Non-canonical resource allocation in heterotrophically growing Thermoanaerobacter kivui

Abstract

Allocation of resources in the costly proteome reflects trade-offs between cellular functions. For example, proteome composition of Escherichia coli is significantly regulated by growth rate. An increasing anabolic, especially ribosomal, proteome fraction correlates with a decreasing catabolic proteome fraction at faster growth, which then leads to changes in catabolism. Our systems-level studies of the thermophilic acetogen Thermoanaerobacter kivui when growth rate is varied over two orders of magnitude revealed a different strategy: proteome allocation is only partially controlled by growth rate, and metabolic rates are primarily controlled posttranslationally. At slower growth, ribosome numbers are controlled by rRNA concentrations with an excess of ribosomal proteins. Composition of the catabolic proteome is uncoupled from catabolic rates as indicated by flux analysis. This study adds to the understanding of acetogenic Clostridia, which are of interest for biotechnological processes in a carbon-neutral economy, and points to a complex landscape of microbial ecophysiological strategies.

Fig. 1. Growth of T. kivui in anoxic, glucose-limited chemostats at different growth rates.

For reference, data from exponentially growing batch cells (orange) are included. Cultures were in steady state, and growth rate was considered equivalent to dilution rate under these conditions. a overview of catabolism in T. kivui and carbon atom patterns in degradation of ¹³C₁ glucose (labeled carbon represented as open circles). Black, metabolites; red, electron carriers (only shown in reduced state); blue, enzymes and pathways. b biomass yield as dry cell weight per mol ATP. As dry cell weight was calculated from OD₆₀₀ using a factor from batch cultures instead of individual measurements for each culture, this is only an approximation to calculate biomass yields. However, dry cell weight was calculated to include biomass of lysed cells as determined by analyzing DNA in the culture supernatant. ATP production is calculated from the amount of extracellular acetate, assuming an ATP yield of 2.9 mol ATP per 3 mol extracellular acetate. c maintenance energy, estimated from the slope of the linear regression of $\frac{1}{Y}$ versus $\frac{1}{\mu }$; d coarse-grained proteome fractions of catabolic (open symbols) and anabolic (filled symbols) proteome sectors; e steady state glucose concentration. Values indicated as 0 mM do not contain detectable glucose (detection limit of 0.05 mM); f fraction of acetate carbon in total carbon of products; g carbon balance as percent of total carbon of glucose degraded that was found in products and biomass. Red, chemostats; orange, exponentially growing cells from batch cultures. EMP Embden-Meyerhoff-Parnas pathway, PFOR pyruvate ferredoxin oxidoreductase, WLP Wood-Ljungdahl pathway, DCW dry cell weight. Source data are provided as a Source Data file.

Fig. 2. Mass fractions of proteome sectors in T. kivui cells grown with glucose in chemostat reactors at different growth rates.

For reference, data from exponentially growing batch cells (orange) are included. Asterisks indicate significantly changing sectors (P < 0.05, Benjamini-Hochberg false discovery rate correction, for exact P-values see Supplementary Data 2—P-values sectors). Datapoints, individual reactors; lines, linear regressions; shading, confidence intervals using the linear regression as center. Linear regressions were used to calculate significantly changing proteins and are depicted as guidance for trends in data with scatter, without implying a linear relationship. Red, chemostats; orange, exponentially growing cells from batch cultures. Source data are provided as a Source Data file.

Fig. 3. Catabolic proteome of T. kivui grown in glucose-limited chemostats at different growth rates.

For reference, data from exponentially growing batch cells (orange) are included. a proteome fraction of glycolysis (EMP and PFOR); b proteome fraction of WLP; c ratio of the proteome fractions of WLP and glycolysis sectors; d turnover in glycolysis normalized to pathway proteins; e turnover in WLP normalized to pathway proteins. Red, chemostats; orange, exponentially growing cells from batch cultures. WLP Wood-Ljungdahl pathway. Source data are provided as a Source Data file.

Fig. 4. Catabolism and proteomic changes of T. kivui grown in glucose-limited chemostats at different growth rates.

Relative protein abundance compared to exponentially growing reference (ordinate) against growth rate (abscissa) on a linear scale. 2–2.5 acetate per glucose is excreted; the rest is present in biomass. Datapoints, reactors, lines, linear regressions, shading, and confidence intervals using the linear regression as the center. Colored graphs, significant (P < 0.05, Benjamini-Hochberg false discovery rate correction, for exact P-values see Supplementary Data 3—slopedata-reliable) linear changes with growth rate; gray graphs, no significant change; text, not quantified reliably or detected. Linear regressions show trends in data with scatter. GK glucokinase, PGI phosphoglucose isomerase, PFK phosphofructokinase, FBA fructose-1,6-bisphosphate aldolase, TPI triosephosphate isomerase, GAPDH glyeraldehyde-3-phosphate dehydrogenase, PGK phosphoglyerate kinase, PGM phosphoglyerate mutase, ENO enolase; PK pyruvate kinase; PFOR pyruvate:ferredoxin oxidoreductase; PTA phosphotransacetylase, ACK acetate kinase, HDCR hydrogen-dependent carbon dioxide reductase, FTHFS formyltetrahydrofolate synthetase, MTHFC methenyltetrahydrofolate cyclohydrolase, MTHFD methylenetetrahydrofolate dehydrogenase, CODH carbonmonoxide dehydrogenase, ACS acetyl-CoA synthase; MET methyl transferase, CFeSP corrinoid FeS protein. Protein locus tags are abbreviated. Source data are provided as a Source Data file.

