Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: Temperature downshifts enhanced biofilm formation in the majority of Pseudomonas aeruginosa clinical and environmental isolates. A , biofilm biomass of 52 clinical P. aeruginosa isolates, P. aeruginosa PAO1, and four environmental isolates following culturing statically at RT and 37 °C. M1: ATCC 27853. M35: PA150663 . M54: PA152541 . B , dot plot summarizing the biofilm patterns of P. aeruginosa isolates at RT and 37 °C. Black : environmental isolates. The two dash lines represent the threshold of 2-fold. Blue : strains formed two-fold or more biofilm at RT relative to 37 °C. Red : strains formed two-fold or more biofilm at 37 °C relative to RT. See related for bar graph with statistical analysis. C , biofilm biomass of ATCC 27853, PAO1, PA150663 , and PA152541 at a temperature gradient from 15 °C to 42 °C at 48 h. D , biofilm formation of P. aeruginosa ATCC 27853 and PAO1 at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01 (based on Student’s t test).

Article Snippet: A , SEM images of ATCC 27853 biofilm formed at RT ( upper ) and 37 °C ( lower ) on LB agar.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: Psl is primarily responsible for the enhanced biofilm formation at RT. A , SEM images of ATCC 27853 biofilm formed at RT ( upper ) and 37 °C ( lower ) on LB agar. Scale bar represents 1 μm. B , maximal biomass of ATCC 27853 and its isogenic Δ pslD, Δ pslF , and Δ pslFpslD mutants at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. C , relative activity of the chromosomal P psl - lux transcriptional reporter in ATCC 27853 collected at half maximal biofilm at RT and 37 °C, respectively. Data are represented as mean ± SD, n = 3 independent experiments. D , Western blot of PslD-3FLAG in ATCC 27853 cultured at RT and 37 °C. Mid-log phase cells were collected for the analysis ( C–D ), that is, static cultures harvested at 24 h at RT and 8 h at 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. E , relative Psl production of ATCC 27853 at RT and 37 °C as detected by immunoblot with Psl-specific antibody. Left panel, half peak biofilm time points (24h at RT and 8 h at 37 °C). Right panel, peak time points (48h at RT and 24 h at 37 °C). Data are represented as mean ± SD, n = 2 independent experiments. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01. ns, not significant (based on Student’s t test). SEM, scanning electron microscopy.

Article Snippet: A , SEM images of ATCC 27853 biofilm formed at RT ( upper ) and 37 °C ( lower ) on LB agar.

Techniques: Activity Assay, Western Blot, Cell Culture, Electron Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: Enhanced Psl production at RT was mediated by the c-di-GMP–FleQ pathway. A , relative maximal biofilm biomass of ATCC 27853 and its isogenic Δ fleQ , Δ rpoS , and Δ amrZ mutants at RT. Data are represented as mean ± SD, n = 3 independent experiments. B , relative activity of the chromosomal P psl - lux transcriptional fusion reporter collected at half maximal biofilm in ATCC 27853 and its isogenic Δ fleQ mutant at RT. Data are represented as mean ± SD, n = 3 independent experiments. C , intracellular c-di-GMP level at half maximal biofilm culture of ATCC 27853 at RT and 37 °C. Data are represented as mean ± SD, n = 2 independent experiments. D , dynamic biofilm formation of ATCC 27853 and its isogenic Δ wspF strain at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. E , relative maximal biofilm biomass of ATCC 27853 and its isogenic Δ wspF strain at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01. ns, not significant (based on Student’s t test). c-di-GMP, cyclic di-guanosine 3′,5′-monophosphate.

Article Snippet: A , SEM images of ATCC 27853 biofilm formed at RT ( upper ) and 37 °C ( lower ) on LB agar.

Techniques: Activity Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: The DGC SiaD is primarily responsible for the elevated c-di-GMP content and enhanced Psl production at RT in ATCC 27853. A , relative maximal biofilm biomass of 28 isogenic mutants relative to the ATCC 27853 WT at RT. Blue , genes identified in RNA-seq analysis. B , relative maximal biofilm formation of ATCC 27853 and its isogenic Δ siaD strain at RT and 37 °C. C , relative maximal biofilm formation of ATCC 27853, isogenic Δ siaD mutant, and Δ siaD mutant complemented with rhamnose-inducible expression of siaD at 48 h at RT. VC, the pJM253 vector control. D – F , relative activity of the chromosomal P psl - lux transcriptional fusion reporter ( D ), Psl level in biofilm matrix ( E ), and intracellular c-di-GMP level ( F ) in ATCC 27853 and its isogenic Δ siaD mutant at RT. Midlog phase static cells harvested at 24 h at RT were collected for the analysis in ( C–F ). Data are represented as mean ± SD. (A–E), n = 3 independent experiments. ( F ), n = 2 independent experiments. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01. ns, not significant (based on Student’s t test). c-di-GMP, cyclic di-guanosine 3′,5′-monophosphate; DGC, diguanylate cyclase.

