Abstract
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. Pseudomonas putida 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. WspR belongs to the wsp operon comprising seven genes (wspA-wspF and wspR). The wsp operon contained six promoters, including one major start promoter (PwspA) and five internal promoters (PwspB, PwspC, PwspD, PwspF, and PwspR), and tetracycline promoted the activity of PwspA. The stress-response sigma factor RpoS directly bound to PwspA and positively regulated its activity under tetracycline stress. Moreover, RpoS was required for tetracycline to induce PwspA activity and promote biofilm formation. Our results enrich the transcriptional regulation of the wsp operon and reveal the mechanism by which tetracycline promotes biofilm formation in P. putida.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 Pseudomonas putida 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 P. putida, resulting in elevated c-di-GMP levels, which leads to increased biofilm formation. The wsp operon contains one major promoter and five internal promoters, and RpoS directly binds to the major promoter to promote its activity.