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M.C.S., A.K., and M.C.S., M.C.S. research; research; performed designed V.M. T.J.H. and and T.J.H., A.K., M.C.S., contributions: Author sol h omrta shresdfreeg osrainby conservation energy for harnessed is that the a former the of only establishment is the to leads of complex Ech ra neeti imdclrsac agtn h lcdto of elucidation a the is targeting there research feed- Therefore, biomedical in acids. the interest fatty from metabo- great saturated acids further “unhealthy” fatty to or the unsaturated stock for animal “healthy” responsible also of the are conversion They by microorganisms. other acids, resorbed by fatty lized short-chain either Their to 9). are sugars (8, convert which ruminants to of is role microbiome physiological the in players abundant to us use led and This using produce organism. 7). sites, indeed (6, coupling organisms complex both Ech these the whether clusters and investigate gene Rnf activ- both number Rnf the possess a and encoding that astonishingly Ech suggested butyrivibrios evidence have rumen Bioinformatic to of cell. shown one were in there that ity far, acetogens So (5). no Ech-containing or are Rnf- either as classified been in Na complex Rnf The (2–4). the fuels and the complex) and is the (Rnf acetogens enzymes, (Fd) of respiratory Ferredoxin bioenergetics distinct ATP. 2 in of carrier Under form electron the source. central in mechanism electron energy chemiosmotic conserve a an on to as rely acetyl- they (heterotrophic) conditions, autotrophic sugars reductive as the such as fix to such use pathway gases (acetyl-CoA) Acetogens A coenzyme bacteria. acetogenic rsn drs:Mcoilg itcnlg,Isiueo ln cecsand Sciences [email protected] Plant Email: addressed. be of may correspondence Institute whom To Biotechnology, & Universit Microbiology Microbiology, address: Present onig yteEhcmlx n h Na complex. the and complex, Ech H the The by pumps. ion distinct 2 by synthases established H ATP a 2 by energy employing driven are for by that circuits anaer- achieved ion is strictly to different This a synthase, 2 conservation. that uses ATP bacterium show the rumen we obic machine, Here, ATP. rotary intracellular electrochem- a make This fuels membrane. potential transport cytoplasmic the ical the to across membrane ions the chain at respiratory of flow a electron on couples relies that It con- microorganisms. energy of for mode servation central a is mechanism chemiosmotic The Significance ¨ h uyiiro,sc as such butyrivibrios, The + H rdet hc ul Na a fuels which gradient, + a dpnetAPsnhs.I atclr ctgn have acetogens particular, In synthase. ATP -dependent ioh .Hackmann J. Timothy , H NSlicense.y PNAS 2 nteohrhand, other the on kivui, Thermoanaerobacter tHmug 20 abr,Germany.y Hamburg, 22609 Hamburg, at ¨ rC attohc rognccompounds organic or (autotrophic) CO or + Fd rNa or 2− suouyiiroruminis Pseudobutyrivibrio ctbceimwoodii Acetobacterium :H + r mn h most the among are ruminis, P. + + rdet h ie rdetis gradient mixed The gradient. + b dpnetAPsnhs.The synthase. ATP -dependent n okrM Volker and , xdrdcae(c complex) (Ech oxidoreductase H rdeti omdb h Rnf the by formed is gradient . Fd y + 2− n Na and https://www.pnas.org/lookup/suppl/ NSLts Articles Latest PNAS :NAD + CO rdeti formed is gradient + 2 + rdet u it but gradient, sn inorganic using oxidoreductase uller ¨ salse a establishes a,2 smodel as | f7 of 1

