Hydrogen production by Sulfurospirillum species enables syntrophic interactions of Epsilonproteobacteria

Hydrogen-producing bacteria are of environmental importance, since hydrogen is a major electron donor for prokaryotes in anoxic ecosystems. Epsilonproteobacteria are currently considered to be hydrogen-oxidizing bacteria exclusively. Here, we report hydrogen production upon pyruvate fermentation for free-living Epsilonproteobacteria, Sulfurospirillum spp. The amount of hydrogen produced is different in two subgroups of Sulfurospirillum spp., represented by S. cavolei and S. multivorans. The former produces more hydrogen and excretes acetate as sole organic acid, while the latter additionally produces lactate and succinate. Hydrogen production can be assigned by differential proteomics to a hydrogenase (similar to hydrogenase 4 from E. coli) that is more abundant during fermentation. A syntrophic interaction is established between Sulfurospirillum multivorans and Methanococcus voltae when cocultured with lactate as sole substrate, as the former cannot grow fermentatively on lactate alone and the latter relies on hydrogen for growth. This might hint to a yet unrecognized role of Epsilonproteobacteria as hydrogen producers in anoxic microbial communities.

The study includes proteome experiments of cells grown with pyruvate and cells grown with pyruvate and the electron acceptor fumarate. Why were no proteome experiments done with cells in different stages of adaptation of growth on pyruvate. As growth of S. multivorans on pyruvate becomes faster upon subculturing, proteome or transcriptome analysis would be an ideal way to get insight into what is changing.
In the manuscript two media are mentioned, one for the pure culture experiments and one for the coculture experiments. The medium for S. multivorans (ref 30) is essentially different from the M. voltae medium (ref 32). The latter medium contains a large number of organic compounds, which needs to be considered in the experimentation and in the interpretation of the results. By the way, in the publication of Whtman et al different media are presented; it is not clear which of these medium was used in the current study. From the description it is also not clear if both the pure culture and the coculture experiment described in Figure 6 were done in the M. voltae medium. It is poorly described how these cocultures were constructed and maintained. What is the inoculum size of each and how is subculturing procedure of the coculture? What are the growth properties of a pure culture of S. multivorans in the M. voltae medium? Is the observed aggregate formation caused by syntrophic growth or by the composition of the medium? This needs further description and clarification.
The syntrophic behaviour is just discussed as a result of interspecies hydrogen transfer. The option of interspecies formate transfer or direct electron transfer is ignored. This is a shortcoming, especially because M. voltae is able to use both hydrogen and formate. The authors mention that the bacterium does not have a pyruvate formate lyase, and that this would make a role of formate less likely. However, formate can also be formed from electrons derived from lactate oxidation and pyruvate oxidation. A proteome or transcriptome analysis of cells of S. multivorans grown in pure culture with pyruvate, lactate with fumarate and in coculture with lactate is recommended. Such an experiment would avoid to draw conclusions that are biased. It would also allow to get insight into which enzyme is (enzymes are) involved in lactate oxidation to pyruvate. When lactate would be oxidized by an NAD-dependent enzyme (SMUL_0438), it is also not clear how NADH is oxidized coupled to hydrogen formation. The possible involvement of formate dehydrogenase (Smul-0438) should be considered.
Hydrogenase activity measurements were done with methylviologen and benzylviologen. Is there any activity with NADH or NAD in the hydrogen formation and hydrogen oxidation reaction, respectively. Was it possible to detect formate dehydrogenase activity in cell extracts?
Minor point: In the introduction it is mentioned that hydrogen production was never shown for Epsilonbacteria. This statement is not completely true. Hydrogen production was shown for Sulfurospirillum carboxydovorans (Jensen and Finster (2005) Antonie van Leeuwenhoek 87: 339-353. This is reference 28 of the manuscript.
Reviewer #2 (Remarks to the Author): The authors show that some Sulfurspirillium spp., which had previously been viewed only as hydrogen oxidizers, produce hydrogen during pyruvate fermentation. In addition, S. multivorans was shown to grow syntrophically on lactate when a hydrogenotrophic methanogen, Methanococcus voltae, was present. Enzyme assays reveal that hydrogen-producing activity was present in the membrane fraction and likely cytoplasmically oriented. Hydrogen production activity was correlated to hydrogenase 4 (Hyf) based on proteomic and transcriptional analyses. Hyf gene cluster was detected in genomes of other Sulfurspirillium and in some Campylobacter species.
