Fig. 1: Identification of genes involved in the regulation of IncX3 plasmid conjugation.

a Genetic map of the pHNYX644 plasmid. This plasmid belongs to the IncX3 incompatibility group and contains carbapenemase gene bla_NDM-5 and bleomycin-resistant gene ble-MBL. Colors are coded by function as follows: blue, conjugation; cyan, prfaH (a gene that activates conjugative transfer); yellow, phns (a gene that inhibits conjugative transfer); red, replication; orange, antibiotic resistance; gray, transposase; white, unknown; partitioning and maintenance, pink. b Conjugation rates of pHNYX644∆rs00140, pHNYX644∆00145(phns), pHNYX644∆rs00175, pHNYX644∆rs00245(prfaH), pHNYX644∆rs00240, pHNYX644∆rs00260, pHNYX644∆rs00270, pHNYX644∆rs00275, pHNYX644∆rs00285, pHNYX644∆rs00290, and pHNYX644∆rs00300. Individual values were obtained from n = 3 independent conjugation assays and represented by black dots; bars represent the mean. Statistical analyses were performed using t-test with Welch’s correction. The p-values are shown when making comparisons with the wild-type (WT). The asterisk denotes that the transfer rate fell beneath the detectable threshold (~1\(\times\)10⁻¹⁸). c The effect of chromosomal homologs RfaH and H-NS on the IncX3 plasmid conjugation. WT represents the wild-type pHNYX644 plasmid; ∆prfaH represents the pHNYX644∆prfaH plasmid;∆phns represents the pHNYX644∆phns plasmid. Complementation assays were conducted by expressing prfaH, chromosomal rfaH, phns, and chromosomal hns with their corresponding native promoters on pHSG575 plasmids, and the resulting plasmids were named P-prfaH, P-chr-rfaH, P-phns, and P-chr-hns, respectively. Conjugation assays were performed with n = 3 biological replicates. Statistical comparisons of ∆phns with ∆phns/P-phns and ∆phns/P-chr-hns were performed using one-way ANOVA with Dunnett’s T3 correction. Statistical comparison of ∆prfaH/P-prfaH with WT was performed using t-test with Welch’s correction. d RT-qPCR analysis of the effect of PrfaH on the expression of virB genes. WT (wild type): BW25113/pHNYX644, ∆prfah: BW25113/pHNYX644∆prfah, ∆prfah/pHSG575-prfaH: BW25113/pHNYX644∆prfah + pHSG575-prfaH. RT-qPCR experiments were performed with n = 3 biological replicates. The statistical comparison of differences between the two groups were performed using t-test with Welch’s correction. e PrfaH enhances the expression of virB operon but not as a promoter activator. The promoter region of virB1 or prfaH was fused with a promoterless lacZ gene in pHGR01 plasmid. The +1 represents the first nucleotide of the open reading frame of prfaH or virB1. Dashed arrows without color annotation indicate gene deletions. IGR represents the intergenic region between rs000240 and virB1. β-Galactosidase activity for each construct was detected in the absence or presence of PrfaH. f The effect of the potential termination signal ahead of virB1 on the function of PrfaH. IGRm1 represents IGR lacking m1 region(gray); IGRm2 represents IGR lacking m2 region(purple); IGRm3 represents IGR harboring GG-AA mutations (brown font); IGRm4 represents deletion of the IGR fragments. All \({{\rm{\beta }}}\)-Galactosidase assays were performed with n = 3 biological replicates. The t-test with Welch’s correction was used for comparison of differences between the two groups.