Fig. 5. Glucose catabolism of T. kivui grown in glucose-limited chemostats at different growth rates.

a–e glucose catabolism in chemostats during steady-state cultivation; f–j glucose catabolism capacities of T. kivui cells grown in chemostats at different growth rates with unlimited access to glucose after stopping medium flow (converted to batch system). (a) and (f), rates of glucose consumption; (b) and (g), rates of acetate production; c and h, ratio of moles of extracellular acetate found in the supernatant per mole of glucose degraded; (d) and (i), ratio of ¹³C-labeled acetate produced from ¹³C₁ glucose; (e) and (j), specific rates of acetate produced via WLP and via glycolysis as determined by addition of ¹³C₁-labeled glucose and NMR analysis of acetate, gray line indicates equal rates via both pathways. For (a–e), red, chemostats. For (f–j), red, metabolic rates after addition of 20 mM glucose without changes of the gas phase; open blue, addition of 50% N₂ at glucose spiking; filled blue, addition of 50% H₂ at glucose spiking; gray, during cultivations in chemostats as shown in (a–e). WLP Wood-Ljungdahl pathway. Source data are provided as a Source Data file.

Fig. 6. Composition of T. kivui cells grown in a glucose-limited chemostat at different growth rates.

For reference, data from exponentially growing batch cells (orange) are included. a RNA content per biomass in μg per OD₆₀₀ and ml; b number of chromosome copies per cell in μg per OD₆₀₀ and ml; c protein content per biomass in µg per OD₆₀₀ and ml; d RNA/protein ratio as a measure of ribosome content; e RNA/protein ratio in comparison to other organisms. Red, chemostat cells; orange, exponentially growing cells from batch cultures; green triangles, M. maripaludis; blue squares, E. coli (filled, batch cultures; open, chemostat cultures); black squares, Corynebacterium glutamicum (batch cultures). Source data are provided as a Source Data file.

Fig. 7. Ribosome concentrations as well as translation rates in T. kivui cells grown with glucose in chemostat reactors at different flow rates.

For reference, data from exponentially growing batch cells (orange) are included. a proteome fraction of ribosomal proteins in T. kivui; b ribosome concentration in T. kivui cells calculated from the amount of ribosomal proteins (filled circles) or total RNA content (open circles, these numbers were calculated assuming all detected RNA is rRNA and are therefore likely over-estimations); c proteome fractions of ribosomal proteins in comparison to other microorganisms; d translation rates based on ribosome numbers derived from RNA concentrations (assuming 85% of total RNA is rRNA, as found for M. maripaludis); e translation rates from (d) in comparison to other microorganisms, all translation rates were calculated as the average rates across all ribosomes as for T. kivui using the following equation: $\frac{\mathrm{protein}\mathrm{per}\mathrm{cell}\times \mathrm{mass}\mathrm{rRNA}}{\mathrm{RNA}\mathrm{per}\mathrm{cell}\times \mathrm{ratio}\mathrm{of}\mathrm{rRNA}\mathrm{in}\mathrm{total}\mathrm{RNA}\times \mathrm{mass}\mathrm{amino}\mathrm{acid}\times \mathrm{doubling}\mathrm{time}}$ using the RNA/protein ratio to determine protein per cell/RNA per cell, the weight of the respective rRNAs in each organism, ratio of rRNA as described in the legend, an average amino acid weight of 109 g mol⁻¹, and doubling time in s. Red, chemostats; orange, exponentially growing cells from batch cultures; green triangles, M. maripaludis, translation rates calculated similar to T. kivui; blue squares, E. coli (filled, batch cultures; open, chemostat culture, assuming 85% rRNA); black squares, Corynebacterium glutamicum (filled, batch cultures, also assuming 85% rRNA as found for E. coli). Source data are provided as a Source Data file.

Fig. 8. Cell shape of T. kivui cells grown in glucose-limited chemostats at different growth rates.

For reference, data from exponentially growing batch cells (orange) are included. a cell length; b cell width; c cell volume; d surface area/volume ratio; e cell volume in comparison to other organisms; f representative microscopy images of T. kivui cells grown at 0.002 h⁻¹; g representative microscopy images of T. kivui cells grown at 0.07 h⁻¹. For (a–c), n = 39–1055 (Cellular dimensions are mean values of ≥39 cells measured from microscopy images as indicated in Supplemental Data 1), symbols indicate mean values, error bars indicate standard deviation. For (f–g), 25 microscopy images were acquired for each datapoint and representative images from the slowest and fastest growth rates are shown. Red, chemostats; orange, exponentially growing cells from batch cultures; green triangles, M. maripaludis; blue squares, E. coli. Source data are provided as a Source Data file.

Fig. 9. Macromolecular densities in T. kivui cells grown in glucose-limited chemostats at different growth rates as well as the distribution of proteins among the cytoplasm and envelope compared to E. coli.

For reference, data from exponentially growing batch cells (orange) are included. a Cytoplasmatic density of macromolecules (protein, RNA, DNA per cell volume); b cytoplasmic protein concentration in comparison to E. coli; c proteome mass fraction of envelope proteins compared to E. coli; d protein density in the membrane of T. kivui and in the inner membrane of E. coli. Red, chemostats; orange, exponentially growing cells from batch cultures; blue squares, E. coli; gray, T. kivui chemostat with unusually low cell counts per OD₆₀₀, which affect the calculation of intracellular macromolecule densities. Due to the outlier in cell counts, we would consider this datapoint an outlier (Supplementary Fig. 17). Source data are provided as a Source Data file.