MICROBIOLOGY butyrivibrios to decrease the amount of unhealthy fatty acids Growth and Product Formation of P. ruminis Is Stimulated in dairy and meat (10). The substrate spectrum of the model by Na+ organism P. ruminis is restricted to several C6 and C5 sug- To gain insights into the bioenergetics of the organism, first ars (11). This requires the presence of 2 distinct metabolic investigations targeted the growth behavior of P. ruminis in modules: the Embden–Meyerhof–Parnas pathway (EMPP) for the presence or absence of Na+. When cultivated on 50 mM degradation of C6 compounds and the pentose–phosphate path- D-glucose in the presence of Na+, P. ruminis grew with a dou- way (PPP) for degradation of C5 compounds. The scope of this bling time of 2.2 h and reached a final OD600 of 3.3 after work was to analyze whether the Rnf and Ech complexes indeed 24 h (Fig. 2). When Na+ was omitted from the medium (resid- form active enzymes and to elucidate their physiological role ual Na+ concentration was 1 mM, and NaCl was substituted in P. ruminis. with equal amounts of KCl to provide the same ionic strength), cells only started to grow after a significant lag phase of 20 h Genetic Blueprint of 2 Respiratory Systems in P. ruminis (Fig. 2). The subsequent doubling time decreased almost 5- The decryption of rumen butyrivibrio genome sequences led to fold to 10.3 h, and the final OD600 decreased 2-fold to 1.6. the identification of several species within these genera that con- The same trend was observed for cultivations on 50 mM D- tain both rnf and ech clusters (6). In the model organism P. xylose: The doubling time decreased 4-fold (4.1 and 17.0 h), and ruminis, the ech cluster is composed of 6 subunits (Fig. 1A) that the final OD600 decreased more than 2-fold (2.6 to 1.1) in the share high sequence similarity with the core Ech complex found absence of Na+ (Fig. 2). The high stimulatory effect of Na+ on in the methanogens Methanosarcina barkeri or Methanosarcina growth thus indicates that Na+ could serve as a coupling ion in mazei, but also the Ech complex of Caldanaerobacter subterraneus P. ruminis, but the organism is not strictly dependent on Na+ subspecies (subspec.) tengcongensis (12). According to the amino for growth. acid sequence of the large hydrogenase subunit (EchE), it is clas- Both glucose and xylose were always completely consumed sified as a group 4 [NiFe] hydrogenase subgroup 4e (13, 14), just under all growth conditions, and the metabolic products were like in C. subterraneus subspec. tengcongensis. The cluster is pre- lactate, butyrate, acetate, formate, and molecular H2. When P. ceded by a gene encoding a putative [FeFe] hydrogenase. This ruminis grew on glucose with Na+, 99% of the carbon was recov- hydrogenase is predicted to be cytoplasmic (13) and the only ered (SI Appendix, Table S2) in the form of 59 mM lactate (59%), other putative hydrogenase found in the genome. Downstream 19 mM butyrate (25%), 15 mM acetate (10%), and 16 mM for- of the cluster are hyp genes that encode the [NiFe] hydrogenase mate (5%) (SI Appendix, Table S1). The cultures also produced + maturation machinery (15). 15 mmol/L medium molecular H2. In the absence of Na , cells The rnf cluster comprises 6 genes (rnfCDGEAB) that are very only produced about half as much butyrate (10 mM) and also similar to the rnf operon in A. woodii (16) (Fig. 1B). The putative less formate (13 mM), but 3-fold the amount of molecular H2 Rnf complex could be Na+-dependent as in A. woodii or H+- (49 mmol/L medium), leading to a decreased carbon recovery dependent as in Clostridium ljungdahlii (17). of 88%. A very similar trend was observed for cells grown on Further inspection revealed that P. ruminis also harbors 2 xylose. A total of 89 or 79% carbon was recovered as lactate (50 atpase clusters (atpase1 and atpase2). Inspection of the genes or 49%), butyrate (23 or 16%), acetate (11 or 10%), and for- + encoding these 2 F1FO ATP synthases showed that they share mate (5 or 4%) in cultivations with or without Na (SI Appendix, high sequence similarities, but there are 2 apparent differences. Fig. S1 and Table S1). Again, H2 production increased more Firstly, the gene encoding the δ subunit of the F1FO ATP than 2-fold from 20 to 44 mmol/L medium in the absence of synthase is missing in the atpase2 cluster (Fig. 1D). This sub- Na+. The metabolite profiling clearly revealed that less carbon unit is responsible for linking the α subunit of the F1 complex was recovered in the form of the metabolites and that electrons with the peripheral stalk, and its role could be compensated were discarded as molecular hydrogen when cells grew in the by the larger α subunit of the ATPase2. Secondly, the c sub- absence of Na+. unit harbors a typical Na+ binding motif in the atpase1, whereas the atpase2 does not. Thus, one F1FO ATP synthase could Expression Levels of Energy Conserving Systems in P. ruminis exploit the µeH + (ATPase2) and the other the µeNa+ (ATPase1) To shed some light on the involvement of the 4 energy- (Fig. 1 G and H). conserving complexes under different physiological conditions,

A echA echB C D echE F Locus tag IE20DRAFT_1835-1840 1 kb

B rnfC rnfD rnfG rnfE rnfA rnfB Locus tag IE20DRAFT_0757-0752

C a c b Locus tag IE20DRAFT_1580-1573 D a c b Locus tag IE20DRAFT_1697-1703 E H+ F Na+ G Na+ H H+ periplasm G

EchB EchA RnfA RnfE RnfD c c a c c c a c cc cc

γ γ cytoplasm RnfB EchD C EchE RnfC b b Fd2- F [NiFe] [flavin] Fd2- Fd + + Fd 2H H2 NAD NADH

Fig. 1. 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.

