Changes in the genetic requirements for microbial interactions with increasing community complexity
- University of California, San Diego, United States
- Jacobs School of Engineering, University of California San Diego, United States
Figures
Figure 1 with 7 supplements
Identification of genes important for growth of E. coli alone on cheese curd agar (Figure 1—source data 1).
(A) The pooled E. coli RB-TnSeq library Keio_ML9 (Wetmore et al., 2015) was grown alone on cheese curd agar (CCA). Gene fitness values were calculated for 3298 genes at days 1, 2, and 3 along with a …
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Figure 1—source data 1
RB-TnSeq analysis of E. coli’s growth alone on 10% cheese curd agar, pH7.
- https://doi.org/10.7554/eLife.37072.012
Figure 1—figure supplement 1
Pipeline of RB-TnSeq experiment using the E. coli Keio M9 library: from experimental set-up to normalized gene fitness and t-score calculation.
Calculation of gene fitness from the Illumina sequencing files was performed using the Perl script BarSeqTest.pl from (Wetmore et al., 2015). The detailed description of the pipeline can be found in …
Figure 1—figure supplement 2
Quantification of free amino acids in CCA.
Free amino acid characterization and quantification have been carried out by the Proteomics and Mass Spectrometry Facility of the Donald Danforth Plant Science Center and each analysis has been …
Figure 1—figure supplement 3
Comparison of individual growth of enterobactin biosynthesis mutants on LB and CCA.
5% tetrazolium solution (100 uL/L of medium) was added to the media to color colonies and make them visible on CCA. We selected 4 mutants of enterobactin biosynthesis from the Keio collection. 5 μL …
Figure 1—figure supplement 4
RB-TnSeq experiments using the P. psychrophila JB418 library.
(A) We grew P. psychrophila JB418 alone, in pairwise conditions with H. alvei, G. candidum or P. camemberti and with the full community on CCA. (B) Using the transposon library of P. psychrophila …
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Figure 1—figure supplement 4—source data 1
RB-TnSeq analysis of P. psychrophila’s growth alone, in pairwise conditions and with the community on 10% cheese curd agar, pH7.
- https://doi.org/10.7554/eLife.37072.008
Figure 1—figure supplement 5
Competitive assays of 25 mutants of the Keio collection (Baba et al., 2006).
Competition assays between single knockouts and the wild-type strain have been carried out for 25 strains associated with genes identified as important for E. coli growth using RB-TnSeq (Significant …
Figure 1—figure supplement 6
Map of the JB418_ECP1 transposon library generated in P. psychrophila JB418.
We built a barcoded-transposon library in the cheese isolate P. psychrophila JB418. 272,329 insertions were mapped to the genome and 143,491 barcodes were located in the central region of a gene. …
Figure 1—figure supplement 7
Quality assessment of all RB-TnSeq experiments.
For each RB-TnSeq experiment different parameters are calculated to assess the quality of each RB-TnSeq experiment. If all quality parameters are met, then the gene fitness values can be further …
Figure 2 with 2 supplements
E. coli genes with negative fitness during growth in pairwise conditions (Figure 2—source data 1).
(A) We grew E. coli in pairwise conditions on CCA with either H. alvei, G. candidum or P. camemberti. Asterisks indicate significant differences in growth of E. coli as compared to growth alone at …
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Figure 2—source data 1
RB-TnSeq analysis of E. coli’s growth in pairwise conditions on 10% cheese curd agar, pH7.
- https://doi.org/10.7554/eLife.37072.016
Figure 2—figure supplement 1
E. coli and community member growth curves alone, in pairwise conditions or during community growth.
Each graph represents the growth over time of E. coli, H. alvei, G. candidum, or P. camemberti alone, in pairwise growth or with the community. Assays have been performed in triplicates. Dunnett's …
Figure 2—figure supplement 2
Comparison of the genes important for E. coli growth alone, in each pairwise condition or with the community.
