Figure 1.
Biofilm formation by K. pneumoniae AJ218.
Biofilm formation by K. pneumoniae AJ218 wild-type, including isogenic mutants and strains harboring trans-complementing plasmids. Biofilm formation was determined using the static microtiter plate assay following incubation in M63B1-GCAA minimal media (supplemented with 1% glycerol and 0.3% casamino acids) for 24 h under static conditions. Results are expressed as a percentage of the biofilm produced by the wild-type AJ218 strain, which is set to 100%. All values represent the mean of four replicate sample wells for each strain performed in two independent experiments. The error bars represent the standard deviation. Statistical significance between AJ218 wild-type and isogenic mutants was analyzed by one-way ANOVA and Tukey HSD post-hoc comparisons are reported, where *** = P<0.001.
Table 1.
Identification of genetic loci participating in K. pneumoniae AJ218 biofilm formation.
Figure 2.
The mrkABCDF and mrkHIJ loci in K. pneumoniae AJ218.
(A) Genetic organization of the mrkABCDF and mrkHIJ gene clusters from K. pneumoniae strains AJ218, NTUH-K2044 (GenBank Ref: AP006725), MGH 78578 (GenBank Ref: CP000647) and 342 (GenBank Ref: CP000964). (B) RT-PCR analysis of mrkHIJ transcription. PCR amplicon products of either RNA (−), reverse transcribed DNA (+) or genomic DNA (gDNA) were visualized on a 1% agarose gel. The mrkH-I product was generated with primer mrkI-R and amplified with primers mrkH-F and mrkI-R. The mrkI-J product was generated with primer mrkJ-R and amplified with primers mrkI-F and mrkJ-R.
Figure 3.
Flow-cell-cultivated biofilm formation by K. pneumoniae AJ218 after 4 days.
(A) Confocal laser scanning micrographs of biofilms formed by K. pneumoniae AJ218 wild-type, ΔmrkH, ΔmrkH [pACYC184] and ΔmrkH [pMrkH] strains. Biofilms were stained with Syto64 to visualize cells and are shown in green as maximum intensity volume rendered projections. (B) COMSTAT analysis of biofilm biomass. The error bars represent the standard deviation. Statistical significance between AJ218 wild-type and ΔmrkH strains was analyzed by one-way ANOVA and Tukey HSD post-hoc comparisons are reported, where *** = P<0.001.
Figure 4.
Conservation of PilZ-, EAL- and GGDEF-domain proteins in K. pneumoniae.
Multiple sequence alignment of the (A) PilZ domain of MrkH, (B) EAL domain of MrkJ and (C) GGDEF domain of YfiN from K. pneumoniae AJ218 and other experimentally studied proteins, generated by ClustalW2 [92] and formatted with ESPript [93]. Residues showing strict identity are written in white characters and highlighted in red. Similarity across groups is indicated with black bold characters and highlighted in yellow. Residues required for c-di-GMP binding in the PilZ domain which form two conserved motifs, including the putative catalytic active site residues within the EAL and GGDEF domain, are marked with an asterisk. Protein names and organisms are as follows: MrkH, MrkJ, YfiN: K. pneumoniae AJ218; BcsA: Gluconacetobacter xylinus NBRC 3288, YcgR: E. coli K-12; YhjH, AdrA: Salmonella Typhimurium LT2; Alg44, PA4608, RocR, WspR: Pseudomonas aeruginosa PA14; VCA0042, VieA, CdgA: Vibrio cholerae O395; DgrA, CC3396, PleD: Caulobacter cresentus CB15.
Figure 5.
Type 3 fimbriae expression by K. pneumoniae AJ218.
(A) Mannose resistant Klebsiella-like hemagglutination (MR/K HA) assays using human erythrocytes. MR/K HA titer is expressed as the lowest concentration (CFU/mL) of bacteria causing a visible agglutination reaction. Values represent the mean of three independent experiments. The error bars represent the standard deviation. Statistical significance between AJ218 wild-type and isogenic mutants was analyzed by one-way ANOVA and Tukey HSD post-hoc comparisons are reported, where *** = P<0.001. (B) Cell lysates were prepared from the indicated strains and analyzed by SDS-PAGE and immunoblotting with anti-MrkA antiserum. The MrkA pilin monomer (which migrates at approximately 21 kDa) is labeled.
