https://doi.org/10.5061/dryad.xsj3tx9pn

The Supplementary Information consist of:

• 6 Supplementary Tables - Supplementary Tables 1 to 6 in .xlsx format.
• 7 Supplementary Network Images - PDF files reporting all networks discussed in the study with a greater resolution.
• 3 Supplementary Data - 1 file in .xlsx format (Supp. Data 1. Developmental time WT-deltaPxr-deltaPxrA.xlsx), 1 file in .csv format (Supp. Data 2. M. xanthus gene annotation from UniProt.csv), and 1 image in .pdf format (Supp. Data 3. Original images of the blot membrane). These data are directly relevant to results present in the main figures.
• 1 Supplementary Code - folder containing 1 R script (app.R), two data frames in .xlsx format (data.xlsx and dev_data.xlsx)
• Supplementary Figure Raw Data - 2 files in .xlsx format (Supp. Fig. 1a and Supp. Fig. 1b). These file are relative to the two figure panels indicated in their name.

Description of the data and file structure

Supplementary Tables

Supp. Table. 1. Differential expression between WT and Δpxr. Colours refer to the sign of expression and significance levels.This data consists of 13 columns:

1. transcript_id: id of individual annotated transcripts.
2. base_mean: the average of the normalized count values, dividing by size factors, taken over all samples.
3. log2FC: log2 of the fold change between the two treatments.
4. StdErr: standard error to the expression values.
5. Wald.T: Wald T Statistics on the two groups.
6. p.val: p-value.
7. p.adj: adjusted p-value for multiple comparisons.
8. MXAN_id: Gene ID reported as MXAN_number
9. gene.name: either an alternative gene name or the MXAN_number.
10. Function: predicted function of the gene product.
11. COG_name: Name of individual Clusters of Orthologous Genes to which a given genes have been annotated.
12. COG_cat: Category of individual Clusters of Orthologous Genes to which a given gene have been annotated.
13. COG_des: Designation/description of individual Clusters of Orthologous Genes to which a given genes have been annotated.

Supp. Table. 2. List of the enriched operons and differentially expressed genes associated with them. Genes previously associated with multicellular development are shown in bold (ref. 7–9). This data consists of 4 columns:

1. Operon ID: M. xanthus operon ID number
2. DE genes per operon: number of genes differentially expressed over the total number of genes included in a given operon.
3. p: adjusted p-value for multiple comparisons.
4. MXAN gene IDs: gene name reported as MXAN_numbers.

Supp. Table. 3. List of enriched COG categories and the differentially expressed genes associated with them. Red and blue MXAN gene IDs indicate decreased and increased transcript levels, respectively. This data consists of 4 columns:

1. Term: specific name identifying a given COG.
2. COG definition: specific description of the COG term.
3. p: adjusted p-value for mutliple comparisons.
4. MXAN gene IDs: gene name reported as MXAN_numbers.

Supp. Table. 4. List of all enriched GO terms and the differentially expressed genes associated with them (also refer to Fig. 2d). This data consist of 8 columns:

1. GO term: GO term identity
2. Cat.: GO category.
3. Description: Description of the GO term.
4. p: p-value for the GO term.
5. FDR: false discovery rate (0.05) for mutliple comparisons.
6. DK1622 genes background: number of DK1622 annoteted genes to match a given GO term.
7. #genes: numer of differentially expressed genes associated to a given GO term.
8. Gene names: gene names reported as MXAN_numbers.

Supp. Table. 5. List of all developmental genes found in M. xanthus. This data consist of 10 columns:

1. MXAN_RS: MXAN Ref Sequence ID.
2. MXAN_id: MXAN_number.
3. log2FC: log2 of the fold change between the two treatments.
4. p.adj: adjusted p-value for multiple comparisons.
5. dev_ in McLoon et al. 2021: associated with development in McLoon et al. 2021. DG = development gene; no-DG non developmental gene.
6. dev_ in Sharma et al. 2021: associated with development in Sharma et al. 2021.
7. dev_ in Muñoz-Dorado et al. 2019: associated with development in Muñoz-Dorado et al. 2019.
8. in 3 all studies: gene associated with development in all three studies.
9. potential Dev Gene: gene associated with development in at least one study.
10. gene.name: standard gene name.

Supp. Table. 6. List reporting the potential target sites bound by Pxr generated using TargetRNA3 (Tjaden B. 2023)ref.51.

