Recent origin of iron oxidation in extant microbial groups and low clade fidelity of iron metabolisms

Recent origin of iron oxidation in extant microbial groups and low clade fidelity of iron metabolisms

ABSTRACT Reduced iron was abundant in Earth's surface environments before their oxygenation, so iron oxidation could have been a common metabolism on the early Earth. Consequently, modern microbial iron oxidation is sometimes seen as a holdover from an earlier biosphere, but the continuity of involved lineages or the metabolic process itself has not been verified. Modern neutrophilic iron oxidizers use cytochrome-porin Cyc2 as the initial electron acceptor in iron oxidation. With the protein as a proxy for the metabolism, we performed a phylogenetic analysis of Cyc2 to understand the evolutionary history of this microbial iron oxidation pathway. In addition to known iron oxidizers, we identified Cyc2 orthologs in gammaproteobacterial endosymbionts of lucinid bivalves. These bivalves have a robust fossil record and rely on seagrass meadows that only appear in the Cretaceous, providing a valuable time calibration in the evolutionary history of Cyc2. Our molecular clock analysis shows that extant sampled Cyc2 diversity has surprisingly recent common ancestry, and iron oxidation metabolisms in Gallionellaceae, Zetaproteobacteria, and photoferrotrophic Chlorobi likely originated in the Neoproterozoic or the Phanerozoic via multiple transfer events. The groups responsible for microbial iron oxidation have thus changed over Earth history, possibly reflecting the instability of niches with sufficient reduced iron. We note that frequent transfer and changing taxonomic distribution may be a general pattern for traits which are selected sporadically across space and time. Based on iron metabolism and other processes, we explore this concept of a trait's "clade fidelity" (or lack thereof) and establish its evolutionary importance.

IMPORTANCE Bacteria can oxidize iron to produce energy. As there was plenty of reduced iron available on the early Earth and there is only a little today, it was sometimes thought that bacteria that oxidize iron today are a small remnant of a larger group that used to do it. We studied the evolutionary history of the iron oxidation pathway that modern bacteria use, and we found that they developed that pathway relatively recently: whatever did it in the past is no longer around today. It would probably be hard for any group of organisms to keep doing iron oxidation over billions of years since iron availability is so variable: they are likely to go extinct or lose this ability at some point. We suggest this as a general trend in evolution that traits which are only sporadically useful are commonly lost-and then re-invented or re-distributed-or the trait will go extinct.

Microbial iron oxidation has been proposed as a common metabolism on the early Earth, given the availability of reduced iron in surface environments before their widespread oxygenation. Banded iron formations present a particularly voluminous body of geological evidence for the abundance of reduced iron once dissolved in the ocean, and photoferrotrophy-one form of microbial iron oxidation-is often implicated in the origin of these formations, given the lack of oxidative power available to oxidize large quantities of iron chemotrophically. As BIFs are a quantitatively important presence in the geological record until the Paleoproterozoic, involvement of photoferrotrophy in their creation would suggest a dominant role for that metabolism in early biological productivity. Consequently, modern microbial iron oxidation is sometimes seen as a holdover from a much earlier biosphere, yet any continuity of involved lineages or even the metabolic process itself has not been verified. Here, we study the phylogenetic history of Cyc2, a key enzyme in modern microbial iron oxidation, in order to understand the history and antiquity of this pathway as present in modern organisms.

Cytochrome-porin Cyc2 is used by chemolithotrophic Gallionellaceae and Zetaproteobacteria as well as photoferrotrophic Chlorobi as the initial electron acceptor in their iron oxidation pathway: it takes electrons from dissolved iron two outside the cell and passes them down an electron transport chain which eventually reaches the terminal oxidase. Since known ecologically important neutrophilic iron oxidizers have Cyc2 and it is highly expressed in environments with a high degree of microbial iron oxidation, it has been proposed as a reliable genomic marker for this metabolism. This allows us to study the phylogenetic history of this protein as a proxy for the history of microbial iron oxidation as observed in modern neutrophilic taxa.

Some previous studies have already considered the possible role of photoferrotrophic Chlorobi in the context of the inference that photoferrotrophy was an important metabolism in the Earth's past. For example, Thompson et al. propose that they were important players in biogeochemical cycles in the Precambrian, but without explicitly dating the clade or their iron-oxidizing metabolism. Other efforts have estimated the age of photoferrotrophic Chlorobi via molecular dating, with varying results: Ward and Shih found that this clade postdates three hundred million years, while Magnabosco et al. ran analyses with multiple calibration sets, which all suggest an age of around one billion years. Now that Cyc2 has been identified as the initial electron acceptor in photoferrotrophic Chlorobi as well as other, chemotrophic iron oxidizers, we attempt to date the Cyc2 phylogeny directly as a proxy for the age of the underlying microbial iron oxidation process, rather than estimating the age of currently iron-oxidizing groups on the species tree. The Cyc2 phylogenetic history will include the photoferrotrophic Chlorobi and offer an estimate for the age of photoferrotrophy in that clade, which is not necessarily the same as the age of that clade, as the metabolism may not always have been inherited vertically. However, our approach also extends the history of Cyc2-dependent microbial iron oxidation backward to the last common ancestor of modern Cyc2 sequences, potentially further into the past than any extant group performing iron oxidation today.

MATERIALS AND METHODS

Cyc2 sequence collection and alignment

The Cyc2 sequence in Chlorobium phaeoferrooxidans strain KB zero one as identified by reference was used to query the National Center for Biotechnology Information non-redundant protein database for homologous Cyc2 sequences using BLASTp.

Two different data sets of Cyc2 orthologs were assembled, differing in the breadth of the sampling strategy. A broad sample includes the first five hundred BLAST hits returned by the search, exhaustively covering the sequence space in and around the main clades of neutrophilic iron oxidizers as well as including any clades containing possible calibration points in the broader protein family. This large sequence set showed multiple suspected misalignments on visual inspection, especially within very distantly related sequences, calling for the cross-validation of results against a smaller data set including only the descendants of the last common ancestor of Cyc2 sequences in the modern groups of neutrophilic iron oxidizers: in particular, chemolithotrophic Gallionellaceae and Zetaproteobacteria as well as photoferrotrophic Chlorobi. That narrow sample is a subset of the broad sample comprising one hundred nine sequences (equivalent to what prior work on Cyc two in references five and six has called cluster one and already identified as corresponding to neutrophilic iron oxidation), and a visual inspection of the resulting alignment showed no obvious misalignments. The alignments were made using MAFFT seven point two four five, with the automatic choice of alignment algorithm ("mafft -- auto") selecting L-INS-i.