Article

Microbial iron oxide respiration coupled to

sulfide oxidation

https://doi.org/10.1038/s41586-025-09467-0 Song-Can Chen1,2,3 ✉, Xiao-Min Li4,5, Nicola Battisti3, Guoqing Guan3,6, Maria A. Montoya3,
Jay Osvatic7,8, Petra Pjevac3,7, Shaul Pollak3, Andreas Richter4, Arno Schintlmeister3,
Received: 3 October 2024
Wolfgang Wanek4, Marc Mussmann3 ✉ & Alexander Loy3,7 ✉
Accepted: 28 July 2025

Published online: xx xx xxxx

Microorganisms have driven Earth’s sulfur cycle since the emergence of life1–6, yet the
Open access sulfur-cycling capacities of microorganisms and their integration with other element
Check for updates cycles remain incompletely understood. One such uncharacterized metabolism is the
coupling of sulfide oxidation with iron(iii) oxide reduction, a ubiquitous environmental
process hitherto considered to be strictly abiotic7,8. Here we present a comprehensive
genomic analysis of sulfur metabolism across prokaryotes, and reveal bacteria that
are capable of oxidizing sulfide using extracellular solid phase iron(iii). Based on a
phylogenetic framework of over hundred genes involved in dissimilatory transformation
of sulfur compounds, we recorded sulfur-cycling capacity in most bacterial and
archaeal phyla. Metabolic reconstructions predicted co-occurrence of sulfur compound
oxidation and iron(iii) oxide respiration in diverse members of 37 prokaryotic
phyla. Physiological and transcriptomic evidence demonstrated that a cultivated
representative, Desulfurivibrio alkaliphilus, grows autotrophically by oxidizing
dissolved sulfide or iron monosulfide (FeS) to sulfate with ferrihydrite as an
extracellular iron(iii) electron acceptor. The biological process outpaced the abiotic
process at environmentally relevant sulfide concentrations. These findings expand
the known diversity of sulfur-cycling microorganisms and unveil a biological mechanism
that links sulfur and iron cycling in anoxic environments, thus highlighting the
fundamental role of microorganisms in global element cycles.

The global biogeochemical sulfur cycle is largely driven by bacteria dissimilatory reduction of oxidized sulfur compounds such as sul-
and archaea that have evolved an array of enzymatic mechanisms for fate, is a major biogeochemical process. Sulfur oxidation replenishes
diverse dissimilatory sulfur redox transformations (hereafter called sulfate and contributes to the sulfur cycle in diverse ecosystems12–14.
sulfur microorganisms). Sulfur transformations are linked to redox Microorganisms oxidize reduced sulfur compounds by utilizing light or
reactions of other elements, such as carbon, oxygen, iron and nitro- chemical oxidants, including oxygen, nitrate and manganese oxides15.
gen1–3. The intertwined electron transfer reactions mediated by sulfur Ferric oxyhydroxides and oxides (hereafter called iron(iii) oxides)
microorganisms in diverse ecosystems represent a metabolic network represent one of the largest pools of oxidants on the Earth’s surface16.
on the global scale that profoundly impacts biogeochemistry, climate The interaction with iron(iii) oxides shapes the concentration of free
and redox state on the Earth’s surface over geological timescales4–6. sulfide and the cycling of sulfur in various anoxic environments, such
Functional gene and genome-centric surveys have expanded the known as marine sediments, wetlands and aquifers7,12,17–19. However, current
diversity of specific guilds of sulfur microorganisms beyond culti- biogeochemical models consider the reaction of sulfide with iron(iii)
vated representatives, and predicted previously unrecognized roles of oxides as purely abiotic, producing mainly elemental sulfur (S(0)) and
uncultivated sulfur microorganisms in biogeochemical cycles across poorly crystalline FeS7,8. Although geochemical studies have hinted at
ecosystems9–11. However, the microbial genomic signatures for the microbial oxidation of sulfide to sulfate with iron(iii) oxides20–22, micro-
full spectrum of dissimilatory sulfur metabolisms, including oxida- organisms and reactions that catalyse this process thus far remained
tion of reduced sulfur compounds (hereafter called sulfur oxidation), uncharacterized.
has not been explored. Moreover, most genome-based predictions of Here we report the wide distribution of dissimilatory sulfur-
sulfur-related metabolisms lack support from experimental evidence. transforming potential across the bacterial and archaeal tree of life,
Among the various transformation processes catalysed by sulfur including many previously unsuspected microbial taxa. By genome-
microorganisms, the re-oxidation of sulfide, which occurs mainly by based reconstruction of the metabolism of potential sulfur-oxidizing

1
State Key Laboratory of Soil Pollution Control and Safety, Zhejiang University, Hangzhou, China. 2MOE Key Laboratory of Environment Remediation and Ecological Health, College of Environmental
and Resource Sciences, Zhejiang University, Hangzhou, China. 3Division of Microbial Ecology, Centre for Microbiology and Environmental Systems, University of Vienna, Vienna, Austria.
4
Division of Terrestrial Ecosystem Research, Centre for Microbiology and Environmental Systems Science, University of Vienna, Vienna, Austria. 5Key Laboratory of Urban Environment and
Health, Ningbo Observation and Research Station, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China. 6Doctoral School in Microbiology and Environmental Science,
University of Vienna, Vienna, Austria. 7Joint Microbiome Facility of the Medical University of Vienna and the University of Vienna, Vienna, Austria. 8Division of Clinical Microbiology, Department
of Laboratory Medicine, Medical University of Vienna, Vienna, Austria. ✉e-mail: [email protected]; [email protected]; [email protected]

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Article
a Total no. of GTDB genomes Total no. of S-metabolizing microorganisms
Species 31,910 (52.8%) Species 16,839 (33.0%)

Genus 9,428 (54.2%) Genus 5,109 (46.7%)

Family 2,600 (58.3%) Family 1,515 (58.1%)

Order 1,034 (67.2%) Order 695 (59.6%)

Class 379 (75.7%) Others Class 287 (60.3%) Cultured

S-metabolizing microorganisms Uncultured
Phylum 149 (80.5%) Phylum 120 (59.2%)

b Halobacteriota
Thermoproteota

Archaea
Methanobacteriota
Thermoplasmatota
Asgardarchaeota**
Hydrothermarchaeota**
Proteobacteria
Actinobacteriota
Bacteroidota
Firmicutes
Firmicutes_A
Desulfobacterota
Cyanobacteria
Campylobacterota Gene category
Firmicutes_B DMS cycling
Planctomycetota
Myxococcota SQ metabolism
Spirochaetota
Bdellovibrionota Inorganic S metabolism
Deinococcota
Firmicutes_C
Fusobacteriota
Aquificota Cultivation status
Firmicutes_F
Deferribacterota Cultured
Tectomicrobia
Chrysiogenetota Uncultured

Bacteria
Moduliflexota
Thermodesulfobiota
Chloroflexota**
Acidobacteriota** log10(no. of genomes)
Verrucomicrobiota**
Nitrospirota** 1
Gemmatimonadota**
Firmicutes_D** 2
Omnitrophota** 3
Methylomirabilota**
Elusimicrobiota**
Firmicutes_E**
Eremiobacterota**
Armatimonadota**
Nitrospinota**
SAR324**
Firmicutes_G**
Margulisbacteria**
KSB1**
Dormibacterota**
Marinisomatota**
Patescibacteria**
Desulfobacterota_B**
dddD
dddL
dddP
dddQ

dmoA
dmsA
dsoB
dsyB
mddA

msmA

mtsA
sfnG
ssuD
tmm

yihQ

doxD
doxDA
fccB
sdo
sHdrB1
soeA
sorA

soxB

tsdA
aprA
asrA
dsrA
dsrB
mccA

phsA
shyB
sudA
ttrA
mmtN

sqr

npsr
otr
dddX

mtoX
dddW

sorT
dddY

Fig. 1 | Distribution of sulfur-cycling potential across bacterial and archaeal of points indicates the number of genomes encoding a particular sulfur-cycling
phyla. a, Left, number of GTDB taxa carrying at least one of 42 sulfur-cycling gene (with log transformation). Sulfur-cycling guilds lacking any cultivated
marker genes. Right, among sulfur-metabolizing microorganisms, a substantial representatives in a phylum are depicted in less opaque colour. Double asterisks
fraction is exclusively represented by uncultured taxa. b, Distribution of 42 indicate microbial phyla with no or few cultivated representatives. Genes
sulfur-cycling marker genes across archaeal and bacterial phyla. The top 50 of associated with broad sulfur-cycling functional categories are shown with
120 phyla with the highest number of sulfur-cycling genes are shown. The size different colours. DMS, dimethylsulfide; SQ, sulfoquinovose.

microorganisms, we further revealed sulfur-based electron transfer The resulting phylogenies and clade-specific HMMs enabled us to
pathways that could facilitate anaerobic sulfur oxidation coupled effectively and accurately retrieve homologues of sulfur-cycling pro-
to the reduction of extracellular iron(iii) oxide in diverse bacterial teins from genomes and curate their function within a phylogenetic
and archaeal taxa. Physiological and transcriptomic evidence of the context (Supplementary Fig. 2).
predicted process in a cultivated representative, D. alkaliphilus, con- Using this resource, we systematically queried a subset of 42 sulfur-
tributes to the evolving view on the biological coupling of the biogeo- cycling enzymes against representative genomes of all bacterial and
chemical sulfur and iron cycles. archaeal species from the Genome Taxonomy Database (GTDB)23. The
selected enzymes are key markers for specific, mostly dissimilatory
sulfur metabolisms (Supplementary Table 1). More than half of the
Sulfur-metabolizing bacteria and archaea species, representing 120 (80.5%) of 149 known bacterial and archaeal
We first established a computational framework to more accurately phyla, encoded at least one sulfur-cycling marker protein (Fig. 1). Most
predict dissimilatory sulfur metabolism from bacterial and archaeal genomes that encode a marker protein (for example, DsrA) also encode
genomes (Supplementary Fig. 1). To this end, we performed phyloge- additional proteins (for example, DsrB, DsrC, DsrE and DsrF) associated
netic analyses of 116 proteins that are involved in diverse sulfur redox with the respective marker protein for coordinated enzymatic function
transformations, and developed hidden Markov models (HMMs) (Supplementary Fig. 3). The occurrence of sulfur-cycling capability
for all monophyletic clades that corresponded to functional homo- in most bacterial and archaeal phyla reflects the deep integration of
logues of experimentally validated proteins (Supplementary Text). sulfur redox processes into microbial metabolism since the origin

2 | Nature | www.nature.com
 a Iron(III) b Iron(III) c Iron(III)
− oxides − oxides S2O32− oxides
HS HS

DsrAB OmcS
Sqr
AprAB OmcZ MtrCAB Sox MtrCAB
FccBA
Sat PCC
Fe(II) Fe(II) Fe(II)
SO42− S(0) SO42−
Burkholderiaceae
Sulfurifustaceae
Desulfurivibrionaceae Rhodoferax spp.
Thiohalomonadaceae
Ectothiorhodospiraceae

20 9HS− + 8Fe(OH)3 + 7H+ 20 3HS− + 2Fe(OH)3 + 3H+ 20 S2O32− + 8Fe(OH)3 + 14H+

SO42− + 8FeS + 20 H2O S(0) + 2FeS + 6H2O 2SO42− + 8Fe(II) + 19H2O
ΔGr (kJ mol–1 per electron)

ΔGr (kJ mol–1 per electron)

ΔGr (kJ mol–1 per electron)

0 0 0

–20 –20 –20

–40 –40 –40

4 6 8 10 4 6 8 10 4 6 8 10
pH pH pH

Fig. 2 | Three potential metabolic pathways for coupling sulfur oxidation assuming two different environmental settings. Marine ecosystem conditions
with reduction of iron(iii) oxides in a single bacterium. Top, genomic (red line): ionic strength (I) = 0.7 M, T = 25 °C, [total dissolved sulfide] = 100 μM,
reconstruction predicts that dissimilatory reduction of iron(iii) oxides is [SO42−] = 28 mM, [S2O32−] = 1 μM, [Fe2+] = 10 μM; freshwater ecosystem conditions
coupled with oxidation of sulfide to sulfate (reaction 1; a), oxidation of sulfide (black line): I = 0.001 M, T = 25 °C, [total dissolved sulfide] = 100 μM, [SO42−] =
to elemental sulfur (reaction 2; b) and oxidation of thiosulfate to sulfate 100 μM, [S2O32−] = 1 μM, [Fe2+] =10 μM. The red and black lines overlap in c. Sox,
(reaction 3; c). Key enzymes from Desulfobacterota and Gammaproteobacteria thiosulfate-oxidizing system; OmcS and OmcZ, outer membrane multi-haem
taxa are indicated as examples. Bottom, Gibbs free energy (∆Gr) of the iron- c-type cytochromes; PCC, porin–cytochrome complex.
dependent sulfur oxidation reactions across a range of pH was calculated,

of life2,5,6. In this context, 5,561 species with sulfur-cycling potential, monophyletic clade, including D. alkaliphilus, which operates DsrAB in
affiliated to 71 phyla, were represented exclusively by genomes from reverse during sulfide oxidation to sulfate27 (Supplementary Fig. 6). The
so far uncultured microorganisms (Fig. 1). This highlights the broad same genomes also contain multiple homologues of Geobacter-type
taxonomic distribution of sulfur-cycling potential and underscores cytochromes, which are necessary for dissimilatory iron(iii) reduc-
that many of the microorganisms that we identified here as capable tion (Supplementary Fig. 5), including extracellular OmcS and porin–
of sulfur-cycling remain uncultured and poorly characterized. Our cytochrome complex28–30 (OmaB–OmbB–OmcB). The interactions of
overview of putative sulfur microorganisms provides a foundation these enzymes would establish a conduit channeling sulfide-derived
for more detailed metabolic predictions and experimental validation. electrons to insoluble extracellular substrates such as iron(iii) oxides.
The second option involves a sulfide:quinone oxidoreductase (Sqr)
and FccBA for sulfide oxidation, and a multi-haem protein complex
Microbial S oxidation with iron(iii) oxide (MtrCAB) for extracellular respiration of iron(iii) oxides (Supplemen-
We next screened the genomes of the identified sulfur microorganisms tary Fig. 7). Homologues of Sqr, FccBA and MtrCAB were detected in
for potential electron transfer metabolisms that facilitate anaerobic two uncultured Rhodoferax species. The Sqr is affiliated to type I Sqr
sulfur oxidation by reducing iron(iii) oxides, a microbial physiology of that has a physiological role in sulfide-based energy transduction,
potentially broad biogeochemical importance in marine and terrestrial as described in Aquifex aeolicus31 and Rhodobacter capsulatus32. The
ecosystems24. In contrast to coupling of elemental, zero-valent sulfur MtrCAB homologue encoded in the Rhodoferax genome is closely
oxidation to dissolved iron(iii) reduction by acidophiles in highly acidic related to MtrCAB from the known iron(iii) reducer Rhodoferax ferrire-
environments25,26 (pH < 3), no microorganism has been shown to gain ducens33,34. These genetic systems could support an energy metabolism
energy from coupled reduction of solid phase extracellular iron(iii) that couples iron reduction with dissimilatory oxidation of sulfide to
oxides and oxidation of reduced sulfur compounds. We identified elemental sulfur (reaction 2). Additionally, MtrCAB was detected in
co-occurring genetic features for dissimilatory sulfur oxidation and multiple known thiosulfate oxidizers (Supplementary Fig. 5), includ-
extracellular iron(iii) reduction metabolisms across 37 bacterial and ing cultivated members within Burkholderiaceae, Sulfurifustaceae,
archaeal phyla (Supplementary Text and Supplementary Figs. 4 and Thiohalomonadaceae and Ectothiorhodospiraceae. This suggests a
5). Reconstruction of sulfur and iron energy conservation pathways third option of extracellular iron(iii)-dependent thiosulfate oxidation
revealed three metabolic options (Fig. 2). The first option couples (reaction 3). Beyond Desulfobacterota and Proteobacteria, members
iron(iii) reduction to the oxidation of sulfide to sulfate (reaction 1). The from 35 other microbial phyla also have the potential to catalyse reac-
underlying metabolic pathway was found in, for example, members of tions 1–3 via different gene combinations (Supplementary Text and
Desulfurivibrionaceae, whose genomes encode a full suite of enzymes Supplementary Figs. 4 and 5).
(sulfate adenylyltransferase (Sat), adenosine-5′-phosphosulfate Calculation of the Gibbs free energy showed that all three reactions
reductase (AprAB) and dissimilatory sulfite reductase (DsrAB)) for could provide energy to microorganisms, even in neutral to moder-
the canonical sulfate reduction pathway (Supplementary Fig. 5). The ately alkaline conditions (Fig. 2). For example, iron(iii)-dependent
DsrAB sequences of Desulfurivibrionaceae form a well-supported sulfide oxidation under natural settings (that is, freshwater and marine

