An alternative resource allocation strategy in the chemolithoautotrophic archaeon Methanococcus maripaludis

Abstract

Most microorganisms in nature spend the majority of time in a state of slow or zero growth and slow metabolism under limited energy or nutrient flux rather than growing at maximum rates. Yet, most of our knowledge has been derived from studies on fast-growing bacteria. Here, we systematically characterized the physiology of the methanogenic archaeon Methanococcus maripaludis during slow growth. M. maripaludis was grown in continuous culture under energy (formate)-limiting conditions at different dilution rates ranging from 0.09 to 0.002 h^-1, the latter corresponding to 1% of its maximum growth rate under laboratory conditions (0.23 h^-1). While the specific rate of methanogenesis correlated with growth rate as expected, the fraction of cellular energy used for maintenance increased and the maintenance energy per biomass decreased at slower growth. Notably, proteome allocation between catabolic and anabolic pathways was invariant with growth rate. Unexpectedly, cells maintained their maximum methanogenesis capacity over a wide range of growth rates, except for the lowest rates tested. Cell size, cellular DNA, RNA, and protein content as well as ribosome numbers also were largely invariant with growth rate. A reduced protein synthesis rate during slow growth was achieved by a reduction in ribosome activity rather than via the number of cellular ribosomes. Our data revealed a resource allocation strategy of a methanogenic archaeon during energy limitation that is fundamentally different from commonly studied versatile chemoheterotrophic bacteria such as E. coli.

Keywords: maintenance energy; methanogen; proteome allocation; ribosome activity; slow growth.

Fig. 1.

Growth yield and maintenance energy estimates. Values are shown based on observed cell densities (black diamonds) and corrected for rate of cell lysis (gold diamonds). Shaded areas depict 5% confidence level intervals. (A) Apparent molar growth yield (black diamonds) significantly correlated logarithmically with dilution rate (y = 0.20ln(x) + 1.47, R² = 0.95, P value < 0.01). The cell lysis-corrected yield (gold diamonds) shows a less steep decline with growth rate (y = 0.15ln(x) + 1.38, R² = 0.90, P value < 0.01). (B) Double reciprocal graph of yield and growth rate. The slope of the linear regression is used to estimate energy spent on maintenance. Using cellular growth yield, maintenance energy costs seem to be nonlinear over different growth rates and are approximated using two separate linear regressions at higher (y = 1.61x + 106, R² = 0.96, P value < 0.01) and at lower growth rates (y = 0.51x + 213, R² = 0.87, P value < 0.01). Using cell lysis-corrected yield, the slopes of the linear relationships in the plot are less steep (y = 0.80x + 117, R² = 0.78, P value < 0.05 at higher growth rates; y = 0.29x + 162, R² = 0.66, P value < 0.05 at lower growth rates). (C) Estimated fraction of available energy used for maintenance.

Fig. 2.

High-level overview of proteome composition at different growth rates. Sum of mass fractions of proteins organized into functional sectors, showing data from chemostat-grown cells (filled diamonds) and an exponential phase batch culture sample (open diamonds). (A) Proteome allocation to the two major proteome sectors, anabolism and catabolism. (B) Proteome allocation to subsystems comprising >2% of the total proteome in at least one sample. Dotted lines show linear regressions.

Fig. 3.

Methanogenesis rates at different growth rates. Specific rates of methane formation during chemostat growth (black diamonds) linearly correlate with dilution rate (y = 208.14x + 1.59, R² = 0.98, P value < 0.01; shaded area depicts 5% confidence level interval for predictions from linear model). Specific methanogenesis rates of cells transferred from continuous to batch culture with new substrate (150 mM formate) with (gold circles) or without (gray circles) inhibition of new protein synthesis by pseudomonic acid.

Fig. 4.

Macromolecular composition and cell size of M. maripaludis at different growth rates, showing data from chemostat-grown cells (filled diamonds) and an exponential phase batch culture sample (open diamonds). (A) Protein (black diamonds), RNA (gold diamonds), and DNA content (gray diamonds) of M. maripaludis at different growth rates. (B) Cell diameter of M. maripaludis at different growth rates determined by microscopy (black diamonds) and flow cytometry (gold diamonds). (C) Density plot of cell diameters determined by microscopy, averaged for different dilution rate: fast (F, 0.082 to 0.090 h⁻¹), medium (M, 0.027 to 0.029 h⁻¹), slow (S, 0.009 to 0.010 h⁻¹) and very slow (XS, 0.002 to 0.004 h⁻¹) and shown by dashed vertical lines.

Fig. 5.

Ribosome activity. Polysome fraction (black diamonds) correlated logarithmically with dilution rate (y = 11.54 ln(x) + 76, R² = 0.93, P value < 0.01), showing data from chemostat-grown cells (filled diamonds) and an exponential phase batch culture sample (open diamonds). Shaded areas depict 5% confidence level intervals.