wild type wt c glutamicum atcc 13032 strain  (ATCC)

Journal: bioRxiv

Article Title: Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from Escherichia coli

doi: 10.1101/2021.04.01.438067

Figure Lengend Snippet: a – Growth curve (solid lines) and specific fluorescence (dotted lines) of the wt (grey) and SM34 [(bL19-EYFP uS2-PAmCherry), green] strains from a BioLector cultivation in BHI+GLU medium. Shown is the mean and standard deviation of biological triplicates. b – DNA and ribosome distribution of exponential and stationary C. glutamicum SM30 (bL19-EYFP) cells cultivated on BHI+GLU. DNA was stained with SYTOX Orange (red). Scale bar = 1 µm.

Article Snippet: The strains used in this study were the C. glutamicum wild-type (wt) strain (ATCC 13032) or its derivatives MB001(DE3), a prophage-free strain which allows for IPTG-inducible T7-RNA polymerase-dependent gene expression [ ] and EVO5, a laboratory evolved faster-growing strain [ ].

Techniques: Fluorescence, Standard Deviation, Staining

Journal: bioRxiv

Article Title: Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from Escherichia coli

doi: 10.1101/2021.04.01.438067

Figure Lengend Snippet: a – Examples of SMLM images taken from cells cultivated in different media and at different stages of growth. C. glutamicum SM34 was cultivated in different media (indicated on the left) enabling increasing exponential growth rates ordered from top to bottom. Samples were taken either in mid-exponential or in stationary growth phase (24 h). Shown are the transmission image (grey) and the corresponding rendered SMLM image (colour) where each dot represents a PAmCherry localization. Higher density regions are shown in yellow whereas darker colours denote lower density regions. Scale bar = 1 µm. b – PAmCherry counts (number of ribosomes) determined by SMLM quantification and cell volume are plotted for every individual cell analysed. Cell volume was calculated for every cell following the formula: V = π* W 2 ( L − W /3)/4 . Each growth condition is colour coded. c – The mean cell length (SEM is within 2-7% and approximately the symbol size) and width (SEM is smaller than 2%) is shown for each growth condition (same colour code as in b ) and plotted against the corresponding growth rate.

Article Snippet: The strains used in this study were the C. glutamicum wild-type (wt) strain (ATCC 13032) or its derivatives MB001(DE3), a prophage-free strain which allows for IPTG-inducible T7-RNA polymerase-dependent gene expression [ ] and EVO5, a laboratory evolved faster-growing strain [ ].

Techniques: Transmission Assay

Journal: bioRxiv

Article Title: Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from Escherichia coli

doi: 10.1101/2021.04.01.438067

Figure Lengend Snippet: C. glutamicum SM34 was cultivated in different media and conditions yielding different growth rates. Cell samples taken during mid-exponential or in stationary growth phase were imaged by SMLM. a – PAmCherry counts (number of ribosomes) per cell volume determined by SMLM quantification for each growth condition tested. Quantification from exponentially growing cultures are represented by black diamonds whereas those from stationary cultures are shown as grey squares. b – The values from exponentially growing cultures, shown in a , are plotted against the growth rates of the respective cultures (black diamonds). A stationary sample was included as reference for zero growth rate (grey square). c – Comparison of the R/P ratio measurements (grey circles) to the SMLM data shown in b . Shown are the mean and standard deviation calculated for each condition to which a segmental linear regression was applied. In panel c , standard deviation was omitted for the SMLM data set to improve clarity.

Article Snippet: The strains used in this study were the C. glutamicum wild-type (wt) strain (ATCC 13032) or its derivatives MB001(DE3), a prophage-free strain which allows for IPTG-inducible T7-RNA polymerase-dependent gene expression [ ] and EVO5, a laboratory evolved faster-growing strain [ ].

Techniques: Comparison, Standard Deviation

Journal: bioRxiv

Article Title: Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from Escherichia coli

doi: 10.1101/2021.04.01.438067

Figure Lengend Snippet: In addition, at low growth rates C. glutamicum presents very low translation elongation rates and a high amount of active ribosomes. a – R/P ratio or ribosomal fraction of C. glutamicum (blue) compared to that of E. coli cultivated at either 30 °C (light grey) or 37 °C (dark grey). E. coli data was extracted from the literature, for 30 °C [ , ] and for 37 °C [ , , , ] b – Translation elongation rate for C. glutamicum MB001(DE3) pMKEx2- eyfp growing at various growth rates. Depicted are the mean values and standard deviation from at least 2 independent repeats with 3 technical replicates each. c – Active ribosome fraction calculated for different growth rates (Supplementary Note 4) for C. glutamicum (blue) compared to that of E. coli cultivated at 37 °C (grey) extracted from Dai et al. . Solid lines in a , b and c were derived from model simulations.

Article Snippet: The strains used in this study were the C. glutamicum wild-type (wt) strain (ATCC 13032) or its derivatives MB001(DE3), a prophage-free strain which allows for IPTG-inducible T7-RNA polymerase-dependent gene expression [ ] and EVO5, a laboratory evolved faster-growing strain [ ].

Techniques: Standard Deviation, Derivative Assay

Journal: bioRxiv

Article Title: Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from Escherichia coli

doi: 10.1101/2021.04.01.438067

Figure Lengend Snippet: Under good nutrient conditions there is no shortage of translation precursors. The majority of ribosomes are active and translation elongation reaches close to maximum rates, thus faster growth is achieved by increasing the ribosome fraction of the proteome. This gives rise to the well-established linear Rb/µ correlation . In less optimal conditions, translation precursors become limiting triggering ppGpp synthesis. In some bacteria, this leads to the downregulation of ribosome production as well inhibition of several GTP-dependent enzymes involved in translation such as the initiation factor 2 (IF2) and ribosome biogenesis factors . The decrease in ribosome abundance, translation initiation and ribosome assembly can all lead to a lower elongation rate. In addition, lack or low abundance of aminoacyl-tRNAs can cause ribosome pausing that if not resolved results in abortive translation events [ , ]. These events lead to premature translation termination of stalled ribosomes and subsequent degradation of the partially translated peptide(s) . Ribosomes involved in abortive translation events are then considered inactive since they do not produce the intended protein. Alternatively, ribosomes can also be kept in an inactive form through ribosome hibernation mechanisms . Significant ribosome inactivation in E. coli frees substrates needed for translation and allows substantial translation rates during slow growth . However, under the conditions examined in this study, we found that ribosome hibernation probably does not play an important role in C. glutamicum . Instead, we show via a model-based approach that the depletion of translation precursors such as aminoacylated-tRNAs during slow growth is able to reproduce the experimentally determined very low elongation rates measured. Towards lower growth rates, when nutrients are limiting, there is then a balance between the fraction of active ribosomes and their translation elongation rate. Under this regime ribosomes exist in excess to allow for quick growth recovery once conditions improve. How different organisms manage this balance appears to be different and may depend on the environmental niches in which they evolved.

Article Snippet: The strains used in this study were the C. glutamicum wild-type (wt) strain (ATCC 13032) or its derivatives MB001(DE3), a prophage-free strain which allows for IPTG-inducible T7-RNA polymerase-dependent gene expression [ ] and EVO5, a laboratory evolved faster-growing strain [ ].

Techniques: Bacteria, Inhibition