2 of 7 | www.pnas.org/cgi/doi/10.1073/pnas.1914939117 Schoelmerich et al. Downloaded by guest on October 2, 2021 Downloaded by guest on October 2, 2021 coleihe al. et Schoelmerich MgCl mM 5 4 and and acid, DTE, maleic mM mM 100 4 Tris ·HCl, NaCl, mM mM 100 Na 10 containing initial Mes, buffer mM in measured 50 was containing hydrolysis buffer in Na measured (−) was without activity 4 or and (+) (DTE), with xylose dithioerythritol or glucose on grown 3. Fig. Na mM 97 containing medium complex in vated 2. Fig. Na n em ob oedmnn hnEhutlclsrahthe reach superior cells a play until might Ech ATPase2 grow than Moreover, dominant they phase. level, stationary more transcript when late on be phase based to way, growth seems Either new Rnf glucose. a not impor- but to more xylose, adjust on are cells that when homeostasis) tant pH (e.g., may processes It cellular phase. midexponential the the that in be lower but phase, stationary the atpase2 with xylose, the on than grown expression cells in the that observed exception was trend similar A late mid- the the or In early S3). The the the Table in only Appendix, phase, highest (SI stationary was phase clusters growth all exponential of Generally, phase. expression growth the the of dependence in glucose on growth the of levels clusters. transcript the analyzed we (circle). + is,w sesdterltv xrsino h lsesduring clusters the of expression relative the assessed we First, ech oe ybl ntepeec f5 MDguoe(qae rD-xylose or (square) D-glucose mM 50 of presence the in symbol) (open rnf n,Eh n Taeatvt nmmrnsof membranes in activity ATPase and Ech, Rnf, rwhof Growth + lse,adthe and cluster, xrsinwssrrsnl lovr ihi h early the in high very also surprisingly was expression lse a tlat2fl oehgl xrse than expressed highly more 2-fold least at was cluster ocnrto nteNa the in concentration H + dpnetAPsnhs sipratfrother for important is synthase ATP -dependent .ruminis P. atpase2 atpase1 atpase eaui tp . n upeetdwt 30 with supplemented and 7.5 pH at resazurin µM ntedpnec fNa of dependence the in lse hwda pt 0fl higher 20-fold to up an showed cluster ech .The S3). Table Appendix , (SI cluster + lse a tl ihyexpressed. highly still was cluster lseswr iial expressed. similarly were clusters fe ufrws68 was buffer -free rnf, + and ech, fildsmo)o mM 1 or symbol) (filled ,adAPs (C ATPase and (B), Ech (A), Rnf ruminis. P. + + µM). t37 at el eeculti- were Cells . atpase ◦ C,1 MNC,4mM 4 NaCl, mM 10 Tris·HCl, mM 50 containing buffer in measured was activity Rnf (A) C. gene IAppendix, (SI nm 340 at absorbance S3 in Fig. increase an as observed regeneration a of in addition Fd Upon reduced system. provide to Acs/CODH the by dized 37 at atmosphere (CO) monoxide washed (30 Subsequently, Fd with 18). mented (4, of previously membranes described as woodii, from (Acs/CODH) dehydrogenase monoxide from synthase/carbon Fd, reduced Fd Rnf, purified the of we donor electron To physiological ultracentrifugation. or the via provide with fraction cytoplasmic xylose, a and or membrane glucose either Na on without com- phase energy-conserving growth of the extracts exponential crude of prepared biochemistry we plexes, the elucidate To in Activity Rnf Na Na of the absence of role the the that Ech that Na bly notion a the as fostering act time, could same the at down-regulated Na with grown that cells in expression the but and (5.1-fold), regulated Na without of expression tive rw nguoeo yoei h rsneo Na of presence the in xylose or glucose on grown . ih16m/gad9 0 3 r2 Um tp .,6.5, 5.5, pH at mU/mg 29 or 83, 80, pH 9.5. at 9, or optimal and 8.5, was mU/mg omitted, activity 136 was The with formation. preparation component 7.5 NADH the 1 show on where not depending controls, did mU/mg, Negative 160 3A). to (Fig. 110 of range the ietasrp ee fclsgonwt Na S3 with Table grown and cells rela- S2 of the to level compared transcript 1.7) of tive the factor the a whereas (by and down-regulated 2.0-fold, 5.3), slightly and up-regulated 7.9 was of factor cluster a (by up-regulated highly Na without glucose Na on of dependence the than other purposes conservation. for energy probably metabolism, xylose during role d 30 Fd, µM h pcfi n ciiyo ebae rprdfo cells from prepared membranes of activity Rnf specific The et easse h eaieepeso ftecutr in clusters the of expression relative the assessed we Next, atpase1 ech + ). eaui tp . n upeetdwt 30 with supplemented and 6.0 pH at resazurin µM dpnetRfcmlx utemr,tedt suggest data the Furthermore, complex. Rnf -dependent H a prgltdi h bec fNa of absence the in up-regulated was + + c/OH 340 Acs/CODH, µM + -dependent a lgtydw-euae 13fl)cmae to compared (1.3-fold) down-regulated slightly was : h rd xrc a ute eaae noa into separated further was extract crude The . ech + ciiywsmaue nmmrnspeae rmcells from prepared membranes in measured was activity ) H .ruminis P. .ruminis P. . + .Asmlrtedwsosre o h rela- the for observed was trend similar A ). 2 a lou-euae (1.8-fold), up-regulated also was ech tp . ih(e)o ihu pn)5m Na mM 5 (pink) without or (red) with 7.5 pH at upta ae vrterl fteproba- the of role the over takes that pump + )adAsCD (30 Acs/CODH and µM) -dependent lsrdu pasteurianum Clostridium and + + F the , neetnl,we el eegrown were cells when Interestingly, . NAD eeicbtdi sa ufrsupple- buffer assay in incubated were 1 atpase2 F rnf ebae,ad4m NAD mM 4 and membranes, µg O ech T ytae2mycompensate may 2 synthase ATP hnclswr rw nxylose on grown were cells when + 4m) AHfrainwas formation NADH mM), (4 F n the and ◦ 1 a prgltd(1.7-fold), up-regulated was .C a otnosyoxi- continuously was CO C. F O .ruminis P. + h aaceryshow clearly data The . NSLts Articles Latest PNAS T ytae1i the in 1 synthase ATP atpase2 + )udracarbon a under µg) Fig. Appendix, (SI n acetyl-CoA and rw ni the until grown rnf + rnf and , lseswere clusters d (C Fd. µM lse was cluster a down- was + + rnf atpase1 Ech (B) . a in was | + f7 of 3 ATP ) was (the A.