Interestingly, it took many transfers for Sulfurspirillium spp. to ferment pyruvate and produce hydrogen effectively. Some Sulfurspirillium spp. produce mainly acetate, hydrogen, and carbon dioxide from pyruvate while other produce less hydrogen due to their ability to produce fermentation products such as succintate. The work on fermentation products was done well but the results are not surprising.
Specific comments: 1. The authors concluded that some members of the Epsilonproteobacteria should be considered to be hydrogen producers in their natural habitats. It is likely that some will grow syntrophically on lactate as shown for S. multivorans. However, the ecological importance of hydrogen production from pyruvate is not clear. It took many transfers in the laboratory before Sulfurspirillium spp. effectively fermented pyruvate and produced hydrogen. This argues that the ecological role of these organisms is not pyruvate fermentation. In general, Sulfurspirillium spp. do not oxidize or ferment carbohydrates so fermentative hydrogen production from pyruvate derived from sugars is not likely. 2. The authors provided proteomic data in supplemental tables excel files, but these data should also be deposited in a database such as PRIDE.
Minor comments: 1. p. 33. Spell out genus name here. 2. Line 109. I would state that the gas volume was adjusted to standard temperature and pressure. 3. Line 110. Was medium used for the adaptation experiment the same as that described on lines 94-95? 4. Line 181. manufacturer's 5. Line 231. There is no period at the end of the sentence making me wonder if there is some text missing. 6. Lines 244-246. This statement seems out of place as the data are discussed in the next section. 7. Lines 259-261. Cite Fig. 2 8. Line 330-331, periplasmically oriented and cytoplasmically oriented would be better wording, as both are located in the cytoplasmic membrane. 9. Line 350-445. Fig. 5 needs to be cited. The order of topics discussed could be improved. I suggest discussion of the data on hydrogenases together with the data on Hyf first (lines 385-403) to make it clear that Hyf is the one involved in hydrogen production. One can then discuss pyruvate/energy metabolism and anabolism.

Reviewers' comments:
Reviewer #1 (Remarks to the Author): The manuscript by Kruse et al describes hydrogen production by Sulfurospirillum spp. and their ability to grow in syntrophic association with methanogens.
This study makes an important contribution to science, especially as hydrogen production and the ability to grow in syntrophic association with methanogens thus far is a property associated with bacteria belonging to the deltaproteobacteria and the Firmicutes. When this ability can be unequivocally linked to other phylogenetic groups, this is an important step forward. The manuscript contains a set of solid experiments, but in my opinion some extra experiments and editorial changes are needed to make the story more complete and conclusive.
---We thank the reviewer very much for seeing the importance in our work! We performed several extra experiments in turn to your constructive criticism and we think that the manuscript is strongly enhanced after revision. We just could not follow several of the reviewers points on formate, since we never, under any condition, detected formate in our experiments. However we added some discussion on that as well (see below).

The following main points are for the authors to consider:
The introduction gives the impression that hydrogen is the only interspecies electron transfer compound in syntrophic methanogenic communities. Formate transfer and direct electron transfer need to be mentioned in the introduction.
---We now added "Besides H 2 , also formate, similarly formed during fermentative metabolism, is an important electron carrier in e.g. syntrophic fatty acid-degrading methanogenic consortia 59 .", and mentioned H 2 AND formate at the other points in the introduction.

The study includes proteome experiments of cells grown with pyruvate and cells grown with pyruvate and the electron acceptor fumarate. Why were no proteome experiments done with cells in different stages of adaptation of growth on pyruvate. As growth of S. multivorans on pyruvate becomes faster upon subculturing, proteome or transcriptome analysis would be an ideal way to get insight into what is changing.
