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Energy conservation involving 2 respiratory circuits
Edited by Caroline S. Harwood, University of Washington, Seattle, WA, and approved November 27, 2019 (received for review August 28, 2019)

Significance
The chemiosmotic mechanism is a central mode of energy conservation for microorganisms. It relies on a respiratory chain that couples electron flow at the membrane to the transport of ions across the cytoplasmic membrane. This electrochemical potential fuels a rotary machine, the ATP synthase, to make intracellular ATP. Here, we show that a strictly anaerobic rumen bacterium uses 2 different ion circuits for energy conservation. This is achieved by employing 2 ATP synthases that are driven by a
Abstract
Chemiosmosis and substrate-level phosphorylation are the 2 mechanisms employed to form the biological energy currency adenosine triphosphate (ATP). During chemiosmosis, a transmembrane electrochemical ion gradient is harnessed by a rotary ATP synthase to phosphorylate adenosine diphosphate to ATP. In microorganisms, this ion gradient is usually composed of
Chemiosmosis and substrate-level phoshorylation (SLP) are responsible for the formation of the energy currency of any cell, adenosine triphosphate (ATP). During chemiosmosis, a transmembrane electrochemical ion gradient is established by an electron-transport chain (ETC), in which exergonic electron flow is coupled to vectorial ion transport out of the cell. The chemiosmotic gradient is harnessed by highly conserved rotary machines, the ATP synthases (1). These ATP-forming
Anaerobic microorganisms also use the chemiosmotic mechanism for ATP formation, and some even solely depend on it for energy conservation (2). The electrons derived from catabolite breakdown are channeled via a membrane-integral ETC onto alternative electron acceptors, such as nitrate, sulfate,
The butyrivibrios, such as P. ruminis, are among the most abundant players in the microbiome of ruminants (8, 9). Their physiological role is to convert sugars to short-chain fatty acids, which are either resorbed by the animal or further metabolized by other microorganisms. They are also responsible for the conversion of “healthy” unsaturated fatty acids from the feedstock to “unhealthy” saturated fatty acids. Therefore, there is a great interest in biomedical research targeting the elucidation of butyrivibrios to decrease the amount of unhealthy fatty acids in dairy and meat (10). The substrate spectrum of the model organism P. ruminis is restricted to several
Genetic Blueprint of 2 Respiratory Systems in P. ruminis
The decryption of rumen butyrivibrio genome sequences led to the identification of several species within these genera that contain both rnf and ech clusters (6). In the model organism P. ruminis, the ech cluster is composed of 6 subunits (Fig. 1A) that share high sequence similarity with the core Ech complex found in the methanogens Methanosarcina barkeri or Methanosarcina mazei, but also the Ech complex of Caldanaerobacter subterraneus subspecies (subspec.) tengcongensis (12). According to the amino acid sequence of the large hydrogenase subunit (EchE), it is classified as a group 4 [NiFe] hydrogenase subgroup 4e (13, 14), just like in C. subterraneus subspec. tengcongensis. The cluster is preceded by a gene encoding a putative [FeFe] hydrogenase. This hydrogenase is predicted to be cytoplasmic (13) and the only other putative hydrogenase found in the genome. Downstream of the cluster are hyp genes that encode the [NiFe] hydrogenase maturation machinery (15).
Genetic arrangement (A–D) and hypothetical models (E–H) for complexes involved in energy conservation in P. ruminis. Dark red, membrane-integral; green, cytoplasmic; blue, hydrogenase; yellow, nicotinamide binding module; purple, periplasmic; orange, coupling ion binding subunits.
The rnf cluster comprises 6 genes (rnfCDGEAB) that are very similar to the rnf operon in A. woodii (16) (Fig. 1B). The putative Rnf complex could be Na+-dependent as in A. woodii or
Further inspection revealed that P. ruminis also harbors 2 atpase clusters (atpase1 and atpase2). Inspection of the genes encoding these 2
Growth and Product Formation of P. ruminis Is Stimulated by Na+
To gain insights into the bioenergetics of the organism, first investigations targeted the growth behavior of P. ruminis in the presence or absence of Na+. When cultivated on 50 mM d-glucose in the presence of Na+, P. ruminis grew with a doubling time of 2.2 h and reached a final
Growth of P. ruminis in the dependence of Na+. Cells were cultivated in complex medium containing 97 mM Na+ (filled symbol) or 1 mM Na+ (open symbol) in the presence of 50 mM d-glucose (square) or d-xylose (circle).
