Journal: mSphere

Article Title: The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms

doi: 10.1128/msphere.00505-22

Figure Lengend Snippet: Dispersion of P. aeruginosa biofilms is dependent on AmrZ. Biofilms were grown in 5-fold diluted VBMM in continuous flow biofilm tube reactors. Dispersion was induced after 5 days of growth via the addition of (A and B) sodium nitroprusside (as a source of nitric oxide) or (C and D) glutamate to the growth medium. The effluent from tube reactors was collected for 24 min at 1 min intervals, and the absorbance was determined by spectrophotometry at 600 nm. Brief spikes in the absorbance of the effluent are indicative of positive dispersion responses. The dispersion of biofilms formed by dt amrZ mutants (A and C) and chromosomally complemented (B and D) was assessed relative to that of wild-type biofilms. The chromosomally complemented strain was grown with 0.1% arabinose to induce the expression of amrZ . The plots shown are representative of at least 3 biological replicates, each of which consisted of 8 technical replicates. (E) Representative confocal images of biofilms formed by the indicated strains, grown for 5 days under continuous flow conditions. Scale bar, 100 μm. COMSTAT was used to quantitatively determine the biofilm biomass (F) and biofilm thickness (G) of PAO1 and the dt amrZ mutant strains. ns, not significant, as determined using a Student’s t test.

Article Snippet: For the visualization of the biofilm architecture, the biofilms were grown in flow cells (glass surface, BioSurface Technologies) at a flow rate of 0.2 mL/min.

Techniques: Dispersion, Spectrophotometry, Expressing, Mutagenesis

Journal: mSphere

Article Title: The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms

doi: 10.1128/msphere.00505-22

Figure Lengend Snippet: Overexpression of amrZ does not coincide with dispersion events. (A) Biofilms formed by dt amrZ harboring the empty vector pHERD20T or an arabinose-inducible amrZ construct cloned into pHERD20T were grown in biofilm tube reactors in 5-fold diluted VBMM. The growth medium was supplemented with 8 μg/mL carbenicillin for the plasmid maintenance of pHERD20T. After 5 days of growth, arabinose was added to the growth medium at a concentration of 1% to induce the expression of amrZ . Effluent from the biofilms was collected for 90 min at 1 min intervals, and the absorbance was subsequently recorded at 600 nm. (B) Dispersion response of 5-day-old biofilms by PAO1 harboring an arabinose-inducible bdlA _G31A construct cloned into pJN05 after the addition of 1% arabinose. Differently colored lines represent individual dispersion responses from at least 3 biological replicates.

Article Snippet: For the visualization of the biofilm architecture, the biofilms were grown in flow cells (glass surface, BioSurface Technologies) at a flow rate of 0.2 mL/min.

Techniques: Over Expression, Dispersion, Plasmid Preparation, Construct, Clone Assay, Concentration Assay, Expressing

Journal: mSphere

Article Title: The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms

doi: 10.1128/msphere.00505-22

Figure Lengend Snippet: Dependency of several known or hypothetical matrix-degrading enzymes and matrix components on AmrZ. qRT-PCR experiments were performed on 3-day-old biofilm cells grown in biofilm tube reactors with 5-fold diluted VBMM. The transcript abundance values of genes obtained from dt amrZ mutant biofilms were compared to those of the wild-type for reference, whereas the plasmid-complemented strain (dt amrZ /pHERD- amrZ ) was compared to an empty vector control (dt amrZ /pHERD20T). Plasmid-complemented and empty vector strains were grown in the presence of 0.1% arabinose and 8 μg/mL carbenicillin for plasmid maintenance. cysD was used as the housekeeping gene. Statistical analysis was performed using a two-tailed t test (*, P < 0.05). Error bars represent the standard deviation.

Article Snippet: For the visualization of the biofilm architecture, the biofilms were grown in flow cells (glass surface, BioSurface Technologies) at a flow rate of 0.2 mL/min.

Techniques: Quantitative RT-PCR, Mutagenesis, Plasmid Preparation, Control, Two Tailed Test, Standard Deviation

Journal: mSphere

Article Title: The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms

doi: 10.1128/msphere.00505-22

Figure Lengend Snippet: Overexpression of genes encoding matrix-degrading enzymes does not restore the dispersion response by dt amrZ biofilms. Biofilms were grown for 5 days in biofilm tube reactors with 5-fold diluted VBMM prior to the induction of gene expression. The growth medium was supplemented with 8 μg/mL carbenicillin for maintenance of the pMJT-1 plasmid and 2 μg/mL gentamicin for the pJN105 plasmid. The expression of genes of interest was induced by the addition of 1% arabinose to the growth medium. Effluent from the biofilms was collected for 90 min at 1 min intervals and the absorbance was subsequently recorded at 600 nm. Response of biofilms formed by (A) PAO1 and (B) dt amrZ harboring the empty vector pJN105 or expressing endA in response to the arabinose-induced gene expression of endA . Response of biofilms formed by (C) PAO1 and (D) dt amrZ harboring the empty vector pMJT-1 or expressing pelA in response to the arabinose-induced gene expression of pelA . Response of biofilms formed by (E) dt amrZ harboring the empty vectors pMJT-1 and pJN105 or coexpressing pelA and endA or pslG and endA after addition to arabinose to induce gene expression. Differently colored lines represent individual dispersion responses from at least 3 biological replicates.