1. MXAN_RS: MXAN Ref Sequence ID.
2. MXAN_ID: MXAN_number.
3. Energy: estimate of the energy from the predicted RNA-DNA interaction.
4. P: p-value.
5. Probability: the probability that there is a regulatory interaction between the sRNA and target.
6. sRNA start: start location on the sRNA sequence.
7. sRNA stop: stop location on the sRNA sequence.
8. T. start: start location on the target sequence.
9. T. stop: stop location on the target sequence.
10. log2 Fold: log2 fold change in the transcriptional expression of the gene target in the experimental condition.
11. adj. p-val: adjusted p-value for multiple comparisons associated with gene expression levels.

Supplementary Network Images

STRINGdB Networks complete - Detailed image reporting all obtained networks.

STRINGdB Network with DE levels - Detailed image of the main network reporting genes coloured coded as in Fig. 2d according to their transcript levels.

STRINGdB Network with GO - Detailed image of the main network reporting genes coloured coded as in Fig. 2d according to their GO.

STRINGdB Network - All Dev Genes - Detailed image reporting the main obtained network with all identified developmental genes from all three consulted studies.

STRINGdB Network - Sharma et al. Dev Genes - Detailed image reporting the main obtained network with developmental genes identified in Sharma et al. 2021 study.

STRINGdB Network - McLoon et al. Dev Genes - Detailed image reporting the main obtained network with developmental genes identified in McLoon et al. 2021 study.

STRINGdB Network - Muñoz-Dorado et al. Dev Genes - Detailed image reporting the main obtained network with developmental genes identified in Muñoz-Dorado et al. 2019 study.

Supplementary Data

Supp. Data 1. M. xanthus gene annotation from UniProt.csv- List of all annotated M. xanthus genes with their associated function obtained from UniProt. This table consist of 7 columns:

1. New_MXAN: MXAN Referece Sequence IDs.
2. Old_MXAN: MXAN_number
3. Protein_ID: protein IDs from Uniprot.
4. Function: annotated protein or transcript function.
5. COG_name: Name of individual Clusters of Orthologous Genes to which a given genes have been annotated.
6. COG_cat: Category of individual Clusters of Orthologous Genes to which a given gene have been annotated.
7. COG_des: Designation/description of individual Clusters of Orthologous Genes to which a given genes have been annotated.

Supp. Data 2. Developmental time WT, ΔPxr, PxrA-.xlsx - Raw data for the fruting body counts over time for WT, Δpxr and pxrA-. This table consist of 4 columns:

1. id: frame id.
2. sample: name or id for each individual samples + replicate.
3. slice: sequencial imaging frame id.
4. time: conversion of the slice to hours.

Supp. Data 3. Original images of the blot membrane - Raw image of the uncut membrane used to estimate FruA aboundancy.

Supp. Fig. 1a. Spore counts of the strain GJV1(WT), pPxr1, pPxrJ all relative to the spore counts of the Δpxr strain. This table consist of 3 main columns:

1. sample: strain name
2. rep: biological replicate
3. spore counts rel. to delta-pxr: spore count ratio relative to Δpxr.

Additional columns:
4. p-values: p-values calcualated using a t-test with Welch's correction relative to the pairwise comparisons reported next to them.
5. Sig: NS = non-significant, S = significance.
6. adj.pval Bonf: reference to estimate significance values base on Bonferroni p-value adjustment relevant to the 2 or 3 pariwise comparisons.

Supp. Fig. 1b. Developmental time WT, Δpxr, dPpxr.xlsx - Raw data for the fruting body counts over time for WT, Δpxr and pxrA-. This table consist of 4 main columns:

1. sample: strain name
2. rep: biological replicate
3. time: time expressed in hours from the beginning of the experiment.
4. FB counts: number of fruiting bodies counted on the plate per time point.

Additional columns:
5. mean: mean FB counts obtained calculated from column 4.
6. sem: standard error of the mean calculated from column 4.

Supplementary Code

The app.R file runs on R software. The authors were using R 4.2.2 to create these script. The two data frames data.xlsx and dev_data.xlsx correspond to the Supp. Table 1 and Supp. Table 5, respectively. The output can be visulasied on Shinyapp.io at the following link: https://jzego7-marco.shinyapps.io/rnaseq_app

Changes after review 10.11.2024

Two new raw data sets are included. Specifically Supp. Fig. 1a and Supp. Fig 1b which contain data relevant to the two indicated supplementary figures (see decription).