Both glucose and xylose were always completely consumed under all growth conditions, and the metabolic products were lactate, butyrate, acetate, formate, and molecular
Expression Levels of Energy Conserving Systems in P. ruminis
To shed some light on the involvement of the 4 energy-conserving complexes under different physiological conditions, we analyzed the transcript levels of the rnf, ech, and atpase gene clusters.
First, we assessed the relative expression of the clusters during growth on glucose in dependence of the growth phase. Generally, the expression of all clusters was highest in the early or midexponential growth phase (SI Appendix, Table S3). In the late stationary phase, only the ech cluster was still highly expressed. The rnf cluster was at least 2-fold more highly expressed than the ech cluster, and the atpase clusters were similarly expressed. A similar trend was observed in cells grown on xylose, with the exception that the atpase2 cluster showed an up to 20-fold higher expression than the atpase1 cluster (SI Appendix, Table S3). The atpase2 expression was surprisingly also very high in the early stationary phase, but lower in the midexponential phase. It may be that the
Next, we assessed the relative expression of the clusters in the dependence of Na+. Interestingly, when cells were grown on glucose without Na+, the ech and the atpase2 clusters were highly up-regulated (by a factor of 7.9 and 5.3), and the atpase1 cluster was up-regulated 2.0-fold, whereas the rnf cluster was slightly down-regulated (by a factor of 1.7) compared to the relative transcript level of cells grown with Na+ (SI Appendix, Fig. S2 and Table S3). A similar trend was observed for the relative expression of ech and rnf when cells were grown on xylose without Na+: ech was also up-regulated (1.8-fold), rnf was down-regulated (5.1-fold), and atpase2 was up-regulated (1.7-fold), but atpase1 was slightly down-regulated (1.3-fold) compared to the expression in cells grown with Na+. The data clearly show that ech was up-regulated in the absence of Na+, and rnf was down-regulated at the same time, fostering the notion that Ech could act as a
Rnf Activity in P. ruminis
To elucidate the biochemistry of the energy-conserving complexes, we prepared crude extracts of P. ruminis grown until the exponential growth phase on either glucose or xylose, with or without Na+. The crude extract was further separated into a membrane and a cytoplasmic fraction via ultracentrifugation. To provide the physiological electron donor of the Rnf, reduced Fd, we purified Fd from Clostridium pasteurianum and acetyl-CoA synthase/carbon monoxide dehydrogenase (Acs/CODH) from A. woodii, as described previously (4, 18). Subsequently, washed membranes of P. ruminis were incubated in assay buffer supplemented with Fd (30 μM) and Acs/CODH (30 μg) under a carbon monoxide (CO) atmosphere at 37 °C. CO was continuously oxidized by the Acs/CODH to provide reduced Fd in a regeneration system. Upon addition of
The specific Rnf activity of membranes prepared from cells grown on glucose or xylose in the presence of Na+ was in the range of 110 to 160 mU/mg, depending on the preparation (Fig. 3A). Negative controls, where 1 component was omitted, did not show NADH formation. The activity was optimal at pH 7.5 with 136 mU/mg and 9, 80, 83, or 29 mU/mg at pH 5.5, 6.5, 8.5, or 9.5.
