Deletion of the moeA gene in Flavobacterium IR1 drives structural color shift from green to blue and alters polysaccharide metabolism
- Hoekmine BV, Netherlands
- Institute of Biodiversity, Faculty of Biological Sciences, Cluster of Excellence Balance of the Microverse, Friedrich Schiller University Jena, Germany
- Laboratory of Microbiology, Wageningen University & Research, Netherlands
- CIIMAR, Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leixões, Portugal
- Department of Biomedical and Clinical Sciences, Faculty of Medicine and Health Sciences, Linköping University, Sweden
- Yusuf Hamied Department of Chemistry, University of Cambridge, United Kingdom
- Max Planck Institute of Colloids and Interfaces, Germany
- Theoretical Biology and Bioinformatics, Science4Life, Utrecht University, Netherlands
Figures
Figure 1
Phylogenetic analysis of MoCo operon.
(A) Schematics of the putative molybdopterin synthesis operon in the IR1 genome. In blue, the target gene: moeA. (B) Phylogenetic tree of the 16 S ribosomal RNA gene, showing IR1 and other seven selected strains. (C) Synteny and homology visualization of genes that are putatively involved in molybdopterin synthesis. Spacers indicated with // represent stretches longer than 5 kb on the same contig, which may encode unshown genes. Whitespaces separate different contigs. (D) Presence of SC in the selected strains and its SC score based on the SC classifier software (Zomer et al., 2024). The suggested cut-off value (0.68) for presence of SC is shown as a dashed vertical line.
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Figure 1—source data 1
Synteny coordinates of MoCo operon.
- https://cdn.elifesciences.org/articles/105029/elife-105029-fig1-data1-v1.txt
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Figure 1—source data 2
Table containing presence or absence of the MoCo synthesis genes in different bacterial genomes.
- https://cdn.elifesciences.org/articles/105029/elife-105029-fig1-data2-v1.csv
Figure 2
Optical phenotype of IR1 WT and ΔmoeA.
(A) Colonies of IR1 WT and IR1 ΔmoeA grown on agar plates with three different nutrient conditions: ASWB, ASWBLow, and MM. (B) Schematic of how the colony image was taken. It shows the position of the incident light and the camera as X, Y, and Z coordinates (X,Y, Z). The colony is positioned at position (0,0,0), the light source at (0,–1,1), and the camera at (1,0,1). The red dotted line represents the light direction, the blue dotted line represents the camera direction, and the red and blue lines represent the position of the light and the camera.
Figure 3
ΔmoeA colonies grown on ASWB and photographed from different angles.
The location of the camera is shown in the bottom right of each panel, following the scheme on Figure 2B. The camera coordinates are (A) (0,–1,1.1), (B) (1,-1,1), (C) (1,–1,0.36), (D) (1,0,0.36), (E) (1,0,0.18), and (F) (0.58,1,1). The light was always positioned at (0,–1,1).
Figure 4
Influence of different polysaccharides in the colony color and diameter of IR1 WT and ΔmoeA.
(A) Colonies of IR1 WT and ΔmoeA are grown for 2 days with 1% of three different polysaccharides: Artificial Sea Water Black with Kappa-Carrageenan instead of agar (ASWBKC), ASWB with agar and Fucoidan (ASWBF), and ASWB with agar Starch (ASWBS). All the photos were taken from position (1,0,1), following the scheme on Figure 2B. (B) Colony diameter in centimeters of IR1 WT and ΔmoeA grown on different media after 6 days, as mean ± standard deviation of three biological replicates. ns (non significance), * (p-value < 0.05), ** (p-value < 0.001), *** (p-value < 0.0001).
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Figure 4—source data 1
Colony diameter data of IR1 WT and ΔmoeA when growing on different polysaccharides.
- https://cdn.elifesciences.org/articles/105029/elife-105029-fig4-data1-v1.xlsx
Figure 5
Goniometry analysis of IR1 WT and ΔmoeA strains grown as a film layer on ASWBKC medium.
Specular reflection analysis of (A) WT, and (B) ΔmoeA, and scattering (light illumination with an angle of 60o) of (C) WT, and (D) ΔmoeA. The dotted lines represent the values of the grating equation.
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Figure 5—source data 1
Specular reflectivity, optical data for IR1 WT.
- https://cdn.elifesciences.org/articles/105029/elife-105029-fig5-data1-v1.zip
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Figure 5—source data 2
Optical scattering data for IR1 WT.
- https://cdn.elifesciences.org/articles/105029/elife-105029-fig5-data2-v1.zip
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Figure 5—source data 3
Specular data for the optical properties of colonies of IR1 ΔmoeA.
- https://cdn.elifesciences.org/articles/105029/elife-105029-fig5-data3-v1.zip
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Figure 5—source data 4
Scattering data for ΔmoeA.
- https://cdn.elifesciences.org/articles/105029/elife-105029-fig5-data4-v1.zip
Figure 6
Images taken with a KEYENCE microscope using full coaxial light at the edge of the colony of IR1 WT and ΔmoeA growing on ASWB.
