Ribosome pausing in amylase producing Bacillus subtilis during long fermentation

Figures

• 

  Fig. 1. Growth and amylase production during repeated batch fermentation. B. subtilis BY212 containing the amyM overexpression plasmid pBW212 was grown in rich medium for 88 h at 30 °C under continuous shaking. Glucose was added at 16, 40 and 64 h (1% final concentration)

• 

  Fig. 2. Comparison of mupirocin ribosome profiling data by counting from either 3’ or 5’ end of RPF reads. (A) Schematic diagram for calculating the position of the first nucleotide in the ribosomal A-site codon from an RPF fragment from mupirocin treated cells when counting from either the 3’ or 5’ end of RPFs. In this example the ribosomal A-site contains the isoleucine codon ATC. (B) A-site distance distribution analysis. Average RPF reads of calculated A-sites for every nucleotide upstream and downstream of isoleucine codons. 0 indicates the first nucleotide of the calculated A-site of isoleucine codons. The A-site was determined by counting from either the 3’ end (offset 12 nt) or 5’ end (offset 14 nt). The advantage of this method is that it also indicates whether the offset used (12 and 14 nt) gives the best score

• 

  Fig. 3. Ribosome pause sites in amyM. (A) Upper profile: ribosome profile of amyM mRNA after 16 h growth. Peaks of more than 150 RPF reads are numbered. Lower profile: unprotected mRNA control sample. The signal peptide of AmyM spans amino acids 1–33. The first peaks, P1 and P2, are located before the start codon (B) Sequence details of the 12 ribosome pause sites. Ribosome density, based on RPF read numbers representing A-site codons (first nucleotide position), are shown in the first two lines, representing the 2 biological replicates (Ribo 1 and Ribo 2). The 3de line shows the read numbers from the unprotected mRNA control sample (mRNA). Red boxes indicate the Shine Dalgarno sequence and start codon. A-site codons of the peaks are bold and underlined. Peak 6 is marked by an asterisk since the high mRNA read numbers suggests this peak does not reflect a ribosome pause site

• 

  Fig. 4. Different protection at translation initiation sites. (A) Ribosome density based on RPF read numbers at the translation initiation site of amyM and several genes coding for abundant proteins. Red boxes indicate Shine Dalgarno sequences and start codons. (B) Average ribosome density around start codons by assigning the A-site from either the 3’ or 5’ end, using an offset of 12 nt and 14 nt, respectively

• 

  Fig. 5. Correlation between codon frequency and codon pause score. Scatter plots showing the correlation between A site codon pause score and genome wide codon frequency at 16 h. Genome wide ribosome frequencies of all the 61 codons are shown in Table S3. Glutamate codons (gaa, gag) and aspartate codons (gac, gat) are indicated. Regression line and R2 value are indicated. Red dots are codons at the main ribosome pause sites of amyM (Fig. 3). Biological replicates are shown in Fig. S5A

• 

  Fig. 6. Ribosome pause sites in amyM mRNA after 64 h fermentation. Ribosome profiles of amyM after 16 h and 64 h growth under repeated batch fermentation conditions. The signal peptide of AmyM spans amino acids 1–33. Main ribosome pause peaks are numbered, and new peaks are indicated in red. Profiles of the biological replicate are shown in Fig. S7. RFP read numbers and sequence details at the peak positions, including those of the biological replicates, are shown in Fig. S8

• 

  Fig. 7. Correlation between ribosome occupancy and mRNA abundance. Correlation of ribosome occupancy and mRNA abundance of genes at 16 h and 64 h growth. Genes coding for the 100 most abundant proteins in B. subtilis are indicated in blue. amyM shown by the encircled white dot. Notice the shift towards lower relative mRNA reads in the 64 h sample

• 

  Fig. 8. Ribosome profiles of toxin encoding genes displaying strong ribosome pause sites. Ribosome profiles of the toxin encoding epeX, sdpC, skfA genes, and the unknown genes yuzK and yhfH after 16 h growth. The amino acid size of the related proteins are indicated between brackets. Biological replicates are shown in Fig. S10