---At first, the adaptation was a coincidental observation and we did not want to focus too much on it. Nonetheless we think it is a very interesting observation and are thankful for the reviewer to convince us to do additional experiments. We now compared the proteome of adapted cells with that of not adapted cells. The new results are included in the proteome results part. A new supplemental table showing the proteome results is added. We did not observe a significant change in any of the proteins linked directly to fermentative physiology. However, we found a higher abundance of some proteins also more abundant in the proteomes of S. multivorans and S. cavolei during fermentative metabolism compared to the respiratory metabolism. Especially the very high abundance and upregulation of a putative aldehyde oxidoreductase under fermentation was observed. We now included a short paragraph on this and three other proteins more abundant in the adapted cells and in fermentative metabolism in the results (L506-521, Table 2) and discussion part (L601 -618) . Whether these proteins are directly involved in fermentative metabolism or contribute to e.g. stress response is subject to further investigations which in our eyes exceed the scope of the current manuscript. Subculturing was done by transferring 10% (v/v) of the co-culture into freshly prepared media for 10 subcultures before the growth experiment and electron microscopy was performed. The basal medium contained per L 0.1 g isoleucine, 0.1 g leucine, 0.34 g KCl, 4 g MgCl 2 · 6 H 2 O, 3.45 g MgSO 4 · 7 H 2 O, 0.25 g NH 4 Cl, 0.14 CaCl 2 · 2 H 2 O, 0.14 g K 2 HPO 4 , 5 g NaCl. After boiling and cooling to room temperature under N 2 , following separately autoclaved anoxic solutions were added (per L basal medium): 10 mL vitamin solution, 10 mL trace element solution (see below), 0.2% (w/v) Casamino Acids (Difco Laboratories, Detroit, Michigan, USA), 10 mL sulfide solution (Na 2 S · 9 H 2 O, 50 g L -1 ) and 10 mL cysteine solution (Lcysteine-HCl, 50 g L -1 ). Final pH was adjusted to 7.0 with 2.5 g L -1 NaHCO 3 . The vitamin solution contained per L 2 mg D(+) biotin, 2 mg folic acid, 10 mg pyridoxamine-2HCl, 5 mg thiamine-HCl · 2 H 2 O. 5 mg riboflavin, 5 mg nicotinic acid, 5 mg Ca-D(+) pantothenate, 0.1 mg vitamin B 12 , 5 mg 4-aminobenzoic acid, 5 mg lipoic acid. The trace element solution contained per L 1.5 g nitriloacetic acid (firstly added, and pH adjustment to 6.5 with KOH), 3  We also added the following sentence to Figure 6: "Pure S. multivorans and the co-culture were cultivated in a slightly modified medium originally optimized for M. voltae as described in the methods section" The main organic compounds in this medium are the amino acids leucin and isoleucine as well as casamino acids, we clarified this by adding "A medium optimized for M. voltae was used for the coculture (see Methods section). This medium contained a number of organic substances (i.e. the amino acids leucin and isolecuin, as well as casamino acids) not present in the medium of S. multivorans and several controls with S. multivorans pure cultures were performed to exclude any unprecedented growth effects potentially caused by this medium." -see also next point. In the discussion, the following sentence was added: "Since M. voltae is not able to thrive in minimal medium 32 , we cultivated the coculture not in the medium used for S. multivorans cultivation, but in an optimized medium for M. voltae which included several organic substances. The influence of this medium on S. multivorans cultivation was negligible, so that the observed lactate deprivation and aggregate formation can be unequivocally linked to the syntrophic co-culture."

What are the growth properties of a pure culture of S. multivorans in the M. voltae medium? Is the observed aggregate formation caused by syntrophic growth or by the composition of the medium? This needs further description and clarification.
---Additional experiments showed that growth of S. multivorans in the M. voltae medium in pure cultures was very similar to that in the "original" medium. We added the sentence "A medium optimized for M. voltae 32 was used for both co-culture and S. multivorans pure culture (see also Methods section). The growth behavior of the latter in this medium with pyruvate and fumarate and pyruvate alone as substrates was similar to that in medium originally used for cultivation of S. multivorans." The aggregate formation is not observed in pure cultures of S. multivorans in this medium. We added the sentence and a corresponding figure: "Aggregates were not observed for S. multivorans pure cultures in the used media (Supplementary Figure 16)."

