Journal: bioRxiv

Article Title: Global characterization of the cyclic-di-GMP regulome by bi-directionally evolving a social trait in Pseudomonas fluorescens

doi: 10.1101/2022.05.10.491292

Figure Lengend Snippet: A) Colony morphologies of the ancestral mucoid (M, left) and dry (D, middle) strains after 4 days of growth. Arrows indicate the emergence of spreading fans that contain the respective parent strain and a new mutant strain with the opposite phenotype. The spreading phenotype is readily produced by artificially mixing M and D strains (right, scale bar = 10mm). B) Phylogeny of strains in select lineages colored by phenotype and corresponding colony morphologies are shown in . The numbers indicate the IDs of the evolved strains as shown in . Each strain was isolated from naturally emerging fans from their respective parent colony with the opposite phenotype. This phylogeny depicts the bidirectional transitions between mucoid and dry states which we show are c-di-GMP dependent. C) C-di-GMP levels of evolved strains. C-di-GMP was quantified through LC-MS/MS and is reported as nM per OD600. Samples are categorized by phenotype and we report the triplicate mean ± SD for each phenotypic classification: mucoid n=14 and dry n=21 (*** Student’s t-test p < 0.001).

Article Snippet: 1mL of 0.5 OD 600 culture was centrifuged at 15,000 RCF for 30 seconds, and cell pellets were resuspended in 100uL of ice cold acetonitrile/methanol/water (40:40:20 (v/v/v)) extraction buffer supplemented with a final concentration of 0.01% formic acid and 25nM c-di-GMP-Fluorinated internal standard (InvivoGen CAT: 1334145-18-4).

Techniques: Mutagenesis, Produced, Isolation, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Global characterization of the cyclic-di-GMP regulome by bi-directionally evolving a social trait in Pseudomonas fluorescens

doi: 10.1101/2022.05.10.491292

Figure Lengend Snippet: Evolved strains are classified as either D (orange) or M (blue) based on their colony morphology. Corresponding motility data, mutation data, and LC-MS/MS c-di-GMP quantification data are summarized in .

Article Snippet: 1mL of 0.5 OD 600 culture was centrifuged at 15,000 RCF for 30 seconds, and cell pellets were resuspended in 100uL of ice cold acetonitrile/methanol/water (40:40:20 (v/v/v)) extraction buffer supplemented with a final concentration of 0.01% formic acid and 25nM c-di-GMP-Fluorinated internal standard (InvivoGen CAT: 1334145-18-4).

Techniques: Mutagenesis, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Global characterization of the cyclic-di-GMP regulome by bi-directionally evolving a social trait in Pseudomonas fluorescens

doi: 10.1101/2022.05.10.491292

Figure Lengend Snippet: Each evolved strain is classified either as M (blue) or D (orange) based on their mucoid or dry-wrinkly colony phenotypes, respectively. Motility data and LC-MS/MS c-di-GMP quantification data for each strain are reported in .

Article Snippet: 1mL of 0.5 OD 600 culture was centrifuged at 15,000 RCF for 30 seconds, and cell pellets were resuspended in 100uL of ice cold acetonitrile/methanol/water (40:40:20 (v/v/v)) extraction buffer supplemented with a final concentration of 0.01% formic acid and 25nM c-di-GMP-Fluorinated internal standard (InvivoGen CAT: 1334145-18-4).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Global characterization of the cyclic-di-GMP regulome by bi-directionally evolving a social trait in Pseudomonas fluorescens

doi: 10.1101/2022.05.10.491292

Figure Lengend Snippet: The Wsp system in P. aeruginosa responds to surface contact to ultimately produce c-di-GMP. (CH3 = methyl group, P= phosphoryl group).

Article Snippet: 1mL of 0.5 OD 600 culture was centrifuged at 15,000 RCF for 30 seconds, and cell pellets were resuspended in 100uL of ice cold acetonitrile/methanol/water (40:40:20 (v/v/v)) extraction buffer supplemented with a final concentration of 0.01% formic acid and 25nM c-di-GMP-Fluorinated internal standard (InvivoGen CAT: 1334145-18-4).

Techniques:

Journal: bioRxiv

Article Title: Global characterization of the cyclic-di-GMP regulome by bi-directionally evolving a social trait in Pseudomonas fluorescens

doi: 10.1101/2022.05.10.491292

Figure Lengend Snippet: C-di-GMP was quantified via LC-MS/MS and is reported as the triplicate mean ± SD. The charts capture the independently evolved strains within the five selected lineages shown in . D strains are shown in orange and M strains are shown in blue. The numerical IDs correspond to , and mutations reflect the affected amino acid residues, except frameshift (FS) and promoter region (PR) mutations refer to the specific nucleotides.