MICROBIOLOGY To consolidate the notion that the Rnf complex is Na+- with or without Na+. Indeed, Ech activity was 362 or 468% dependent, we analyzed the activity in membranes prepared higher in cells cultivated without Na+, with specific activities from cells grown on glucose or xylose with or without Na+. of 11.30 ± 0.95 or 9.00 ± 0.39 mU/mg, as opposed to 3.13 ± Indeed, Rnf activity was only 28 or 30% in cells grown without 1.22 or 1.93 ± 0.22 in cells grown with Na+ on glucose Na+, with specific activities of 44.14 ± 4.19 or 51.68 ± 24.53 and xylose, respectively (n = 4; ±SD) (Fig. 3B). The activ- + mU/mg, as opposed to 159.83 ± 20.58 or 175.05 ± 21.40 in ity (either NaDt:Fd:H or H2:MV oxidoreductase activity) in cells grown with Na+ on glucose and xylose, respectively (n = 4; membranes of glucose-pregrown cells (with Na+) could not ±SD) (Fig. 3A). Furthermore, the activity in membranes of be stimulated by 20 mM NaCl in the assay. The observa- glucose-pregrown cells (with Na+) was 1.5-fold stimulated by tion that ech expression and Ech activity was higher in cells 20 mM NaCl in the assay. The observation that rnf expression grown without Na+ and that Na+ did not affect the activity and Rnf activity was higher in cells grown with Na+ than with- are strong indications that the Ech complex is part of the H+ out, as well as the observed slight stimulation of Rnf activity by circuit. + Na , are strong indications that the Rnf complex is part of the + Na+ circuit. ATPase Activity Is Stimulated by Na To assess the ATP synthases biochemically, we prepared mem- Ech Activity in P. ruminis branes of P. ruminis grown on glucose or xylose with or without Next, we assessed hydrogenase activity in cell-free extracts of Na+ and measured ATP hydrolysis activity. P. ruminis. Just like the Rnf complex, Ech is typically fueled In Na+-free buffer (68 µM Na+), the ATPase activity was by Fd2− (2, 19, 20). However, the Fd2− regeneration system 253 or 139% higher in cells grown without Na+, with spe- proved unsuitable to assess Ech activity, since CO is a potent cific activities of 265.23 ± 100.81 or 242.65 ± 38.92 mU/mg as inhibitor of hydrogenases (SI Appendix, Table S4). Hydrogenase opposed to 104.57 ± 25.27 or 173.75 ± 39.99 in cells grown activity was not detected by using either nicotinamide NADH with Na+ on glucose and xylose, respectively (n = 4; ±SD) or NADPH (4 mM) as reductant (at neither pH 6.0 nor 7.5) (Fig. 3C). The higher ATPase activity in cells grown under (SI Appendix, Table S4). The reverse reaction of Ech was mea- Na+-deprived conditions can be explained by a higher abun- surable by using the artificial dye methylviologen (MV) as the dance of the H+-dependent ATP synthase 2, which was cor- electron acceptor and molecular hydrogen as the electron donor. roborated by the higher expression of atpase2 under the same + This H2-dependent:MV oxidoreductase activity was detected conditions. In Na -containing buffer (20 mM NaCl), ATPase with specific activities of 243 or 52 mU/mg at pH 7.5 or 6.0. activity was generally 3- to 5-fold higher in all membrane + However, since the physiological direction should be H2 evolu- preparations than in Na -free buffer. This can be explained by tion rather than oxidation, we searched for alternative assays. the presence of the Na+-dependent ATPase1, which becomes An alternative enzyme that provides Fd2− is the pyruvate:Fd active once its coupling ion is available. The activity was still oxidoreductase (PFOR). We identified this enzyme activity in 197% higher in membranes prepared from glucose-grown cells crude extracts of P. ruminis. The assay contained crude extract without Na+, with specific activities of 867.43 ± 284.30, as (100 to 350 µg), CoA (200 µM), and Fd (60 µM), and forma- opposed to 440.00 ± 62.44 in cells grown with Na+ (n = tion of Fd2− was detected at 430 nm upon addition of pyruvate 