Fig. 8: The conjugation regulation of IncX3 plasmids and its role on plasmids persistence under different nutritional conditions.

a Model of conjugation regulation of IncX3 plasmids. The expression of PrfaH is directly repressed by H-NS (colored by orange). The prfaH and virB genes are co-transcribed. The PrfaH activates the expression of virB cluster via its transcriptional antitermination activity. RNA polymerase (RNAP) (colored by gray) paused at the ops site (colored by red) upstream of the ATG codon of prfaH. Then, PrfaH was recruited by this ops signal and binds with RNAP. The RNAP-PrfaH complex can further recruit ribosomes. Through the mechanism of transcription and translation coupling, this complex enhances the expression of virB cluster by resisting the potential termination signals. Deletion of prfaH completely abolished the conjugation of the IncX3 plasmid. b The PrfaH-H-NS regulon’s role in the persistence of IncX3 plasmids under LB or M9 medium conditions. The plasmid-free strain represents the E. coli BW25113 without plasmids. The phns⁺ prfaH⁺ strain represents the E. coli BW25113 with the wild-type IncX3 plasmid. The phns⁻ prfaH⁺ strain represents the E. coli BW25113 with the phns-deficient IncX3 plasmid. The phns⁻ prfaH^- strain represents the E. coli BW25113 harboring the IncX3 plasmid with deletion both of phns and prfaH. The fitness and conjugation ability of each strain were shown in the diagram. PrfaH is the master activator for the conjugation of IncX3 plasmids. However, overactivation of the conjugative transfer system usually impairs the fitness of the host strain. The IncX3 plasmids encoded H-NS maintain the host fitness by directly repressing the expression of prfaH. So, the phns⁺ prfaH⁺ strain could persist stably in the environment (nutrition-rich or poor condition) for the long term. The phns-deficient plasmids possess the highest transfer ability but incur a high cost on their host. Their persistence is highly dependent on the growth environment. Under nutrition-rich conditions (e.g., LB broth), these plasmids could quickly invade large plasmid-free populations. However, under conditions of nutritional deprivation (e.g., M9 medium), the contribution of high-frequency conjugative transfer to plasmid persistence is counteracted by the high fitness cost.