xtension  (Oxford Instruments)

Journal: bioRxiv

Article Title: A network perspective on the role of c-di-GMP-associated protein complexes in biofilm formation

doi: 10.64898/2026.05.12.724550

Figure Lengend Snippet: A selected number of genes encoding in-network CDG proteins and their interacting partners, as well as out-of-network CDGs were targeted by CRISPRi and subjected to biofilm and motility phenotypes. A - Swarming assay, imaging, and calculation of swarm areas. NTC, non-targeting control; TC, targeted positive control. B - Biofilm pellicle assay and imaging by confocal microscopy. Pellicles at the liquid-air interface were peeled out onto circular glass coverslips and mounted on a microscopy chamber containing transparent media. Biofilms were stained with FilmTracer TM green fluorescent dye prior to volume imaging using confocal microscopy. Parameters were extracted by image analysis using Imaris software. C - Formation of submerged biofilm at pegs (MBEC TM ). Cells were released from the pegs by sonication and counted using flow cytometry. D - Real-time monitoring of amyloid fibers and extracellular matrix production in the presence of optotracer using fluorometry (EbbaBiolight680).

Article Snippet: Images were processed using the Biofilm Analysis XTension package of IMARIS (Bitplane, South Windsor, CT, United States).

Techniques: Imaging, Control, Positive Control, Confocal Microscopy, Microscopy, Staining, Software, Sonication, Flow Cytometry

Journal: bioRxiv

Article Title: A network perspective on the role of c-di-GMP-associated protein complexes in biofilm formation

doi: 10.64898/2026.05.12.724550

Figure Lengend Snippet: The analyzed phenotypes were biofilm biovolume (BV), biomass (BM) mean thickness (MS), maximum height (MX), roughness (R), submerged biofilm at pegs (SBP), and production of amyloids (AMF), and swarming motility (SW). The targeted proteins (rows on the heatmap) are split into six groups by k-means clustering. Groups are indicated by colors (left side) and distance is Pearson correlation. The extent of phenotype change is indicated by the side bar, blue for decrease and red for increase.

Article Snippet: Images were processed using the Biofilm Analysis XTension package of IMARIS (Bitplane, South Windsor, CT, United States).

Techniques:

Journal: bioRxiv

Article Title: A network perspective on the role of c-di-GMP-associated protein complexes in biofilm formation

doi: 10.64898/2026.05.12.724550

Figure Lengend Snippet: The analyzed phenotypes were biofilm biovolume (BV), biomass (BM), mean thickness (MS), maximum height (MX), roughness (R), submerged biofilm at pegs (SBP), production of amyloids (AMF), and swarming motility (SW). Statistical significance was calculated using cumulative hypergeometric distributions. All P-values are reported in Table S6. A-Phenotypic enrichment within interaction modules comprising a CDG bait and its direct interaction partners. 1 = significant enrichment for gene partners that increase the phenotype; −1 = significant enrichment for gene partners that decrease the phenotype; and 0 = no enrichment. Note that a value of ‘no enrichment’ does not indicate a lack of change in the phenotype, rather it indicates that the observed frequency of change is equivalent to random chance given the distribution of phenotypes across all knockdowns. B-Phenotypic enrichment by annotation category. C-Phenotypic effects of in-network versus out-of-network knockdowns. Split heatmaps illustrating -Top: phenotypic decrease or increase for knockdown of genes encoding CDG proteins from the PPI network (CDGs IN Network, orange) versus the CDGs not present in the network (CDGs OUT-Network, blue); - Middle: Gene knockdowns of DipA interacting partners (DipA-IP CDGs, orange) versus all other CDGs (blue); - Bottom: Knockdown of all genes present in the network (IN Network, orange) versus those outside of the network (OUT Network, blue).

Article Snippet: Images were processed using the Biofilm Analysis XTension package of IMARIS (Bitplane, South Windsor, CT, United States).

Techniques: Knockdown

Journal: bioRxiv

Article Title: A network perspective on the role of c-di-GMP-associated protein complexes in biofilm formation

doi: 10.64898/2026.05.12.724550

Figure Lengend Snippet: A-Principal component analysis plot with K-means clustering of biofilm pellicle and swarming motility phenotypes. Note: the less robust SBP phenotype was excluded from this analysis; WspR (PFLU1225) is here missing due to the lack of AMF data). Clusters are indicated by colors (1= green, 2= blue and 3= red). Purple arrows indicate the eigenvectors associated with the biofilm and swarming phenotypes. B-Pearson correlation analysis between Log 2 fold change of the biofilm and motility phenotypes (BV, MX, MS, R, SW, and AMF). Positive and negative correlations are indicated by red and blue shades, respectively. The genes were ordered to group the most correlated. “In-network” vs “out-network CDG genes are indicated by orange and blue asterisks, respectively. The vertical bar represents the color legend of the correlation coefficients. The size of the dot also reflects the value of the correlation coefficient. White asterisks indicate the statistical significance levels (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Left: edge-weighted network of significant correlations. Each node represents a CDG gene from the PPI network (orange) or outside the network (blue).

Article Snippet: Images were processed using the Biofilm Analysis XTension package of IMARIS (Bitplane, South Windsor, CT, United States).

Techniques:

Journal: bioRxiv

Article Title: A network perspective on the role of c-di-GMP-associated protein complexes in biofilm formation

doi: 10.64898/2026.05.12.724550

Figure Lengend Snippet: A-dipA interaction module. Pearson correlation matrix for Log2FC in biofilm and motility phenotypes (BV, MX, MS, R, SW, and AMF). Positive and negative correlations are indicated in shades of red and blue respectively. Genes were ordered from the highest to lowest correlation with dipA. The color intensity and size of the circles are proportional to the correlation coefficients. The vertical bar shows the color legend of the correlation coefficients. Significantly correlated positive and negative clusters are indicated by red and blue shades, respectively. White asterisks indicate the levels of statistical significance (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Domain composition is indicated (GGDEF, open triangle; GGDEG/EAL, open square). B-Principal component analysis plot with K-means clustering of genes based on biofilm and motility phenotypes. Clusters are indicated by colors (1 red, 2 blue and 3 black). The eigenvectors associated with the biofilm and swarming phenotypes are shown in purple. CDG proteins containing the GGEEF domain (filled triangle) or dual GGDEF/EAL domain (filled square). Open circles are non-CDG partners. C-PFLU5127 interaction module. Pearson correlation matrix for Log2FC in biofilm and motility phenotypes (BV, MX, MS, R, SBP, and AMF). D-BifA (PFLU4858) interaction module. Pearson correlation matrix for Log 2 fold change in biofilm and motility phenotypes (BV, MX, MS, R, and SW). E-Alg44 (PFLU0988) interaction module. Pearson correlation matrix for Log 2 fold change in biofilm and motility phenotypes (BV, MX, MS, R, and SW). Partners with similar functional annotations are framed in red (correlated) or blue (anti-correlated).

Article Snippet: Images were processed using the Biofilm Analysis XTension package of IMARIS (Bitplane, South Windsor, CT, United States).

Techniques: Functional Assay