Figure 6.
Quantitative RT-PCR analysis of mrkA RNA levels.
Fold differences in mrkA transcript expression levels compared to K. pneumoniae AJ218 wild-type levels are shown for the indicated K. pneumoniae strains. The mrkA transcription expression was normalized to rpoD concentrations. Values represent the mean of reactions performed in triplicate. The error bars represent the standard deviation.
Figure 7.
Transcriptional analysis of the mrkA regulatory region.
(A) The nucleotide sequence of the mrkA regulatory region is shown. The numbering on the left of the sequence (in brackets) is relative to the transcriptional start site of mrkA. The numbering on the right of the sequence is relative to the start codon of the mrkA coding sequence. The transcriptional start site is marked with an angled arrow and the putative -35 and -10 regions of the mrkA promoter are indicated and underlined. (B) Promoter activities of various mrkA-lacZ transcriptional fusions in the MrkH– (MC4100 containing pACYC184) and MrkH+ (MC4100 containing pMrkH) E. coli backgrounds are shown as specific activities of β-galactosidase (Miller) units which are the mean values from three independent assays, with variation <15%. Fold activation (Fold act.) is the specific activity of β-galactosidase of the MrkH+ strain divided by that of the MrkH− strain. The numbers shown above the various mrkA fragments are relative to the start site of transcription (angled arrows) and the lengths of the various mrkA fragments are not to scale. (C) The effect of MrkH on transcription of the mrkA promoter was analyzed by a CAT assay in three isogenic K. pneumoniae backgrounds: MrkH− (ΔmrkH + pACYC184-KmR), MrkH+ haploid (wild-type + pACYC184-KmR), and multi-copy MrkH+ (ΔmrkH + pACYC184-KmR-mrkH). Specific CAT activities are the averages of three independent assays and the standard deviation values are shown. Values in brackets are fold activation (for details, see above).
Figure 8.
Mapping the start site of transcription of the mrkA promoter by primer extension.
Total cellular RNA was purified from E. coli MC4100 strains containing pMrkH with either pMU2385 (control) or mrkA-lacZ-2. The RNA samples were then hybridized with 32P-labelled primer Px1mrkARev. Primer extension was performed using AMV reverse transcriptase in the presence of dNTPs. GA Ladder: GA sequence ladder prepared using the mrkA PCR fragment generated using primer pairs 32P-Px1mrkARev and mrk295F. Lane 1: control experiment using RNA from E. coli MC4100 strain containing pMrkH and pMU2385. Lane 2: experiment using RNA from E. coli MC4100 strain containing pMrkH and mrkA-lacZ-2. The positions corresponding to 32P-Px1mrkARev primer and the extension product are marked.
Figure 9.
Analysis of the binding of MrkH-8×His to the mrkA regulatory region by EMSA.
The 32P-labelled PCR fragment containing the mrkA regulatory region was generated using primer pairs 32P-Px1mrkARev and mrk295F. The mrkA fragment was mixed with varying amounts of the purified MrkH-8×His protein (from 0 to 500 nM) in the absence or presence of c-di-GMP (200 µM). Following incubation at 30°C for 20 min, the samples were analyzed on native polyacrylamide gels. The right-hand panel shows control reactions with approximately 100-fold molar excess of the unlabeled (cold) mrkA promoter fragment (specific competitor DNA), used to demonstrate the specificity of the c-di-GMP-mediated MrkH binding to the mrkA promoter region. The unbound DNA (F) and protein-DNA complexes (C1, C2 and C3) are marked.
Figure 10.
Analysis of a PilZ-domain mutation on MrkH-mediated transcriptional activation, biofilm formation and type 3 fimbriae expression.