(A) Venn diagram of genes with negative fitness during growth alone or in each pairwise condition. The genes are detailed in Figure 2—source data 1. (B) Venn diagram of genes with negative fitness …
Figure 3
Comparison of E. coli genes with negative fitness within the community and in pairwise conditions (Figure 3—source data 1).
(A) Using the E. coli RB-TnSeq library, we identified genes required to grow with the community (H. alvei + G. candidum + P. camemberti). During growth with the community, we identified a total of …
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Figure 3—source data 1
RB-TnSeq analysis of E. coli’s growth with the community on 10% cheese curd agar, pH7.
- https://doi.org/10.7554/eLife.37072.018
Figure 4 with 1 supplement
Differential expression analysis of E. coli during interactive and non-interactive growth conditions (Figure 4—source data 1).
We used RNASeq to investigate E. coli gene expression at three timepoints (1, 2 and 3 days) during growth on CCA alone, in pairwise conditions (with H. alvei, G. candidum or P. camemberti) and with …
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Figure 4—source data 1
Differential expression analysis of E. coli’s growth in pairwise and with the community versus growth alone.
- https://doi.org/10.7554/eLife.37072.021
Figure 4—figure supplement 1
RNASeq analysis of E. coli gene expression during growth alone and in pairwise conditions.
We used RNASeq to investigate E. coli gene expression at three timepoints (1, 2 and 3 days) during growth on CCA alone, in pairwise conditions (with H. alvei, G. candidum or P. camemberti) and with …
Author response image 1
Investigation of parameters potentially influencing variability in gene fitness calculation.
Here, we compare (i) the number of insertion mutants/gene, (ii) the number of counts/gene in the T0 sample and (iii) the variability associated with insertion location, for genes with fitness values …
Author response image 2
Insertion location and variability of gene fitness values.
Upper-part: Quality parameters generated along with gene fitness calculation indicate low variability in gene fitness values associated with insertion location. Lower-part: Examples of insertion …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Library, strain background (Escherichia coli K12) | Keio collection | PMID: 16738554 | CGSC, RRID:SCR_002303 | Collection of 3,818 E. coli knockout strains |
| Library, strain background (Escherichia coli K12) | Keio_ML9 | PMID: 25968644 | RB-TnSeq library of E. coli K12 BW25113 (152,018 pooled insertion mutants) | |
| Library, strain background (Pseudomonas psychrophila) | JB418_ECP1 | this paper | RB-TnSeq library generated in the P. psychrophila JB418 strain isolated from cheese (272,329 pooled insertion mutants) | |
| Strain, strain background (Escherichia coli K12) | Keio ME9062 | PMID: 16738554 | CGSC#: 7636 | Parent strain of the Keio collection mutants. Also referred as E. coli K12 BW25113 |
| Strain, strain background (Hafnia alvei) | Hafnia alvei JB232 | this paper | Strain isolated from cheese | |
| Strain, strain background (Geotrichum candidum) | Geotrichum candidum | Danisco - CHOOZIT | GEO13 LYO 2D | Industrial starter for cheese production |
| Strain, strain background (Penicillium camemberti) | Penicllium camemberti | Danisco - CHOOZIT | PC SAM 3 LYO 10D | Industrial starter for cheese production |
| Strain, strain background (P. psychrophila) | Pseudomonas psychrophila JB418 | this paper | Strain isolated from cheese | |
| Strain, strain background (E. coli) | E. coli APA766 | PMID: 25968644 | donor WM3064 which carries the pKMW7 Tn5 vector library containing 20 bp barcodes | |