Article Snippet: A , SEM images of ATCC 27853 biofilm formed at RT ( upper ) and 37 °C ( lower ) on LB agar.

Techniques: RNA Sequencing, Mutagenesis, Expressing, Plasmid Preparation, Control, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: Temperature downshift from 37 °C to RT induces biofilm formation in ATCC 27853 through the SiaABCD signaling module. A , schematic diagram of the SiaABCD signaling and functional pathway. B , relative maximal biomass of ATCC 27853 and its isogenic Δ siaA , Δ siaB, Δ siaC, and Δ siaD mutants at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. C , maximal biomass of ATCC 27853 supplemented with a vector control (VC) or rhamnose-inducible siaB at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. D , maximal biomass of ATCC 27853 and its isogenic mutants carrying siaC alleles encoding a phosphorylation-deficient (SiaC T68A ) or phosphomimetic (SiaC T68D ) mutant at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. E , phosphorylation status of SiaC-3FLAG at RT and 37 °C detected by Phos-tag SDS-PAGE. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01. ns, not significant (based on Student’s t test).

Article Snippet: A , SEM images of ATCC 27853 biofilm formed at RT ( upper ) and 37 °C ( lower ) on LB agar.

Techniques: Functional Assay, Plasmid Preparation, Control, Phospho-proteomics, Mutagenesis, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: Temperature downshifts induce distinct membrane perturbations to activate the SiaABCD signaling module. A , 2D graph plotted from output signals of two channels of the PI-BactD probe, fluorescence increase (ΔS/S 0 ), and fluorescence radiometric changes (I 483 /I 375 ), in ATCC 27853 and PAO1 at RT and 37 °C. Each assay was conducted in six replicates. B , schematic diagram showing the domain structures of WT SiaA, WT CusS, and CusS-SiaA chimeric variant. C , maximal biomass of ATCC 27853 WT strain, Δ siaA mutant, and Δ siaA mutant carrying chromosomally integrated CusS-SiaA chimeric variant at RT. Data are represented as mean ± SD, n = 3 independent experiments. D , upper panel, Log2 relative area of membrane lipid groups of ATCC 27853 and PAO1 as detected by lipidomics analysis (RT/37 °C). Orange and green : differentially changed in ATCC 27853 relative to PAO1 in response to temperature shift. Black and blue : synchronously changed in ATCC 27853 and PAO1 in response to temperature shift. Gray : similar levels at RT and 37 °C. Lower panel, a table summarizing the membrane perturbations upon temperature downshift and SDS treatment. E , dynamic biofilm formation of ATCC 27853 harboring either a vector control (VC) or rhamnose-inducible fabAB construct at RT and 37 °C. VC, the pJM253 vector control. Data are represented as mean ± SD, n = 3 independent experiments. F , pellicle formation of ATCC 27853 at RT and 37 °C collected at half-maximal biofilm stage, that is, 24h at RT and 8h at 37 °C, OD 600 ∼0.4. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01 (based on Student’s t test).

Article Snippet: A , SEM images of ATCC 27853 biofilm formed at RT ( upper ) and 37 °C ( lower ) on LB agar.

Techniques: Membrane, Fluorescence, Variant Assay, Mutagenesis, Plasmid Preparation, Control, Construct

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: A model to illustrate the activation of SiaABCD and its mediated biofilm enhancement in ATCC 27853 in response to temperature downshifts. Temperature downshift from 37 °C to RT causes membrane perturbations in ATCC 27853, which induce SiaA phosphatase activity to promote the formation of SiaC–SiaD complex and DGC activity of SiaD. The activated Sia system regulates psl production via c-di-GMP–FleQ–Psl pathway. Increased Psl production enhanced biofilm formation of ATCC 27853 at RT through enhancing both surface attachment and biofilm matrix development. c-di-GMP, cyclic di-guanosine 3′,5′-monophosphate; DGC, diguanylate cyclase.

Article Snippet: A , SEM images of ATCC 27853 biofilm formed at RT ( upper ) and 37 °C ( lower ) on LB agar.

Techniques: Activation Assay, Membrane, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: Temperature downshifts enhanced biofilm formation in the majority of Pseudomonas aeruginosa clinical and environmental isolates. A , biofilm biomass of 52 clinical P. aeruginosa isolates, P. aeruginosa PAO1, and four environmental isolates following culturing statically at RT and 37 °C. M1: ATCC 27853. M35: PA150663 . M54: PA152541 . B , dot plot summarizing the biofilm patterns of P. aeruginosa isolates at RT and 37 °C. Black : environmental isolates. The two dash lines represent the threshold of 2-fold. Blue : strains formed two-fold or more biofilm at RT relative to 37 °C. Red : strains formed two-fold or more biofilm at 37 °C relative to RT. See related for bar graph with statistical analysis. C , biofilm biomass of ATCC 27853, PAO1, PA150663 , and PA152541 at a temperature gradient from 15 °C to 42 °C at 48 h. D , biofilm formation of P. aeruginosa ATCC 27853 and PAO1 at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01 (based on Student’s t test).