(A) Amino acids within the conserved PilZ domain are shown and residues in red are those known to be critical for c-di-GMP binding in other studies [50]. As indicated, the MrkH mutation carries an arginine to alanine change at position 113. β-galactosidase assays were performed with E. coli MC4100 strains: MrkH− (carrying reporter plasmid mrkA-lacZ-2 and pACYC184), MrkH+ (carrying mrkA-lacZ-2 and pMrkH) and MrkH:113R-A (carrying mrkA-lacZ-2 and pMrkH:113R-A). The specific β-galactosidase activities are as follows: MrkH−: 11±0.9; MrkH+: 3760±186 and MrkH:113R-A: 16±1.5. Values represent the mean of three replicate samples. (B) Static biofilm formation assay by the indicated K. pneumoniae strains. Values represent the mean of four replicate sample wells for each strain performed in two independent experiments. (C) MR/K HA assay of the indicated K. pneumoniae strains. Values represent the mean of three independent experiments. The error bars represent the standard deviation. Statistical significance between MrkH+ and other strains (β-galactosidase assay) was analyzed by the Van der Waerden test, and significance between AJ218 wild-type and ΔmrkH strains (biofilm and MR/K HA assays) was analyzed by one-way ANOVA. Tukey HSD post-hoc comparisons are reported, where *** = P<0.001, * = P<0.05.
Figure 11.
Analysis of EAL- and GGDEF-domain mutations on MrkH-mediated transcriptional activation, biofilm formation and type 3 fimbriae expression.
(A) Amino acids within the conserved EAL and GGDEF domains of MrkJ and YfiN were substituted with alanine residues. β-galactosidase assays were performed with: E. coli MC4100 strain MrkH+ (carrying reporter plasmid mrkA-lacZ-2 and pMrkH), including pBR322-derived vectors pBMrkJ (wild-type), pBMrkJ:36ECL-AAA, pBYfiRNB (wild-type) or pBYfiRNB:328DEF-AAA. Values represent the mean of three replicate samples. (B) Static biofilm formation assay by the indicated K. pneumoniae strains. Values represent the mean of four replicate sample wells for each strain performed in two independent experiments. (C) MR/K HA assay of the indicated K. pneumoniae strains. Values represent the mean of three independent experiments. The error bars represent the standard deviation. Statistical significance between MrkH+ (wild-type) and other strains (β-galactosidase assay), as well as AJ218 wild-type and isogenic mutant strains (MR/K HA assay) was analyzed by the Van der Waerden test. Significance between AJ218 wild-type and isogenic mutant strains (biofilm assay) was analyzed by one-way ANOVA. Tukey HSD post-hoc comparisons are reported, where *** = P<0.001, * = P<0.05.
Figure 12.
MrkJ displays strong phosphodiesterase activity.
C-di-GMP hydrolysis by purified MrkJ was analyzed by HPLC. The formation of 5′-pGpG was monitored at various time-points and the c-di-GMP-specific phosphodiesterase RocR [94] was used as the positive control.
Figure 13.
Model of c-di-GMP-mediated control of type 3 fimbriae expression and biofilm formation in K. pneumoniae.
Signals resulting in increased/decreased intracellular concentration of c-di-GMP, via changes in the relative activities of DGCs (carrying a GGDEF domain [YfiN]) and PDEs (carrying an EAL domain [MrkJ]), direct the DNA-binding activity of the c-di-GMP receptor MrkH (carrying a PilZ domain). High c-di-GMP levels promote biofilm formation through MrkH:c-di-GMP-dependent transcriptional activation of the mrkABCDF operon encoding type 3 fimbriae. The major pilin subunit MrkA is bound by the MrkB chaperone in the periplasm to activate it for polymerization by the usher translocase MrkC. Once sufficiently elongated, the fimbriae would emerge through the extracellular capsule layer (represented by grey shading surrounding the cell). Conversely, low c-di-GMP levels promote biofilm dispersal and the planktonic state through a decrease in activated MrkH:c-di-GMP availability. Whether the LuxR-like regulator MrkI acts on mrkABCDF gene expression, or the expression of other factors influencing type 3 fimbriae polymerization or capsule polysaccharide production remains to be determined.
Table 2.
Bacterial strains and plasmids used in this study.