4; ±SD) (Fig. 3C). However, membranes prepared from (10 mM). The specific activity was 200 to 300 mU/mg. The result- xylose-pregrown cells without Na+ possessed a lower specific ing PFOR-fueled H2 evolution activity in crude extracts was activity (86%) with 745.45 ± 293.37 mU/mg as opposed to 1.5 to 4.0 mU/mg, depending on the preparation (SI Appendix, 870.73 ± 194.25 mU/mg in cells grown with Na+ (n = 4; Table S4). The activity could be increased by a factor of 1.6, ±SD). This last observation may be explained by the slight 2.4, or 1.9 when doubling the amount of Fd (120 µM), CoA up-regulation of the atpase1 gene in cells grown on xylose (400 µM), or pyruvate (20 mM), respectively. The highest activ- with Na+ (while there was a slight down-regulation in cells ities and easiest assay conditions to measure Ech, however, grown on glucose with Na+). The data revealed that there were to use sodium dithionite (10 mM NaDt) as reductant (SI was ATPase activity in Na+-free buffer, which most likely Appendix, Fig. S4). This activity was stimulated by Fd with activ- reflects the H+-dependent ATPase2. This activity was higher ities of 3.25 ± 0.92 or 10.65 ± 2.05 mU/mg without or with in cells grown without Na+, matching the expression data for Fd (n = 2; ±SD). Thus, the characteristic reaction of the Ech atpase2. Moreover, the stimulation of ATPase activity by NaCl 2− + complex, Fd -dependent H2 formation, was detected in crude demonstrated the presence of the Na -dependent ATPase1, extracts and membranes of P. ruminis, and NaDt:Fd:H+ oxi- and the activity values matched the expression patterns for doreductase activity was used to determine Ech activity for atpase1. subsequent analyses. To dissect the Na+-dependence of the ATPase1 further, we To assess the localization of the hydrogenase, the membranes measured the activity in the presence of different concentrations + + + were washed, and hydrogenase activity was monitored in the of Na , K and Li (Fig. 4A). The determined KM value for membranes and supernatant. The crude extract exhibited a total Na+ was 543 µM. Lithium chloride could partially substitute the activity (Utot ) of 936 mU, and membranes and cytoplasm con- stimulatory effect of NaCl, whereas KCl showed no stimulatory tained 888 and 254 mU. Washing the membranes once or twice effect on ATP hydrolysis activity. Furthermore, the stimulatory resulted in a decrease in the Utot with 167 or 75 mU and effect of NaCl on ATP hydrolysis was investigated at different 198 or 101 mU in the respective supernatant fractions. That pH values. At the physiological pH value 7.5, the ATPase activ- activity remained in the membranes even after 2 washing steps ity was stimulated up to 6-fold in the presence of NaCl with a suggests that membrane-bound Ech was indeed present and specific activity of 209 mU/mg without Na+ and 1,303 mU/mg active. The loss of activity is likely a result of the dissociation of with 2.5 mM added NaCl (Fig. 4B). The same stimulatory effect the hydrophilic hydrogenase module from the hydrophobic Ech was observed at pH 6.5 with specific activities of 203 and 1,200 core, as described for other multisubunit respiratory enzymes (4, mU/mg without or with 2.5 mM added NaCl (the actual Na+ con- 21–23). Ech is the only membrane-bound hydrogenase encoded centration of the buffer was 203 µM). The stimulation by NaCl by P. ruminis. A 2nd hydrogenase is encoded, but it is cytoplasmic was, however, abolished at pH 5.5 (the actual Na+ concentra- (see above). Retention of hydrogenase activity in the membranes tion of the buffer was 260 µM). This indicates an inactivation thus indicates activity of Ech. of the Na+-dependent ATP synthase in the absence of Na+, Finally, we assessed Ech activity in washed membranes of leaving only residual activity due to the H+-dependent ATP P. ruminis prepared from cells cultivated on glucose or xylose synthase.