Article Snippet: Scanning electron microscopy analysis of ATCC 27853 biofilms formed at RT and 37 °C showed that, the biofilm formed at RT displayed an enhanced level of matrix production relative to 37 °C ( A ).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: Psl is primarily responsible for the enhanced biofilm formation at RT. A , SEM images of ATCC 27853 biofilm formed at RT ( upper ) and 37 °C ( lower ) on LB agar. Scale bar represents 1 μm. B , maximal biomass of ATCC 27853 and its isogenic Δ pslD, Δ pslF , and Δ pslFpslD mutants at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. C , relative activity of the chromosomal P psl - lux transcriptional reporter in ATCC 27853 collected at half maximal biofilm at RT and 37 °C, respectively. Data are represented as mean ± SD, n = 3 independent experiments. D , Western blot of PslD-3FLAG in ATCC 27853 cultured at RT and 37 °C. Mid-log phase cells were collected for the analysis ( C–D ), that is, static cultures harvested at 24 h at RT and 8 h at 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. E , relative Psl production of ATCC 27853 at RT and 37 °C as detected by immunoblot with Psl-specific antibody. Left panel, half peak biofilm time points (24h at RT and 8 h at 37 °C). Right panel, peak time points (48h at RT and 24 h at 37 °C). Data are represented as mean ± SD, n = 2 independent experiments. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01. ns, not significant (based on Student’s t test). SEM, scanning electron microscopy.

Article Snippet: Scanning electron microscopy analysis of ATCC 27853 biofilms formed at RT and 37 °C showed that, the biofilm formed at RT displayed an enhanced level of matrix production relative to 37 °C ( A ).

Techniques: Activity Assay, Western Blot, Cell Culture, Electron Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: Enhanced Psl production at RT was mediated by the c-di-GMP–FleQ pathway. A , relative maximal biofilm biomass of ATCC 27853 and its isogenic Δ fleQ , Δ rpoS , and Δ amrZ mutants at RT. Data are represented as mean ± SD, n = 3 independent experiments. B , relative activity of the chromosomal P psl - lux transcriptional fusion reporter collected at half maximal biofilm in ATCC 27853 and its isogenic Δ fleQ mutant at RT. Data are represented as mean ± SD, n = 3 independent experiments. C , intracellular c-di-GMP level at half maximal biofilm culture of ATCC 27853 at RT and 37 °C. Data are represented as mean ± SD, n = 2 independent experiments. D , dynamic biofilm formation of ATCC 27853 and its isogenic Δ wspF strain at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. E , relative maximal biofilm biomass of ATCC 27853 and its isogenic Δ wspF strain at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01. ns, not significant (based on Student’s t test). c-di-GMP, cyclic di-guanosine 3′,5′-monophosphate.

Article Snippet: Scanning electron microscopy analysis of ATCC 27853 biofilms formed at RT and 37 °C showed that, the biofilm formed at RT displayed an enhanced level of matrix production relative to 37 °C ( A ).

Techniques: Activity Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: The DGC SiaD is primarily responsible for the elevated c-di-GMP content and enhanced Psl production at RT in ATCC 27853. A , relative maximal biofilm biomass of 28 isogenic mutants relative to the ATCC 27853 WT at RT. Blue , genes identified in RNA-seq analysis. B , relative maximal biofilm formation of ATCC 27853 and its isogenic Δ siaD strain at RT and 37 °C. C , relative maximal biofilm formation of ATCC 27853, isogenic Δ siaD mutant, and Δ siaD mutant complemented with rhamnose-inducible expression of siaD at 48 h at RT. VC, the pJM253 vector control. D – F , relative activity of the chromosomal P psl - lux transcriptional fusion reporter ( D ), Psl level in biofilm matrix ( E ), and intracellular c-di-GMP level ( F ) in ATCC 27853 and its isogenic Δ siaD mutant at RT. Midlog phase static cells harvested at 24 h at RT were collected for the analysis in ( C–F ). Data are represented as mean ± SD. (A–E), n = 3 independent experiments. ( F ), n = 2 independent experiments. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01. ns, not significant (based on Student’s t test). c-di-GMP, cyclic di-guanosine 3′,5′-monophosphate; DGC, diguanylate cyclase.

