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1.
Figure 2

Figure 2. YfiN is a diguanylate cyclase.. From: YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa.

A) A truncated YfiN lacking the transmembrane and periplasmic parts has DGC activity in vitro. C-di-GMP production was observed on TLC plates over time against a positive control (DgcA). The c-di-GMP peak is marked with an arrow. B) YfiN binding of c-di-GMP. Elution and wash fractions from YfiN purification were run on an HPLC column and the absorbance at 254 nm plotted. The elution fraction contains a large c-di-GMP peak, presumably bound by YfiN, and not seen in the wash fraction.

Jacob G. Malone, et al. PLoS Pathog. 2010 March;6(3):e1000804.
2.
Figure 5

Figure 5. Downstream components of the YfiBNR system.. From: YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa.

A) Colony morphologies of mutants lacking potential downstream targets of the YfiBNR system grown on LB Congo Red agar. 1 =  PA01, 2 =  ΔyfiR, 3 =  ΔyfiR pelG::Tn, 4 =  ΔyfiR ΔpslAB, 5 =  ΔyfiR ΔpslAB pelG::Tn, 6 =  ΔyfiR cupA4::Tn, 7 =  ΔyfiR cupB4::Tn, 8 =  ΔyfiR cupC2::Tn. B) Attachment of mutants lacking potential downstream targets of the YfiBNR system, relative to PA01. C) YfiBNR effects on downstream gene transcription. Values are expressed as the percentage of luminescence reporter gene expression in ΔyfiR or ΔyfiBNR, as compared to PA01 wild type. The control strain contains pME6032-luxCDABE. Transcription of pel is massively up-regulated in ΔyfiR, and down regulated in ΔyfiBNR.

Jacob G. Malone, et al. PLoS Pathog. 2010 March;6(3):e1000804.
3.
Figure 7

Figure 7. YfiN-mediated SCVs contribute to persistence in vitro and in vivo.. From: YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa.

A) Tobramycin survival assay with PA01 wild type (open triangles) and ΔyfiR mutant (open circles). ΔyfiR suppressor colonies are shown as red circles. Statistically significant differences between PA01and ΔyfiR are marked with asterisks (** = p<0.01, * = p<0.05). B) Single infections with PA01 wild type (black symbols) and ΔyfiR mutant (open symbols). Catheter samples (circles) are presented as cfu/ml, tissue samples (squares) as cfu/mg tissue. Statistically significant differences are marked with an asterisk (* = p<0.05). C) Competition experiments with PA01 wild type and ΔyfiR mutant. The graph shows the ratio of wild type to ΔyfiR mutant colonies recovered from catheters (C, circles) and tissue (T, squares) after four and eight weeks, respectively. Values above 1 indicate greater numbers of wild type colonies, values below 1 greater numbers of SCVs.

Jacob G. Malone, et al. PLoS Pathog. 2010 March;6(3):e1000804.
4.
Figure 1

Figure 1. The YfiBNR system of Pseudomonas aeruginosa.. From: YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa.

A) Organization of the yfiBNR operon. Transposon insertions in yfiR inducing the SCV phenotype are marked with grey triangles. B) Domain organization of the Yfi proteins. S.S. (YfiR) denotes an export signal sequence, vertical grey bars (YfiN) represent transmembrane helices, and OMP (YfiB) denotes the OmpA/Pal-like protein fold. The HAMP and GGDEF domains of YfiN are labeled. C) Colony morphology of the yfiR::Tn mutants grown on LB Congo-red agar. The morphology of the non-polar yfiR deletion is indistinguishable from the yfiR::Tn strains. D) Over-expression of the phosphodiesterase PA5295 abolishes surface attachment and SCV morphology of the ΔyfiR mutant. PA5295-AAL denotes an active site mutant of PA5295. Attachment levels are expressed relative to PA01. The graph shows a representative of two independent experiments.

Jacob G. Malone, et al. PLoS Pathog. 2010 March;6(3):e1000804.
5.
Figure 6

Figure 6. YfiN-mediated SCVs resist nematode predation and phagocytosis.. From: YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa.

