Type 3 Fimbriae and Biofilm Formation Are Regulated by the Transcriptional Regulators MrkHI in Klebsiella pneumoniae

Department of Microbiology, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
Journal of bacteriology (Impact Factor: 2.81). 05/2011; 193(14):3453-60. DOI: 10.1128/JB.00286-11
Source: PubMed


Klebsiella pneumoniae is an opportunistic pathogen which frequently causes hospital-acquired urinary and respiratory tract infections. K. pneumoniae may establish these infections in vivo following adherence, using the type 3 fimbriae, to indwelling devices coated with extracellular matrix components. Using
a colony immunoblot screen, we identified transposon insertion mutants which were deficient for type 3 fimbrial surface production.
One of these mutants possessed a transposon insertion within a gene, designated mrkI, encoding a putative transcriptional regulator. A site-directed mutant of this gene was constructed and shown to be deficient
for fimbrial surface expression under aerobic conditions. MrkI mutants have a significantly decreased ability to form biofilms
on both abiotic and extracellular matrix-coated surfaces. This gene was found to be cotranscribed with a gene predicted to
encode a PilZ domain-containing protein, designated MrkH. This protein was found to bind cyclic-di-GMP (c-di-GMP) and regulate
type 3 fimbrial expression.

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Available from: Jeremiah Johnson, Aug 14, 2014
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    • "Some PDE domain proteins do not exhibit PDE-specific enzyme activity (Römling, 2009), so we studied whether FimK possesses PDE activity. As shown in Fig. 5(a), the purified recombinant FimK exhibited considerably less bis- PNPP catalytic activity than the recombinant MrkJ, for which PDE activity has been reported (Johnson et al., 2011). Recombinant clones expressing EIL fimK and AIL fimK containing the C-terminal EIL domain of FimK and the domain with critical E245 residue replaced with alanine, respectively, were also generated and the corresponding overexpressed proteins were purified. "
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    ABSTRACT: Klebsiella pneumoniae CG43, a heavy encapsulated liver abscess isolate, mainly expresses type 3 fimbriae. Type 1 fimbriae expression was only apparent in CG43S3ΔmrkA (the type 3 fimbriae-deficient strain). The expression of type 1 fimbriae in CG43S3ΔmrkA was reduced by deleting the fimK gene, but was unaffected by removing the 3'end of fimK encoding the C-termial EIL domain (EILfimK). Quantitative reverse-transcription PCR and promoter activity analysis showed that the putative DNA binding region at the N-terminus, but not the C-terminal EIL domain, of FimK positively affects transcription of type 1 fimbrial major subunit, fimA. An electrophoretic mobility shift assay demonstrated that the recombinant FimK could specifically bind to fimS, that is located upstream of fimA and contains a vegetative promoter for the fim operon, also reflecting possible transcriptional regulation. EILfimK was shown to encode a functional phosphodiesterase (PDE) via enhancing motility in Escherichia coli JM109 and in vitro using PDE activity assays. Moreover, EILfimK exhibited higher PDE activity than FimK implying that the N-terminal DNA binding domain may negatively affect the PDE activity of FimK. FimA expression was detected in CG43S3 expressing EILfimK or AILfimK suggesting that FimA expression is not directly influenced by the c-di-GMP level. In summary, FimK influences type 1 fimbriation by binding to fimS at the N-terminal domain, and thereafter, the altered protein structure may activate C-terminal PDE activity to reduce the intracellular c-di-GMP level.
    Full-text · Article · May 2013 · Microbiology
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    • "The pLN126_33 cassette also encodes a highly truncated gene from the downstream region of mrkF. In K. pneumoniae this region contains the mrkHIJ control cassette [38], [39], where mrkJ encodes an EAL-domain phosphodiesterase shown to regulate expression of mrkABCDF through cleavage of the signalling molecule cyclic-di-GMP [40]. The existence of three different lengths of mobile mrkABCDF cassettes suggests a general course of evolution for adaptive loads that result from the recruitment of chromosomal gene cassettes. "
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    ABSTRACT: Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids) into a laboratory strain (Escherichia coli Genehogs®) for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54) and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33) were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.
    Full-text · Article · Aug 2012 · PLoS ONE
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    • "Although the formation of biofilms by K. pneumoniae is still not fully understood, several key determinants have been identified such as pili, polysaccharides, quorum sensing and transport and regulatory proteins [7-13]. More recently, it has been shown that c-di-GMP controls type 3 fimbria expression and biofilm formation in K. pneumoniae by binding to and modulating the activity of the transcriptional regulator MrkH [14,15]. The second messenger c-di-GMP is known to play a key role in several cellular functions as well as in biofilm formation in bacteria where it modulates the transition between planktonic and sessile lifestyles. "
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    ABSTRACT: Klebsiella pneumoniae can be found in environmental habitats as well as in hospital settings where it is commonly associated with nosocomial infections. One of the factors that contribute to virulence is its capacity to form biofilms on diverse biotic and abiotic surfaces. The second messenger Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a ubiquitous signal in bacteria that controls biofilm formation as well as several other cellular processes. The cellular levels of this messenger are controlled by c-di-GMP synthesis and degradation catalyzed by diguanylate cyclase (DGC) and phophodiesterase (PDE) enzymes, respectively. Many bacteria contain multiple copies of these proteins with diverse organizational structure that highlight the complex regulatory mechanisms of this signaling network. This work was undertaken to identify DGCs and PDEs and analyze the domain structure of these proteins in K. pneumoniae. A search for conserved GGDEF and EAL domains in three sequenced K. pneumoniae genomes showed that there were multiple copies of GGDEF and EAL containing proteins. Both single domain and hybrid GGDEF proteins were identified: 21 in K. pneumoniae Kp342, 18 in K. pneumoniae MGH 78578 and 17 in K. pneumoniae NTUH-K2044. The majority had only the GGDEF domain, most with the GGEEF motif, and hybrid proteins containing both GGDEF and EAL domains were also found. The I site for allosteric control was identified only in single GGDEF domain proteins and not in hybrid proteins. EAL-only proteins, containing either intact or degenerate domains, were also identified: 15 in Kp342, 15 in MGH 78578 and 10 in NTUH-K2044. Several input sensory domains and transmembrane segments were identified, which together indicate complex regulatory circuits that in many cases can be membrane associated. The comparative analysis of proteins containing GGDEF/EAL domains in K. pneumoniae showed that most copies were shared among the three strains and that some were unique to a particular strain. The multiplicity of these proteins and the diversity of structural characteristics suggest that the c-di-GMP network in this enteric bacterium is highly complex and reflects the importance of having diverse mechanisms to control cellular processes in environments as diverse as soils or plants and clinical settings.
    Full-text · Article · Jul 2012 · BMC Microbiology
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