The syntrophic behaviour is just discussed as a result of interspecies hydrogen transfer. The option of interspecies formate transfer or direct electron transfer is ignored. This is a shortcoming, especially because M. voltae is able to use both hydrogen and formate. The authors mention that the bacterium does not have a pyruvate formate lyase, and that this would make a role of formate less likely.
However, formate can also be formed from electrons derived from lactate oxidation and pyruvate oxidation. A proteome or transcriptome analysis of cells of S. multivorans grown in pure culture with pyruvate, lactate with fumarate and in coculture with lactate is recommended. Such an experiment would avoid to draw conclusions that are biased. It would also allow to get insight into which enzyme is (enzymes are) involved in lactate oxidation to pyruvate. When lactate would be oxidized by an NADdependent enzyme (SMUL_0438), it is also not clear how NADH is oxidized coupled to hydrogen formation. The possible involvement of formate dehydrogenase (Smul-0438) should be considered.
---Since we never detected formate in any of the HPLC measurements of any culture (and concluding from standard measurements with formate we should have been able to detect it), we did not consider the possibility of interspecies formate transfer and still think that it is unlikely. This holds especially true, since the fermentation balance is completely even in both Sulfurospirillum spp. To make more clear we didn't detect formate which is therefore obviously not a metabolite produced during fermentation we now wrote "No other organic acids such as formate, or alcohols e.g. ethanol, were dected". We would include direct electron transfer as a possibility if we would have found either 1) structures for DIET in the electron micrographs or 2) one of the archetypical proteins (e.g. the large Geobacter-like outer surface cytochromes or any pili) encoded in one of the Sulfurospirillum spp. genomes. Both were not the case, so we did not include it in the manuscript. We added a discussion on both: "The interspecies electron carrier in the syntrophic relationship of S. multivorans and M. voltae is hydrogen based on the metabolite analysis of pure S. multivorans cultures, where only hydrogen was found as a product of pyruvate fermentation. Direct interspecies electron transfer is also unlikely to proceed in this syntrophic interaction, since besides none of the typical nanowire structures were visible in electron microscopy, no pili or extracellular cytochromes 60 , usually used for interspecies electron transfer of e.g. Shewanella spp. and Geobacter spp. 61 were found in the genome of any Sulfurospirillum spp." We could also not measure any NAD(P)(H)-dependent lactate oxidation or production, which is also stated in the manuscript, we added the complete methods in the supplement (Line 476 "…lack of pyridine dinucleotide-dependent lactate-oxidizing or pyruvate-reducing activities in cell extracts of S. multivorans (data not shown, methods described in Supplementary Note 2"). There is also evidence in the literature that such an enzyme is not involved in lactate metabolism of the closely related Campylobacter jejuni (see reference 50, "Two respiratory enzyme systems in Campylobacter jejuni NCTC 11168 contribute to growth on L-lactate").
However, the reviewer is right in that proteomics could give helpful insights into the involvement of lactate oxidating enzymes. Therefore we investigated the proteome of lactate-cultivated cells in comparison to the proteome of cells grown with pyruvate, both with fumarate as electron acceptor. The results are now added as supplementary excel file and added to the results text (L.484-494). However, we could not find an upregulation of the putative lactate-oxidizing enzymes, therefore we suggest a substrate-independent expression of those, which is in line with the observation of the corresponding enzymes in C. jejuni (Ref 50). We included a brief discussion as follows: "A role of the S. multivorans "lactate dehydrogenase" in respiratory lactate oxidation was also unlikely as its low abundance in the proteome of lactate-grown cells suggest. Instead, lactate is likey oxidizied in S. multivorans by orthologs of the NAD + -independent enzymes recruited by C. jejuni for lactate oxidation, a flavin Fe-S cluster-containing enzyme and a three-partite lactate utilization protein 50 . These proteins were shown to be not substrate-inducible in C. jejuni, which is in line with the observed similar abundance of both proteins in lactate and pyruvate-cultivated S. multivorans cells." Hydrogenase activity measurements were done with methylviologen and benzylviologen. Is there any activity with NADH or NAD in the hydrogen formation and hydrogen oxidation reaction, respectively. Was it possible to detect formate dehydrogenase activity in cell extracts?
