Positive Autoregulation ofmrkHIby the Cyclic Di-GMP-Dependent MrkH Protein in the Biofilm Regulatory Circuit of Klebsiella pneumoniae

Abstract

ABSTRACTKlebsiella pneumoniaeis an important cause of nosocomial infections, primarily through the formation of surface-associated biofilms to promote microbial colonization on host tissues. Expression of type 3 fimbriae byK. pneumoniaefacilitates surface adherence, a process strongly activated by the cyclic di-GMP (c-di-GMP)-dependent transcriptional activator MrkH. In this study, we demonstrated the critical importance of MrkH in facilitatingK. pneumoniaeattachment on a variety of medically relevant materials and demonstrated the mechanism by which bacteria activate expression of type 3 fimbriae to colonize these materials. Sequence analysis revealed a putative MrkH recognition DNA sequence (“MrkH box”; TATCAA) located in the regulatory region of themrkHIoperon. Mutational analysis, electrophoretic mobility shift assay, and quantitative PCR experiments demonstrated that MrkH binds to the cognate DNA sequence to autoregulatemrkHIexpression in a c-di-GMP-dependent manner. A half-turn deletion, but not a full-turn deletion, between the MrkH box and the −35 promoter element rendered MrkH ineffective in activatingmrkHIexpression, implying that a direct interaction between MrkH and RNA polymerase exists.In vivoanalyses showed that residues L260, R265, N268, C269, E273, and I275 in the C-terminal domain of the RNA polymerase α subunit are involved in the positive control ofmrkHIexpression by MrkH and revealed the regions of MrkH required for DNA binding and transcriptional activation. Taken together, the data suggest a model whereby c-di-GMP-dependent MrkH recruits RNA polymerase to themrkHIpromoter to autoactivatemrkHexpression. Increased MrkH production subsequently drivesmrkABCDFexpression when activated by c-di-GMP, leading to biosynthesis of type 3 fimbriae and biofilm formation.IMPORTANCEBacterial biofilms can cause persistent infections that are refractory to antimicrobial treatments. This study investigated how a commonly encountered hospital-acquired pathogen,Klebsiella pneumoniae, controls the expression of MrkH, the principal regulator of type 3 fimbriae and biofilm formation. We discovered a regulatory circuit whereby MrkH acts as a c-di-GMP-dependent transcriptional activator of both the gene cluster of type 3 fimbriae and themrkHIoperon. In this positive-feedback loop, whereby MrkH activates its own production,K. pneumoniaehas evolved a mechanism to ensure rapid MrkH production, expression of type 3 fimbriae, and subsequent biofilm formation under favorable conditions. Deciphering the molecular mechanisms of biofilm formation by bacterial pathogens is important for the development of innovative treatment strategies for biofilm infections.