Article Snippet: Scanning electron microscopy analysis of ATCC 27853 biofilms formed at RT and 37 °C showed that, the biofilm formed at RT displayed an enhanced level of matrix production relative to 37 °C ( A ).

Techniques: RNA Sequencing, Mutagenesis, Expressing, Plasmid Preparation, Control, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: Temperature downshift from 37 °C to RT induces biofilm formation in ATCC 27853 through the SiaABCD signaling module. A , schematic diagram of the SiaABCD signaling and functional pathway. B , relative maximal biomass of ATCC 27853 and its isogenic Δ siaA , Δ siaB, Δ siaC, and Δ siaD mutants at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. C , maximal biomass of ATCC 27853 supplemented with a vector control (VC) or rhamnose-inducible siaB at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. D , maximal biomass of ATCC 27853 and its isogenic mutants carrying siaC alleles encoding a phosphorylation-deficient (SiaC T68A ) or phosphomimetic (SiaC T68D ) mutant at RT and 37 °C. Data are represented as mean ± SD, n = 3 independent experiments. E , phosphorylation status of SiaC-3FLAG at RT and 37 °C detected by Phos-tag SDS-PAGE. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01. ns, not significant (based on Student’s t test).

Article Snippet: Scanning electron microscopy analysis of ATCC 27853 biofilms formed at RT and 37 °C showed that, the biofilm formed at RT displayed an enhanced level of matrix production relative to 37 °C ( A ).

Techniques: Functional Assay, Plasmid Preparation, Control, Phospho-proteomics, Mutagenesis, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: Temperature downshifts induce distinct membrane perturbations to activate the SiaABCD signaling module. A , 2D graph plotted from output signals of two channels of the PI-BactD probe, fluorescence increase (ΔS/S 0 ), and fluorescence radiometric changes (I 483 /I 375 ), in ATCC 27853 and PAO1 at RT and 37 °C. Each assay was conducted in six replicates. B , schematic diagram showing the domain structures of WT SiaA, WT CusS, and CusS-SiaA chimeric variant. C , maximal biomass of ATCC 27853 WT strain, Δ siaA mutant, and Δ siaA mutant carrying chromosomally integrated CusS-SiaA chimeric variant at RT. Data are represented as mean ± SD, n = 3 independent experiments. D , upper panel, Log2 relative area of membrane lipid groups of ATCC 27853 and PAO1 as detected by lipidomics analysis (RT/37 °C). Orange and green : differentially changed in ATCC 27853 relative to PAO1 in response to temperature shift. Black and blue : synchronously changed in ATCC 27853 and PAO1 in response to temperature shift. Gray : similar levels at RT and 37 °C. Lower panel, a table summarizing the membrane perturbations upon temperature downshift and SDS treatment. E , dynamic biofilm formation of ATCC 27853 harboring either a vector control (VC) or rhamnose-inducible fabAB construct at RT and 37 °C. VC, the pJM253 vector control. Data are represented as mean ± SD, n = 3 independent experiments. F , pellicle formation of ATCC 27853 at RT and 37 °C collected at half-maximal biofilm stage, that is, 24h at RT and 8h at 37 °C, OD 600 ∼0.4. Asterisks indicate statistical significance. ∗, p < 0.05. ∗∗∗, p < 0.01 (based on Student’s t test).

Article Snippet: Scanning electron microscopy analysis of ATCC 27853 biofilms formed at RT and 37 °C showed that, the biofilm formed at RT displayed an enhanced level of matrix production relative to 37 °C ( A ).

Techniques: Membrane, Fluorescence, Variant Assay, Mutagenesis, Plasmid Preparation, Control, Construct

Journal: The Journal of Biological Chemistry

Article Title: Temperature downshifts induce biofilm formation in Pseudomonas aeruginosa through the SiaABCD signal and functional module

doi: 10.1016/j.jbc.2025.111086

Figure Lengend Snippet: A model to illustrate the activation of SiaABCD and its mediated biofilm enhancement in ATCC 27853 in response to temperature downshifts. Temperature downshift from 37 °C to RT causes membrane perturbations in ATCC 27853, which induce SiaA phosphatase activity to promote the formation of SiaC–SiaD complex and DGC activity of SiaD. The activated Sia system regulates psl production via c-di-GMP–FleQ–Psl pathway. Increased Psl production enhanced biofilm formation of ATCC 27853 at RT through enhancing both surface attachment and biofilm matrix development. c-di-GMP, cyclic di-guanosine 3′,5′-monophosphate; DGC, diguanylate cyclase.

Article Snippet: Scanning electron microscopy analysis of ATCC 27853 biofilms formed at RT and 37 °C showed that, the biofilm formed at RT displayed an enhanced level of matrix production relative to 37 °C ( A ).

Techniques: Activation Assay, Membrane, Activity Assay