Chlorobaculum tepidum Modulates Amino Acid Composition in Response to Energy Availability, as Revealed by a Systematic Exploration of the Energy Landscape of Phototrophic Sulfur Oxidation

Abstract

Microbial sulfur metabolism, particularly the formation and consumption of insoluble elemental sulfur (S⁰), is an important biogeochemical engine that has been harnessed for applications ranging from bioleaching and biomining to remediation of waste streams. Chlorobaculum tepidum, a low-light-adapted photoautolithotrophic sulfur-oxidizing bacterium, oxidizes multiple sulfur species and displays a preference for more reduced electron donors: sulfide > S⁰ > thiosulfate. To understand this preference in the context of light energy availability, an "energy landscape" of phototrophic sulfur oxidation was constructed by varying electron donor identity, light flux, and culture duration. Biomass and cellular parameters of C. tepidum cultures grown across this landscape were analyzed. From these data, a correction factor for colorimetric protein assays was developed, enabling more accurate biomass measurements for C. tepidum, as well as other organisms. C. tepidum's bulk amino acid composition correlated with energy landscape parameters, including a tendency toward less energetically expensive amino acids under reduced light flux. This correlation, paired with an observation of increased cell size and storage carbon production under electron-rich growth conditions, suggests that C. tepidum has evolved to cope with changing energy availability by tuning its proteome for energetic efficiency and storing compounds for leaner times.

Importance: How microbes cope with and adapt to varying energy availability is an important factor in understanding microbial ecology and in designing efficient biotechnological processes. We explored the response of a model phototrophic organism, Chlorobaculum tepidum, across a factorial experimental design that enabled simultaneous variation and analysis of multiple growth conditions, what we term the "energy landscape." C. tepidum biomass composition shifted toward less energetically expensive amino acids at low light levels. This observation provides experimental evidence for evolved efficiencies in microbial proteomes and emphasizes the role that energy flux may play in the adaptive responses of organisms. From a practical standpoint, our data suggest that bulk biomass amino acid composition could provide a simple proxy to monitor and identify energy stress in microbial systems.

FIG 1

Schematic of the energy landscape of phototrophic sulfur oxidation. The energy landscape of phototrophic sulfur oxidation is constructed from three factors at three levels, i.e., (i) electron donor identity (sulfide, S⁰, or thiosulfate), (ii) light flux (5, 20, or 35 μmol photons m⁻² s⁻¹), and (iii) duration of batch culture (10, 18, or 26 h).

FIG 2

Calibration of indirect protein measurements against AAA. Values from BCA and Bradford assays are plotted versus direct protein quantitation by AAA for WH samples (A) and EX samples (B) along with the linear least-squares regression. Insets show low-concentration regions. The solid black identity reference line indicates equality between indirect and AAA protein determinations. The vertical error bars for indirect assay measurements represent the standard errors of triplicate determinations; horizontal error bars on AAA measurements represent propagation of the pooled standard error for four replicated AAA analyses.

FIG 4

Light level affects variation in amino acid abundance according to biosynthetic cost. (A) Mean values of the normalized difference from the average composition are plotted as a function of light. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Amino acids are arranged in order of increasing biosynthetic cost (ATP required per amino acid) using values for phototrophic bacteria (45). The average amino acid biosynthetic cost for C. tepidum biomass (21.8 ATP molecules per amino acid) is indicated by the vertical dashed line. Error bars represent the standard error of the mean within each light level. (B) Box-and-whisker plot representation of the distribution of the measured ∼P_avg values for low, medium, and high light levels.

FIG 3

Effects of the electron donor and acetate depletion on cell volume and storage carbohydrate accumulation across cultures at different growth rates. Box-and-whisker plots of culture-averaged log-transformed cell volumes (A) and total culture carbohydrate (B) normalized to culture protein binned by growth rate and classified by electron donor identity and acetate (Ac) availability. The number of cultures in each grouping is indicated above the box. Acetate was fully depleted by the 26-h time point for S⁰- and thiosulfate-grown cultures at the medium (20 μmol photons m⁻² s⁻¹) and high (35 μmol photons m⁻² s⁻¹) light levels.