Nature | www.nature.com | 3
Article
sediment) yields −20 to −40 kJ per mole electron, which is considered upon successive supply of 1 mM sulfide, whereas the sterile control
sufficient to support microbial growth35. Overall, these findings suggest followed the predicted trend (Fig. 3d,e). The faster rate indicates that
wider occurrence of sulfur oxidation-dependent energy metabolisms additional processes consumed the dissolved sulfide. We suggest dis-
across microbial taxa than previously recognized and provide test- similatory iron(iii) reduction fuelled by reduced sulfur species oxi-
able hypotheses regarding the physiology of uncultured and cultured dation40 as an additional process that rapidly precipitates dissolved
microorganisms. sulfide by producing excessive Fe(ii) (Fig. 3f). Depending on the sulfur
speciation in our incubation, the sulfur compounds supporting biologi-
cal iron(iii) reduction include dissolved sulfide, FeS and S(0). Their
Physiology and transcriptome oxidation, together with S(0) disproportionation by D. alkaliphilus
We aimed to experimentally validate the genome-predicted potential contributes to the observed sulfate formation.
to oxidize sulfide with iron(iii) oxide in D. alkaliphilus, which encodes To probe for direct oxidation of dissolved sulfide to sulfate by D. alka-
the genomic repertoire for reaction 1. We prioritized validatation of liphilus, we measured sulfate formation upon sulfide addition (1 mM)
this predicted sulfide metabolism given the pervasive co-occurrence to ferrihydrite at higher temporal resolution. We observed biphasic
of sulfide and iron(iii) oxides across a wide range of ecosystems7,18,36. sulfate formation dynamics within 24 h (Fig. 3g). Within the first 70 min,
D. alkaliphilus has a versatile sulfur metabolism. It grows by dispro- when dissolved sulfide was available (phase I), sulfate was formed at
portionation of elemental sulfur or by coupling sulfide oxidation with an average rate of 0.69 fmol cell−1 h−1. However, the rate decreased by
nitrate reduction27,37 but was not known to reduce iron(iii) oxides. approximately tenfold (0.061 fmol cell−1 h−1) after the depletion of dis-
Here we expand the known physiology of D. alkaliphilus by showing its solved sulfide (phase II). To measure the effect of poorly soluble sulfur
capability to reduce iron(iii) oxides (ferrihydrite) with formate, poorly species S(0) and FeS on sulfate accumulation rates, we first allowed
crystalline FeS or dissolved sulfide as electron donor. Initial incubation sulfide and ferrihydrite to react for 70 min, after which dissolved sulfide
of D. alkaliphilus with ferrihydrite and formate led to simultaneous has been completely transformed into FeS and S(0), and only then
formate consumption and Fe(ii) production, with the following stoichi- inoculated cells. Here we also observed a low sulfate formation rate
ometry: HCOO− + 2Fe(iii) → CO2 + 2Fe(ii) + H+. This verified its capacity (0.047 fmol cell−1 h−1) comparable to the one in the sulfide-free phase II
to reduce extracellular solid iron(iii) oxides (Fig. 3a). (Fig. 3g). These results demonstrated that the presence of poorly solu-
We then fed D. alkaliphilus FeS as electron donor and ferrihydrite ble sulfur species alone (such as S(0) and FeS) was insufficient to sustain
as electron acceptor (Fig. 3b). Over a five-day incubation period, we the sulfate formation rate observed at phase I. We thus attribute the
observed the concurrent production of sulfate and Fe(ii). By contrast, sulfide-dependent, high sulfate formation rate of D. alkaliphilus in
Fe(ii) production was not detected in autoclaved controls, indicat- phase I to direct biological sulfide oxidation with ferrihydrite.
ing that FeS was not chemically oxidized by ferrihydrite to form more To further show that D. alkaliphilus directly oxidizes dissolved sulfide
oxidized sulfur species38, such as S(0). This observation excludes the with iron(iii) oxide, we tracked sulfide kinetics in the culture incubated
possibility of sulfate formation by microbial disproportionation of with a low concentration of dissolved sulfide (approximately 50 µM)
S(0). Sulfate formation by FeS oxidation with residual nitrate from the and ferrihydrite. We hypothesized that the low sulfide concentration
inoculum was also excluded, as addition of FeS alone (that is, without reduces its chemical reaction rate with ferrihydrite, enabling a clear
ferrihydrite addition) did not result in sulfate accumulation. The slight readout of the biological sulfide transformation process. Indeed,
increase of sulfate in ferrihydrite-only controls is probably owing to sulfide was readily consumed by the ferrihydrite-amended culture
residual sulfide from the inoculum. After accounting for sulfur transfor- down to 10 µM within 25 min, whereas 20–30 µM of sulfide remained
mations observed in control incubations, the calculated ratio of sulfate in controls lacking cells. Repetitive addition of sulfide showed the
to Fe(ii) formation was close to the predicted stoichiometry of 1:8 for same recurring pattern. The culture rapidly consumed sulfide and its
reaction 1 (Fig. 3b), indicating that D. alkaliphilus oxidizes FeS-sulfide to estimated rate constant of removal was significantly higher than of
sulfate by reducing ferrihydrite. The slight deviation from the expected abiotic controls (P < 0.01, Student’s t-test; Extended Data Fig. 1). This
stoichiometry is likely to result from reverse electron flow for carbon pattern was observed reproducibly in independent incubations with
fixation (Supplementary Text and Supplementary Figs. 8 and 9). different cell densities (Extended Data Fig. 1). By ruling out alternative
Finally, to examine whether D. alkaliphilus oxidizes free sulfide with biogeochemical processes that support accelerated sulfide consump-
iron(iii) as electron acceptor, we incubated the culture with ferrihy- tion (Supplementary Text), we concluded that D. alkaliphilus partici-
drite and supplied 1 mM dissolved sulfide daily (Fig. 3c). This led to pates in sulfide transformation using ferrihydrite. Repeated supply
progressive accumulation of sulfate, S(0), and Fe(ii). In sterile controls, (n = 8) of sulfide showed the culture transformed more than 90% of
S(0) and Fe(ii) accumulated, but not sulfate. Similarly, sulfate was not spiked sulfide to sulfate with negligible S(0) formation, while produc-
formed in biotic controls lacking either ferrihydrite or sulfide (Fig. 3c, ing an excessive amount of Fe(ii) (Extended Data Fig. 2). In the abiotic
Supplementary Fig. 10 and Supplementary Text). These results showed control, however, S(0)—but not sulfate—was the main oxidized sulfur
that sulfate was formed in a biological process requiring ferrihydrite. compound and Fe(ii) accumulated to much lower levels (Extended Data
We reasoned that biological oxidation of dissolved sulfide with ferrihy- Fig. 2). These results indicate that respiration of ferrihydrite with sulfide
drite contributed to sulfate formation, as evidenced by: (1) exclusion of by D. alkaliphilus outpaces the chemical reaction rate between sulfide
sulfur disproportionation as the only source of sulfate; (2) a biphasic and ferrihydrite at environmentally relevant low sulfide concentrations.
production of sulfate following the supply of dissolved sulfide; and To show that coupled sulfide and iron(iii) oxides metabolism sup-
(3) accelerated sulfide consumption with cells compared to abiotic ports growth, we monitored the cell density of D. alkaliphilus during
controls. We consider these lines of physiological evidence separately 4- to 13-day incubation with ferrihydrite and sulfide. We observed two-
in the following three paragraphs. fold to threefold increases in cell numbers for ferrihydrite-incubated
A plausible source of sulfate in our incubation is microbial dispro- cultures periodically amended with either 1 mM dissolved sulfide,
portionation of S(0), derived from the chemical oxidation of sulfide approximately 50 µM dissolved sulfide or FeS (Fig. 4a–c). In compari-
by ferrihydrite12,39. Under this canonical biogeochemical scenario, con- son, incubation of D. alkaliphilus with dissolved sulfide and nitrate
sumption of dissolved sulfide is controlled by the availability of reac- led to a 5–6 fold increase of cell number over 3 days (Fig. 4d and Sup-
tive sites on the surface of ferrihydrite7, and the rate should decrease plementary Text). The calculated specific growth rate was higher in
over time with periodic supply of sulfide due to surface saturation and ferrihydrite cultures with dissolved sulfide (0.288 ± 0.015 day−1 at 1 mM
production of sulfide by S(0) disproportionation. However, tracing the and 0.436 ± 0.074 day−1 at 50 µM) compared to those with solid phase
kinetics of dissolved sulfide removal revealed accelerated consumption FeS (0.089 ± 0.005 day−1) (Supplementary Fig. 11), probably owing to

4 | Nature | www.nature.com
 a b 2.0

HCl-extractable Fe(II) (mM)

Treatment
8:1

Fe(II) formation (mM)

Concentration (mM)

Cells + Fh + formate 2.0

3 1.5
20

Sulfate (mM)
Cells + Fh
Treatment
Cells + formate 1.8 2 1.0 Cells + Fh + FeS
10 Killed cells + Fh + FeS
Metabolite Cells + Fh
Fe(II) 1.6 1 0.5
Cells + FeS
0 Formate
0
0 2 5 8 10 12 0 2 4 6 0 2 4 6 0 0.1 0.2 0.3 0.4
Time (day) Time (day) Time (day) Sulfate formation (mM)
c

Cline-extractable sulfide (mM)

3.0 HCl-extractable Fe(II) (mM) 1.00 6

10 0.75
Sulfate (mM)

S(0) (mM)
2.5
0.50
2.0 5
2
0.25
1.5 0
0 0 Treatment
−
0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 Cells + Fh + HS
Time (day) Time (day) Time (day) Time (day) Killed cells + Fh + HS−
Cells + Fh
d Day 0 Day 1 Day 2 Day 3 3.00 Cells + HS
−

Rate constant (min−1)

Dissolved sulfide (mM)

1.00
0.9
0.30
0.6
0.10
0.3
0.03
0
0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 1 2 3
Time (min) Time (day)

e f g
I II
S(0) disproportionation −
Biological oxidation of HS /FeS 2.4
Sulfate (mM)
Chemical HS− oxidation Treatment
2.2
Cells + Fh + HS−
Fe(III) oxides
Cells + Fh + S(0)/FeS
2.0
Fe(II)
1.8

FeS HS
− 70 400 800 1,200
FeS HS− Time (min)
h 60

Fe(II)
Sulfide (μM)

2− Fe(II) 40
S(0) SO4 Treatment
S(0) SO42− −
Cells + Fh + HS
Fe(III) oxides 20 Killed cells + Fh + HS−
Fe(III) oxides

0
0 5 10 15 20 25
Time (min)

Fig. 3 | D. alkaliphilus is capable of reducing ferrihydrite and oxidizing biogeochemical scenario of sulfate formation considering chemical sulfide
sulfide or FeS. a, D. alkaliphilus oxidizes formate (triangles) while reducing oxidation to elemental sulfur (S(0)), followed by bacterial disproportionation.
ferrihydrite (Fh) to Fe(ii) (circles). No Fe(ii) or formate consumption occurred f, Updated scenario considering the contribution of sulfate formation from
in the absence of Fh or formate. b, Left and middle, D. alkaliphilus couples microbial oxidation of sulfide or FeS with iron oxides. g, Biphasic sulfate
oxidation of poorly crystalline FeS to sulfate with reduction of Fh. Sulfate and formation by D. alkaliphilus during 24 h incubation with ferrihydrite and sulfide.
Fe(ii) formation were not detected in abiotic controls or in cultures with FeS Phases I and II differ by sulfide availability. Phase I is shown with a larger scale on
or Fh alone. Right, the ratio of sulfate to Fe(ii) formation (solid line) is close to the x axis for visualization. A control for phase II was conducted by inoculating
predicted 1:8 stoichiometry (dashed line). c, Sulfate, HCl-extractable Fe(ii), cells after the chemical reaction between sulfide and ferrihydrite (cells + Fh +
elemental sulfur and Cline-extractable sulfide during the incubation of S(0)/FeS). h, Faster consumption of a low concentration (approximately 50 µM)
D. alkaliphilus cultures with Fh and daily spike of sulfide (1 mM). Parallel of sulfide by D. alkaliphilus compared with chemical controls lacking cells.
incubations with killed cells, or living cells without Fh or sulfide served as Three spikes of sulfide were supplied at 1.5 h intervals. Results with different
controls. Arrows indicate sulfide spike. d, Kinetics of dissolved sulfide and cell density are shown in Extended Data Fig. 1. In a–d,g,h, triplicate cultures
first-order rate constant in the incubations described in c. e, The canonical (n = 3) were used for each incubation condition.

the limited solubility of FeS. By contrast, no or only minor increases with ferrihydrite to the low energy field and higher maintenance
in cell density were detected in the dissolved sulfide-only, FeS-only energy requirements at alkaline pH (Supplementary Text). To fur-
and ferrihydrite-only controls (Fig. 4a,b). These results demonstrate ther explore the association between growth and carbon fixation,
that D. alkaliphilus is capable of utilizing sulfide or FeS together with we supplemented parallel cultures with 13C-bicarbonate (10 atom%),
ferrihydrite for growth. We attribute the overall restricted growth ferrihydrite, and sulfide or FeS. Carbon isotope composition analysis

Nature | www.nature.com | 5
Article
a c 11 50 μM HS– e
HS– 3.5 Cells + HS– + Fh + 13C–HCO3–

10 Killed cells + HS– + Fh + 13C–HCO3–

Bulk biomass 13C atom%

No. of cells (×107 ml–1)

No. of cells (×107 ml–1)

3.0
9
Cells + Fh + HS– Cells + Fh + 50 μM HS–
8 P = 0.04
Cells + Fh 2.5
*
Cells + HS– 7
6 2.0

5 1.5
4

2 3 1.0
0 1 2 3 4 0 1 2 3 4 0 2 4 6
Time (day) Time (day) Time (day)

b FeS d HS– HS– f 1.6 Cells + FeS + Fh + 13C–HCO3–

8 30 Killed cells + FeS + Fh + 13C–HCO3–

Bulk biomass 13C atom%

Dissolved sulfide (mM)
No. of cells (×107 ml–1) Cells + HS– + NO3–
No. of cells (×107 ml–1)

