MrKH, a novel c-di-GMP-dependent transcriptional activator, controls klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression

Wilksch, JJ; Yang, J; Clements, A; Gabbe, JL; Short, KR; Cao, H; Cavaliere, R; James, CE; Whitchurch, CB; Schembri, MA; Chuah, MLC; Liang, ZX; Wijburg, OL; Jenney, AW; Lithgow, T; Strugnell, RA

MrKH, a novel c-di-GMP-dependent transcriptional activator, controls klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression

Wilksch, JJ Yang, J Clements, A Gabbe, JL Short, KR Cao, H Cavaliere, R

James, CE Whitchurch, CB

Schembri, MA Chuah, MLC Liang, ZX Wijburg, OL Jenney, AW Lithgow, T Strugnell, RA

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Publication Type:: Journal Article
Citation:: PLoS Pathogens, 2011, 7 (8)
Issue Date:: 2011-08-01

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Field	Value	Language
dc.contributor.author	Wilksch, JJ	en_US
dc.contributor.author	Yang, J	en_US
dc.contributor.author	Clements, A	en_US
dc.contributor.author	Gabbe, JL	en_US
dc.contributor.author	Short, KR	en_US
dc.contributor.author	Cao, H	en_US
dc.contributor.author	Cavaliere, R https://orcid.org/0000-0002-4709-5081	en_US
dc.contributor.author	James, CE	en_US
dc.contributor.author	Whitchurch, CB https://orcid.org/0000-0003-2296-3791	en_US
dc.contributor.author	Schembri, MA	en_US
dc.contributor.author	Chuah, MLC	en_US
dc.contributor.author	Liang, ZX	en_US
dc.contributor.author	Wijburg, OL	en_US
dc.contributor.author	Jenney, AW	en_US
dc.contributor.author	Lithgow, T	en_US
dc.contributor.author	Strugnell, RA	en_US
dc.date.available	2011-06-24	en_US
dc.date.issued	2011-08-01	en_US
dc.identifier.citation	PLoS Pathogens, 2011, 7 (8)	en_US
dc.identifier.issn	1553-7366	en_US
dc.identifier.uri	http://hdl.handle.net/10453/37917
dc.description.abstract	Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices. © 2011 Wilksch et al.	en_US
dc.relation.ispartof	PLoS Pathogens	en_US
dc.relation.isbasedon	10.1371/journal.ppat.1002204	en_US
dc.subject.classification	Virology	en_US
dc.subject.mesh	Fimbriae, Bacterial	en_US
dc.subject.mesh	Biofilms	en_US
dc.subject.mesh	Klebsiella pneumoniae	en_US
dc.subject.mesh	Phosphoric Diester Hydrolases	en_US
dc.subject.mesh	Phosphorus-Oxygen Lyases	en_US
dc.subject.mesh	Bacterial Proteins	en_US
dc.subject.mesh	Escherichia coli Proteins	en_US
dc.subject.mesh	DNA, Bacterial	en_US
dc.subject.mesh	Cyclic GMP	en_US
dc.subject.mesh	Gene Expression Regulation, Bacterial	en_US
dc.subject.mesh	Gene Deletion	en_US
dc.subject.mesh	Amino Acid Sequence	en_US
dc.subject.mesh	Protein Binding	en_US
dc.subject.mesh	Plasmids	en_US
dc.subject.mesh	Molecular Sequence Data	en_US
dc.subject.mesh	Transcriptional Activation	en_US
dc.title	MrKH, a novel c-di-GMP-dependent transcriptional activator, controls klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression	en_US
dc.type	Journal Article
utslib.citation.volume	8	en_US
utslib.citation.volume	7	en_US
utslib.for	0605 Microbiology	en_US
utslib.for	1107 Immunology	en_US
utslib.for	1108 Medical Microbiology	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - ithree - Institute of Infection, Immunity and Innovation
utslib.copyright.status	open_access
pubs.issue	8	en_US
pubs.publication-status	Published	en_US
pubs.volume	7	en_US

Abstract:

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices. © 2011 Wilksch et al.

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http://hdl.handle.net/10453/37917