Positive autoregulation of mrkHI by the cyclic di-GMP-dependent MrkH protein in the biofilm regulatory circuit of Klebsiella pneumoniae.
Klebsiella pneumoniae is 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 by K. pneumoniae facilitates 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 facilitating K. pneumoniae attachment 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 the mrkHI operon. Mutational analysis, electrophoretic mobility shift assay, and quantitative PCR experiments demonstrated that MrkH binds to the cognate DNA sequence to autoregulate mrkHI expression 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 activating mrkHI expression, implying that a direct interaction between MrkH and RNA polymerase exists. In vivo analyses 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 of mrkHI expression 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 the mrkHI promoter to autoactivate mrkH expression. Increased MrkH production subsequently drives mrkABCDF expression when activated by c-di-GMP, leading to biosynthesis of type 3 fimbriae and biofilm formation.


OBJECTIVE
Bacterial 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 the mrkHI operon. In this positive-feedback loop, whereby MrkH activates its own production, K. pneumoniae has 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.
Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia.
Jason W H
JW
Tan
Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia wilkschj@unimelb.edu.au.
Jonathan J
JJ
Wilksch
Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia.
Dianna M
DM
Hocking
Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia.
Nancy
N
Wang
Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia.
Yogitha N
YN
Srikhanta
Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia.
Marija
M
Tauschek
Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Trevor
T
Lithgow
Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia.
Roy M
RM
Robins-Browne
Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia.
Ji
J
Yang
Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia.
Richard A
RA
Strugnell
D001422:Bacterial Adhesion; D001665:Binding Sites; D018441:Biofilms / Q000254:growth & development; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D010861:Fimbriae, Bacterial / Q000502:physiology; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D006706:Homeostasis; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D014157:Transcription Factors; C062025:bis(3',5')-cyclic diguanylic acid; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit; D006152:Cyclic GMP