Bacterial conjugation.

E. coli C600[R388/plv-dCas9-R(0–6)] was transformed with the aforementioned recombinant plasmid pINT-cassette, yielding donor E. coli C600[R388/pINT-cassette/plv-dCas9-R(0–6)]. Since plasmids R388, plv-dCas9-R(0–6), and pINT-cassette possess distinct replication origins, they are compatible with each other in E. coli. To examine the HGT of ARGs from the above donor strain to the recipient E. coli J53, conjugation experiments (17) were performed by the method of filter mating as described previously (35). The donors were incubated overnight in LB broth at 37°C and then diluted 100-fold with fresh medium containing 25 μg/ml CM, 25 μg/ml kanamycin (KAN), and 32 μg/ml TMP. After 4 h of cultivation, 2 μM aTc and 0.5 mM IPTG were simultaneously added into the donor to facilitate the integration of ARG cassettes into the R388 class 1 integron upon CRISPRi induction. Each donor received only 0.5 mM IPTG for use as a reference. The recipient was cultivated overnight in LB medium and then diluted 100-fold with fresh medium containing 100 μg/ml sodium azide (NaN₃). When the OD₆₀₀ reached 1.0, the donor and recipient strains were pelleted and resuspended with sterile phosphate-buffered saline (PBS). Subsequently, the donor and recipient strains were mixed at a ratio of 3:1. After the mixture was pelleted and resuspended in 20 μl of PBS, filter mating was carried out by spotting the mixture onto a 0.45-μm-pore-size filter (Millipore) on an LB plate. After 16 to 18 h of conjugation, cells were harvested by vigorously vortexing the filter in 1 ml of aseptic PBS. The mixture was serially diluted using PBS and plated onto LB agar containing 1,024 μg/ml SUL and 100 μg/ml NaN₃ for screening total transconjugants with R388 plus those with R388-aadA1 or R388-aadB. As the genes aadA1 and aadB confer resistance to streptomycin (STR) and gentamicin (GEN), respectively, the mixture was transferred to an LB plate containing 1,024 μg/ml SUL, 25 μg/ml STR, and 100 μg/ml NaN₃ to screen for transconjugant E. coli J53(R388-aadA1). In parallel, the mixture was plated onto an LB plate containing 1,024 μg/ml SUL, 25 μg/ml GEN, and 100 μg/ml NaN₃ to screen for transconjugant E. coli J53(R388-aadB). The HGT rate of aadA1 or aadB was defined as the ratio of transconjugants with R388-aadA1 or R388-aadB to total transconjugants. The donor and recipient strains were independently screened on triple-resistance plates to exclude the effects of spontaneous mutation on the mating assay. To unravel whether ARGs could move from the donor to the recipient, a conjugation experiment was first performed between the recipient E. coli J53 and the control donor E. coli C600(R388/plv-dCas9-R0/pINT-cassette) without CRISPRi induction. The genetic identity of transconjugants with R388-aadA1 or R388-aadB was investigated by PCR using the specific primers Integration-CX-F/aadA1-CX-R to amplify intI1-attI-aadA1 in transconjugant E. coli J53(R388-aadA1) and Integration-CX-F/aadB-CX-R to amplify intI1-attI-aadB in transconjugant E. coli J53(R388-aadB). All primers used for sequencing are listed in Table S2.