1.5
Cells + Fh + FeS Sulfide P = 0.04
1.4 *
Cells + Fh 20
6 1.0
Cells + FeS

1.2
10 0.5
4

0 0 1.0
0 5 10 0 1 2 0 2 4 6
Time (day) Time (day) Time (day)

g h i
7.0
1.11

Single cell 13C atom%

5.0
1.08
Day 0

3.0
1.05

1.0 (n = 81)
1.02

7.0

6
Single cell 13C atom%

5.0

Day 5 4

3.0

1.0
12 14
C N – 13
C atom% 0 (n = 80)

Fig. 4 | D. alkaliphilus grows autotrophically with ferrihydrite and sulfide (1 mM). f, Bulk 13C abundance increased (P = 0.003; ANOVA) over 6 days in living
or FeS. a, Mean cell density (n = 3) increased significantly (P = 2.18 × 10−8; ANOVA) cultures (n = 3) incubated with 10% 13C-bicarbonate, ferrihydrite, and two spikes
over 4 days of incubation with ferrihydrite and daily addition of sulfide (1 mM). of FeS (approximately 1 mM). e,f, Asterisks denote significant differences
b, Mean cell density (n = 3) increased significantly (P = 3.79 × 10 −10; ANOVA) over (P < 0.05; two-sided Student’s t-test) on day 6. Dashed lines indicate natural 13C
13 days of incubation with ferrihydrite and periodic spikes of FeS (approximately levels. a–f, Arrows indicate sulfide or FeS addition. g,h, Nano-scale secondary
1 mM). c, Mean cell density (n = 3) increased significantly (P = 1.84 × 10 −8; ion mass spectrometry (NanoSIMS) images showing cellular biomass (g; 12C14N–)
ANOVA) over 5 days of incubation with ferrihydrite and periodic addition of and 13C content (h) of cells at day 0 and 5. Colour scale refers to 13C atom%. Scale
approximately 50 µM sulfide. d, Mean cell density (n = 3) increases significantly bars correspond to 5 μm. i, Dot plots displaying the 13C content of individual
(P = 5.53 × 10−11; ANOVA) alongside sulfide consumption over 3 days of incubation cells analysed in a representative NanoSIMS field view at day 0 (n = 81 cells) and
with sulfide and nitrate (4 mM). e, Bulk 13C abundance (atom%) increased 5 (n = 80 cells). Box plots show the median, the 25th and 75th percentiles of 13C
(P = 2 × 10 −4; ANOVA) over 6 days in living cultures (n = 3; orange circles) content of cells; whiskers extend to 1.5 times the interquartile range from the
incubated with 10% 13C-bicarbonate, ferrihydrite, and daily addition of sulfide first and third quartiles.

6 | Nature | www.nature.com
 RPMK Percentile
768–10,000 90–99 Haem c
Tad pili Type IV pili
320–768 75–90 b Haem b
138–320 50–75 Y1
Electron flow OmcA-like OmcS-like
Flp 58–138 25–50 Proton flow MHC Fe(II) MHC
Upregulation in FeSoxFeR/SoxFeR/FoxFeR vs SoxNR/Sdisp PilA
Upregulation in FeSoxFeR/SoxFeR/SoxNR vs FoxFeR e
– 10x Fe(III) 6x

Outer
CpaC Porin PilQ membrane
CpaB CpaB 8x W W
TadD TadD e
– P P

Periplasmic
6x 7x 12x
MHCs
– O O
e
HS– S(0) HSn–
H+ H+ N N
TadG TadG 3x
Qred Qred b Qred
Cytoplasmic
TadB TadC Sqr YeeE DsrMKJOP CbcL QmoABC Atp PilC membrane
Qox Qox Qox PilD
PilM
e-
CpaF CpaE Rhd B T
ADP ATP
TusA

–
HS DsrAB AprAB Sat
DsrC DCT SO32– APS SO42–
–
e
PPi ATP Cytoplasm

Fig. 5 | Schematic metabolic model of D. alkaliphilus for sulfide oxidation reduction, respectively. The identifier, full name, RPKM, statistical significance
with iron(iii) oxides based on comparative transcriptomics. D. alkaliphilus of differential transcription and genomic arrangement for each gene are
uses a reversed canonical dissimilatory sulfate reduction (Dsr) pathway to provided in Extended Data Fig. 3 and Supplementary Tables 2–5. Electron flow
oxidize sulfide to sulfate (blue protein labels). Sulfide reacts with DsrC to form and proton translocation are indicated by red arrows. Black dotted arrows
DsrC trisulfide (DCT), which is oxidized stepwise to sulfate via DsrAB, AprAB indicate putative sulfur redox reactions with unclear enzymology. APS,
and Sat. The reducing equivalents are transferred to iron(iii) oxides via EET adenosine-5′-phosphosulfate, Qox, oxidized menaquinone; Qred, reduced
mechanisms (green protein labels) facilitated by MHCs. D. alkaliphilus also has menaquinone; FoxFeR, incubation of D. alkaliphilus under formate-oxidizing
genes encoding type IV pili (grey protein labels). The tight adhesion (Tad) pilus and ferrihydrite-reducing conditions; FeSoxFeR, incubation of D. alkaliphilus
may drive the cell adherence to the surface of solid iron oxides and/or FeS. The under FeS-oxidizing and ferrihydrite-reducing conditions; Sdisp, incubation of
transcription level of each gene during MISO is indicated by mean reads per D. alkaliphilus under S(0) disproportionation conditions; SoxFeR, incubation
kilobase of transcript per million mapped reads (RPKM) of four replicates of D. alkaliphilus under sulfide-oxidizing and ferrihydrite-reducing conditions;
corresponding to the colour in the legend. Blue and green dots show significantly SoxNR, incubation of D. alkaliphilus under sulfide-oxidizing and nitrate-
increased gene transcription during sulfide/FeS oxidation and ferrihydrite reducing conditions.

of bulk biomass and single cells revealed enrichment of 13C in living After the transfer of sulfide-derived electrons to the membrane
cells after five-day incubation (Fig. 4e–i), reflecting a chemoauto- quinone pool via the proposed sulfide oxidation pathways, their flow
trophic lifestyle of D. alkaliphilus when growing on ferrihydrite and to extracellular ferrihydrite is central to the activities and energy con-
dissolved sulfide or FeS. servation mode of MISO. We propose that this extracellular electron
After providing physiological evidence for microbial reduction of transfer (EET) is facilitated by a network of multi-haem cytochromes
extracellular iron(iii) being coupled to sulfide oxidation (MISO) in (MHCs) at the inner membrane, periplasmic space and outer mem-
D. alkaliphilus, we revealed the differential activity of candidate genes brane, similar to mechanisms shown for the model iron(iii)-respiring
of this metabolism by comparative transcriptomics under various bacteria Geobacter sulfurreducens and Shewanella oneidensis42–44. Dur-
ferrihydrite-amended and ferrihydrite-free growth conditions. Sulfide ing MISO growth, D. alkaliphilus showed upregulated transcription of
oxidation is most probably achieved by the reversal of the canonical 13 out of 46 predicted MHC genes compared with nitrate-reducing and
dissimilatory sulfate reduction pathway (Fig. 5 and Extended Data S(0)-disproportionation growth conditions (Extended Data Fig. 4).
Fig. 3), as proposed for D. alkaliphilus growing under nitrate-reducing The most prominent MHC, upregulated by more than 100-fold (Sup-
conditions27 and its close relatives, Electrothrix cable bacteria, which plementary Fig. 12), was predicted to be extracellular, and to share
grow by oxygen-dependent sulfide oxidation41. All genes required for structural homology and haem arrangement with OmcS from Geo-
this pathway had substantial transcription in D. alkaliphilus during bacter (Extended Data Fig. 5). This OmcS homologue may facilitate
MISO, and many showed significant upregulation (adjusted P value EET to iron oxides (Fig. 5, Supplementary Text and Supplementary
(Padj) < 0.05) compared with cultures grown with formate as electron Fig. 13), as observed in G. sulfurreducens28,45. Another differentially
donor. Additionally, D. alkaliphilus may oxidize sulfide in a two-step transcribed MHC, also predicted to be extracellular, is homologous to
process in which sulfide is initially converted into zero-valent sulfur in OmcA, which is involved in anaerobic iron reduction in Shewanella46.
the periplasm, which is then either disproportionated or transported The remaining MHCs are predicted to be located in the periplasm or
into the cytoplasm to enter the reverse Dsr pathway27. Multiple genes associated with the cytoplasmic membrane. One of the latter includes
proposed for this pathway were transcribed and/or upregulated during a homologue of a cytochrome bc complex CbcL that is essential for
MISO growth, including those encoding Sqr, the sulfur-transferring the growth of G. sulfurreducens on iron(iii) oxides47. As proposed for
membrane protein YeeE, the rhodanese Rhd and the sulfur transferase Geobacter, the periplasmic MHCs may facilitate EET by channelling
TusA. electrons to the extracellular MHCs30; the CbcL homologue may pass

Nature | www.nature.com | 7
Article
electrons from the quinone pool to periplasmic MHCs, while generat- biogeochemical model could help explain the observed widespread
ing a proton motive force via a scalar mechanism47. The involvement geochemical pattern of sulfide removal and sulfate formation in anoxic,
of these MHCs in the reduction of iron(iii) oxides was further sup- iron-rich marine, freshwater and terrestrial environments14,20,22,58–60
ported by their increased transcription in D. alkaliphilus grown with (Supplementary Text). A notable example of such phenomena is the
formate and ferrihydrite (Extended Data Fig. 4). D. alkaliphilus also occurrence of high oxidation rates of sulfide, produced from dissimila-
upregulated transcription of genes for tight adherence (Tad) type IV tory sulfate reduction, back to sulfate in anoxic layers of marine sedi-
pilus assembly proteins during iron(iii) reduction. Transcription of ments, where typical oxidants for sulfide oxidation, such as oxygen
pilA, encoding the main component of another type IV pilus, was high and nitrate, have been depleted, leaving iron(iii) oxides as the primary
across all growth conditions but not increased during iron(iii) reduc- oxidants20. On the basis of a first estimate, MISO could account for 1–7%
tion. These proteins were suggested to have a role in EET by forming of total sulfide oxidation to sulfate in marine sediments on a global
electrically conductive pili48 or facilitating the surface attachment and/ scale, owing to the large flux of reactive iron from terrestrial runoff to
or the secretion of extracellular MHCs49–52. Under MISO growth condi- the sea (Supplementary Text). The MISO metabolism may therefore
tions, genes involved in Wood Ljungdahl pathways ranked among the be globally important in modulating the oxidation and reduction of
top 30% highly transcribed genes (Supplementary Fig. 14), suggesting sulfur, with broader implications for Earth’s biogeochemical cycles
a role in autotrophic carbon fixation. and climate.

Ecological and biogeochemical impacts Online content

Experimental validation of MISO in D. alkaliphilus expands the known Any methods, additional references, Nature Portfolio reporting summa-
physiologies of bacteria, and broadens the diversity of microbial sulfide ries, source data, extended data, supplementary information, acknowl-
oxidation pathways beyond what was proved possible in the absence edgements, peer review information; details of author contributions
of oxygen. Unlike oxidation of S(0) with dissolved iron(iii) by acido- and competing interests; and statements of data and code availability
philes26, MISO relies on solid phase iron(iii) oxides, which represent are available at https://doi.org/10.1038/s41586-025-09467-0.
the most prevalent form of iron in natural habitats with mildly acidic
to mildly alkaline pH. Our data suggested that MISO utilizes an elabo-
1. Canfield, D. E. & Farquhar, J. in Fundamentals of Geobiology 49–64 (John Wiley & Sons,
rate multi-haem apparatus to conduct EET to iron(iii) oxides, similar 2012).
to iron-reducing model organisms. The proposed sulfide-fuelled EET 2. Fike, D. A., Bradley, A. S. & Rose, C. V. Rethinking the ancient sulfur cycle. Annu. Rev. Earth
Planet. Sci. 43, 593–622 (2015).
model provides a mechanistic basis for several underexplored sulfide
3. Wasmund, K., Mußmann, M. & Loy, A. The life sulfuric: microbial ecology of sulfur cycling
oxidation observations, including those driven by manganese oxides15, in marine sediments. Environ. Microbiol. Rep. 9, 323–344 (2017).
coupled with electricity generation in microbial fuel cells53–55, and 4. Falkowski, P. G., Fenchel, T. & Delong, E. F. The microbial engines that drive Earth’s
biogeochemical cycles. Science 320, 1034–1039 (2008).
supporting direct interspecies electron transfer to methanogens56,57.
5. Mateos, K. et al. The evolution and spread of sulfur cycling enzymes reflect the redox
The genomic potential to perform MISO appears as a conserved trait state of the early Earth. Sci. Adv. 9, eade4847 (2023).
within the Desulfurivibrionaceae family (Extended Data Fig. 6). Des- 6. Canfield, D. E. & Raiswell, R. The evolution of the sulfur cycle. Am. J. Sci. 299, 697–723
(1999).
ulfurivibrionaceae members with the MISO gene set occupy a wide
7. Poulton, S. W. Sulfide oxidation and iron dissolution kinetics during the reaction of
range of habitats in marine (for example, sediments and hydrothermal dissolved sulfide with ferrihydrite. Chem. Geol. 202, 79–94 (2003).
vents), freshwater (for example, aquifers and lake sediments), terres- 8. Canfield, D. E. Reactive iron in marine sediments. Geochim. Cosmochim. Acta 53, 619–632
(1989).
trial (for example, wetlands and soils) and engineered (for example,
9. Anantharaman, K. et al. Expanded diversity of microbial groups that shape the dissimilatory
microbial fuel cells) environments. These observations suggest that sulfur cycle. ISME J. 12, 1715–1728 (2018).
MISO-performing Desulfurivibrionaceae members are biogeochemi- 10. Müller, A. L., Kjeldsen, K. U., Rattei, T., Pester, M. & Loy, A. Phylogenetic and environmental
diversity of DsrAB-type dissimilatory (bi)sulfite reductases. ISME J. 9, 1152–1165 (2015).
cally relevant in diverse ecological contexts. Given the presence of 11. Diao, M. et al. Global diversity and inferred ecophysiology of microorganisms with the
genomic potential for MISO in microbial lineages beyond Desulfurivi- potential for dissimilatory sulfate/sulfite reduction. FEMS Microbiol. Rev. 47, fuad058
brionaceae (Fig. 2, Supplementary Fig. 4 and Supplementary Text), we (2023).
12. Jørgensen, B. B., Findlay, A. J. & Pellerin, A. The biogeochemical sulfur cycle of marine
envisage an even broader environmental distribution of MISO capacity. sediments. Front. Microbiol. 10, 849 (2019).
This study adds a new dimension to the understanding of the inter- 13. Canfield, D. E. et al. A cryptic sulfur cycle in oxygen-minimum-zone waters off the Chilean
play of sulfur and iron biogeochemistry in anoxic environments. coast. Science 330, 1375–1378 (2010).
14. Pester, M., Knorr, K.-H., Friedrich, M. W., Wagner, M. & Loy, A. Sulfate-reducing
Although re-oxidation of sulfide with iron(iii) oxides has previously microorganisms in wetlands - fameless actors in carbon cycling and climate change.
been regarded to be a strictly chemical reaction, we demonstrate here Front. Microbiol. 3, 72 (2012).
that MISO can not only outperform chemical oxidation of sulfide at 15. Henkel, J. V. et al. A bacterial isolate from the Black Sea oxidizes sulfide with manganese(IV)
oxide. Proc. Natl Acad. Sci. USA 116, 12153–12155 (2019).
environmentally relevant concentrations, but also acts on solid phase 16. Hayes, J. M. & Waldbauer, J. R. The carbon cycle and associated redox processes through
FeS-sulfide, which known to be chemically inert towards iron(iii) time. Philos. Trans. R. Soc. B 361, 931–950 (2006).
oxides38. In this context, FeS, which is widespread in anoxic environ- 17. Jørgensen, B. B. & Nelson, D. C. in Sulfur Biogeochemistry—Past and Present 63–81
(Geological Society of America, 2004).
ments, offers a distinct ecological niche for MISO bacteria, largely 18. Hansel, C. M. et al. Dominance of sulfur-fueled iron oxide reduction in low-sulfate
devoid of competition from chemical oxidation. Thermodynamic mod- freshwater sediments. ISME J. 9, 2400–2412 (2015).
elling indicates that MISO is exergonic over a range of ecologically 19. Flynn, T. M., O’Loughlin, E. J., Mishra, B., DiChristina, T. J. & Kemner, K. M. Sulfur-mediated
electron shuttling during bacterial iron reduction. Science 344, 1039–1042 (2014).
relevant pH values and substrate conditions. This contrasts with canoni- 20. Findlay, A. J., Pellerin, A., Laufer, K. & Jørgensen, B. B. Quantification of sulphide oxidation
cal reduction processes of iron(iii) oxides, which become thermody- rates in marine sediment. Geochim. Cosmochim. Acta 280, 441–452 (2020).
namically unfavourable at alkaline pH19. MISO is likely to overcome such 21. Michaud, A. B. et al. Glacial influence on the iron and sulfur cycles in Arctic fjord sediments
(Svalbard). Geochim. Cosmochim. Acta 280, 423–440 (2020).
energy limitation by exploiting highly reactive sulfide that maintains 22. Elsgaard, L. & Jørgensen, B. B. Anoxie transformations of radiolabeled hydrogen sulfide in
its reducing power over a wide pH range. The free energy yielded from marine and freshwater sediments. Geochim. Cosmochim. Acta 56, 2425–2435 (1992).
MISO is sufficient to support microbial growth and power sulfate for- 23. Parks, D. H. et al. GTDB: an ongoing census of bacterial and archaeal diversity through a
phylogenetically consistent, rank normalized and complete genome-based taxonomy.
mation at a rate resembling that of sulfate reduction in environments Nucleic Acids Res. 50, D785–D794 (2022).
such as marine sediments (Supplementary Text). These unique fea- 24. Raiswell, R. & Canfield, D. E. The iron biogeochemical cycle past and present. Geochem.
tures suggest that MISO bacteria may directly facilitate environmental Perspect. 1, 1–220 (2012).
25. Osorio, H. et al. Anaerobic sulfur metabolism coupled to dissimilatory iron reduction in
sulfate formation from sulfide or FeS with iron(iii) oxides, bypassing the extremophile Acidithiobacillus ferrooxidans. Appl. Environ. Microbiol. 79, 2172–2181
the need for S(0) disproportionation. Including MISO in the existing (2013).