Rnf, Ech, and ATPase activity in membranes of P. ruminis. Rnf (A), Ech (B), and ATPase (C) activity was measured in membranes prepared from cells grown on glucose or xylose with (+) or without (−) Na+ at 37 °C. (A) Rnf activity was measured in buffer containing 50 mM Tris⋅HCl, 10 mM NaCl, 4 mM dithioerythritol (DTE), and 4 μM resazurin at pH 7.5 and supplemented with 30 μM Fd, 30 μM Acs/CODH, 340 μg membranes, and 4 mM
To consolidate the notion that the Rnf complex is Na+-dependent, we analyzed the activity in membranes prepared from cells grown on glucose or xylose with or without Na+. Indeed, Rnf activity was only 28 or 30% in cells grown without Na+, with specific activities of 44.14 ± 4.19 or 51.68 ± 24.53 mU/mg, as opposed to 159.83 ± 20.58 or 175.05 ± 21.40 in cells grown with
Ech Activity in P. ruminis
Next, we assessed hydrogenase activity in cell-free extracts of P. ruminis. Just like the Rnf complex, Ech is typically fueled by
To assess the localization of the hydrogenase, the membranes were washed, and hydrogenase activity was monitored in the membranes and supernatant. The crude extract exhibited a total activity (
Finally, we assessed Ech activity in washed membranes of P. ruminis prepared from cells cultivated on glucose or xylose with or without Na+. Indeed, Ech activity was 362 or 468% higher in cells cultivated without Na+, with specific activities of 11.30 ± 0.95 or 9.00 ± 0.39 mU/mg, as opposed to 3.13 ± 1.22 or 1.93 ± 0.22 in cells grown with
ATPase Activity Is Stimulated by Na+
To assess the ATP synthases biochemically, we prepared membranes of P. ruminis grown on glucose or xylose with or without Na+ and measured ATP hydrolysis activity.
In Na+-free buffer (68 μM Na+), the ATPase activity was 253 or 139% higher in cells grown without Na+, with specific activities of 265.23 ± 100.81 or 242.65 ± 38.92 mU/mg as opposed to 104.57 ± 25.27 or 173.75 ± 39.99 in cells grown with
To dissect the Na+-dependence of the ATPase1 further, we measured the activity in the presence of different concentrations of Na+,
Stimulation of ATP hydrolysis activity by monovalent salts and by Na+ at different pH values. ATP hydrolysis was measured in buffer containing (A) 100 mM Tris⋅HCl, 10 mM maleic acid, and 5 mM
Occurrence of Ech, Rnf, and ATPases in Microbial Genomes
After demonstrating that there is a
We found that 13 organisms encoded all components of these circuits (Fig. 5 and Dataset S1). These all belong to the phylum Firmicutes and the class Clostridia. Two were Pseudobutyrivibrio (P. ruminis and Pseudobutyrivibrio xylanivorans). Most of the remainder were of the genus Clostridium (n = 8).
Several organisms may have 2 bioenergetic circuits. Genomes of n = 2,925 bacteria and archaea were searched for genes encoding Ech, Rnf, and ATP synthases. The Venn diagram shows the number of organisms encoding each combination of these enzymes. Enzymes were encoded if genes for all subunits were found. The ATPases were distinguished into Na+- and
More organisms encoded “incomplete” circuits. For example, 70 organisms encoded
Discussion
This work demonstrates the existence of 2 bioenergetic circuits in a strictly anaerobic bacterium: a Na+ circuit involving Rnf in conjunction with a Na+-dependent ATP synthase and a
The rumen is home to about 200 bacterial species that can be cultured (26), with many more that are uncultured (27). P. ruminis is part of the “core bacterial microbiome,” and its community structure is greatly influenced by the diet of its host (8, 28). In the intestinal tract and the rumen, it has been shown that butyrate and other short-chain organic acids have an inhibitory effect on inflammation (29, 30). This symbiosis between host and microbe may be better when Na+ is abundant, since we demonstrated an increase in microbial fitness and thus production of butyrate in the model rumen bacterium P. ruminis.
