, A. R. P., Kim, J. S., V&#225;zquez-Torres, A., Rather, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+RelA+in+Acinetobacter+baumannii.">Characterization of RelA in Acinetobacter baumannii.</a> J Bacteriol. 202 (12), e00045 (2020).</li><li>Williams, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Acinetobacter+baumannii+copper+resistance+reveals+a+role+in+virulence.">Characterization of Acinetobacter baumannii copper resistance reveals a role in virulence.</a> Front Microbiol. 11, 16 (2020).</li></ol>

The authors have nothing to disclose.

Even though this procedure for the chromosomal insertion of an inducible single-copy gene expression system in A. baumannii is technically straightforward and not labor-intensive, there are a few important steps that need to be emphasized. First, preparation of the competent cells needs to be done on ice as much as possible as the cells become fragile during the replacement of the media with ice-cold water. Ideally, the centrifugation steps are performed at 4 &#176;C, but centrifugation at room temperature is ac...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL PCR tube</td>
			<td>VWR</td>
			<td>20170-012</td>
			<td>For colony boil preparations and PCR reactions</td>
		</tr>
		<tr>
			<td>1.5 mL microfuge tubes</td>
			<td>Sarstedt</td>
			<td>72-690-301</td>
			<td>General use</td>
		</tr>
		<tr>
			<td>13-mL culture tubes, Pyrex</td>
			<td>Fisher</td>
			<td>14-957K</td>
			<td>Liquid culture vessels</td>
		</tr>
		<tr>
			<td>6x DNA loading buffer</td>
			<td>Froggabio</td>
			<td>LD010</td>
			<td>Agarose gel electrophoresis sample loading dye</td>
		</tr>
		<tr>
			<td>Acetic acid, glacial</td>
			<td>Fisher</td>
			<td>351271-212</td>
			<td>Agarose gel running buffer component</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Bioshop</td>
			<td>AGR003</td>
			<td>Solid growth media</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>BioBasic</td>
			<td>D0012</td>
			<td>Electrophoretic separation of PCR reaction products; used at a concentration of 0.8&#8211;2%</td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis unit</td>
			<td>Fisher</td>
			<td>29-237-54</td>
			<td>Agarose gel electrophoresis; separation of PCR reaction products</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Fisher</td>
			<td>50841231</td>
			<td>Selective media</td>
		</tr>
		<tr>
			<td>Culture tube closures</td>
			<td>Fisher</td>
			<td>13-684-138</td>
			<td>Stainless steel closure for 13-mL culture tubes</td>
		</tr>
		<tr>
			<td>Deoxynucleotide triphosphate (dNTP) set</td>
			<td>Biobasic</td>
			<td>DD0058</td>
			<td>PCR reaction component; supplied as 100 mM each dATP, dCTP, dGTP, dTTP; mixed and diluted for 10 mM each dNTP</td>
		</tr>
		<tr>
			<td>Dry bath/block heater</td>
			<td>Fisher</td>
			<td>88860023</td>
			<td>Isotemp digital dry bath for boil preparations</td>
		</tr>
		<tr>
			<td>Electroporation cuvettes</td>
			<td>VWR</td>
			<td>89047-208</td>
			<td>2 mm electroporation cuvettes with round cap</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Cole Parmer</td>
			<td>940000009</td>
			<td>110 VAC, 60 Hz electroporator</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Fisher</td>
			<td>BP102-1</td>
			<td>Visualization of PCR reaction products and DNA marker in agarose gel</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>VWR</td>
			<td>CA-EM4050</td>
			<td>Agarose gel running buffer component</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Biobasic</td>
			<td>GB0217</td>
			<td>For the preparation of selective media</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>G33</td>
			<td>Preparation of bacterial stocks for long-term storage in an ultra-low freezer</td>
		</tr>
		<tr>
			<td>Incubator (shaking)</td>
			<td>New Brunswick Scientific</td>
			<td>M1352-0000</td>
			<td>Excella E24 Incubator Shaker for liquid culture growth</td>
		</tr>
		<tr>
			<td>Incubator (static)</td>
			<td>Fisher</td>
			<td>11-690-550D</td>
			<td>Isotemp Incubator Oven Model 550D for solid (LB agar) culture growth</td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>Sarstedt</td>
			<td>86.1562.050</td>
			<td>Streaking colonies onto agar plates</td>
		</tr>
		<tr>
			<td>Inoculation spreader</td>
			<td>Sarstedt</td>
			<td>86.1569.005</td>
			<td>Spreading of culture onto agar plates</td>
		</tr>
		<tr>
			<td>Lysogeny broth (LB) broth, Lennox</td>
			<td>Fisher</td>
			<td>BP1427</td>
			<td>Liquid growth media (20 g/L: 5 g/L sodium chloride, 10 g/L tryptone, 5 g/L yeast extract)</td>
		</tr>
		<tr>
			<td>Microfuge</td>
			<td>Fisher</td>
			<td>75002431</td>
			<td>Sorvall Legend Micro 17 for centrifugation of samples</td>
		</tr>
		<tr>
			<td>Mini-centrifuge</td>
			<td>Fisher</td>
			<td>S67601B</td>
			<td>Centrifugation of 0.2 mL PCR tubes</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>SPL Life Sciences</td>
			<td>10090</td>
			<td>For solid growth media (agar plates): 90 x 15 mm</td>
		</tr>
		<tr>
			<td>Pipettes&#160;</td>
			<td>Mandel</td>
			<td>Various</td>
			<td>Gilson single channel pipettes (P10, P20, P200, P1000)</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Biorad</td>
			<td>1645050</td>
			<td>PowerPac Basic power supply for electrophoresis</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>IDT</td>
			<td>NA</td>
			<td>PCR reaction component; specific to gene of interest; prepared at 100 &#956;M as directed on the product specification sheet</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BioBasic</td>
			<td>SB0498</td>
			<td>For the preparation of counterselective media for removal of the pFLP2ab plasmid from transformed A. baumannii</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>FroggaBio</td>
			<td>T-500</td>
			<td>PCR reaction component; polymerase supplied with a 10x buffer</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Biorad</td>
			<td>1861096</td>
			<td>Model T100 for PCR</td>
		</tr>
		<tr>
			<td>Toothpicks</td>
			<td>Fisher</td>
			<td>S24559</td>
			<td>For patching colonies onto agar plates</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>Agarose gel running buffer component</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>Millipore Sigma</td>
			<td>ZLXLSD51040</td>
			<td>MilliQ water purification system: ultra pure water for media and solution preparation, and cell washing</td>
		</tr>
		<tr>
			<td>Wide range DNA marker</td>
			<td>Biobasic</td>
			<td>M103R-2</td>
			<td>Size determination of PCR products on an agarose gel</td>
		</tr>
		<tr>
			<td>Wooden inoculating sticks</td>
			<td>Fisher</td>
			<td>29-801-02</td>
			<td>Inoculating cultures with colonies from agar plates</td>
		</tr>
	</tbody>
</table>