8 | Nature | www.nature.com
26. Malik, L. & Hedrich, S. Ferric iron reduction in extreme acidophiles. Front. Microbiol. 12, 46. Johs, A., Shi, L., Droubay, T., Ankner, J. F. & Liang, L. Characterization of the decaheme
818414 (2021). c-type cytochrome OmcA in solution and on hematite surfaces by small angle x-ray
27. Thorup, C., Schramm, A., Findlay, A. J., Finster, K. W. & Schreiber, L. Disguised as a sulfate scattering and neutron reflectometry. Biophys. J. 98, 3035–3043 (2010).
reducer: Growth of the Deltaproteobacterium Desulfurivibrio alkaliphilus by sulfide 47. Antunes, J. M. A., Silva, M. A., Salgueiro, C. A. & Morgado, L. Electron flow from the
oxidation with nitrate. mBio 8, e00671-17 (2017). inner membrane towards the cell exterior in Geobacter sulfurreducens: Biochemical
28. Wang, F. et al. Structure of microbial nanowires reveals stacked hemes that transport characterization of cytochrome CbcL. Front. Microbiol. 13, 898015 (2022).
electrons over micrometers. Cell 177, 361–369.e10 (2019). 48. Walker, D. J. et al. Electrically conductive pili from pilin genes of phylogenetically diverse
29. Shi, L., Fredrickson, J. K. & Zachara, J. M. Genomic analyses of bacterial porin-cytochrome microorganisms. ISME J. 12, 48–58 (2018).
gene clusters. Front. Microbiol. 5, 657 (2014). 49. Wang, F., Craig, L., Liu, X., Rensing, C. & Egelman, E. H. Microbial nanowires: type IV pili or
30. Portela, P. C. et al. Widespread extracellular electron transfer pathways for charging cytochrome filaments? Trends Microbiol. 31, 384–392 (2023).
microbial cytochrome OmcS nanowires via periplasmic cytochromes PpcABCDE. 50. Wang, Y. et al. Structural mechanisms of Tad pilus assembly and its interaction with an
Nat. Commun. 15, 2434 (2024). RNA virus. Sci. Adv. 10, eadl4450 (2024).
31. Nübel, T., Klughammer, C., Huber, R., Hauska, G. & Schütz, M. Sulfide:quinone 51. Gu, Y. et al. Structure of Geobacter pili reveals secretory rather than nanowire behaviour.
oxidoreductase in membranes of the hyperthermophilic bacterium Aquifex aeolicus Nature 597, 430–434 (2021).
(VF5). Arch. Microbiol. 173, 233–244 (2000). 52. Sonani, R. R. et al. Tad and toxin-coregulated pilus structures reveal unexpected diversity
32. Schütz, M., Maldener, I., Griesbeck, C. & Hauska, G. Sulfide-quinone reductase from in bacterial type IV pili. Proc. Natl Acad. Sci. USA 120, e2316668120 (2023).
Rhodobacter capsulatus: requirement for growth, periplasmic localization, and extension 53. Sun, M. et al. Microbial communities involved in electricity generation from sulfide
of gene sequence analysis. J. Bacteriol. 181, 6516–6523 (1999). oxidation in a microbial fuel cell. Biosens. Bioelectron. 26, 470–476 (2010).
33. Finneran, K. T., Johnsen, C. V. & Lovley, D. R. Rhodoferax ferrireducens sp. nov., a 54. de Rink, R. et al. Continuous electron shuttling by sulfide oxidizing bacteria as a novel
psychrotolerant, facultatively anaerobic bacterium that oxidizes acetate with the strategy to produce electric current. J. Hazard. Mater. 424, 127358 (2022).
reduction of Fe(iii). Int. J. Syst. Evol. Microbiol. 53, 669–673 (2003). 55. Ter Heijne, A., de Rink, R., Liu, D., Klok, J. B. M. & Buisman, C. J. N. Bacteria as an electron
34. Baker, I. R., Conley, B. E., Gralnick, J. A. & Girguis, P. R. Evidence for horizontal and vertical shuttle for sulfide oxidation. Environ. Sci. Technol. Lett. 5, 495–499 (2018).
transmission of Mtr-mediated extracellular electron transfer among the bacteria. mBio 56. Jung, H., Baek, G. & Lee, C. Magnetite-assisted in situ microbial oxidation of H2S to S0
13, e02904–e02921 (2021). during anaerobic digestion: A new potential for sulfide control. Chem. Eng. J. 397, 124982
35. Nixon, S. L., Bonsall, E. & Cockell, C. S. Limitations of microbial iron reduction under (2020).
extreme conditions. FEMS Microbiol. Rev. 46, fuac033 (2022). 57. Jung, H., Yu, H. & Lee, C. Direct interspecies electron transfer enables anaerobic oxidation
36. Jakobsen, R. & Postma, D. Redox zoning, rates of sulfate reduction and interactions with of sulfide to elemental sulfur coupled with CO2-reducing methanogenesis. iScience 26,
Fe-reduction and methanogenesis in a shallow sandy aquifer, Rømø, Denmark. Geochim. 107504 (2023).
Cosmochim. Acta 63, 137–151 (1999). 58. Holmkvist, L., Ferdelman, T. G. & Jørgensen, B. B. A cryptic sulfur cycle driven by iron in
37. Sorokin, D. Y., Tourova, T. P., Mussmann, M. & Muyzer, G. Dethiobacter alkaliphilus the methane zone of marine sediment (Aarhus Bay, Denmark). Geochim. Cosmochim.
gen. nov. sp. nov., and Desulfurivibrio alkaliphilus gen. nov. sp. nov.: two novel Acta 75, 3581–3599 (2011).
representatives of reductive sulfur cycle from soda lakes. Extremophiles 12, 431–439 59. Mikucki, J. A. et al. A contemporary microbially maintained subglacial ferrous ‘ocean’.
(2008). Science 324, 397–400 (2009).
38. Schippers, A. & Jørgensen, B. B. Oxidation of pyrite and iron sulfide by manganese 60. Och, L. M. et al. New insights into the formation and burial of Fe/Mn accumulations in
dioxide in marine sediments. Geochim. Cosmochim. Acta 65, 915–922 (2001). Lake Baikal sediments. Chem. Geol. 330-331, 244–259 (2012).
39. Finster, K. Microbiological disproportionation of inorganic sulfur compounds. J. Sulfur
Chem. 29, 281–292 (2008). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
40. Sun, J., Wei, L., Yin, R., Jiang, F. & Shang, C. Microbial iron reduction enhances in-situ published maps and institutional affiliations.
control of biogenic hydrogen sulfide by FeOOH granules in sediments of polluted urban
waters. Water Res. 171, 115453 (2020). Open Access This article is licensed under a Creative Commons Attribution
41. Kjeldsen, K. U. et al. On the evolution and physiology of cable bacteria. Proc. Natl Acad. 4.0 International License, which permits use, sharing, adaptation, distribution
Sci. USA 116, 19116–19125 (2019). and reproduction in any medium or format, as long as you give appropriate
42. Santos, T. C., Silva, M. A., Morgado, L., Dantas, J. M. & Salgueiro, C. A. Diving into the credit to the original author(s) and the source, provide a link to the Creative Commons licence,
redox properties of Geobacter sulfurreducens cytochromes: a model for extracellular and indicate if changes were made. The images or other third party material in this article are
electron transfer. Dalton Trans. 44, 9335–9344 (2015). included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
43. Kappler, A. et al. An evolving view on biogeochemical cycling of iron. Nat. Rev. Microbiol. to the material. If material is not included in the article’s Creative Commons licence and your
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44. Lovley, D. R. & Holmes, D. E. Electromicrobiology: the ecophysiology of phylogenetically need to obtain permission directly from the copyright holder. To view a copy of this licence,
diverse electroactive microorganisms. Nat. Rev. Microbiol. 20, 5–19 (2022). visit http://creativecommons.org/licenses/by/4.0/.
45. Filman, D. J. et al. Cryo-EM reveals the structural basis of long-range electron transport in
a cytochrome-based bacterial nanowire. Commun. Biol. 2, 219 (2019). © The Author(s) 2025