---We did not detect any hydrogenase activity with NADH, which is corroborated by the lack of any typical NAD ( ---True, we overlooked this here since we mainly searched for fermentative hydrogen production. We added now "Sulfurospirillum carboxydovorans was shown to produce minor amounts of hydrogen, which was finally consumed again, upon CO oxidation 28 ." Also, we deleted "exclusively" in front of H2 oxidizing bacteria and added "fermentative"to "hydrogen production was never observed".

Reviewer #2 (Remarks to the Author):
The authors show that some Sulfurspirillium spp., which had previously been viewed only as hydrogen oxidizers, produce hydrogen during pyruvate fermentation. In addition, S. multivorans was shown to grow syntrophically on lactate when a hydrogenotrophic methanogen, Methanococcus voltae, was present. Enzyme assays reveal that hydrogen-producing activity was present in the membrane fraction and likely cytoplasmically oriented. Hydrogen production activity was correlated to hydrogenase 4 (Hyf) based on proteomic and transcriptional analyses. Hyf gene cluster was detected in genomes of other Sulfurspirillium and in some Campylobacter species.
Interestingly, it took many transfers for Sulfurspirillium spp. to ferment pyruvate and produce hydrogen effectively. Some Sulfurspirillium spp. produce mainly acetate, hydrogen, and carbon dioxide from pyruvate while other produce less hydrogen due to their ability to produce fermentation products such as succintate. The work on fermentation products was done well but the results are not surprising.
Specific comments: 1. The authors concluded that some members of the Epsilonproteobacteria should be considered to be hydrogen producers in their natural habitats. It is likely that some will grow syntrophically on lactate as shown for S. multivorans. However, the ecological importance of hydrogen production from pyruvate is not clear. It took many transfers in the laboratory before Sulfurspirillium spp. effectively fermented pyruvate and produced hydrogen. This argues that the ecological role of these organisms is not pyruvate fermentation. In general, Sulfurspirillium spp. do not oxidize or ferment carbohydrates so fermentative hydrogen production from pyruvate derived from sugars is not likely.
---We agree with the author in that the amount of pyruvate in natural habitats is most likely rather low and thus its ecological role as substrate is limited (despite cell lysis could provide some pyruvate also in natural habitats). Therefore we also follow the reviewer that lactate is the main substrate in natural habitats. This we underline in our manuscript by setting up the co-cultures of S. multivorans with M. voltae with lactate instead of pyruvate, which was not possible for pure S. multivorans cultures. This we underline in the manuscript by adding "The inability of Sulfurospirillum spp. to use lactate as sole substrate in pure cultures is most probably due to the thermodynamically unfavorable lactate oxidation to pyruvate upon H 2 production. To test the ecological significance of our observation, we established a lactate-conuming syntrophic partnership of S. multivorans with a hydrogen-consumer, Methanococcus voltae. " However, to reveal the details of the fermentative physiology of Sulfurospirillum spp., we looked at pyruvate fermentation.
One reason for the relatively long adaptation process could be that the bacteria are cultivated usually for a very long time in a respiratory mode, in our laboratory as well as in cell culture collections. We added the following sentence to discuss this: "The low adaptation to fermentative metabolism in Sulfurospirillum might also be an effect of the long-term respiratory cultivation of the organisms in our laboratory and cell culture collections. Whether Sulfurospirillum spp. in natural habitats show an equally long adaptation time to fermentative metabolism is likely dependent on the type and concentration of electron donor and electron acceptor in the environment." Indeed the low amount of hydrogen produced by not fermentatively adapted S. multivorans cells could hinder the syntrophic relationship. Therefore we performed additional experiments with S. multivorans cells not adapted to pyruvate fermentation and cultivated them with Methanococcus voltae. To our surprise, the growth/lactate consumption was not much slower (see Supplementary Figure 16) than with the adapted cells, which points towards a higher ecological relevance of fermentation of Sulfurospirillum. Therefore we added a sentence to the discussion in L 691, "Both, S. multivorans cells adapted and not adapted to pyruvate fermentation supported growth of the co-culture, which strengthens our suggested role of Sulfurospirillum spp. as H 2 producers in anaerobic food webs." However, of course we agree with the reviewer in that fermentation is only one of many lifestyles of