P. ruminis generates most of its ATP from substrate-level phosphorylation in the EMPP or PPP and from butyrate production (Fig. 6 and SI Appendix, Fig. S7). The chemiosmotic gradient is only a 2nd mode of energy conservation, but nevertheless essential for all sorts of cellular processes, including transport, motility, and ion and pH homeostasis. The experiments presented herein demonstrate that P. ruminis uses both a Na+ and a
Redox-balanced models for glucose metabolism in P. ruminis involving 2 chemiosmotic circuits. Models for metabolism in cells grown with (A) or without (B) Na+ are shown. The numbers indicate the moles of substance degraded or produced, as determined in this study (bold) or calculated. In brackets are the actual measured
When cells grow on glucose or xylose, these sugars are converted to pyruvate via the EMPP or PPP at the gain of ATP and reducing equivalents in the form of NADH (Fig. 6 and SI Appendix, Fig. S7). Pyruvate is metabolized further to lactate by the lactate dehydrogenase at the cost of NADH, and this was in fact the main product measured under all growth conditions. Pyruvate is also converted to acetyl-CoA via PFOR (at the gain of
Under Na+-rich conditions, electrons are shuttled into both circuits to regenerate NADH by the Rnf complex or discard excess electrons in the form of
Under Na+-deprived conditions, there is an excess of reducing equivalents in the form of NADH when cells are grown on glucose (Fig. 6B). The lack of Na+ severely slows down the Na+ circuit and thus the interconversion of NADH and Fd pools. The
Putting this into perspective of the physiological conditions, feeding large amounts of grain can reduce the pH in the rumen to well below 5.5 (33), which could lead to an inactivation of ATPase1 (Fig. 4B) and concomitantly a shutdown of the Na+ circuit. However, normally the Na+ concentration in the rumen is at least 60 mM and can be up to 500 to 800 mM (34). Therefore, both circuits are finetuned to ensure microbial fitness of P. ruminis and ultimately of the host, by providing health-promoting organic acids. Moreover, higher ATP pools may contribute to biohydrogenation of unsaturated fatty acids (35) via a membrane-associated oxidoreductase (36). This process is tied to the provision of reducing equivalents, but neither to ATP formation nor changed when
Besides the “core microbiome,” the rumen is also home to methanogenic archaea and acetogenic bacteria (37⇓–39), and both Rnf and Ech have been identified and characterized in these groups of strict anaerobes (2, 3, 40). The
It is likely that rumen butyrivibrios are not the only groups of organisms that use both Ech and Rnf for separate chemiosmotic circuits. Intriguingly, acetogens were postulated to rely either on Rnf or Ech (5), but the decryption of more genomes revealed a cooccurrence of rnf and ech genes in some (14). It will be particularly interesting to investigate in these organisms, which depend on the chemiosmotic gradient for energy conservation (acetogens and methanogens), whether they also use 2 circuits for energy conservation.
Material and Methods
Experimental procedures for cultivation of the organism; cell harvest and preparation of crude extracts and membranes; measurement of Ech, Rnf, and ATP hydrolysis activity; determination of relative transcript levels; determination of metabolites, pH, and Na+ concentrations; and the search for Ech, Rnf, and ATases in microbial genomes are described in SI Appendix, SI Materials and Methods.
Data Availability Statement.
Data discussed in the paper are available in Datasets S1–S3.
Acknowledgments
This work was supported by a European Research Network grant from the Bundesministerium für Bildung und Forschung. Additional support was from Agriculture and Food Research Initiative Competitive Grant 2018-67015-27495/Project Accession No. 1014959 and Hatch Project Accession No. 1019985 from the US Department of Agriculture National Institute of Food and Agriculture. M.C.S. is a recipient of a Claussen–Simon–Stiftung (Germany) Fellowship.
Footnotes
↵1Present address: Microbiology & Biotechnology, Institute of Plant Sciences and Microbiology, Universität Hamburg, 22609 Hamburg, Germany.
- ↵2To whom correspondence may be addressed. Email: vmueller{at}bio.uni-frankfurt.de.
Author contributions: M.C.S., A.K., T.J.H., and V.M. designed research; M.C.S., A.K., J.D., and T.J.H. performed research; M.C.S., A.K., J.D., T.J.H., and V.M. analyzed data; and M.C.S. and V.M. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1914939117/-/DCSupplemental.
Published under the PNAS license.
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