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Article
Methods
Sulfur-cycling genes in bacterial and archaeal genomes
Phylogenetic framework and HMMs of sulfur-cycling proteins To provide a comprehensive overview of sulfur metabolism across
To discern sulfur-cycling genes or proteins from their functionally bacteria and archaea, the phylogeny-derived HMMs were searched
divergent homologues, phylogenetic analysis was conducted for 116 against all genomes in GTDB release 95 (ref. 23) using hmmsearch
sulfur-cycling proteins (Supplementary Table 1). For each protein with the --cut_ga option. Each retrieved homologue was then searched
family, sequences of enzymes with biochemically validated functions, against the full set of phylogeny-derived HMMs using hmmscan with
including related sequences of enzymes with divergent functions --cut_ga, and annotated as the HMM showing the highest score. For
(outgroups), were identified by literature surveys and recovered from initial screening, a subset of genes (n = 42) was selected as markers for
SwissProt61. Additional homologues of experimentally validated pro- specific sulfur metabolisms if the gene: (1) has been widely recognized
teins in KEGG prokaryotic genomes were retrieved using KEGG BLAST as a marker for a specific sulfur metabolism, (2) encodes a catalytic
Search (https://www.genome.jp/tools/blast/; E value: 10−4). Distant subunit essential for the activity of enzymatic complex; or (3) on its
homologues that did not align properly with biochemically charac- own confers a specific sulfur redox transformation (see justification
terized proteins (alignment length covered <50% of query and target for each of selected genes in Supplementary Table 1). The retrieved
length) were removed. The resulting homologues were de-replicated homologues were further curated using our reference phylogeny
using CD-HIT v4.8.1 (ref. 62), with longest sequences retained as rep- of sulfur proteins. Specifically, the GTDB homologues were aligned
resentatives. For computational efficiency, different clustering iden- with sequences contained in our reference phylogeny using Muscle.
tity thresholds (75–95%) were chosen for de-replication to ensure the A maximum-likelihood tree was reconstructed from the alignment
total number of representative sequences for phylogenetic analysis trimmed by TrimAl. The tree was overlaid with biochemical informa-
did not exceed 500. The genome context of the representative KEGG tion and data on the genomic context of sulfur genes, and visualized
homologues was analysed by retrieving genes located in a distance of using ggtree77. The physiological role of the GTDB homologues was
fewer than n genes (n = 7–15), followed by annotation using biochemi- interpreted on the basis of their evolutionary relationship with bio-
cally characterized gene clusters based on BLAST analysis63. All repre- chemically validated proteins and genome context. To predict the dis-
sentative KEGG homologues were further aligned with biochemically similatory iron(iii) reduction potential, GTDB genomes were screened
validated proteins and outgroups using Muscle v3.8.1551 (ref. 64). for marker genes involved in EET on the basis of homology search and/
Poorly aligned regions were excised using TrimAl v1.4.rev15 (ref. 65). or motif analysis. Homologues of iron(iii) reduction genes with estab-
Protein phylogeny was inferred from the trimmed alignment using lished HMMs in FeGenie database (https://github.com/Arkadiy-Garber/
FastTree v2.1.7 (ref. 66) with -wag and -gamma options. Statistical FeGenie/tree/master/hmms/iron/iron_reduction) were retrieved using
support for each branch of the tree was estimated by nonparametric hmmsearch from HMMER v3.2.1, with cut-off recommended by FeGenie
bootstrap (n = 100). (https://github.com/Arkadiy-Garber/FeGenie/blob/master/hmms/
Information on reference sequences from biochemically verified iron/HMM-bitcutoffs.txt). Additionally, homologues of MmcA gene,
proteins (for example, ingroup/outgroup, conserved residues or motif) which is involved in dissimilatory iron(iii) reduction in Methanosar-
and genomic contexts of all homologues were mapped on the tree. To cina acetivorans78, were extracted using BLASTP on the basis of an
identify monophyletic, orthologous clades within each tree, interior e-value of 10−4. The outer membrane MHCs responsible for EET with
nodes of the annotated tree were scrutinized using the following cri- metal oxides in anaerobic methanotrophs79 and putative electroac-
teria: (1) bootstrap support over 70%; (2) presence of at least one bio- tive bacteria80 were recognized on the basis of the following: (1) the
chemically verified ingroup protein and absence of outgroup proteins; presence of four or more haem-binding motifs (CXXCH); and (2) their
and (3) consistent gene neighbouring patterns and biochemical traits predicted outer membrane or extracellular localization, as determined
(thatis, catalytic residues and PFAM domain composition) among its by DeepProLoc v1.0 (ref. 81).
members. All descendants of the identified clade were regarded as
functional orthologs of the biochemically verified protein. If possible, Annotation and metabolic reconstruction of specific sulfur-
existing definitions of orthologous clades from previous phyloge- cycling microbial lineages
netic analysis of sulfur-cycling proteins was preserved, including the The genomes of microbial lineages of interest were downloaded
well-recognized clades in the phylogeny of DsrAB10 and Sqr67. For pro- from the GTDB database. The protein-coding genes were predicted
teins for which the biochemically validated ingroup proteins formed from the genome using Prodigal v2.6.3 with default setting. The
polyphyletic groups, multiple monophyletic clades were proposed predicted genes were annotated using KoFam70, PFAM72, and the
to fulfill our criteria. EggNOG82 database. Additional metabolic pathways were predicted
To leverage our phylogenetic framework for large-scale homol- using HMMs (Supplementary Table 6) downloaded from dbCAN83,
ogy searches, sequences from the defined monophyletic clades of metabolicHMM73, CANT-HYD84, MicRhoDE85 and FeGenie86. For
sulfur-cycling proteins were used to build HMMs. A cut-off that opti- HMM-based annotation, the HMMs were used as queries to search
mizes the sensitivity and specificity of homology search was calculated against microbial genomes using hmmsearch from HMMER v3.2.1,
for each HMM using receiver operating curve (ROC)68. This cut-off was with cut-off recommended by each database (-T, -domT or -cut_ga
embedded in the HMMER profile HMM file as the gathering thresh- options). The cellular localization of the protein was predicted using
old of the model (HMMER User’s Guide, p. 108; ref. 69). The perfor- Signalp v6.0 (ref. 87). The completeness of the KEGG metabolic path-
mance of the newly developed HMMs was compared with that of six way was calculated on the basis of the definition of each module.
published sets of HMMs for sulfur metabolism genes, including those The KEGG module is defined with a logic expression of K numbers
from KoFam70, TIGRFAM71, PFAM72, metabolicHMM73, DiSCo74, Teng that records the composition of enzymes in the pathway. A particu-
et al.75 and HMS-S-S76. This was accomplished by querying each HMM lar metabolic module was considered to be present in the genome
against the phylogeny-curated protein dataset using hmmsearch in when: (1) the diagnostic/marker genes of the module were detected;
HMMER v3.2.1 with a predefined cut-off (http://hmmer.org/). The per- and (2) the overall completeness of the pathway module was >70%.
formance of the various HMM sets in detecting sulfur-cycling genes and The environmental distribution of the GTDB species was retrieved
proteins was assessed in terms of specificity, sensitivity, and F score by searching their GTDB species name in the Sandpiper database88.
(Supplementary Text). F score balancing both precision and recall The occurrence of the GTDB species across biomes was downloaded
of the homology detection was calculated using F score = 2 × (preci- as CSV from Sandpiper (https://sandpiper.qut.edu.au/) and further
sion × recall) / (precision + recall). visualized with R v4.1.0.
 conditions in 500 ml Schott bottles27, with 2 mM Na2S 9H2O and 1.2 mM
Thermodynamic modelling KNO3 (Sigma Aldrich). This yielded a culture with an optical density at
The Gibbs free energy associated with iron(iii)-dependent sulfur oxida- 600 nm (OD600) of ~0.040, corresponding to a cell density of ~1.3 × 108
tion at environmentally relevant conditions was estimated by following cells per ml. To test alternative growth modes, five incubation experi-
the guidelines described previously89. In brief, the actual Gibbs free ments were conducted, each supplemented with different electron
energy of reaction (ΔGr) was calculated using: donors and acceptors (details provided below). For all experiments,
regularly maintained cultures (30 ml) that have been depleted in sulfide
ΔGr = ΔGr0 + RT lnQ r (< 100 µM) and nitrate (< 10 µM) were used as inoculum. Incubations
were set up in 60 ml serum bottles and sealed with butyl rubber stop-
where ΔGr0 refers to the standard Gibbs free energy of reaction, given pers in the anaerobic chamber (N2:H2 = 95:5). Each culture was then
in kJ mol−1; R and T are the universal gas constant (8.314 J K−1mol−1) and flushed with pure N2 to remove H2 in the headspace, and incubated
the temperature in Kelvin, respectively; and Qr is the reaction quotient. in the dark at room temperature. All incubations, abiotic and biotic
ΔGr0 values were calculated from the values of the standard Gibbs free controls from each experiment were set up in triplicates.
energy of formation (ΔGf0) of reactants and products (Supplementary (1) Incubations with sulfide and nitrate. The incubations were set up by
Table 7). Values of Qr were determined from the activity (ai) and the supplying 2 mM sulfide and 2 mM nitrate to 30 ml pre-growns cells
stoichiometric coefficient (vi) of the ith chemical species involved in in 60 ml serum bottles. Sulfide and nitrate was spiked using syringes
the reaction using: flushed with pure N2. The growth was monitored by phase-contrast
microscopy and by the measurement of sulfide and sulfate over 3
Qr = ∏ aiv i
days.
(2) Incubations with elemental sulfur. The incubations were initiated
The activity of the solvent (that is, pure water) and the solids (that is, by adding 0.1 g elemental sulfur in 3 ml MilliQ water (Sigma Aldrich)
ferrihydrite and FeS) were taken to be 1. The activity of dissolved ions to each of the serum bottles, followed by autoclaving at 110 °C for
was related to the concentration (Ci) using: 60 min. After sterilization, 30 ml pre-grown cells were inoculated into
the S(0) suspension (approximately 94 mM) and incubated under
ai = γi × Ci /Ci 0 an N2 atmosphere for 15 days. Microbial activity was monitored by
measuring sulfide and/or sulfate.
where γi denotes the activity coefficient; Ci0 represents the standard (3) Incubations with ferrihydrite and formate. Synthetic ferrihydrite
state concentration (usually 1 M). γi for cations (that is, Fe2+) and anions (0.2 g) was ground into fine particles with an agate mortar and pes-
(that is, HS−, S2O32− and SO42−) in solutions of different ionic strength tle before being added to the culture. Assuming ferrihydrite has a
were retrieved from Amend et al.89. Sulfide speciation in aqueous phase composition92 Fe(OH)3, the final concentration of Fe(iii) was ap-
across a range of pH was determined from the pH, and pKa1 (7.04) and proximately 62 mM. Formate was spiked anoxically using a syringe
pKa2 (11.96) of hydrogen sulfide. to a final concentration of 10 mM. To test the coupling of ferrihydrite
reduction and formate oxidation, parallel cultures were set up with
Synthesis of ferrihydrite and poorly crystalline FeS either ferrihydrite or formate alone. The culture activity was moni-
Synthetic ferrihydrite was prepared by titrating 1 M NaOH (Sigma tored by measuring total Fe(ii) and formate concentrations over a
Aldrich) into 0.1 M aqueous solution of FeCl3 6H2O (Carl Roth) under 15 day incubation.
vigorous stirring until pH 7.5 was reached, as described90. The suspen- (4) Incubations with ferrihydrite and poorly crystalline FeS. Synthetic
sion was centrifuged (Centrifuge 5804 R, Eppendorf) at 4 °C, 12,857g ferrihydrite (0.2 g) was supplied to the cultures as described in the
and the ferrihydrite nanoparticles were washed thoroughly with incubation (3). To amend poorly crystalline FeS, 1 ml stock solution
deionized water to remove traces of chloride. The pellets were then of freshly prepared FeS (30 mM) was anoxically spiked to the cultures
freeze-dried (Alpha 1-4 LSCbasic, Christ) and stored at −20 °C for no using syringes, resulting in a final FeS concentration of 1 mM. Abiotic
longer than 3 weeks before use. The mineralogy was determined by controls were prepared using 30 ml autoclaved cells as inoculum to
LabRAM HR800 Raman microscope (Horiba Jobin-Yvon) equipped test for chemical reactions between ferrihydrite and poorly crystal-
with a 532-nm neodymium-yttrium aluminium garnet laser and either line FeS. Biotic controls amended with either ferrihydrite or FeS
300 or 600 grooves/mm diffraction grating. Iron monosulfide (FeS; were set up to assess the impacts of residual sulfide and/or nitrate on
30 mM) was prepared by mixing equal volume of 60 mM aqueous solu- culture activity. Cultures were sampled daily over 5 days for sulfate
tion of Na2S.9H2O (Acros Organics) with 60 mM aqueous solution of and total Fe(ii) measurements.
FeCl2.4H2O (Sigma Aldrich) in an anaerobic chamber (Coy Lab) with 95% (5) Incubations with ferrihydrite and dissolved sulfide. The cultures
N2 and 5% H2 (O2 < 1 ppm) atmosphere. The initially precipitated FeS is were prepared similarly as incubation (4), but with dissolved sulfide
often designated as ‘amorphous FeS’ or ‘poorly crystalline FeS’91. The replacing FeS. Due to the rapid chemical reaction between dissolved
dissolved sulfide in the FeS stock is less than 50 µM. The FeS solution sulfide and ferrihydrite, dissolved sulfide was anoxically spiked daily
was freshly prepared and used on the same day. at a concentration of 1 mM using N2-flushed syringes. Abiotic controls
and the sulfide-only biotic controls received dissolved sulfide at the
Cultivation of D. alkaliphilus DSM 19089 same concentration and frequency. To trace the transformation of
D. alkaliphilus (DSM 19089, ATH2) was purchased from the German S and Fe over 5 days, subsamples were taken daily for measurement
Collection of Microorganisms and Cell Cultures GmbH (DSMZ). The of S(0), total Fe(ii), sulfate, and Cline-extractable sulfide before
bacterium was cultivated at room temperature in an alkaline mineral the addition of sulfide. The consumption of dissolved sulfide in the
medium (pH 9.3) containing 3 g NaCl (Carl Roth), 0.25 g K2HPO4 (Merck), cultures was monitored by sampling at 2, 10, 20, 35, 50 and 70 min
6.5 g Na2CO3 (Carl Roth), and 15 g NaHCO3 (Sigma Aldrich) per liter of after the spike of sulfide. The kinetics of sulfide consumption were
medium. After autoclaving, the medium was cooled down under N2 modelled as a first-order reaction. The rate constant was estimated
atmosphere and supplemented aseptically with 1 ml liter−1 of following using the exponential decay model in the drm() function from the
components (all stored under anoxic conditions): 4 M NH4Cl (Sigma drc R package93. To compare sulfate formation patterns with and
Aldrich), 1 M MgCl2 (Sigma Aldrich), trace element solution, Se-W solu- without sulfide, cultures incubated with ferrihydrite and sulfide
tion, and four different vitamin solutions (DSMZ medium 1104). The were sampled for sulfate measurement following two phases after
culture was routinely grown under nitrate-reducing, sulfide-oxidizing the 1st sulfide spike. During phase I, detectable sulfide was present
Article