Article Snippet: For the visualization of the biofilm architecture, the biofilms were grown in flow cells (glass surface, BioSurface Technologies) at a flow rate of 0.2 mL/min.

Techniques: Over Expression, Dispersion, Gene Expression, Plasmid Preparation, Expressing

Journal: mSphere

Article Title: The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms

doi: 10.1128/msphere.00505-22

Figure Lengend Snippet: Overexpression of pslG dispersed biofilms formed by dt cdrA does not restore the dispersion response by dt amrZ biofilms. Biofilms were grown for 5 days in in biofilm tube reactors with 5-fold diluted VBMM prior to the induction of gene expression. The growth medium was supplemented with 8 μg/mL carbenicillin for the maintenance of the pMJT-1 plasmid. Expression of pslG was induced by the addition of 1% arabinose to the growth medium. Effluent from the biofilms was collected for 90 min at 1 min intervals, and the absorbance was subsequently recorded at 600 nm. (A) Dispersion profile of biofilms by PAO1 in response to the arabinose-induced gene expression of pslG. (B) Dispersion profile of biofilms by dt cdrA vector control and dt cdrA /pMJT- pslG in response to the arabinose-induced gene expression of pslG. (C) Dispersion profile of biofilms by dt amrZ vector control and dt amrZ /pMJT- pslG in response to the arabinose-induced gene expression of pslG. Differently colored lines represent individual dispersion responses from at least 3 biological replicates.

Article Snippet: For the visualization of the biofilm architecture, the biofilms were grown in flow cells (glass surface, BioSurface Technologies) at a flow rate of 0.2 mL/min.

Techniques: Over Expression, Dispersion, Gene Expression, Plasmid Preparation, Expressing, Control

Journal: mSphere

Article Title: The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms

doi: 10.1128/msphere.00505-22

Figure Lengend Snippet: The hyperdispersive phenotype of PAO1/pJN- bdlA _G31A is dependent on AmrZ. (A) Transcript abundance of bdlA and amrZ , as determined by qRT-PCR. RNA isolated from 5-day-old biofilms formed by the indicated strains were used, and the fold change in transcript abundance was determined relative to the transcript abundance values of wild-type biofilms. cysD was used as the housekeeping gene. The experiments were done in triplicate, and the standard deviation is shown. An asterisk denotes a statistically significant difference ( P < 0.05) relative to the PAO1 control strain, as determined using a one-way ANOVA, followed by Dunnett’s post hoc test. (B) The domain structure of BdlA. Cleavage occurs between the two PAS domains, resulting in PASa and PASb-TarH. Using C-terminally tagged BdlA, only intact BdlA and the cleaved PASb-TarH fraction are detectable via an immunoblot analysis using anti-V5 antibodies. (C) Image of an immunoblot showing the abundance of intact BdlA and cleaved C-terminally tagged BdlA_V5/His present in total cell extracts obtained from 3-day-old biofilms by ΔbdlA , dt amrZ , and dt amrZ /pHERD- amrZ . The respective strains harbor a chromosomally inserted, C-terminally tagged BdlA_V5/His under the control of its own promoter. The biofilms were grown in 5-fold diluted VBMM. Intact and cleaved BdlA was detected using anti-V5 antibodies. The experiments were carried out in triplicate, and a representative image is shown. (D) Biofilms formed by PAO1 and dt amrZ harboring an arabinose-inducible bdlA _G31A construct cloned into pJN105 were grown as biofilms in tube reactors in 5-fold diluted VBMM. The growth medium was supplemented with 2 μg/mL gentamicin for the plasmid maintenance of pJN105. After 5 days of growth, arabinose was added to the growth medium at a concentration of 1% to induce the expression of bdlA _G31A. Effluent from the biofilms was collected for 90 min at 1 min intervals, and the absorbance was subsequently recorded at 600 nm. The individual dispersion responses from at least 3 biological replicates are indicated by colored lines. Each biological replicate consisted of 4 technical replicates.

Article Snippet: For the visualization of the biofilm architecture, the biofilms were grown in flow cells (glass surface, BioSurface Technologies) at a flow rate of 0.2 mL/min.