These are frames at 0 min, 30 min, and 60 min from the respective 1 hr time-lapse videos. The blue arrows indicated the motility of a group of cells, and the yellow arrows indicated the forming of circular ‘vortex’ patterning and movement.
Figure 7
Volcano plots of the peptides identified in (A) the intracellular protein analysis, and in (B) the extracellular protein analysis.
Some of the most regulated proteins are shown in the plots. The horizontal dashed lines represent the cut-off value for the p-value (2), and the vertical dashed lines represent the cut-off value for the fold change (–1 and 1).
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Figure 7—source data 1
Cellular proteomics data for IR1 WT and ΔmoeA.
- https://cdn.elifesciences.org/articles/105029/elife-105029-fig7-data1-v1.xlsx
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Figure 7—source data 2
Extracellular proteomics data for IR1 WT and ΔmoeA.
- https://cdn.elifesciences.org/articles/105029/elife-105029-fig7-data2-v1.xlsx
Figure 8
Putative operons or gene clusters with differentially expressed proteins identified in (A) intracellular and (B) extracellular proteomic analyses, based on function and proximity.
To the left of each operon are the accession numbers of the translated proteins, to the right is the predicted function. Gene or protein names are indicated. Genes are colored based on the fold change of the encoded proteins. The black bars show the scale in kilobase pairs (kbp). TP: transporter, hyp: hypothetical protein, GA: glycoamylase, GHX: glycosyl hydrolase family X, GDP: glycerophosphoryl diester phosphodiesterase, CLB: colibactin biosynthesis, TBDR: TonB-dependent receptor, OMP: outer membrane protein, NTF: nuclear transport factor 2, ABH: alpha/beta hydrolase, TRX: thioredoxin domain-containing protein, XAT: xenobiotic acyltransferase, MT: SAM-dependent methyltransferase, GT-X: glycosyltransferase family X, CF: cell surface protein, SP: secretion protein.
Figure 9
Colonies of IR1 WT (top row) and ΔmoeA (bottom) grown on ASWS.
Iodine vapor was used to dye the starch remaining in the media. The zones of starch degradation are seen as the lighter areas under the colonies. The images were taken at the same 90° angle from the front (left column) and back (right) of the plate.
Tables
Table 1
Analysis of 117 bacterial genomes (87 SC and 30 non-SC) for the presence of the genes involved in molybdopterin cofactor synthesis.
| moeA | mobA | sumT | moaD | moeZ | moaE | moaC2 | moaA | |
|---|---|---|---|---|---|---|---|---|
| SC bacteria | 100% | 87% | 100% | 70% | 100% | 100% | 100% | 100% |
| Non-SC bacteria | 40% | 37% | 63% | 20% | 70% | 40% | 40% | 50% |
Author response table 1
| Protein | Intracellular | Extracellular |
|---|---|---|
| GIdL | 0.34 | -2.22 |
| PorU | 0.67 | 1.15 |
| SprT | 0.69 | - |
Additional files
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Supplementary file 1
Supplementary tables.
a. Bacterial strains used in this study. b. Plasmids used in this study. c. Oligonucleotides used in this study. d. The most downregulated intracellular proteins in the ΔmoeA mutant, and proteins mentioned in the main text and in Figure 8. * Gene name or protein with the designation that gives the most information. “-” means no gene name; hyp: hypothetical; TP: transporters. e. The 5 most upregulated intracellular proteins in the ΔmoeA mutant, and proteins mentioned in the main text and in Figure 8. * Gene name or protein with the designation that gives the most information. “-” means no gene name; hyp: hypothetical; GT-2: glycosyltransferase family 2; GHX: glycosyl hydrolase family X; TRX: thioredoxin domain-containing protein; TBDR: TonB-dependent receptor; OMP: outer membrane protein; CLB: colibactin biosynthesis ABH: alpha/beta hydrolase. f. The most 5 downregulated extracellular proteins in the ΔmoeA mutant, and proteins mentioned in the main text and in Figure 8. * Gene name or protein with the designation that gives the most information. “-” means no gene name; hyp: hypothetical. ** SP: signal peptide; NC: non-classical; -: not secreted. g. The 5 most upregulated extracellular proteins in the ΔmoeA mutant, and proteins mentioned in the main text and in Figure 8. * Gene name or protein with the designation that gives the most information. ABH: alpha/beta hydrolase; SP: secreted protein; CF: cell surface. ** SP: signal peptide; NC: non-classical; -: not secreted.
- https://cdn.elifesciences.org/articles/105029/elife-105029-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/105029/elife-105029-mdarchecklist1-v1.docx
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Deletion of the moeA gene in Flavobacterium IR1 drives structural color shift from green to blue and alters polysaccharide metabolism
eLife 14:RP105029.
https://doi.org/10.7554/eLife.105029.3