| Sequence-based reagent | NEBNext Multiplex Oligos for Illumina (Set 1); NEBNext multiplex Oligos for Illumina (Set 2) | New England Biolabs | NEB #E7335 (lot 0091412);, NEB #E7500 (lot 0071412) | |
| Sequence-based reagent | Nspacer_barseq_pHIMAR; P7_MOD_TS_index3 primers | PMID: 25968644 | Primers for transposon-insertion sites amplication for P. psychrophila RB-TnSeq library characterization | |
| Sequence-based reagent | BarSeq_P1; BarSeq_P2_ITXXX | PMID: 25968644 | Primers for RB-TnSeq PCR (amplification of the barcode region of the transposon) | |
| Commercial assay or kit | NEBNext Ultra DNA Library Prep Kit for Illumina | New England Biolabs | NEB #E7645 | |
| Commercial assay or kit | MinElute purification kit | Qiagen | ID:28004 | |
| Commercial assay or kit | Turbo DNA-free kit | AMBION, Life Technologies | AM1907 | |
| Commercial assay or kit | MEGAclear Kit Purification for Large Scale Transcription Reactions | AMBION, Life Technologies | AM1908 | |
| Commercial assay or kit | Ribo-Zero rRNA removal kit (bacteria); Ribo-Zero rRNA removal kit (yeast) | Illumina | MRZMB126; MRZY1306 | |
| Commercial assay or kit | NEBNextUltraTM RNA Library Prep Kit for Illumina | New England Biolabs | NEB #E7770 | |
| Software, algorithm | Geneious | http://www.geneious.com | ||
| Software, algorithm | Perl | https://www.perl.org/ | ||
| Software, algorithm | R | https://www.r-project.org/ | ||
| Other | MapTnSeq.pl; DesignRandomPool.pl; BarSeqTest.pl | PMID: 25968644 | Perl scripts for RB-TnSeq library characterization and RB-TnSeq analysis - https://bitbucket.org/berkeleylab/feba | |
| Other | DESeq2 | PMID: 25516281 | R package for RNASeq analysis |
Table 1
E. coli strains used during the study.
https://doi.org/10.7554/eLife.37072.022| Experiment | E. coli strain(s) | Reference |
|---|---|---|
| RB-TnSeq | E. coli Keio_ML9 library | (Wetmore et al., 2015) |
| Growth assays | E. coli JW0024 strain (undisrupted mutant) | (Baba et al., 2006) |
| Competition assays | WT: Keio ME9062 Mutants: (Figure 1—figure supplement 5) | (Baba et al., 2006) |
Table 2
Organization of CFU’s quantification for growth assays.
https://doi.org/10.7554/eLife.37072.023| E. coli + H. alvei JB232 | LB (E. coli + H. alvei JB232 CFUs) LB-kanamycin (50 µg/ml) (E. coli CFUs) |
|---|---|
| E. coli + G. candidum | LB-kanamycin:cycloheximide (50 µg/ml and 10 µg/ml) (E. coli CFUs) LB-chloramphenicol (G. candidum CFU’s) |
| E. coli + P. camemberti | LB-kanamycin:cyclohexamide (50 µg/ml and 10 µg/ml) (E. coli CFUs) LB-chloramphenicol (50 µg/ml)(P. camemberti CFU’s) |
| E. coli + Community | LB-cyclohexamide (10 µg /mL) (E. coli and H. alvei JB232 CFU’s), LB-kanamycin:cyclohexamide (50 µg/ml and 10 µg/ml) (E. coli CFU’s) and LB-chloramphenicol (50 µg/ml) (G. candidum and P. camemberti CFU’s) |
Additional files
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Supplementary file 1
Differential expression analysis of E. coli growth in community versus pairwise conditions.
We used RNASeq to investigate changes in E. coli gene expression between growth in the community and in each pairwise conditions (with H. alvei, G. candidum or P. camemberti). Using DESeq2 (Love et al., 2015), we identified up and downregulated genes during growth in community versus growth in each pairwise condition individually. Only genes associated with an adjusted p-value lower than 1% (Benjamini-Hochberg correction for multiple testing) and an absolute log of fold change higher than one were considered differentially expressed.
- https://doi.org/10.7554/eLife.37072.024
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Transparent reporting form
- https://doi.org/10.7554/eLife.37072.025