Techniques: Quantitative RT-PCR, Isolation, Standard Deviation, Control, Western Blot, Construct, Clone Assay, Plasmid Preparation, Concentration Assay, Expressing, Dispersion

Journal: mSphere

Article Title: The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms

doi: 10.1128/msphere.00505-22

Figure Lengend Snippet: Contribution of AmrZ-targets napB and PA1891 in the dispersion response. Biofilms by PAO1, napB ::IS, and PA1891::IS were grown in 5-fold diluted VBMM in continuous flow biofilm tube reactors. (A) qRT-PCR experiments were performed on 5-day-old biofilm cells grown in biofilm tube reactors with 5-fold diluted VBMM. The transcript abundance values of napB and PA1891 obtained from dt amrZ mutant biofilms were compared to those of the wild-type for reference. cysD was used as the housekeeping gene. An asterisk indicates a statistically significantly difference from PAO1 ( P < 0.05), as determined using a one-way ANOVA, followed by Dunnett’s post hoc test. (B–D) Dispersion was induced after 5 days of growth via the addition of sodium nitroprusside as a source of nitric oxide. Effluents from tube reactors of biofilms by (B) P. aeruginosa PAO1, (C) napB ::IS and napB::IS/pMJT- napB , and (D) PA1891::IS and the same strain overexpressing PA1891 were collected for 35 min at 1 min intervals. The absorbance was determined by spectrophotometry at 600 nm. Colored lines represent individual dispersion responses from at least 3 biological replicates. (E) Representative confocal images of biofilms formed by the indicated strains grown for 5 days under continuous flow conditions. Scale bar, 100 μm. Biofilm biomass (F) and biofilm thickness (G) by PAO1 and the respective mutant strains was determined using COMSTAT analysis. ns, not significant, as determined using an ANOVA, followed by Dunnett’s post hoc test.

Article Snippet: For the visualization of the biofilm architecture, the biofilms were grown in flow cells (glass surface, BioSurface Technologies) at a flow rate of 0.2 mL/min.

Techniques: Dispersion, Quantitative RT-PCR, Mutagenesis, Spectrophotometry

Journal: mSphere

Article Title: The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms

doi: 10.1128/msphere.00505-22

Figure Lengend Snippet: The hyperdispersive response of biofilms overexpressing bdlA _G31A is dependent on PA1891 but not napB . Biofilms by napB :: IS , PA1891::IS, and the respective mutant strains harboring an arabinose-inducible bdlA _G31A construct cloned into pJN05 were grown as biofilms in tube reactors in 5-fold diluted VBMM with 2 μg/mL gentamicin for plasmid maintenance. After 5 days of growth, 1% arabinose was added to the growth medium to induce the expression of bdlA _G31A. The effluents from the tube reactors were collected for 90 min, and the absorbance was determined by spectrophotometry at 600 nm. (A) Absorbance of effluents by biofilms formed by napB ::IS/pJN105 and napB ::IS/pJN- bdlA _G31A after the addition of arabinose. (B) Absorbance of effluents by biofilms formed by PA1891::IS/pJN105 and PA1891::IS/pJN- bdlA _G31A after the addition of arabinose. The colored lines represent individual dispersion responses from at least 3 biological replicates, each of which consisted of 2 to 4 technical replicates.

Article Snippet: For the visualization of the biofilm architecture, the biofilms were grown in flow cells (glass surface, BioSurface Technologies) at a flow rate of 0.2 mL/min.

Techniques: Mutagenesis, Construct, Clone Assay, Plasmid Preparation, Expressing, Spectrophotometry, Dispersion

Journal: mSphere

Article Title: The Alginate and Motility Regulator AmrZ is Essential for the Regulation of the Dispersion Response by Pseudomonas aeruginosa Biofilms

doi: 10.1128/msphere.00505-22

Figure Lengend Snippet: Multicopy expression of napB and PA1891 restore the dispersion response by dt amrZ biofilms. (A) Biofilms by dt amrZ harboring an arabinose-inducible napB construct cloned into pMJT-1 were grown in tube reactors in 5-fold diluted VBMM with 8 μg/mL carbenicillin for plasmid maintenance. After 5 days of growth, 1% arabinose was added to the growth medium to induce the expression of napB . After the induction of gene expression, biofilm effluents were collected for 90 min, and the absorbance was determined by spectrophotometry at 600 nm. Biofilms by dt amrZ harboring the empty plasmid pMJT-1 were used as controls. (B) Biofilms by dt amrZ harboring an arabinose-inducible PA1891 construct cloned into pJN105 were grown as biofilms in tube reactors in 5-fold diluted VBMM with 2 μg/mL gentamicin for plasmid maintenance. After 5 days of growth, 1% arabinose was added to the growth medium to induce the expression of PA1891, and biofilm effluents were subsequently collected for 90 min. The absorbance of biofilm effluents was determined by spectrophotometry at 600 nm. Biofilms by dt amrZ harboring an empty vector were used as controls. The colored lines represent individual dispersion responses from at least 3 biological replicates, each of which consisted of 2 to 4 technical replicates.

Article Snippet: For the visualization of the biofilm architecture, the biofilms were grown in flow cells (glass surface, BioSurface Technologies) at a flow rate of 0.2 mL/min.

Techniques: Expressing, Dispersion, Construct, Clone Assay, Plasmid Preparation, Gene Expression, Spectrophotometry