4 of 7 | www.pnas.org/cgi/doi/10.1073/pnas.1914939117 Schoelmerich et al. Downloaded by guest on October 2, 2021 Downloaded by guest on October 2, 2021 n ncnucinwt Na cir- a Na bioenergetic with a a 2 conjunction bacterium: of in anaerobic strictly Rnf existence a the in cuits demonstrates work This Discussion study. further Unmasking for them. goal complete a is Rnf components and remaining that Ech the incom- and than complete truly are other are they pumps circuits that ion these possible also that is possible it is However, plete. It Rnf. or Ech not encoded organisms 70 Na 4. Fig. rdeti nya2dmd feeg osrain u nev- but conservation, al. et energy Schoelmerich of mode 2nd pro- a butyrate only from is and and gradient 6 PPP or (Fig. EMPP duction the in phosphorylation in butyrate of production bacterium thus rumen model and the fitness microbial in increase Na microbe when and better on host be between effect butyrate may symbiosis inhibitory that This an 30). shown have (29, been acids inflammation has organic short-chain it the In other rumen, 28). and (8, the host and its of tract diet intestinal the by influenced greatly is structure (27). uncultured are that nis more many with (26), cultured model the in metabolism. strategy sophisticated bacterium a rumen poses This synthase. ATP genus the of were remainder ruminis were Two (P. Clostridia. class the and Firmicutes and 5 (Fig. circuits analyzed readily be could that (25)]. sequence IMG/M; genome [on a had also in genomes that strains type the are searched genomes These We ATPases. organisms. other in of be may Na circuits a in and a ATPase1 ATPase2 is and there Ech that Genomes demonstrating Microbial in After ATPases and Rnf, Ech, of Occurrence Na 243 165 initial be or The to indicated. determined as was added buffers was the H) ( ( KCl measured. or was (M), ATP 5 phosphate LiCl mM inorganic and 3 of acid, adding formation maleic by dependent mM started was 100 reaction Tris·HCl, The mM membranes. MgCl 50 mM Mops, 5 mM MgCl and 50 mM acid, Mes, maleic mM mM 50 10 (B) Tris·HCl, mM 100 (A) ing AB ATPase activity [mU/mg] H + oeognssecdd“nopee icis o example, For circuits. “incomplete” encoded organisms More .ruminis P. be can that species bacterial 200 about to home is rumen The these of components all encoded organisms 13 that found We ,0 atraadacaafrgnsfrEh n,and Rnf, Ech, for genes for archaea and bacteria ∼2,900 spr fte“oebceilmcoim, n t community its and microbiome,” bacterial “core the of part is 100 200 300 400 500 600 700 + tdfeetp aus T yrlsswsmaue nbfe contain- buffer in measured was hydrolysis ATP values. pH different at 0 p .)(B). 7.5) (pH µM ici novn c oehrwt 2nd a with together Ech involving circuit 01 20 15 10 5 0 tmlto fAPhdoyi ciiyb ooaetslsadby and salts monovalent by activity hydrolysis ATP of Stimulation 2 atcnetain[mM] concentration salt n upeetdwt 70 with supplemented and (H) 7.5 or (•), 6.5 (), 5.5 pH at and edtrie o omntee2 these common how determined we ruminis, P. eeae oto t T rmsubstrate-level from ATP its of most generates .Ms fthe of Most xylanivorans). Pseudobutyrivibrio .ruminis P. .Teealbln otephylum the to belong all These S1). Dataset .Techemiosmotic The S7). Fig. Appendix, SI + H saudn,snew eosrtdan demonstrated we since abundant, is + n Na and - .ruminis. P. oaatisrdx o,adenergy and ion, redox, its adapt to Clostridium + + dpnetAPsnhs and synthase ATP -dependent r20(H55,23(H6.5), (pH 203 5.5), (pH 260 or (A) µM ici opsdo n and Rnf of composed circuit ATPase activity [mU/mg] 1000 1200 1400 + 200 400 600 800 dpnetAPss but ATPases, -dependent H 0 + 01 20 15 10 5 0 (n Na ici opsdof composed circuit egysManual Bergey’s 8). = + + concentration [mM] concentration Pseudobutyrivibrio ici involving circuit + H ocnrto in concentration irs n ATP- and ·DiTris, + 2 -dependent al(), NaCl A) tp . or 7.5 pH at .rumi- P. of µg (24) o l uuiswr on.TeAPsswr itnuse noNa genes into if distinguished were encoded ATPases were The H found. Enzymes encod- were enzymes. organisms subunits these of all for number of the combination shows each and Rnf, diagram ing Ech, Venn encoding The genes for synthases. searched ATP were archaea and bacteria 2,925 5. Fig. aae S1 Dataset h n ope ttegi fmr T n eano NADH, of regain and ATP refuels more This of 6). gain and the 4 at enzymes complex 6; Rnf (Fig. the NADH with Fd and CoA (31), (Bcd/Etf) Fd complex the butyryl–CoA/Etf involves formation electron-bifurcating and butyrate to ATPase1 Since lead production. the pools butyrate NADH by more high harnessed The conservation. is energy for which ATPase2 complex, Ech and Rnf icist eeeaeND yteRfcmlxo discard or complex Na Rnf a of Concomitantly, the form the by in electrons NADH excess regenerate to circuits play. into come circuits and ion catabolism, the the where of is continuation this a allow in to equivalents reoxidized be reducing then and and NADH SLP NADH of from form of ATP the of cost formation the the at to steps used of several gain finally in and is production acetyl-CoA The butyrate and ATP. via for phosphotransacetylase gaining converted kinase, the further acetate by then the acetate active is and to acetyl-CoA present The acetyl-phosphate are quan- organism. enzymes was the both formate in supernatants, and the extracts in crude tified in (at measured con- PFOR was growth via activity this all acetyl-CoA and under to of gain NADH, converted measured the also of product is main cost Pyruvate lactate the the ditions. to fact at ATP further in dehydrogenase and of was metabolized 6 lactate gain is (Fig. the the NADH Pyruvate by at of S7). PPP form Fig. the or Appendix, in EMPP equivalents the reducing via and pyruvate to verted rnpr,mtlt,adinadp oesai.Teexperi- The homeostasis. that Na pH demonstrate a including herein and processes, presented cellular ion ments of and sorts motility, all transport, for essential ertheless Ech + ne Na Under con- are sugars these xylose, or glucose on grow cells When dpnettpsbsdo h mn cdsqec fsubunit of sequence acid amino the on based types -dependent 2− + 7 sgnrtdfo h iutnosrdcino crotonyl– of reduction simultaneous the from generated is eea raim a ae2bonrei icis eoe of Genomes circuits. bioenergetic 2 have may organisms Several n a and