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 the mechanisms behind this resistance to design new and effective therapeutic options. Unfortunately, our ability to investigate these mechanisms in A. baumannii is hindered by the paucity of suitable genetic manipulation tools. Here, we describe methods for utilizing a chromosomal mini-Tn7-based system to achieve single-copy gene expression in an A. baumannii strain that lacks functional RND-type efflux mechanisms. Single-copy insertion and inducible efflux pump expression are quite advantageous, as the presence of RND efflux operons on high-copy number plasmids is often poorly tolerated by bacterial cells. Moreover, incorporating recombinant mini-Tn7 expression vectors into the chromosome of a surrogate A. baumannii host with increased efflux sensitivity helps circumvent interference from other efflux pumps. This system is valuable not only for investigating uncharacterized bacterial efflux pumps but also for assessing the effectiveness of potential inhibitors targeting these pumps.

The chromosomal insertion procedure takes only 2 h total across 3 days to see a result-colonies growing on a selective agar plate (Figure 1A-C). The expected number of colonies on the transformation plate is strain dependent: one may see 20-30 or even hundreds of colonies as insertion of Tn7 at attTn7 sites is specific and efficient9. Patching transformation plate colonies onto selective media (Figure 4A<...

Watch this Scientific Journal Video about Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System at JoVE.com

Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

We describe a facile procedure for the single-copy chromosomal complementation of an efflux pump gene using a mini-Tn7-based expression system into an engineered efflux-deficient strain of Acinetobacter baumannii. This precise genetic tool allows for controlled gene expression, which is key for the characterization of efflux pumps in multidrug resistant pathogens.

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 ...

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.

characterizing-multidrug-efflux-systems-acinetobacter-baumannii-using

Research

JoVE Journal

Immunology and Infection

527 Views.  University of Manitoba. 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 ...

Video: Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism&#8217;s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...