Technical Comments

Response to Comment on "A 3-Hydroxypropionate/4-Hydroxybutyrate Autotrophic Carbon Dioxide Assimilation Pathway in Archaea"

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Science  18 Jul 2008:
Vol. 321, Issue 5887, pp. 342
DOI: 10.1126/science.1158879

Abstract

We proposed that the 3-hydroxypropionate/4-hydroxybutyrate cycle might be important in global carbon cycling based on the abundance of related autotrophic Crenarchaea in the ocean and the high number of gene sequences for a key enzyme of the cycle. Here, we counter the specific criticisms raised by Ettema and Andersson.

We recently elucidated a previously unknown autotrophic CO2 assimilation pathway termed the 3-hydroxypropionate/4-hydroxybutyrate cycle, which operates in the extreme thermophile Metallosphaera sedula (Crenarchaea) (1). This pathway is likely to exist in other autotrophic members of the order Sulfolobales and possibly in some mesophilic Crenarchaea. We proposed that the pathway might be important in global carbon cycling based on the abundance of related autotrophic Crenarchaea in the ocean (2, 3) and the high number of gene sequences for a key enzyme of the cycle, 4-hydroxybutyryl-CoA dehydratase (4HCD), in the Global Ocean Sampling (GOS) database (4).

Ettema and Andersson (5) claim that we predicted the abundance of the pathway in the oceans to be of the same order of magnitude as the Calvin-Benson-Bassham cycle. This is incorrect. Rather, we estimated that the contribution of Crenarchaea to the carbon cycle “should be much lower than that of phototrophs (∼1% of annual marine production).” We are aware that the number of identified gene sequences does not necessarily reflect the relative abundance of the key enzyme or the occurrence of the pathway.

Ettema and Andersson also question the existence of an abundant group of mesophilic autotrophic Crenarchaea in the ocean that may use the proposed new autotrophic cycle. Mesophilic Crenarchaea have been reported to be abundant in the ocean, where they may represent up to 20% of prokaryotes (2), with much higher abundance in the deep water (2, 3). Although the GOS database contains samples from both deep and surface waters, most specimens were collected from surface water environments (4), as Ettema and Andersson rightly note (5). This may result in underrepresentation of archaeal sequences in the GOS database. Indeed, in a 3000-m-deep Mediterranean plankton fosmid library, archaeal sequences represent a much larger fraction of microbial diversity as compared with the GOS database (6). Besides metagenomic data, the widespread occurrence of archaeal lipids in ocean water suggests the ubiquitous presence of mesophilic marine Crenarchaea (7), and carbon isotope analysis of these lipids indicates that they are most likely autotrophs (8, 9). Whether mesophilic marine Crenarchaea use the new pathway remains to be shown. At any rate, the genomes of two autotrophic representatives (Nitrosopumilus and Cenarchaeum spp.) contain the genes for characteristic enzymes of the cycle, while lacking the key genes for other autotrophic pathways.

Ettema and Andersson further argue that 4HCD homologs may function in other pathways. We completely agree. However, we strongly doubt the involvement of this enzyme in phenylacetate catabolism or pyoverdine chromophore biosynthesis as (4-hydroxyphenylacetate) monooxygenase. These putative enzymes can easily be distinguished from 4HCD by sequence comparison. Moreover, it is unlikely that the bearers of the 4HCD gene conduct 4-aminobutyrate fermentation in the oxic seawater, as we noticed (1). On the contrary, its participation in autotrophic CO2 fixation or, in the reverse direction, in acetyl-CoA assimilation, as was discussed in (1), seems to be more plausible. Indeed, we have recently identified in Crenarchaea another autotrophic pathway termed dicarboxylate/4-hydroxybutyrate cycle, in which 4HCD also plays a key role (10, 11).

Analyzing the remainder of the genes encoded by the scaffold on which 4HCD sequences are located, Ettema and Andersson discovered that the representatives of GOS3 cluster of “marine cluster”–1 are likely Archaea, whereas those of GOS1 and GOS2 clusters are likely bacteria. These new insights into the taxonomic origin of the pathway-related sequences and flanking genes provide the basis for future work on the occurrence of the new pathway in nature. It remains unclear whether the 3-hydroxypropionate/4-hydroxybutyrate cycle is restricted to archaeal species. 4HCD may play a role in a similar metabolic process in bacteria, or it may function in a yet unknown pathway. Unfortunately, an unambiguous taxonomic designation of GOS1 and GOS2 sequences is not possible because of the scattered character of the phylogenetic origin of their best hits. In addition, the poor representation of the mesophilic Crenarchaeota in existing genomic databases currently prevents deeper analysis. The sequences of only closely related Cenarchaeum and Nitrosopumilus spp. are available for this possibly third archaeal phylum (12).

Regarding the argument that 4HCD is unlikely to play a role under oxic conditions because of the oxygen sensitivity of this enzyme, we note that 4HCD is not as oxygen sensitive as previously thought and nothing is known about its stabilization (1). Even nitrogenase is active in strict aerobes such as Azotobacter sp. We are indeed aware that the “crenarchaea type-2” 4HCD sequences lack conserved amino acids for the iron-sulfur-cluster pocket and are currently addressing the question of what the function of this gene product may be.

Ettema and Andersson state that we predicted the 3-hydroxypropionate/4-hydroxybutyrate pathway to be operational in Cenarchaeum symbiosum and Archaeoglobus fulgidus. Although we did propose this for C. symbiosum, we did not claim this for Archaeoglobus. Rather, we posed the question, “Which of the potential autotrophic pathways is operating in Archaeoglobus...?”

It is true that candidate genes for some components of the pathway cannot be identified in C. symbiosum and A. fulgidus genomes. However, only the presence of conserved enzymes of the pathway, which all catalyze mechanistically difficult reactions, may be used as an indicator for the pathway in a given organism. As noted in (1), “various alcohol dehydrogenases, aldehyde dehydrogenases, acyl-CoA synthetases, or enoyl-CoA hydratases may fulfill the same function; yet, each superfamily of those enzymes comprises different enzyme families, from which genes may have been recruited.” For instance, for the 3-hydroxypropionate part of the cycle, such key/characteristic enzymes are acetyl-CoA/propionyl-CoA carboxylase and B12-dependent methymalonyl-CoA mutase. They are present in both Metallosphaera and Chloroflexus, although enzymes converting malonyl-CoA to propionyl-CoA cannot simply be identified by database search because they are not homologous or are only distantly related. A telling example is malonyl-CoA reductase (1, 11, 1315). The same arguments hold true for the 4-hydroxybutyrate part, where the 4-hydroxybutyryl-CoA dehydratase reaction is the mechanistically most intriguing one and the enzyme is conserved, whereas succinyl-CoA reductase, succinate semialdehyde reductase, and other enzymes may be quite different (1, 11). Puzzlingly, Ettema and Andersson repeat our comments and warnings almost exactly.

Finally, although we disagree with some of the comments made by Ettema and Andersson, we acknowledge their analysis and are grateful to them for this opportunity to clarify our work.

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