A) SCV resistance to nematode predation. Panel 1 shows C. elegans incubated with PA01 as a food source for 72 hours. Panels 2 to 5 show the corresponding experiments with ΔyfiR (2), ΔyfiR pelG::Tn (3), ΔyfiR ΔpslAB (4) and ΔyfiR pelG::Tn ΔpslAB (5), respectively. Eggs and mature adults were seen with PA01 (1) and the double exopolysaccharide mutant (5) only. Worms and larvae are indicated with black arrows. Insets to panels 1 and 2 show nematodes incubated for three hours with GFP-labeled PA01 or ΔyfiR, respectively. B) Macrophage absorption assay. Values shown are the fraction of J774 macrophages whose lysosomes co-localize with at least one bacterium. White bars show PA01 background strains, dark grey bars denote a ΔyfiR background. C) Surface attachment patterns of ΔyfiR exopolysaccharide mutants. Panels show attachment to polystyrene of GFP-labeled bacteria (green) following three hours growth at 37°C. J774 macrophages are stained with DAPI (blue) and Lysotracker (red).

Jacob G. Malone, et al. PLoS Pathog. 2010 March;6(3):e1000804.
6.
Figure 3

Figure 3. Epistasis and regulation of the YfiBNR system.. From: YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa.

A) Attachment of yfiBNR mutant strains. The bars marked ‘ΔyfiBNR Tn7::’ depict Tn7 complementation strains containing variants of the yfiBNR operon inserted into the att-Tn7 site of ΔyfiBNR. For these, ‘FL’ refers to the full-length yfiBNR operon, ‘A-site’ refers to the yfiN active-site mutant D330A, and ‘I site’ to the yfiN feedback-inhibition-site mutant R319A. Strains without an active copy of yfiN (light grey) display reduced attachment compared to those containing active copies of both yfiN and yfiR (white). Strains missing yfiR but containing yfiN (dark grey) showed a large increase in attachment. Immunoblots show the levels of YfiB and YfiN present in each strain. B) Colony morphology of selected Tn7 complementation strains. Deletion of yfiR induces a small colony variant phenotype. Deletion of yfiB has no effect on morphology, either alone or combined with an yfiR deletion. C) Over-expression of yfiB in trans induces SCV colony morphology and stimulates PA01 attachment in an yfiN- and yfiR-dependent manner. yfiB expression is induced in Tn7 complementation strains, which are labeled as in 3A. The colony morphologies of these strains with and without induction of yfiB expression are shown on the right. D) YfiR and YfiN expressed in trans act antagonistically on PA01 colony morphology and attachment. The X-axis of the graph shows the plasmids present in each case. Colony morphologies with and without induction of yfiN expression are shown on the right. E) The full-length yfiBNR operon (FL) and ΔyfiR Tn7 complementation constructs were inserted into the att-Tn7 site of PA01. Attachment levels for all assays are shown relative to PA01.

Jacob G. Malone, et al. PLoS Pathog. 2010 March;6(3):e1000804.
7.
Figure 4

Figure 4. The YfiBNR complex.. From: YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa.

A) Membrane localization of YfiB and YfiR. Membrane fractionation was carried out with PA01 yfiR-M2. The separate fractions are labeled as follows: C  =  whole cell sample, L  =  cell lysate, S  =  soluble fraction, M  =  membrane fraction. YfiB localizes to the membrane fraction, YfiR-M2 to the soluble fraction. B) Periplasmic localization of YfiR. Periplasmic fractionation was carried out with PA01 yfiR-M2 pAD6Ω, and YfiR-M2 and GFP were detected by immunoblot analysis. GFP localizes to the spheroplast (cytosolic/membrane) fraction, while YfiR-M2 localizes to the periplasmic fraction. C) In-vivo crosslinking of YfiR-M2. Whole cell samples of PA01 yfiR-M2, ΔyfiN yfiR-M2, and ΔyfiBN yfiR-M2 mutant strains were crosslinked by addition of formaldehyde and YfiR-M2 detected by immunoblot analysis. 0  =  sample before crosslinking; 20 =  sample taken 20 min after formaldehyde addition; 20* = 20 min sample, boiled to break crosslinks. Black arrows indicate major bands corresponding in size to an YfiR-M2 monomer (20 kDa) and an YfiR-M2 oligomer (40 kDa), respectively. D) Bacterial two-hybrid analysis of YfiN and YfiR interactions. Positive interactions produce a red color on MacConkey indicator plates. Clockwise from top left, the cartoons denote YfiN-YfiN, YfiR-YfiR and HAMP domain-YfiN interactions. E) A model for YfiBNR function. YfiN is a membrane-localized DGC and is subject to product inhibition and control by YfiR. YfiB activates YfiN, possibly by releasing YfiR-mediated repression. OM and IM refer to the outer and inner membranes, respectively and PG refers to the peptidoglycan layer.

Jacob G. Malone, et al. PLoS Pathog. 2010 March;6(3):e1000804.

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