Bacterial strains, growth media and conditions
Bacterial strains were cultured in Luria-Bertani (LB) media at 37°C unless otherwise stated. E. coli Jke201 was used for plasmid propagation and as a donor strain for conjugation[Cianfanelli et al., 2020], and was cultured in the presence of 100μM of diaminopimelic acid (DAP) (Sigma Aldrich). A. baumannii strains AB5075_UW, AB0057, BAL062 were included in this study as recipient strains (Table 1 and 2).
Chemically competent cells for E. coli Jke201 were prepared using the calcium chloride/MES (2-(N-morpholino) ethanesulfonic acid) method. Briefly, cells were grown to mid exponential phase (OD600nm 0.4–0.6) from overnight cultures in the presence of 15mM MgCl2. Bacterial cultures were chilled on ice for 40 minutes before harvesting. The cell pellets were washed twice in 10ml ice-cold solution A (50mm CaCl2, 10mM MnCl2, 10mM MES at pH 6.3). The cells were then resuspended in 1.5ml solution A containing 15%(v/v) glycerol and 100μl aliquots were either used directly or frozen and stored at -80°C.
Transformation was performed using the heat shock method[Froger, and Hall, 2007]. E. coli transformants were selected in the presence of 100μg/ml ampicillin (Sigma Aldrich). For positive selection of trans-conjugants, a final concentration of 250μg/ml hygromycin (Invitrogen) or 10μg/ml tetracycline (Amresco) or 50μg/ml potassium tellurite (Sigma Aldrich) was used. For counter-selection, fresh low-salt LB plates (10g/L tryptone, 5g/L NaCl, 5g/L yeast extract, 15g/L agar) with 5% sucrose (Sigma Aldrich) were prepared.
Construction of suicide vectors
Suicide vector pALFI was constructed by combining the origin of conjugational transfer (OriT), origin of replication (R6K), and multiple cloning site (MCS) from the SEVA database[Silva-Rocha et al., 2013]. The sacB gene sequence with its promoter region was obtained from pKNG101_Tc[Evans et al., 1997]. pALFI1 was generated by combining the hygromycin (HygR) and ampicillin (AmpR) resistance cassettes and a reporter gene (green fluorescent protein, GFP). The GFP gene, preceded by the amvR promoter[Short et al., 2021] was intended for use as a second selection marker for single crossovers, however the signal was not high enough under normal growth conditions to reliably indicate colonies containing the plasmid. Each segment of the pALFI suicide vector is flanked with unique restriction sites, adopting the SEVA standard [Silva-Rocha et al., 2013] (Figure 1). The plasmid sequence of pALFI1 was submitted for plasmid synthesis (GENEWIZ). Once the pALFI1 plasmid was synthesized, pALFI2 was generated by replacing hygR with tetA with its native regulator tetR from A. baumannii AB0057. pALFI3 was generated by replacing hygR with tpm (thiopurine-S-methyltranferase) conferring resistance to tellurite from pFOKT [Cianfanelli et al., 2020] (Supplementary material S2). The TcR and Tpm markers were cloned using the SwaI/ScaI restriction sites. Plasmids used in this work are listed in Table 2. Primers used to amplify the tetracycline and tellurite resistance cassettes are listed in Table S1.
Cloning the mutant allele into the suicide vector
In order to amplify the knockout fragment containing approximately 700bp upstream and downstream region oftargeted gene, flanking primers with 15-18bp overlap (Table S1) were designed. The upstream and downstream regions were first amplified using the high-fidelity Platinum SuperFi PCR master mix (Invitrogen) and purified from a 1% agarose gel (QiAquick gel extraction kit, Qiagen). The fragments were then spliced together by overlap extension PCR and gel purified. Plasmids were isolated from overnight cultures using Wizard Plus SV miniprep kit (Promega). Plasmid and the knockout fragment were digested using SpeI-HF or BamHI-HF (New England Biolabs) for 2h at 37 °C and gel purified. The restriction digested knockout fragment and plasmid were ligated together using T4 DNA ligase (New England Biolabs) and transformed into E. coli Jke201 using the heat-shock method. After a heat shock, the cells were resuspended in pre-warmed LB broth supplemented with 100μM DAP and incubated at 37°C for 2h before plating on an ampicillin (100μg/ml) selection plate. Transformants were screened by colony PCR using the primers flanking the upstream and downstream regions of the targeted gene (Table S1).
Conjugation, positive selection and counterselection
Recipient A. baumannii strains AB5075_UW, AB0057, and BAL062 were streaked on fresh LB agar plates containing no antibiotics and incubated overnight at 37°C. The donor strain, E. coli JKe201 was streaked on a fresh LB agar plate supplemented with 100μM DAP and 100μg/ml ampicillin and incubated overnight at 37°C. Cells from each plate were resuspended in sterile phosphate buffered saline (PBS) and adjusted to an optical density (OD600nm) of 40 for the donor strain and 20 for the recipient strains. Equal volumes of recipient and donor strains were mixed and spotted on a very dry LB agar plate supplemented with 100μM DAP. The conjugation patch was left to dry and incubated at 37°C for 2h.
For positive selection, a loopful of cells from the conjugation patch was streaked on LB agar plate containing 250μg/ml hygromycin (pALFI1) or 20-100μg/ml potassium tellurite (pALFI3) and incubated at 37°C overnight. The conjugation plate was incubated at 28°C overnight and a loopful of cells was again transferred onto the selective LB agar plates the next day (16-18h). The selection conditions were optimised for each A. baumannii strains to avoid background growth. The single crossover mutants were screened by colony PCR to confirm the presence of resistance cassette.
For counter-selection, at least three trans-conjugant colonies were selected and streaked on freshly prepared low-salt LB agar plates containing 5% sucrose. The plates were incubated at room temperature (RT) for 24-48h until well defined colonies were obtained. The colonies from sucrose plates were patched onto a fresh selective LB agar plate as well as a fresh LB agar plate supplemented with sucrose to confirm the excision of the plasmid backbone. The selection plates were incubated at 37°C and the sucrose plates were incubated at RT. Colonies that grew on the LB sucrose plate but not on the selective LB plate were screened by PCR to identify the knockout mutants. Genomic DNA was extracted from the deletion mutants using the DNeasy ultraclean microbial kit (Qiagen) and mutants were confirmed by genome sequencing. A detailed step by step protocol is included as the supplementary material S1.