Article Snippet: 1mL of 0.5 OD 600 culture was centrifuged at 15,000 RCF for 30 seconds, and cell pellets were resuspended in 100uL of ice cold acetonitrile/methanol/water (40:40:20 (v/v/v)) extraction buffer supplemented with a final concentration of 0.01% formic acid and 25nM c-di-GMP-Fluorinated internal standard (InvivoGen CAT: 1334145-18-4).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Global characterization of the cyclic-di-GMP regulome by bi-directionally evolving a social trait in Pseudomonas fluorescens

doi: 10.1101/2022.05.10.491292

Figure Lengend Snippet: C-di-GMP measurements in triplicate mean ± SD. Raw c-di-GMP values of each mutant and its respective parent are reported in . Causal mutation of each phenotypic shift is indicated on the left. D strains are shown in orange and M strains are shown in blue. (Student’s t-test: ns = non-significant, * = p < 0.05).

Article Snippet: 1mL of 0.5 OD 600 culture was centrifuged at 15,000 RCF for 30 seconds, and cell pellets were resuspended in 100uL of ice cold acetonitrile/methanol/water (40:40:20 (v/v/v)) extraction buffer supplemented with a final concentration of 0.01% formic acid and 25nM c-di-GMP-Fluorinated internal standard (InvivoGen CAT: 1334145-18-4).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Global characterization of the cyclic-di-GMP regulome by bi-directionally evolving a social trait in Pseudomonas fluorescens

doi: 10.1101/2022.05.10.491292

Figure Lengend Snippet: Strains were grown in the chemically defined PMM medium with 0, 50, 100, or 150 uM BCAA supplementation (valine, leucine, isoleucine). C-di-GMP was extracted and quantified via LC-MS/MS in triplicates and plotted as the mean with STD (ANOVA; Tukey’s HSD Post Test: ns = non-significant, * = p < 0.05). M and D refer to the prototypical M and D strains in . The ilvH mutant (D strain 5.7) produces c-di-GMP levels similar to the D strain control and the parA mutant (M strain 5.8) produces c-di-GMP levels similar to the M strain control. These relative patterns are consistent with the c-di-GMP quantification data reported in under nutrient-rich conditions. Exogenous supplementation of BCAA across all concentrations does not significantly impact c-di-GMP production. There is no data for the ilvI mutant at 0 uM BCAA, since this strain fails to grow in PMM unless BCAA is exogenously provided. However, the ilvI mutant (M strain 5.10a) produces c-di-GMP levels comparable to both D and the ilvH mutant (D strain 5.7) with BCAA supplementation.

Article Snippet: 1mL of 0.5 OD 600 culture was centrifuged at 15,000 RCF for 30 seconds, and cell pellets were resuspended in 100uL of ice cold acetonitrile/methanol/water (40:40:20 (v/v/v)) extraction buffer supplemented with a final concentration of 0.01% formic acid and 25nM c-di-GMP-Fluorinated internal standard (InvivoGen CAT: 1334145-18-4).

Techniques: Liquid Chromatography with Mass Spectroscopy, Mutagenesis

Journal: bioRxiv

Article Title: Global characterization of the cyclic-di-GMP regulome by bi-directionally evolving a social trait in Pseudomonas fluorescens

doi: 10.1101/2022.05.10.491292

Figure Lengend Snippet: A) A node-edge plot reveals focal proteins under selection. Each node represents a protein and each edge represents a relationship where one mutation precedes or proceeds another. Orange edges represent M to D shifts and blue edges represent D to M shifts. The relative size of a node and thickness of an edge is proportional to frequencies within the dataset. Proteins that collectively form the ring represent commonly mutated targets in this study and likely have a substantial role in c-di-GMP regulation. Proteins with limited connectivity fall outside the main ring. B) LC-MS/MS validates that proteins with significant network connectivity, but not previously associated with c-di-GMP, have a significant impact on intracellular c-di-GMP levels. C-di-GMP measurements are reported as the triplicate mean ± SD. C-di-GMP values of each mutant and its respective parent are summarized in with the corresponding ID tag. D strains are shown in orange and M strains are shown in blue. (Student’s t-test: * = p < 0.05, ** = p < 0.01).

Article Snippet: 1mL of 0.5 OD 600 culture was centrifuged at 15,000 RCF for 30 seconds, and cell pellets were resuspended in 100uL of ice cold acetonitrile/methanol/water (40:40:20 (v/v/v)) extraction buffer supplemented with a final concentration of 0.01% formic acid and 25nM c-di-GMP-Fluorinated internal standard (InvivoGen CAT: 1334145-18-4).

Techniques: Selection, Mutagenesis, Liquid Chromatography with Mass Spectroscopy

Journal: Frontiers in Immunology

Article Title: New MoDC-Targeting TNF Fusion Proteins Enhance Cyclic Di-GMP Vaccine Adjuvanticity in Middle-Aged and Aged Mice

doi: 10.3389/fimmu.2020.01674

Figure Lengend Snippet:

Article Snippet: Cyclic di-GMP (vaccine-grade) , Invivogen , Cat# vac-nacdg.

Techniques: Enzyme-linked Immunosorbent Assay, Protein Extraction, Lysis, Software