in the culture, and the samples were collected at 0, 11, 21, 37, 53 and through a 0.2 µm membrane, and addition of 1 mM chlorate as the
70 min of the incubation. Phase II, spanning the next 23 h, began internal standard. The standards were prepared by adding defined
once sulfide was depleted, with samples taken at 3, 5.5, 8.33, 12.33, amounts of sulfate (Sigma Aldrich) to the alkaline medium, followed
20.25 and 24 h. As a control for phase II, cells were incubated with by the same treatment procedure as described for samples. The sulfate
chemically sulfidized ferrihydrite. Specifically, 1 mM sulfide was content in the prepared samples/standards was measured using an
firstly added to 0.2 g ferrihydrite (approximately 62 mM) with 30 ml Agilent 7100 capillary electrophoresis system (Agilent Technologies),
autoclaved cultures for chemical reaction. After 70 min, the reaction equipped with a capillary (72 cm × 72 µm internal diameter; Agilent
mixture was centrifuged (12,857g; room temperature) under anoxic Technologies) and a diode array UV-vis detector (DAD). Electrolytes for
conditions, and 30 ml of active cells were inoculated to resuspend anion separation contains 2.25 mM pyromellitic acid (Sigma Aldrich),
the solid phase compounds (for example, FeS and S(0)) produced by 1.6 mM triethanolamine (Sigma Aldrich), 0.75 mM hexamethonium
chemical reaction between sulfide and ferrihydrite. Samples were hydroxide (Sigma Aldrich), and 6.5 mM NaOH97 at pH 7.8 ± 0.1. Anion
collected from cultures for sulfate measurement at the same time separation was implemented at a voltage of −30 kV. The data were
intervals as those in phases I and II. acquired through indirect UV detection at a wavelength of 350 nm with
To test whether the microbial process can outperform the chemical a bandwidth of 60 nm, and a reference wavelength of 245 nm with a
process in transforming sulfide with ferrihydrite, the incubation (5) bandwidth of 10 nm. For the formate measurement, 900 µl MilliQ water
was repeated using ca. 50 µM sulfide instead of 1 mM. In this experi- was added to 100 µl samples/standards (Sigma Aldrich), which were
ment, a small amount of sulfide was spiked three times at 1.5-h intervals then filtered through a 0.2 µm membrane. l-malate (Sigma Aldrich) was
into ferrihydrite-amended cultures, abiotic controls, and sulfide-only added to the filtrate as the internal standard. Organic Acids Buffer for
biotic controls. After each spike, subcultures (~ 0.3 ml) were collected capillary electrophoresis (pH 5.6; Agilent Technologies) was used as
at 2, 5, 10, 15, 20, and 25 min for dissolved sulfide measurements. Two electrolytes, and the separation conditions, including DAD and capil-
biological replicates were performed for each treatment. To verify lary electrophoresis settings, were configured according to manufac-
the reproducibility of the observed sulfide consumption pattern, turer’s instructions. All electropherogram data were analysed with the
incubations were conducted using inocula at different cell densities Agilent ChemStation.
(OD600 of 0.042, 0.075, and 0.086). To quantify the transformation of Elemental sulfur was measured using high performance liquid chro-
spiked sulfide during the incubation, independent cultures were set matography (HPLC). One-hundred microlitres of sample was fixed
up using an inoculum with an OD600 of 0.072 and supplied with eight with 10 µl of 3% w/v zinc acetate. Then, 300 µl chloroform was added,
spikes of sulfide. Ferrihydrite-amended cultures, abiotic controls, and the mixture was shaken at 500 rpm for 1 h. The elemental sulfur in
and sulfide-only controls received ca. 50 µM sulfide at 1.5-h intervals, chloroform phase was then measured using a Dionex UltiMate 3000
whereas ferrihydrite-only biotic controls were spiked with anoxic water. UPLC system, equipped with an UltiMate 3000 pump (0.2 ml min−1),
Three replicate incubations were performed for each treatment. Sub- a column Compartment (25 °C), a column Waters ACCQ-TAG ULTRA
samples were taken every three hours for concentration measurement C18 1.7 µm × 2.1 × 100 mm, and an UltiMate 3000 Variable Wavelength
of S(0), total Fe(ii), sulfate and Cline-extractable sulfide. Detector (UV) (wavelength 254 nm). The isocratic elution with 100%
methanol was applied. With these adjustments, the peak appeared
Chemical analysis of metabolites after 3.4 min. Data were analysed with Dionex Chromeleon software.
To monitor the dynamics of metabolites in the incubation experiments,
subsamples of the culture were taken periodically with sterile syringes Microscopy of D. alkaliphilus incubated with ferrihydrite and
flushed with pure N2 as described above. HCl-extractable Fe(ii) was sulfide
determined by adding 0.1 ml sample aliquots to 0.2 ml 0.75 N HCl. The For scanning electron microscopy (SEM), transmission electron micros-
sample was immediately centrifuged for 15 min at 12,044g. Fe(ii) in copy (TEM) and fluorescence microscopy, cultures incubated with
the resulting 0.5 N HCl was measured using the ferrozine assay. Previ- ferrihydrite and sulfide (daily spike of 1 mM) for 5 days were fixed in
ous studies have shown the 0.5 N HCl treatment allowed quantitative 2% glutaraldehyde or 2.3% formaldehyde, respectively. For SEM imag-
extraction of the solid phase Fe(ii) associated with the surface of iron ing, solid iron phase iron was allowed to settle without centrifugation,
oxides, Fe(ii) from FeS, and the dissolved Fe(ii) in the Fe/S system7,94. carefully washed with MilliQ water, and transferred to 100% ethanol.
Therefore, we referred to HCl-extractable Fe(ii) as total Fe(ii). Samples were then dried using rapid chemical drying with hexam-
Aqueous and total sulfide were determined using spectrophoto- ethyldisilazane and mounted on aluminium stubs with double-sided
metric methods. To measure dissolved sulfide, approximately 0.3 ml sticky carbon tape and sputtered with Gold ( JEOL JFC-2300HR). The
subculture was filtered through a 0.2 µm membrane (CHROMAFIL). images were taken with a Scanning Electron Microscope ( JEOL IT 300
The dissolved sulfide in the filtrate (0.1 ml) was fixed by 0.25 ml 3% LAB6EOL) with Secondary Electron Detector (SED) and Backscattered
w/v zinc acetate dihydrate (Sigma Aldrich), followed by quantification Electron Detector (BED-C) at 20 kV.
using the Cline method95. The filtered sample from the incubation with For TEM imaging, cultures were treated with a solution contain-
ferrihydrite and 1 mM dissolved sulfide showed black colour, indicating ing 50 g l−1 sodium dithionite, 0.2 M sodium citrate and 0.35 M acetic
the formation of FeS particles smaller than 0.2 µm. The sulfide associ- acid (hereafter termed dithionite solution) as previously described98.
ated with this FeS fractionation was approximated as HCl-extractable After dissolution of solid iron phase, cells were pelleted at low speed
Fe(ii), assuming a 1:1 stoichiometry. The total sulfide was determined (2,300g) to minimize shear forces and washed with MilliQ water before
as Cline-extractable sulfide. The Cline reagent contains 6 N HCl that suspending cells in MilliQ water. For negative staining, 4 µl of sam-
dissolves some solid sulfides (for example, freshly formed FeS), and ple was incubated for 1 min on a formvar-filmed and carbon-coated
thus the Cline-extractable sulfide comprises dissolved sulfide and grid (200 mesh, Cu) and excess liquid was removed with a filter paper.
HCl-reactive solid phase sulfide. Total sulfide in the Fe/S system is A drop of stain (2.5% gadolinium acetate) was applied and immedi-
typically determined as acid volatile sulfide. Acid volatile sulfide was ately removed. Samples were examined in a TEM EM 900 N (Zeiss)
not analysed here owing to the large uncertainties inherent to this at 80 kV.
methodology91,96. For fluorescence microscopy, the formaldehyde-fixed cultures were
Sulfate and formate concentrations in the incubations were deter- resuspended and a subsample was filtered onto a 0.2 µm pore size
mined by capillary electrophoresis techniques. Sample preparation polycarbonate membrane (Millipore). Cells on the filter were stained
for sulfate measurement involved fixation of 100 µl subsample with with a 1× SYBR Green solution, and images were acquired using a epif-
10 µl 3% w/v zinc acetate, dilution with 890 ul MilliQ water, filtration luorescence microscope (Zeiss Axio Imager M1 with an AxioCam MRm).
 polycarbonate filters (GTTP type, 0.2 µm pore size, Millipore). The fil-
Monitoring the growth of D. alkaliphilus during incubation ters were gold-coated by physical vapour deposition, utilizing an Agar
experiments B7340 sputter coater (Agar Scientific) equipped with an Agar B7348 film
Growth was monitored by cell counting for cultures incubated under thickness monitor (Agar Scientific) for precise adjustment of the coat-
4 different conditions: (1) ferrihydrite (approximately 62 mM Fe ing thickness (150 nm). The filters were incubated for 2 h in 0.1 M HCl to
equivalent) and periodic spike of approximately 50 µM dissolved remove residual carbonates and then washed twice in MilliQ water and
sulfide (sulfide was spiked 40 times over 5 days, with one spike every then air-dried. Filter sections were attached to antimony-doped silicon
hour and 8 times per day); (2) ferrihydrite (approximately 62 mM Fe wafers (7.1 ×7.1 mm, Active Business Company) with a commercially
equivalent) and daily spike of 1 mM sulfide; (3) ferrihydrite (approxi- available glue (SuperGlue Loctide).
mately 62 mM Fe equivalent) and periodic spike of FeS (approximately NanoSIMS measurements were carried out on a NanoSIMS 50 L
1 mM Fe equivalent); and (4) nitrate (4 mM) and 2 spikes of sulfide instrument (Cameca) at the Large-Instrument Facility for Environ-
at concentration of 1–2 mM. The setup of the cultures and controls mental and Isotope Mass Spectrometry at the University of Vienna.
was the same as described in ‘Cultivation of alkaliphilus DSM 19089’ Prior to data acquisition, analysis areas were pre-conditioned in situ
except that a lower starting cell density (3–5 × 107 cells per ml) was by rastering a high-intensity, slightly defocused Cs+ ion beam for
used. During each of the incubation experiments, subcultures (450 µl) removal of surface adsorbates and establishment of the steady state
were sampled periodically and preserved in 2.3% formaldehyde (final secondary ion signal intensity regime with minimum sample erosion.
concentration). Before counting, 500 µl dithionite solution was added For this purpose, the following sequence of high and extreme low
to 50–100 µl of fixed cells to dissolve the FeS and ferrihydrite parti- Cs+ ion impact energy (EXLIE) was applied: high energy (16 keV) at
cles. After dissolution of solid iron phase (within 10–15 min), 100 µl 100 pA beam current to a fluence of 5 × 1014 ions cm−2; EXLIE (50 eV)
of each sample was diluted in 900 µl of 1× phosphate-buffered saline at 400 pA beam current to a fluence of 5 × 1016 ions cm−2; high energy
(PBS). The suspension was then sonicated using a SONOPULS ultra- to an additional fluence of 2.5 × 1014 ions cm−2. Data were acquired as
sonic homogenizer (Bandelin, Berlin, Germany) at 25% power with a multilayer image stacks by scanning of a finely focused Cs+ primary ion
cycle setting of 2 for a total of 30 s. Cells were subsequently stained beam with 2 pA beam current at approximately 80 nm physical reso-
with SYBR Green 1× (ThermoFisher) and incubated for 10 min at room lution (probe size) over areas between 60 × 60 and 62 × 62 µm2 with
temperature in the dark. Flow cytometric analysis was performed 512 × 512 pixel and 1,024 × 1,024 pixel image resolution and a per-pixel
using a CytoFLEX S flow cytometer (Beckman Coulter) equipped with dwell time of 5 ms and 1.5 ms, respectively. The detectors of the mul-
a blue 488 nm laser. SYBR Green fluorescence was detected using a ticollection assembly were positioned for parallel detection of 12C2−,
12 13 − 12 14 − 31 −
525/40 nm bandpass filter. A fluorescence threshold was applied on C C , C N , P and 32S-secondary ions. Secondary electrons were
the SYBR Green signal to exclude background events. For each sample, detected simultaneously for gaining information about the sample
80–100 µl was measured. Data were gated on SYBR Green–positive morphology and topography. The mass spectrometer was tuned to
cells displaying fluorescence shifts relative to unstained controls to achieve a mass resolving power ((MRP) = M/ΔM) of >10,000 for detec-
identify the target population (Supplementary Fig. 15). Data were tion of C2− secondary ions.
acquired and analysed with the CytExpert 2.6 software (Beckman Measurement data were processed using the WinImage software
Coulter). The specific growth rate (k; day−1) was estimated via linear package provided by Cameca (WinImage V4.8) and the OpenMIMS
regression analysis of ln(Cellt/Cell0) versus time (day) over an appar- plugin in the image processing package ImageJ (V1.54p). Prior to data
ent exponential growth phase. Here, Cellt is the cell concentration (in evaluation, images were corrected for detector dead-time and posi-
cells per ml) at sampling time t (day). tional variations emerging from primary ion beam and/or sample stage
drift. Carbon isotope composition images displaying the 13C/(12C + 13C)
13
C-bicarbonate labelling experiments and isotope analysis isotope fraction, given in atom percent (atom%), were inferred from
To probe for autotrophic carbon fixation during MISO growth con- the C2− secondary ion signal intensity distribution images via per-pixel
ditions, 13C-labelled bicarbonate (98 atom% 13C; Sigma Aldrich) was calculation of 13C12C−/(2 × 12C2− + 12C13C−) intensity ratios. For numeri-
added to ferrihydrite-incubated cultures receiving dissolved sulfide cal data evaluation, regions of interest, referring to individual cells,
(1 mM) or FeS (ca. 1 mM S equivalent), to reach a 10 atom% 13C in the were manually defined on the basis of the 12C14N− and 31P− secondary
inorganic carbon pool. The dissolved sulfide or solid phase FeS were ion maps as indicators of biomass and verified by the topographical/
spiked in the same frequency as for the growth experiment. Abiotic morphological appearance in the secondary electron images. Biomass
controls for each culture were set up using autoclaved inoculum. To aggregates, in which an unambiguous identification of single cells was
detect 13C content in bulk biomass and in single cells, subcultures not feasible, were rejected.
were sampled, fixed by formaldehyde (2.3% final concentration), and Cells were assessed as being significantly enriched in 13C after incu-
analysed using elemental analyser-isotope ratio mass spectrometry bation in the presence of 13C-bicarbonate if (1) the 13C isotope fraction
(EA-IRMS) and NanoSIMS. For EA-IRMS, 1.5 ml of fixed samples that value was higher than the mean plus 3 standard deviations (σ) of the
included ferrihydrite and cells were pelleted by centrifugation and values determined on the cells from the control (on day 0) and (2) the
washed with MilliQ water, followed by overnight treatment by 0.1 M statistical counting error (3σ, Poisson) was smaller than the difference
HCl to remove residual carbonates. The dried cells attached to ferri- between the considered 13C enriched cell and the mean value meas-
hydrite particles were weighed (4–6 mg) and transferred to tin cups. ured on the cells from the control. The Poisson error was calculated
Bulk cell carbon isotope ratios (13C:12C) were measured by EA-IRMS from the secondary ion signal intensities (given in counts per region
(Delta V Advantage) coupled by a ConFlo IV interface to an elemental of interest) via
analyser (EA-Isolink, all Thermo Finnigan). Sample 13C contents were
2
calculated as atomic percentage of 13C in total carbon, following 13C  12 12 13 
σPois = 1/ 2 × C2− + C C− 
atom% = 13C/(13C + 12C) × 100%. The analytical precision of replicate  
analyses of isotopically homogeneous international standards was
  12 
2
12 13  12 13 
2
12 
±0.0001% for 13C atom% measurements. ×   C2−  × C C− +  C C−  × C2− 
    
