Tetracycline induces wsp operon expression to promote biofilm formation in Pseudomonas putida

The overuse and wanton discharge of antibiotics produces a threat to bacteria in the environment, which, in turn, stimulates the more rapid emergence of antibiotic-resistant bacteria. actively forms biofilms to protect the population under tetracycline stress, but the molecular mechanism remains unclear. This study found that tetracycline at sub-minimal inhibitory concentrations increased cyclic diguanylate (c-di-GMP), a second messenger that positively regulates biofilm formation. Four c-di-GMP-metabolizing proteins were found to be involved in the tetracycline-mediated biofilm promotion, including DibA, WspR, PP_3242, and PP_3319. Among them, the diguanylate cyclase WspR displayed the most significant effect on c-di-GMP level and biofilm formation. belongs to the operon comprising seven genes ( and ). The operon contained six promoters, including one major start promoter (P ) and five internal promoters (P , P , P , P , and P ), and tetracycline promoted the activity of P . The stress-response sigma factor RpoS directly bound to P and positively regulated its activity under tetracycline stress. Moreover, RpoS was required for tetracycline to induce P activity and promote biofilm formation. Our results enrich the transcriptional regulation of the operon and reveal the mechanism by which tetracycline promotes biofilm formation in .IMPORTANCEThe overuse and wanton discharge of antibiotics produces a threat to bacteria in the environment, which, in turn, stimulates the more rapid emergence of antibiotic-resistant bacteria. The actively forms biofilm against antibiotic threats, but the mechanism remains unclear. Here, our results showed that tetracycline treatment at sub-minimal inhibitory concentrations could induce the expression of the Wsp system via the sigma factor RpoS in , resulting in elevated c-di-GMP levels, which leads to increased biofilm formation. The operon contains one major promoter and five internal promoters, and RpoS directly binds to the major promoter to promote its activity.