Comparative Genomics on Cultivated and Uncultivated
Freshwater and Marine
“
Candidatus
Manganitrophaceae
”
Species Implies Their Worldwide Reach in Manganese
Chemolithoautotrophy
Hang Yu
,
a
Grayson L. Chadwick
,
a
Usha F. Lingappa
,
a
Jared R. Leadbetter
a
,
b
a
Division of Geological & Planetary Sciences, California Institute of Technology, Pasadena, California, USA
b
Division of Engineering & Applied Science, California Institute of Technology, Pasadena, California, USA
ABSTRACT
Chemolithoautotrophic manganese oxidation has long been theorized but
only recently demonstrated in a bacterial coculture. The majority member of the cocul-
ture,
“
Candidatus
Manganitrophus noduliformans,
”
is a distinct but not yet isolated line-
age in the phylum
Nitrospirota
(
Nitrospirae
). Here, we established two additional
MnCO
3
-oxidizing cultures using inocula from Santa Barbara (California) and Boetsap
(South Africa). Both cultures were dominated by strains of a new species, designated
“
Candidatus
Manganitrophus morganii.
”
The next most abundant members differed in
the available cultures, suggesting that while
“
Ca.
Manganitrophus
”
species have not
been isolated in pure culture, they may not require a speci
fi
c syntrophic relationship
with another species. Phylogeny of cultivated
“
Ca.
Manganitrophus
”
and related meta-
genome-assembled genomes revealed a coherent taxonomic family,
“
Candidatus
Manganitrophaceae,
”
from both freshwater and marine environments and distributed
globally. Comparative genomic analyses support this family being Mn(II)-oxidizing che-
molithoautotrophs. Among the 895 shared genes were a subset of those hypothesized
for Mn(II) oxidation (Cyc2 and PCC_1) and oxygen reduction (TO_1 and TO_2) that
could facilitate Mn(II) lithotrophy. An unusual, plausibly reverse complex 1 containing 2
additional pumping subunits was also shared by the family, as were genes for the
reverse tricarboxylic acid carbon
fi
xation cycle, which could enable Mn(II) autotrophy.
All members of the family lacked genes for nitri
fi
cation found in
Nitrospira
species. The
results suggest that
“
Ca.
Manganitrophaceae
”
share a core set of candidate genes for
the newly discovered manganese-dependent chemolithoautotrophic lifestyle and likely
have a broad, global distribution.
IMPORTANCE
Manganese (Mn) is an abundant redox-active metal that cycles in many
of Earth
’
s biomes. While diverse bacteria and archaea have been demonstrated to
respire Mn(III/IV), only recently have bacteria been implicated in Mn(II) oxidation-de-
pendent growth. Here, two new Mn(II)-oxidizing enrichment cultures originating
from two continents and hemispheres were examined. By comparing the community
composition of the enrichments and performing phylogenomic analysis on the abun-
dant
Nitrospirota
therein, new insights are gleaned on cell interactions, taxonomy,
and machineries that may underlie Mn(II)-based lithotrophy and autotrophy.
KEYWORDS
autotroph, lithotroph, chemolithoautotroph, manganese oxide,
manganese carbonate,
Nitrospirae
,
Nitrospirota
, Mn, Mn
2
1
, Mn(II)
M
embers of the bacterial phylum
Nitrospirota
(formerly
Nitrospirae
) are best known
for performing dif
fi
cult physiologies that exploit the utilization of unusually high
potential electron donors or low potential electron acceptors (1, 2). Cultivated
Editor
Colleen M. Cavanaugh, Harvard
University
Copyright
© 2022 Yu et al. This is an open-
access article distributed under the terms of
the
Creative Commons Attribution 4.0
International license
.
Address correspondence to Hang Yu,
hyu@caltech.edu, or Jared R. Leadbetter,
jleadbetter@caltech.edu.
The authors declare no con
fl
ict of interest.
Received
12 November 2021
Accepted
24 February 2022
Published
14 March 2022
March/April 2022 Volume 13 Issue 2
10.1128/mbio.03421-21
1
RESEARCH ARTICLE
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organisms representing this phylum cluster within 4 clades. Order
Nitrospirales
(for-
merly genus
Nitrospira
) plays an important role in the nitrogen cycle, carrying out nitrite
oxidation (3, 4) and complete ammonium oxidation to nitrate (5, 6). Class
Leptospirilla
(formerly genus
Leptospirillum
) thrive in low-pH environments oxidizing iron (7). Class
Thermodesulfovibria
(formerly genus
Thermodesulfovibrio
) includes high-temperature
dissimilatory sulfate reducers (8), some with the capacity of S disproportionation (9), as
well as uncultivated magnetotactic bacteria (10). Recently, a bacterial coculture was
demonstrated to perform Mn(II) oxidation-dependent chemolithoautotrophic growth
(11). This metabolism was attributed to a member of a previously uncultivated clade of
Nitrospirota
,
“
Candidatus
Manganitrophus noduliformans
”
strain Mn1, given that the mi-
nority member in the coculture,
Ramlibacter lithotrophicus
(
Comamonadaceae
; formerly
within the
Betaproteobacteria
, now within
Gammaproteobacteria
) could be isolated yet
would not oxidize Mn(II) alone (11). Based on 16S rRNA gene phylogeny, relatives of
strain Mn1 were identi
fi
ed around the world and in diverse freshwater ecosystems (11).