For NanoSIMS analysis, 0.1 ml formaldehyde-fixed samples that  
included ferrihydrite and cells were mixed with dithionite solution as
described above and incubated for 2 h. After complete dissolution of On the basis of these two criteria, all individual cells measured in the
13
ferrihydrite, 400 µl of the suspension was transferred onto gold-coated C incubated sample showed a significant enrichment in 13C.
Article
the manufacturer’s instructions. The absolute abundance of transcripts
RNA-seq and transcriptomics from DA_402 and recA were quantified by quantitative PCR using cDNA
D. alkaliphilus cultures grown under five incubation conditions (as as a template. Purified PCR products of gene DA_402 and recA amplified
described in ‘Cultivation of D. alkaliphilus DSM 19089’), each in four from genomic DNA of D. alkaliphilus were used as quantitative PCR
replicates, were used for comparative transcriptomic analysis. Cultures standards. The PCR reactions were prepared in triplicates and run at
(30 ml) showing metabolic activity (for example, Fe(ii) production, 95 °C for 3 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s,
sulfide consumption or production) were collected in the middle to late and 72 °C for 45 s, on the Thermal Cycler with CFX96 Real-Time System
stage of incubation experiments. Cells were collected by centrifuging (Bio-Rad). The RT–qPCR data were acquired and analysed using CFX
(12,857g; room temperature) under anoxic condition using oak ridge Maestro software (Bio-Rad). The transcription level of DA_402 was
tubes (Thermo Fisher Nalgene) with replacement O-rings for sealing compared between treatments after normalization with that of recA.
cap (Thermo Fisher Nalgene). The cell pellets were resuspended with The statistical significance of differential transcription between treat-
1.5 ml supernatant, and distributed to three lysis matrix E tubes (MP ments were determined via Student’s t-test.
Biomedicals), each with approximately 0.5 ml. The collected cells
were immediately frozen with liquid N2, and stored at −80 °C before Structure prediction and phylogenetic analysis of multi-haem
subsequent analysis. The total nucleic acids were extracted follow- c-type cytochromes in D. alkaliphilus
ing a phenol-chloroform protocol as described previously99,100. In D. alkaliphilus proteins with more than one haem-binding motifs
brief, the sample was lysed for 30 s at a speed of 5.5 m s−1, after mixing (CXnCH; n = 2 to 5) were considered MHCs107. The haem-binding
with hexadecyltrimethylammonium bromide extraction buffer and motifs in protein sequences were counted using regex expressions
phenol-chloroform-isoamyl alcohol (25:24:1) (pH 8.0). The aqueous in the Python re package. The subcellular localization of all putative
phase was extracted by centrifugation, and the phenol within was MHCs (n = 46) from D. alkaliphilus was predicted using PSORTb v3.0
removed by mixing with chloroform-isoamyl alcohol (24:1). The total (ref. 108) and DeepLocPro v1.0 (ref. 81). Prediction from DeepLocPro
nucleic acids in the aqueous phase were then precipitated with poly- was used for the proteins for which PSORTb returned ‘Unknown’.
ethylene glycol 6000, followed by centrifugation. The pelleted nucleic The transcription levels of MHCs were compared between different
acids were washed with ice-cold ethanol and dried before resuspension incubation experiments on the basis of RPKM values. The statistical
in diethyl pyrocarbonate-treated water. DNA from the total nucleic significance of differential transcription was assessed as described
acids were removed using the TURBO DNA-free kit (Thermo Fisher in the ‘RNA-Seq and transcriptomics’ chapter. The most highly tran-
Scientific). scribed extracellular MHC (DA_402) during MISO was further selected
RNA-sequencing was performed at the Joint Microbiome Facility of for structure prediction and phylogenetic analysis. The structure of the
the Medical University of Vienna and the University of Vienna ( JMF) DA_402 monomer and oligomer were predicted using AlphaFold2 v2.3.2
under project IDs JMF-2311-14 and JMF-2405-05. Sequencing libraries at the COSMIC2 science gateway. The leading signal peptide, predicted
were prepared from rRNA depleted (Ribo-Zero Plus rRNA Depletion using SignalP 5.0 (ref. 109), was cleaved from the protein sequence
Kit, Illumina) RNA samples (NEBNext Ultra II Directional RNA Library before structure modelling. For comparison, the cryo-EM structure of
Prep Kit for Illumina, New England Biolabs) and sequenced in 2× 100 bp OmcS from G. sulfurreducens was retrieved from the Protein Data Bank
paired-end mode (NextSeq 6000, Illumina), yielding 74.2–303.7 million (PDB) database (6EF8). The protein sequence of DA_402 was aligned
raw reads per sample. Individual read libraries were quality checked to OmcS using the T_coffee alignment tool110. The structure-structure
using fastQC v0.12.1 (http://www.bioinformatics.babraham.ac.uk/ similarity between DA_402 and OmcS was calculated using an online
projects/fastqc/) and quality statistics were merged using multiQC TM-align tool and DaliLite.v5 (ref. 111–113). The haem-binding sites in
v1.21 (ref. 101). Adapters were trimmed and phiX contamination was DA_402 and OmcS were visualized using MacPyMOL v.1.7.4 (https://
removed using BBDuk (part of BBMap v39.06). Reads were k-trimmed pymol.org). The haem was docked to the target haem-binding site in
from the right with a kmer of 21, minimum kmer of 11 and hamming DA_402 using AutoDockTools v1.5.7 (ref. 114) and AutoDock Vina 1.1.2
distance of two along with the tpe and tbo options. Quality trimming (ref. 115). To conduct phylogenetic analysis of DA_402, homologues of
was performed from the right with a q-score of 28 to a minimum of 50 DA_402 were retrieved from the KEGG database using Blastp with an E
bases in length (https://sourceforge.net/projects/bbmap/). The quality value of 0.01. The retrieved homologues were then de-replicated with
filtered reads were aligned to the reference genome of D. alkaliphilus CD-HIT at 70% identity, aligned with Muscle, trimmed with trimAl (--gt
(NC_014216.1) using BBMap with a mapping identity of 99% and with 0.1). The resulting sequence alignment was used to reconstruct the
ambiguous reads assigned to the best location (that is, counted only maximum-likelihood tree using RAxML v8.2.12. The clustering pattern
once for duplicated genes). FeatureCounts (part of SubRead 2.0.6 and decoration of the tree were performed using iTOL v6 (ref. 116).
(ref. 102)) with reverse-stranded and –countReadPairs were used to
generate counts tables with the resulting alignments based on gene Environmental distribution of Desulfurivibrionaceae with
call locations by prodigal v2.6.3 (ref. 103). Counts tables were ana- genomic potential of MISO
lysed using DESeq2 release 3.19 (ref. 104) to calculate the RPKM and to The metabolic potential of members belonging to the Desulfurivibrion-
determine statistical significance of differential transcription between aceae family was analysed using publicly available genomes recov-
treatment groups. All P values are adjusted for multiple comparisons ered from different environments. Metagenome-assembled genomes
using the Benjamini–Hochberg method105. (MAGs) classified as Desulfurivibrionaceae were obtained from GTDB
Quantitative PCR with reverse transcription (RT–qPCR) was per- r214 (n = 121), NCBI (n = 9), JGI IMG (n = 68) and GMGC (n = 7). The envi-
formed to verify the upregulated transcription for the MHC gene ronmental origins of these genomes were retrieved from the metadata
DA_402 under iron-reducing conditions. Primers DA_402_998F in the respective databases (Supplementary Table 8). The taxonomy
(5′-TTCCCAATCGGGGCGAATAC-3′) and DA_402_1081R (5′-TGGCCTCG of collected genomes was assigned using GTDB-tk version 2.3.2 with
GTATAGAGGGTC-3′) were used to target DA_402. Primers recA_79F database release 214 (ref. 117). The phylogenomic tree of Desulfurivi-
(5′-TTCGGCAAAGGCTCCATCAT-3′) and recA_221R (5′-TCCGGCCCA brionaceae was reconstructed from a concatenated alignment of 120
TATACCTCGAT-3′) were used to quantify the transcription level of single-copy genes with FastTree v2.1.10 (ref. 66). The protein-coding
the house-keeping gene recA (DA_1926) encoding the DNA recombi- genes in the genomes were called using Prodigal v2.6.3 (ref. 103), and
nation protein. Primers for both genes were newly designed using the resulting proteomes were screened for proteins involved in dis-
Primer-Blast106. For RT–qPCR, DNA-free RNA was first reverse tran- similatory sulfide oxidation (DsrAB) and iron oxides reduction (that
scribed to cDNA using SuperScript III reverse transcriptase according to is, OmcS, OmcZ, porin–cytochrome complex and OmcE). DsrAB was
detected using HMMs and the phylogenetic framework established (https://github.com/elizabethmcd/metabolisHMM), CANT-HYD
in this study, while proteins involved in dissimilatory iron reduction (https://github.com/dgittins/CANT-HYD-HydrocarbonBiodegrada­
were identified with HMMs from FeGenie86. Additional proteins likely tion), MicRhoDE (http://application.sb-roscoff.fr/micrhode/), Fe­Geneie
involved in dissimilatory reduction of iron oxides—that is, extracellu- (https://github.com/Arkadiy-Garber/FeGenie), NCBI (https://www.
lar MHC DA_402 and PilA—were retrieved from Desulfurivibrionaceae ncbi.nlm.nih.gov/) and JGI IMG (https://img.jgi.doe.gov/). The tran-
genomes by hmmsearch or BLASTP. Homologues of PilA were extracted scriptomic data of D. alkaliphilus cultured under five conditions (each
by searching TIGR02532 HMM model against the Desulfurivibrionaceae with four replicates) have been deposited at the NCBI under BioPro-
genomes using hmmsearch with --cut_ga option. For DA_402, homo- ject PRJNA1165744 (NCBI Sequence Read Archive (SRA) accession:
logues were collected from the Desulfurivibrionaceae genomes using SRX26208148–SRX26208167). HMM models for the 116 sulfur-cycling
BLASTP with an E value of 1e-10, followed by prediction of the subcel- genes have been deposited at Github (https://github.com/Song-
lular localization and counting of haem-binding sites. The extracellular CanChen11/HMMs). Source data are provided with this paper.
homologues containing multi-haem-binding sites (n > 3) were then
placed into a reference tree created through phylogenetic analyses of
DA_402 (see above) with the RAxML evolutionary placement algorithm Code availability
(EPA). The alignment for EPA was generated using MAFFT v7.407 with A custom script to retrieve homologues of sulfur-cycling marker genes
--add option. The homologues that were placed with accumulated prob- from GTDB database has been deposited at Github (https://github.
ability over 0.95 to the OmcS-like clade were considered as functional com/SongCanChen11/HMMs/).
orthologs of DA_402. For visualization purposes, Desulfurivibrion-
aceae genomes (n = 119) encoding both dissimilatory iron and sulfur
61. Boutet, E., Lieberherr, D., Tognolli, M., Schneider, M. & Bairoch, A. UniProtKB/Swiss-Prot.
metabolism were de-replicated on the basis of relative evolutionary Methods Mol. Biol. 406, 89–112 (2007).
divergence (RED). RED was calculated for each internal node of the 62. Fu, L., Niu, B., Zhu, Z., Wu, S. & Li, W. CD-HIT: Accelerated for clustering the next-generation
Desulfurivibrionaceae phylogenomic tree following the procedure sequencing data. Bioinformatics 28, 3150–3152 (2012).
63. Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinformatics 10, 421
described previously118. The tree was then collapsed at a RED value of 0.9 (2009).
and one representative was chosen randomly from the collapsed clades, 64. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput.
Nucleic Acids Res. 32, 1792–1797 (2004).
yielding 53 representative members that were visualized in the tree.
65. Capella-Gutiérrez, S., Silla-Martínez, J. M. & Gabaldón, T. trimAl: A tool for automated
alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973
Statistics and reproducibility (2009).
66. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2—approximately maximum-likelihood
The physiological experiments showing the ability of ferrihydrite-
trees for large alignments. PLoS ONE 5, e9490 (2010).
incubated D. alkaliphilus to oxidize formate (Fig. 3a), FeS (Fig. 3b), 67. Marcia, M., Ermler, U., Peng, G. & Michel, H. A new structure-based classification of
1 mM sulfide (Fig. 3c,d,g) or ~50 µM sulfide (Fig. 3h) were repeated sulfide:quinone oxidoreductases. Proteins 78, 1073–1083 (2010).
68. Orellana, L. H., Rodriguez-R, L. M. & Konstantinidis, K. T. ROCker: Accurate detection and
independently at least three times, all yielding consistent results. The
quantification of target genes in short-read metagenomic data sets by modeling sliding-
sulfide removal kinetic experiments at low sulfide concentration were window bitscores. Nucleic Acids Res. 45, e14 (2017).
replicated independently for two times, and all results are present in 69. Eddy, S. R., Wheeler, T. J. & the HMMER development team. HMMER User’s Guide Version
3.1b2, 108 (Howard Hughes Medical Institute, 2015); http://eddylab.org/software/hmmer3/
the Extended Data Fig. 1. The experiment showing the transformation
3.1b2/Userguide.pdf.
of sulfide and ferrihydrite with periodic supply of ~50 µM sulfide was 70. Aramaki, T. et al. KofamKOALA: KEGG Ortholog assignment based on profile HMM and
performed independently twice, and both yielded similar results. The adaptive score threshold. Bioinformatics 36, 2251–2252 (2020).
71. Haft, D. H. et al. TIGRFAMs: a protein family resource for the functional identification of
growth experiments of ferrihydrite-incubated cells with periodic addi-
proteins. Nucleic Acids Res. 29, 41–43 (2001).
tion of 1 mM sulfide (Fig. 4a), FeS (Fig. 4b) or 50 µM sulfide (Fig. 4c), 72. Finn, R. D. et al. Pfam: the protein families database. Nucleic Acids Res. 42, D222–D230
and the experiment with nitrate and sulfide (Fig. 4d) were conducted (2014).
73. McDaniel, E. A., Anantharaman, K. & McMahon, K. D. metabolisHMM: Phylogenomic
once with three biological replicates per treatment and control. The analysis for exploration of microbial phylogenies and metabolic pathways. Preprint at
13
C-bicarbonate labelling experiment and bulk 13C analysis of cells incu- bioRxiv https://doi.org/10.1101/2019.12.20.884627 (2019).
bated with ferrihydrite and either dissolved sulfide (Fig. 4e) or FeS 74. Neukirchen, S. & Sousa, F. L. DiSCo: a sequence-based type-specific predictor of Dsr-
dependent dissimilatory sulphur metabolism in microbial data. Microb. Genomics 7,
(Fig. 4f) were independently repeated for two times, both yielding 000603 (2021).
comparable outcomes. Samples for NanoSISM analysis were chosen 75. Teng, Z.-J. et al. Biogeographic traits of dimethyl sulfide and dimethylsulfoniopropionate
randomly among biological replicates collected at day 0 and 5, and rep- cycling in polar oceans. Microbiome 9, 207 (2021).
76. Tanabe, T. S. & Dahl, C. HMS-S-S: A tool for the identification of sulphur metabolism-related
resentative field views are present in Fig. 4g–i. Transcriptomic analysis genes and analysis of operon structures in genome and metagenome assemblies. Mol. Ecol.
of cells growing under five different conditions was conducted once, Resour. 22, 2758–2774 (2022).
with four biological replicates per condition (Fig. 5 and Extended Data 77. Yu, G., Smith, D. K., Zhu, H., Guan, Y. & Lam, T. T.-Y. ggtree: an R package for visualization
and annotation of phylogenetic trees with their covariates and other associated data.
Fig. 4). Methods Ecol. Evol. 8, 28–36 (2017).
78. Gupta, D., Chen, K., Elliott, S. J. & Nayak, D. D. MmcA is an electron conduit that facilitates
Reporting summary both intracellular and extracellular electron transport in Methanosarcina acetivorans.
Nat. Commun. 15, 3300 (2024).
Further information on research design is available in the Nature Port- 79. Leu, A. O. et al. Lateral gene transfer drives metabolic flexibility in the anaerobic
folio Reporting Summary linked to this article. methane-oxidizing archaeal family Methanoperedenaceae. mBio 11, e01325 (2020).
80. Garber, A. I., Nealson, K. H. & Merino, N. Large-scale prediction of outer-membrane
multiheme cytochromes uncovers hidden diversity of electroactive bacteria and
underlying pathways. Front. Microbiol. 15, 1448685 (2024).
Data availability 81. Moreno, J., Nielsen, H., Winther, O. & Teufel, F. Predicting the subcellular location of
The GTDB r95 genomes were retrieved from the GTDB repository prokaryotic proteins with DeepLocPro. Bioinformatics 40, btae677 (2024).
82. Huerta-Cepas, J. et al. eggNOG 5.0: A hierarchical, functionally and phylogenetically
(https://data.ace.uq.edu.au/public/gtdb/data/releases/release95/95.0/ annotated orthology resource based on 5090 organisms and 2502 viruses. Nucleic Acids
genomic_files_reps/). The protein structure of OmcS from G. sulfurredu- Res. 47, D309–D314 (2019).
cens was retrieved from the PDB accession 6EF8. Reference sequences 83. Yin, Y. et al. dbCAN: A web resource for automated carbohydrate-active enzyme annotation.
Nucleic Acids Res. 40, W445–W451 (2012).
and gene-specific HMMs mentioned in the Methods were acquired 84. Khot, V. et al. CANT-HYD: A curated database of phylogeny-derived Hidden Markov Models
from SwissProt (https://www.uniprot.org/), KEGG (https://www.kegg. for annotation of marker genes involved in hydrocarbon degradation. Front. Microbiol. 12,
jp/), PFAM (http://pfam.xfam.org/), EggNOG v5.0 (http://eggnog5. 764058 (2021).
85. Boeuf, D., Audic, S., Brillet-Guéguen, L., Caron, C. & Jeanthon, C. MicRhoDE: A curated
embl.de/#/app/home), Sandpiper (https://sandpiper.qut.edu.au/), database for the analysis of microbial rhodopsin diversity and evolution. Database 2015,