o ulls fgnmsadlcstg o genes. for tags locus and genomes of list full a for Rnf Fd Fd + H 1 2− 6 rc odtos lcrn r htldit both into shuttled are electrons conditions, -rich + 2− rvateprvt omt ys.SnePFOR Since lyase. formate pyruvate the via or ) ici o chemiosmosis. for circuit n T.Teeoe h aaoi ot leads route catabolic the Therefore, ATP. and 45 + 3 5 n a and Fd Total: 2925 2− hs euigeuvlnsmust equivalents reducing These . H 51 58 13 H + 2 rdeti salse ythe by established is gradient yteEhcmlx(i.6A). (Fig. complex Ech the by 14 90 NSLts Articles Latest PNAS 75

.ruminis P. a-ATPase Na 332 70 + 1934 ssboth uses H + | -ATPase + See c. f7 of 5 and - None 221 n SI =

MICROBIOLOGY Fig. 6. 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 H2 amounts or the acetyl-CoA moieties recovered as organic acids detected, whereas the numbers outside the brackets result from calculations. The ATP synthase has an assumed rotor stoichiometry of 12/3, and Rnf and Ech are assumed to translocate 1 or 0.5 ions per electron transferred, respectively. Green, carbon compounds; yellow, reducing equivalents; red, energy in the form of ATP. 1, PFOR; 2, pyruvate formate lyase; 3, thiolase; 4, 3-hydroxybutyryl–CoA dehydrogenase; 5, crotonase; 6, Bcd–Etf complex; 7, CoA-transferase; 8, kinase; 9, lactate dehydrogenase; 10, acetate kinase.

