cAMP-CRP promotes ColE1 plasmid replication by reducing RNAI stability through transcriptional repression of hfq

Plasmids play a crucial role in the spread of antibiotic resistance genes (ARGs), with their replication controlled by both host and plasmid-encoded factors. ColE1-type plasmids, prevalent in Enterobacterales, are key carriers of ARGs. Their replication relies on interactions between two plasmid-encoded RNAs, RNAI and RNAII, and the host regulatory factor cAMP-CRP. However, the precise interplay between these RNAs and cAMP-CRP remains elusive. Previous studies have shown that Hfq, a conserved RNA chaperone, stabilizes RNAI to inhibit ColE1 plasmid replication, while cAMP-CRP represses hfq expression. Nevertheless, a direct mechanistic link between these regulatory pathways remains unresolved. Here, we reveal that cAMP-CRP promotes ColE1 plasmid replication by downregulating hfq expression, thereby reducing RNAI stability. First, we examined the effect of carbon sources on ColE1 plasmid replication and observed that glycerol led to higher plasmid yields compared to glucose. Notably, the addition of cAMP reversed the inhibitory effect of glucose on replication. Next, we examined the previously reported role of cAMP-CRP and validated that cAMP-CRP stimulates ColE1 plasmid replication. However, in Δhfq mutants, inactivating crp or the adenylate cyclase cyaA gene did not further decrease plasmid yields, suggesting that cAMP-CRP regulates replication through Hfq. Further analysis revealed that cAMP-CRP reduced RNAI stability by repressing hfq expression, thereby stimulating ColE1 plasmid replication. Importantly, we found that the cAMP-CRP-Hfq-RNAI regulatory axis is conserved among ColE1 plasmids across Enterobacterales. These findings uncover a novel mechanism by which cAMP-CRP regulates ColE1 plasmid replication and provide new insights into potential strategies for ARGs spread.