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KU Leuven | ku leuven · Systems Biology

Here, we determined the relative importance of different transcriptional mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae, by employing an array of experimental techniques under multiple genetic and environmental perturbations. Of the 143 genes tested (21% of the bacterium's annotated proteins), only 55% showed an altered phenotype,...

Transcription is a core process of bacterial physiology, and as such it must be tightly controlled, so that bacterial cells maintain steady levels of each RNA molecule in homeostasis and modify them in response to perturbations. The major regulators of transcription in bacteria (and in eukaryotes) are transcription factors. However, in genome-reduc...

ChIP-seq peaks associated to RNAP (see Methods and Materials and Supp. Methods text for experimental procedures and for the identification of peaks). For each of the peaks, the following information is displayed: peak position (in bps); peak height (in arbitrary units); peak width (in bps covered); peak score, based on the confidence in the intra-p...

Sheet 1: List of RNAseq experiments used in this work. For each sample, we indicate the strain (wt, M129 or mutant), transgene (indicates the gene that was overexpressed or mutated), timeOfGrowth_experimentPerformedAt in h (time of growth after inoculum), medium used, treatment (type of drug/perturbation), perturbant (drug, condition…), finalConcen...

Table S1. Known or Putative TFs and Operon Map of Mycoplasma pneumonia, Related to Experimental Procedures Sheet 1: List of known or putative transcriptional regulators in M. pneumoniae. The last column indicates the name of the strains in which the TF is perturbed (see Table S2) Sheet 2: Manual operon and sub-operon annotation of the M. pneumonia...

Table S3. Analysis of Variations of Transcriptional Read-Through, Related to Figure 4 Sheet 1: Leftmost list: conditions leading to an overall repression of TRT, that is, showing a tendency for having Δdown≤Δup. The average value of Δdown − Δup (third column) is computed over all the TTSs. The list is sorted according to the p values of the bias o...

Oligos used for the RT-qPCR.

Coordination of transcription in bacteria occurs at supra-operonic scales, but the extent, specificity, and mechanisms of such regulation are poorly understood. Here, we tackle this problem by profiling the transcriptome of the model organism Mycoplasma pneumoniae across 115 growth conditions. We identify three qualitatively different levels of co-...

cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the numb...

Summary of structure-energy and statistical properties of mutations in the hydrophobic pocket of BRAF and parameters used for random forest analyses. This table summarizes on sheet 1 for all mutations in the hydrophobic pocket the mutation frequencies in cancer, the average energies predicted using FoldX using active or inactive BRAF template struc...

Many driver mutations in cancer are specific in that they occur at significantly higher rates than – presumably – functionally alternative mutations. For example, V600E in the BRAF hydrophobic activation segment (AS) pocket accounts for >95% of all kinase mutations. While many hypotheses tried to explain such significant mutation patterns, conclusi...

The human respiratory tract pathogen M. pneumoniae is one of the best characterized minimal bacterium. Until now, two main groups of clinical isolates of this bacterium have been described (types 1 and 2), differing in the sequence of the P1 adhesin gene. Here, we have sequenced the genomes of 23 clinical isolates of M. pneumoniae. Studying SNPs, n...

Distinguishing between promoter-like sequences in bacteria that belong to true or abortive promoters, or to those that do not initiate transcription at all, is one of the important challenges in transcriptomics. To address this problem, we have studied the genome-reduced bacterium Mycoplasma pneumoniae, for which the RNAs associated with transcript...

Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome-reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (smORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality...

Background RNA sequencing methods have already altered our view of the extent and complexity of bacterial and eukaryotic transcriptomes, revealing rare transcript isoforms (circular RNAs, RNA chimeras) that could play an important role in their biology. Results We performed an analysis of chimera formation by four different computational approache...

In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expres...

Transcriptome and proteome data. MPNr is the nomenclature used for ribosomal RNAs and MPNt is the nomenclature used for tRNAs. MPNs are the non coding RNAs. (PDF)

Functional enrichment of COG categories for promoter (a) and 5′ UTR regions (b). The functional enrichment was measured by comparing all the function of all the ORFs that have an associated promoter sequence or 5′UTR with those that have these regions methylated. Enrichment is measured by Fisher's test and significant enrichment is considered when...

Methylation in 5′UTR regions. Start and End regions are the genome positions comprising the 5′UTR region. Column named “motifs” indicates the number of motifs identified. Str is the abbreviation for strand and indicates the gene orientation (“+” forward strand, “−” reverse strand). The rest of columns indicate the sequence of the motifs and the gen...

Study of transcription in ORFs containing 5′-CTAT-3′ motifs that showed changes in methylation between 96 and 6 h. First column indicates methylated positions that show an increase of methylation in different phases of growth. Second column shows the strand where the motif is found. Third column indicates the IPD ratio between 6 h and 96 h. Fourth...

a) Table of primers used in this study. b) Table of vectors used to clone and express different putative methyltransferases of M. pneumoniae in ER2796 strain. (PDF)

Heatmap of expression data of 13 genes involved in transcription and replication obtained by RNA-seq. The heatmap shows the correlation in gene expression among these genes, using data from 12 different time points from exponential and stationary growth phases. mpn003 and mpn004 codify for the subunits of the DNA gyrase; mpn198 (M.MpnI); mpn515 and...

ORFs with a 5′-CTAT-3′ motif that changed from non-methylated to methylated state from 6 to 96 h. (PDF)

Functional analysis of genes located in “hot spots of methylation”. COG categories of genes located in enriched regions for 5′-CTAT-3′ (a) and 5′-GAN7TAY-3′/3′-CTN7ATR-5′ (b) motifs have been compared to those categories in whole genome by using Fisher's test. A functional enrichment is considered significant when the Pvalue<0.05. (PDF)

Methylation in promoter sequences. Genomic regions 40 bp upstream from the TSS are considered as putative promoter sequences. This table shows the 197 out 663 ORFs with assigned TSS that showed methylation at the promoter region. First column indicates the ORF name. The rest of columns indicate the sequence of the motifs and the genome positions, a...

Enriched regions for 5′-GAN7TAY-3′/3′-CTN7ATR-5′ (a) and 5′-CTAT-3′ (b) motifs. Table S2c is the legend for the functions assigned to the different COG categories. (PDF)