However, whether or not these relatives share the same Mn(II) oxidation metabolism
was not something that could be gleaned from their rRNA genes.
Mn is the third most abundant redox-active metal in the Earth
’
s crust and is actively
cycled (12
–
14). Microbial reduction of Mn oxides for growth has been demonstrated in
numerous bacterial and archaeal phyla (14
–
18). The notion that microbial oxidation of
Mn(II) with O
2
could serve as the basis for chemolithoautotrophic growth was
fi
rst theor-
ized decades ago (13, 14, 19, 20). This metabolism, while energetically favorable (
D
G
°
9
=
2
68 kJ/mol Mn), poses a biochemical challenge to the cell because of the high average
potential of the two Mn(II)-derived electrons [Mn(II)/Mn(IV), E°
9
=
1
466 mV (11)]. These
electrons would need their redox potential to be lowered by nearly a full volt in order to
reduce the ferredoxin (
E
°
9
=
2
320 to
2
398 mV [21]) employed in their CO
2
fi
xation path-
way (11). This is a larger and more signi
fi
cant mismatch in redox potential than similar
chemolithotrophic metabolisms, such as nitrite or iron oxidation [NO
2
2
/NO
3
2
,E°
9
=
1
433 mV (21); Fe(II)/Fe(III), E°
9
of
;
0 mV (22)]. Based on deduced homology with charac-
terized proteins involved with Fe(II) oxidation or aerobic metabolism, genes for 4 puta-
tive Mn-oxidizing complexes and 5 terminal oxidases were identi
fi
ed in strain Mn1 and
proposed as candidates for energy conservation via electron transport phosphorylation
(11). Remarkably, gene clusters for 3 different complex I exist in strain Mn1 and could
facilitate the otherwise endergonic coupling of Mn(II) oxidation to CO
2
reduction, allow-
ing for autotrophic growth via reverse electron transport, i.e., expending motive force to
drive down electron reduction potential (11). The apparent redundancy of diverse novel
complexes in several members of the family remains puzzling. It seems clear that the
identi
fi
cation and analysis of additional strains and genomes of Mn(II)-oxidizing chemoli-
thoautotrophs could shed light on the complexes essential for this newfound mode of
metabolism.
The ever-increasing number of metagenome-assembled genomes (MAGs) available in
the databases provides for an unprecedented opportunity to learn about the gene con-
tent and potential functions of many uncultured microorganisms. However, cultivation
remains critical to forming interconnections between the genomes of both cultured and
uncultivated microbes and their metabolisms. Here, we successfully established new
enrichment cultures performing chemolithoautotrophic Mn oxidation from two disparate
environmental inoculum sources. By comparing the MAGs of the most abundant organ-
isms present in these enrichments, members of the
Nitrospirota
,aswellas66newlyand
publicly available MAGs in the databases belonging to
Nitrospirota
clades with unexa-
mined metabolisms, we gain insight into a core set of candidate genes for facilitating che-
molithoautotrophic Mn oxidation as well as the phylogenetic and geographic distribution
of known and putatively Mn-oxidizing
Nitrospirota
.
RESULTS
Reproducible cultivation of Mn-oxidizing chemolithoautotrophs.
“
Ca.
Manganitrophus noduliformans
”
strain Mn1 was accidentally enriched in tap water (11).
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Using the de
fi
ned Mn(II) carbonate medium in this previous study (11), new Mn-oxidizing
enrichment cultures were successfully established from two distinct sample sources. One
inoculum was material from a Mn oxide-containing rock surface near Boetsap, Northern
Cape, South Africa (South Africa enrichment), and the other inoculum was material from
an iron oxide microbial mat in Santa Barbara, CA, USA (Santa Barbara enrichment). While
the new enrichments grew in the same de
fi
ned freshwater medium, they exhibited differ-
ent temperature optima. The South Africa enrichments initially grew at 28.5°C, although
they oxidized Mn(II) faster at 32°C, similar to the previous enrichment from the Pasadena
drinking water distribution system (Pasadena enrichment) (11). The Santa Barbara enrich-
ments grew at 28.5°C but not at 32°C. Otherwise, the three enrichment cultures exhibited
similar phenotypes, including the formation of small Mn oxide nodules. These results indi-
cate that the de
fi
ned Mn(II) carbonate medium can successfully be employed during
intentional, directed attempts to cultivate Mn-oxidizing chemolithoautotrophs from
diverse terrestrial and aquatic freshwater environments.
Community analysis of Mn-oxidizing enrichment cultures from three origins.
As was the case with cultures of
“
Ca.
M. noduliformans,
”
repeated attempts to identify sin-
gle colonies of the lithotrophs responsible for Mn oxidation were not successful on an
agar-solidi
fi
ed, de
fi
ned Mn(II) carbonate medium. Sequencing of partial 16S rRNA genes
ampli
fi
ed from the liquid cultures revealed differences in community structures between
the Mn-oxidizing enrichments. The most abundant microorganism from the South Africa
and Santa Barbara enrichments belonged to the same taxon as the previously described
“
Ca.