dbCAN (https://bcb.unl.edu/dbCAN2/download/), metabolicHMM bav080 (2015).
Article
86. Garber, A. I. et al. FeGenie: A comprehensive tool for the identification of iron genes and 112. Holm, L. Benchmarking fold detection by DaliLite v.5. Bioinformatics 35, 5326–5327 (2019).
iron gene neighborhoods in genome and metagenome assemblies. Front. Microbiol. 11, 113. Zhang, Y. & Skolnick, J. Scoring function for automated assessment of protein structure
37 (2020). template quality. Proteins 57, 702–710 (2004).
87. Teufel, F. et al. SignalP 6.0 predicts all five types of signal peptides using protein language 114. Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective
models. Nat. Biotechnol. 40, 1023–1025 (2022). receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).
88. Woodcroft, B. J. et al. Comprehensive taxonomic identification of microbial species in 115. Eberhardt, J., Santos-Martins, D., Tillack, A. F. & Forli, S. AutoDock Vina 1.2.0: new docking
metagenomic data using SingleM and Sandpiper. Nat. Biotechnol. https://doi.org/ methods, expanded force field, and Python bindings. J. Chem. Inf. Model. 61, 3891–3898
10.1038/s41587-025-02738-1 (2025). (2021).
89. Amend, J. P. & LaRowe, D. E. Minireview: Demystifying microbial reaction energetics. 116. Letunic, I. & Bork, P. Interactive Tree of Life (iTOL) v6: Recent updates to the phylogenetic
Environ. Microbiol. 21, 3539–3547 (2019). tree display and annotation tool. Nucleic Acids Res. 52, W78–W82 (2024).
90. Cornell, R. M. & Schwertmann, U. The Iron Oxides: Structure, Properties, Reactions, 117. Chaumeil, P.-A., Mussig, A. J., Hugenholtz, P. & Parks, D. H. GTDB-Tk: A toolkit to classify
Occurrence and Uses (Wiley-VCH, 2003). genomes with the Genome Taxonomy Database. Bioinformatics 36, 1925–1927 (2019).
91. Rickard, D. & Morse, J. W. Acid volatile sulfide (AVS). Mar. Chem. 97, 141–197 (2005). 118. Parks, D. H. et al. A standardized bacterial taxonomy based on genome phylogeny
92. Majzlan, J., Navrotsky, A. & Schwertmann, U. Thermodynamics of iron oxides: part III. substantially revises the tree of life. Nat. Biotechnol. 36, 996–1004 (2018).
Enthalpies of formation and stability of ferrihydrite (∼Fe(OH)3), schwertmannite 119. Cai, L. et al. Tad pilus-mediated twitching motility is essential for DNA uptake and survival
(∼FeO(OH)3/4(SO4)1/8), and ε-Fe2O3. Geochim. Cosmochim. Acta 68, 1049–1059 (2004). of Liberibacters. PLoS ONE 16, e0258583 (2021).
93. Ritz, C., Baty, F., Streibig, J. C. & Gerhard, D. Dose–response analysis using R. PLoS ONE 120. Melville, S., & Craig, L. Type IV pili in Gram-positive bacteria. Microbiol. Mol. Biol. Rev. 77,
10, e0146021 (2015). 323–341 (2013).
94. Poulton, S. W., Krom, M. D. & Raiswell, R. A revised scheme for the reactivity of iron
(oxyhydr)oxide minerals towards dissolved sulfide. Geochim. Cosmochim. Acta 68,
3703–3715 (2004). Acknowledgements The authors thank B. Hausmann, K. Wasmund and T. S. Tanabe for help
95. Cline, J. D. Spectrophotometric determination of hydrogen sulfide in natural waters. with selection of sulfur-cycling target genes; M. Schmid for Raman analyses of ferrihydrite;
Limnol. Oceanogr. 14, 454–458 (1969). J. Ramesmayer for preparation of cDNA libraries; L. Seidl for technical assistance with elemental
96. Wan, M., Shchukarev, A., Lohmayer, R., Planer-Friedrich, B. & Peiffer, S. Occurrence of sulfur analysis; M. Watzka for isotope ratio mass spectrometry analysis; N. Kumar for support in
surface polysulfides during the interaction between ferric (hydr)oxides and aqueous ferrihydrite synthesis; O. Coruh for help with protein structure modelling; K. Schmidt, S. Dürr
sulfide. Environ. Sci. Technol. 48, 5076–5084 (2014). and D. Gruber for SEM and TEM imaging; T. Rattei and his team for maintaining and providing
97. Ehmann, T. et al. Modification and validation of the pyromellitic acid electrolyte for the access to the Life Science Compute Cluster (https://lisc.univie.ac.at/); the Bundesministerium
capillary electrophoretic determination of anions. J. Chromatogr. A 995, 217–226 (2003). für Bildung und Forschung (BMBF)-funded deNBI cloud within German Network for Bioinformatics
98. Thamdrup, B., Finster, K., Hansen, J. W. & Bak, F. Bacterial disproportionation of elemental Infrastructure (de.NBI) (no. 031A532B, 031A533A, 031A533B, 031A534A, 031A535A, 031A537A,
sulfur coupled to chemical reduction of iron or manganese. Appl. Environ. Microbiol. 59, 031A537B, 031A537C, 031A537D and 031A538A) for providing computational resources; and
101–108 (1993). H. Daims for valuable discussion. The drawing of pili was inspired by figures in Cai et al.
99. Hausmann, B. et al. Consortia of low-abundance bacteria drive sulfate reduction-dependent (2021)119 and Melville & Craig (2013)120. This research was funded by the Austrian Science Fund
degradation of fermentation products in peat soil microcosms. ISME J. 10, 2365–2375 (FWF) (grants https://doi.org/10.55776/P31996 and https://doi.org/10.55776/COE7), the EU
(2016). MSCA postdoctoral fellowship (action number 101059607, DatingSuCy) to S.-C.C., the National
100. Griffiths, R. I., Whiteley, A. S., O’Donnell, A. G. & Bailey, M. J. Rapid method for coextraction Natural Science Foundation of China (grants 42307167 and 42021005), and a Chinese
of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA- Scholarship Council fellowship to X.-M.L. For open access purposes, the author has applied a
based microbial community composition. Appl. Environ. Microbiol. 66, 5488–5491 (2000). CC BY public copyright license to any author accepted manuscript version arising from this
101. Ewels, P., Magnusson, M., Lundin, S. & Käller, M. MultiQC: summarize analysis results for submission.
multiple tools and samples in a single report. Bioinformatics 32, 3047–3048 (2016).
102. Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: fast, accurate and scalable read Author contributions S.-C.C., M.M. and A.L. conceived the study and planned experiments,
mapping by seed-and-vote. Nucleic Acids Res. 41, e108 (2013). with help from P.P. and S.P. S.-C.C. performed bioinformatic analyses. S.-C.C., X.-M.L., N.B., G.G.
103. Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site and M.A.M. designed and performed physiological experiments. S.-C.C., N.B., M.M. and M.A.M.
identification. BMC Bioinformatics 11, 119 (2010). quantified cell growth and metabolite concentrations. W.W. contributed to elemental sulfur
104. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion and isotope ratio mass spectrometry analysis. A.S. performed NanoSIMS analysis. S.-C.C. and
for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). M.A.M. extracted RNA from cultures incubated under different conditions. S.-C.C., J.O. and P.P.
105. Haynes, W. in Encyclopedia of Systems Biology (eds Dubitzky, W. et al.) 78–78 (Springer, performed comparative transcriptome analyses. W.W. and A.R. provided essential infrastructure
2013). for metabolite and isotope analyses. S.-C.C. analysed the data. S.-C.C., M.M. and A.L. interpreted
106. Ye, J. et al. Primer-BLAST: a tool to design target-specific primers for polymerase chain the data. S.-C.C. and A.L. wrote the paper, with comments from all other authors.
reaction. BMC Bioinformatics 13, 134 (2012).
107. Deng, X., Dohmae, N., Nealson, K. H., Hashimoto, K. & Okamoto, A. Multi-heme Funding Open access funding provided by University of Vienna.
cytochromes provide a pathway for survival in energy-limited environments. Sci. Adv. 4,
eaao5682 (2018). Competing interests The authors declare no competing interests.
108. Yu, N. Y. et al. PSORTb 3.0: Improved protein subcellular localization prediction with
refined localization subcategories and predictive capabilities for all prokaryotes. Additional information
Bioinformatics 26, 1608–1615 (2010). Supplementary information The online version contains supplementary material available at
109. Almagro Armenteros, J. J. et al. SignalP 5.0 improves signal peptide predictions using https://doi.org/10.1038/s41586-025-09467-0.
deep neural networks. Nat. Biotechnol. 37, 420–423 (2019). Correspondence and requests for materials should be addressed to Song-Can Chen,
110. Notredame, C., Higgins, D. G. & Heringa, J. T-Coffee: a novel method for fast and accurate Marc Mussmann or Alexander Loy.
multiple sequence alignment. J. Mol. Biol. 302, 205–217 (2000). Peer review information Nature thanks Andreas Schramm and the other, anonymous, reviewer(s)
111. Holm, L. & Park, J. DaliLite workbench for protein structure comparison. Bioinformatics for their contribution to the peer review of this work. Peer review reports are available.
16, 566–567 (2000). Reprints and permissions information is available at http://www.nature.com/reprints.
Extended Data Fig. 1 | Sulfide removal rates at low concentration of sulfide by fitting the first-order rate equation to the measured sulfide concentration
(ca. 50 µM) are higher in ferrihydrite-amended D. alkaliphilus cultures changes (see panel a). The center lines and box limits of the boxplot denote
than in abiotic controls. a, Kinetics of dissolved sulfide during 25-min the median, and the 25% and 75% percentile of the estimated rate constant,
incubation with ferrihydrite (ca. 62 mM) in the presence or absence of respectively. The whiskers extend 1.5 times the interquartile range from the
D. alkaliphilus. The incubation experiment was conducted independently with 25th and 75th percentiles. The two-sided Student’s t-test between biotic (n = 6)
three different cell densities, indicated by optical density at 600 nm (OD600) and abiotic (n = 6) treatment was conducted for each cell density. P values were
of the inoculum. Each incubation received three spikes of sulfide at 1.5-h adjusted for multiple comparisons (q-values) using the Benjamini and Hochberg
intervals. Two replicates were performed for each condition. OD600 of 0.042 method. d, Concentration of HCl-extractable Fe(II) before three consecutive
corresponds to an average cell density of 1.38 × 108 cells ml−1. b, Sulfide spikes of sulfide to ferrihydrite with or without D. alkaliphilus. The values
concentrations in the sulfide-only control of the incubation experiments represent the mean of duplicate culture with OD600 of 0.086. Fe(II) concentration
remained constant. The residual sulfide carried over from the inoculum was in both treatments increased with repeated spikes of low conditions of sulfide.
subtracted from the measured sulfide concentration. c, The rate constant of The treatment with cells produced an excessive amount of Fe(II) compared to
sulfide consumption was significantly higher (P < 0.01; two-sided Student’s the abiotic control. Fe(II) before the 1st sulfide spike results from biological or
t-test) in D. alkaliphilus cultures than in abiotic controls. The rate was determined chemical oxidation of sulfide (ca. 50 µM) carried over from the inoculum.
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Extended Data Fig. 2 | Transformation of sulfide during the incubation of used for the incubation experiment had an OD600 of 0.072. b, Kinetics of
D. alkaliphilus with ferrihydrite (ca. 62 mM) and periodic supply (n = 8) of a dissolved sulfide following the first three spikes to ferrihydrite-amended
low concentration of sulfide (ca. 50 µM) at 1.5-h intervals. a, Development culture in a parallel incubation experiment with the same inoculum as in panel a.
of sulfate, HCl-extractable Fe(II), elemental sulfur (S(0)), and Cline-extractable Sulfide measurements from two biological replicates are displayed. The data
sulfide during the incubation experiment (n = 3 replicate cultures). Parallel confirmed that the culture consumed sulfide more rapidly than the abiotic
incubations with living cells without Fh or sulfide, and with killed cells served control. c, Schematic graph showing the flow of spiked sulfide during the 12-h
as controls. The arrows in each panel indicate the addition of sulfide. The red incubation. Sulfur metabolite concentrations derive from data in panel a. All
stars denote sampling from the culture for chemical analysis. The inoculum values are the means of triplicate cultures with standard deviation.
Extended Data Fig. 3 | Genomic arrangement of genes putatively involved adhesion (Tad) type IV pilus. The gene name and locus tag are shown above and
in MISO. a, Genes for reversal of the canonical sulfate reduction pathway. below the gene arrows, respectively. Count of heme-binding motifs (CX 2-5CH)
b, Genes for the up-regulated multi-heme c-type cytochromes (MHCs) during is shown for each MHC in b. Co-localized genes not shown in Fig. 5 are colored
MISO. c, Genes for the PilA-containing type IV pilus. d, Genes for the tight in gray.
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Extended Data Fig. 4 | Up-regulated transcription of 13 multi-heme c-type method. The 13 MHCs showed significantly elevated relative transcription levels
cytochromes (MHCs) in D. alkaliphilus under ferrihydrite-amended (adjusted P < 0.01) in all pairwise comparisons (n = 6) between ferrihydrite-
conditions. Comparative transcriptomics of three ferrihydrite-amended and amended incubations (n = 3) vs. incubations (n = 2) without ferrihydrite. Electron
two ferrihydrite-free growth conditions (n = 4 replicate cultures each). The donors and acceptors for each of the five incubations are indicated above the
heatmap is based on the z-score of reads per kilobase of transcript per million heatmap. Four biological replicates of each incubation are displayed side-by-side.
mapped reads (RPKM) after logarithm transformation. The statistical significance The subcellular localization of MHCs was predicted by Psortb v3.0 and
(P-value) of up-regulation was calculated using the DESeq2 R package, followed DeepLocPro v1.0. PCC MHC, MHC from a gene cluster encoding a putative
by adjustment of P for multiple comparison using the Benjamini and Hochberg porin-cytochrome complex (PCC).
Extended Data Fig. 5 | Sequence alignment and predicted structure of the cryo-EM structure of OmcS28. c, Structural comparison between DA_402 and
most expressed multi-heme c-type cytochrome (DA_402) of Desulfurivibrio OmcS. DA_402 shares significant topological similarity with OmcS, with a
alkaliphilus during MISO. a, Pairwise alignment between DA_402 and the TM-score of 0.56 and DALI-score of 14.3. d, Spatial arrangement of six heme-
OmcS from G. sulfurreducens. Six out of seven predicted heme-binding motifs binding sites in DA_402 (cyan). All heme-binding sites predicted in DA_402
(CX 2-5CH) are aligned with those in OmcS (labeled with numbers in circles). align closely with those in OmcS (yellow), except the site 1’ showing shifted
The seventh heme-binding motif lacks the paired histidine (blue color) for position. e, Predicted heme position of the heme-binding site 1’. Molecular
coordination of heme in the predicted structure of DA_402 and is therefore docking analysis placed the heme (1’) in close proximity to the adjacent heme ②,
not considered in the following analysis. The leading signal peptide in DA_402 with an iron-to-iron distance of 9.1 Å, comparable with the distance (12.5 Å)
and OmcS is not shown. b, Predicted structure of DA_402 oligomer. DA_402 observed in OmcS.
monomers are predicted to polymerize into a filament structure, similar to the
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Extended Data Fig. 6 | Desulfurivibrionaceae members with genomic component (pilin) of the type IV pilus; PCC, porin-cytochrome complex.
potential for MISO are widespread across various ecosystems. b, Phylogeny of DsrB from Desulfurivibrionaceae shown in a, which are
a, Co-occurrence of marker proteins for dissimilatory sulfide oxidation and classified as the reductive-type DsrB putatively involved in sulfide oxidation, as
iron reduction in diverse Desulfurivibrionaceae members recovered from proposed in D. alkaliphilus and in cable bacteria. Known sulfide oxidizers (e.g.,
different ecosystems. Phylogenomic tree of Desulfurivibrionaceae is based on cable bacteria genus Electrothrix) encoding reductive-type DsrBs are indicated
maximum-likelihood analysis of a concatenated alignment of 120 bacterial by red squares. c, Phylogeny of OmcS-like proteins from Desulfurivibrionaceae
single-copy proteins. 119 of 205 Desulfurivibrionaceae genomes encode both shown in a, which form a sister clade of OmcS. The sister clade of OmcS comprise
dissimilatory sulfur and iron metabolisms. For visualization purposes, the extracellular MHCs from known iron reducers (dark green squares) and three
phylogeny includes only 53 de-replicated genomes. D. alkaliphilus is shown in MHCs (DA_402, DA_756, and DA_765) from D. alkaliphilus that are up-regulated
bold and red type. The source ecosystem of each genome is denoted by filled under MISO conditions. All OmcS-like proteins shown in a are predicted to be
circles at the tips of the tree. Environmental origin, NCBI accession, and genus extracellular multiheme c-type cytochromes (MHCs). Blue circles indicate the
classification is shown at each branch tip. Desulfobulbaceae members were numbers of Desulfurivibrionaceae MHC genes that are placed to the tree branches
used as an outgroup. Presence and absence of marker proteins involved in using RAxML EPA algorithm. The scale bar in a, b, and c indicates the substitutions
sulfide oxidation (DsrAB, red squares) and extracellular respiration of iron of amino acids per site. The branches with bootstrap values > 50 are shown with
oxides (green squares) is shown for each genome. Abbreviation: OmcS and black dots in a and b.
OmcZ, two evolutionarily unrelated outer membrane cytochromes; PilA, major