leading to better cellular fitness (reflected in the stimulation of (35) via a membrane-associated oxidoreductase (36). This pro- growth by Na+). cess is tied to the provision of reducing equivalents, but neither Under Na+-deprived conditions, there is an excess of reduc- to ATP formation nor changed when H+ are replaced by an ing equivalents in the form of NADH when cells are grown unsaturated fatty acid as electron acceptor (6). on glucose (Fig. 6B). The lack of Na+ severely slows down the Besides the “core microbiome,” the rumen is also home to Na+ circuit and thus the interconversion of NADH and Fd methanogenic archaea and acetogenic bacteria (37–39), and both pools. The H+ circuit must now maintain the membrane poten- Rnf and Ech have been identified and characterized in these + tial and immediate redox homeostasis. The NAD required for groups of strict anaerobes (2, 3, 40). The H2 evolved from Ech a continuation of catabolism may be regenerated by the Na+ can be scavenged by these members. Furthermore, they could circuit running in reverse: A Na+-pumping ATPase1 fuels the be responsible for the emergence of both rnf and ech clusters in regeneration of NAD+ from NADH oxidation and Fd reduc- several rumen butyrivibrios (6) via horizontal gene transfer (41). tion at the Rnf complex. This hypothesis is supported by the After all, it is the ecological environment comprising a diverse fact that Na+ could not be completely abolished in the Na+- consortium that made the occurrence of Ech and Rnf possible free medium, and such a Na+-gradient-consuming role for the and advantageous for P. ruminis. Rnf complex is described in other metabolisms, such as lactate It is likely that rumen butyrivibrios are not the only groups of metabolism in A. woodii, for example (32). In cells grown on organisms that use both Ech and Rnf for separate chemiosmotic xylose without Na+, the redox-balanced models indicate that the circuits. Intriguingly, acetogens were postulated to rely either on Na+ circuit is not required, because redox balancing is achieved Rnf or Ech (5), but the decryption of more genomes revealed by the Ech complex and the electron flow during product for- a cooccurrence of rnf and ech genes in some (14). It will be mation (SI Appendix, Fig. S7). Under both metabolisms (glucose particularly interesting to investigate in these organisms, which and xylose), the calculated ATP yields are lower in the absence depend on the chemiosmotic gradient for energy conservation of Na+, which is a consequence of the ATP-depleting Na+ cir- (acetogens and methanogens), whether they also use 2 circuits cuit (glucose) or shutdown of the Na+ circuit (xylose), as well as for energy conservation. decreased butyrate production. The latter entails less available redox energy (in the form of Fd2−) to fuel energy conservation Material and Methods via the circuits. Ultimately, the lower theoretical ATP yields in Experimental procedures for cultivation of the organism; cell harvest and combination with very slow redox maintenance is most likely the preparation of crude extracts and membranes; measurement of Ech, Rnf, and ATP hydrolysis activity; determination of relative transcript levels; deter- reason for the severely decreased fitness of P. ruminis grown in + + mination of metabolites, pH, and Na concentrations; and the search for the absence of Na . Ech, Rnf, and ATases in microbial genomes are described in SI Appendix, SI Putting this into perspective of the physiological conditions, Materials and Methods. feeding large amounts of grain can reduce the pH in the rumen to well below 5.5 (33), which could lead to an inactivation Data Availability Statement. Data discussed in the paper are available in of ATPase1 (Fig. 4B) and concomitantly a shutdown of the Datasets S1–S3. + + Na circuit. However, normally the Na concentration in the ACKNOWLEDGMENTS. This work was supported by a European Research rumen is at least 60 mM and can be up to 500 to 800 mM Network grant from the Bundesministerium fur¨ Bildung und Forschung. (34). Therefore, both circuits are finetuned to ensure microbial Additional support was from Agriculture and Food Research Initiative fitness of P. ruminis and ultimately of the host, by providing Competitive Grant 2018-67015-27495/Project Accession No. 1014959 and Hatch Project Accession No. 1019985 from the US Department of Agricul- health-promoting organic acids. Moreover, higher ATP pools ture National Institute of Food and Agriculture. M.C.S. is a recipient of a may contribute to biohydrogenation of unsaturated fatty acids Claussen–Simon–Stiftung (Germany) Fellowship.

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