M. noduliformans
”
(Fig. 1). However, the identities of the next most abundant mem-
bers of the communities differed. The previously described Pasadena enrichment contain-
ing
“
Ca.
M. noduliformans
”
had
Ramlibacter lithotrophicus
as the second most abundant
member throughout the enrichment re
fi
ning process (see Table S1 in the supplemental
material).
R. lithotrophicus
could be isolated from the enrichment using the same de
fi
ned
medium but with other electron donors such as succinate and hydrogen but could not
oxidize Mn(II) as an isolate (11). Organisms belonging to the same taxon as
R. lithotrophi-
cus
were present in the South Africa enrichments, varying from 2 to 28 in rank abundance,
but were not abundant in Santa Barbara enrichments (
,
0.5% relative abundance) (Fig. 1
andTableS1).IntheSouthAfricaenrichments,thesecondmostabundantmembervaried
between a
Pseudomonas
species (
Gammaproteobacteria
), a member of the
Zavarziniales
(
Alphaproteobacteria
),
R. lithotrophicus
,and
Hydrogenophaga
(a
Comamonadaceae
closely
related to
R. lithotrophicus
) (Fig. 1). In the Santa Barbara enrichments, the second most
abundant member was a member of the
Anaerolineaceae
(phylum
Chloro
fl
exi
or
Chloro
fl
exota
; Fig. 1). Changing the incubation temperature did not affect the identities of
the 3 most abundant taxa in the South Africa enrichments (Fig. 1). However, the choice of
nitrogen source in the medium resulted in a shift in community member relative abun-
dances (Fig. 1). Notably, the only other shared organism between South Africa, Santa
Barbara, and Pasadena enrichments with
.
1% relative abundance was a member of
the
Zavarziniales
(Fig. 1 and Table S1). Its relative abundance markedly increased
when the South Africa enrichments were grown in medium with nitrate instead of
ammonia as the nitrogen source. Overall, while the community composition varied
between the Mn-oxidizing enrichments, strains of
“
Ca.
Manganitrophus
”
were consis-
tently the most abundant species in all such cultures.
Expansion of MAGs of cultivated and environmental Mn-oxidizing
Nitrospirota
.
We performed shotgun metagenomic sequencing on two of the new Mn-oxidizing
enrichments in order to gain phylogenetic and functional insights into the newly culti-
vated
“
Ca.
Manganitrophus
”
strains. We reconstructed high-quality metagenome-
assembled genomes (MAGs) (
.
97% completeness,
,
5% contamination) (23) of the
most abundant organism from each metagenome (Table S1). We refer to these MAGs
as strains SA1 and SB1 to indicate that they originated from South Africa and Santa
Barbara, respectively. Both genome and 16S rRNA gene phylogenies con
fi
rmed that
strain SA1 and strain SB1 were related to the previously characterized
“
Ca.
M. noduli-
formans
”
strain Mn1 (Fig. 2). Based on their average nucleotide identities (ANI) and
using 95% ANI as a possible metric for species delineation (24
–
26), strains SA1 and SB1
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FIG 1
Community analysis of manganese-oxidizing enrichment cultures using partial 16S rRNA gene amplicon
sequencing. Taxonomic classi
fi
cation is based on the SILVA small subunit rRNA database v138. Detailed taxon
relative abundances can be found in Table S1.
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were provisionally considered to represent distinct strains of the same species (96%
ANI). Both could be considered a different species than strain Mn1 (94% ANI)
(Table S3). The genome sizes of these 2 new strains were smaller (4.3 Mb) than that of
strain Mn1 (5.2 Mb) (Table S2). The arrangements of homologous regions in strains SA1
and SB1 were similar (Fig. S1a) but were different from that of strain Mn1 (Fig. S1b).
These differences were also observed at the deduced protein level, with strains SA1
and SB1 more closely related to each other than to strain Mn1 (Table S4). These
FIG 2
Phylogenetic analysis of the bacterial phylum
Nitrospirota
. (A) Multilocus phylogram, based on a Bayesian analysis of 5,040 aligned amino acid
positions concatenated from 120 bacterial protein markers. (B) 16S rRNA gene phylogram, based on a Bayesian analysis of 1,508 aligned nucleotide
positions. For both panels A and B, NCBI accession numbers or IMG contig identi
fi
ers for the genome assemblies or 16S sequences are in the node names,
with their source environments shown in parentheses. Two phylograms can be linked by the genomes assemblies that contain 16S rRNA genes, with
environmental metagenomes in brown and manganese-oxidizing enrichment cultures in red. Previously described taxonomic groups based on GTDB
taxonomic classi
fi
cations and the proposed taxonomic groups are grouped by color.
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variations in the proteins were not concentrated in one genomic region but instead
scattered throughout the genome (Fig. S1c). Further,
de novo
gene clustering showed
that strains SA1 and SB1 shared more genes with each other than with strain Mn1
(Fig. S1d). Altogether, our results support strains SA1 and SB1 as distinct species, which
we designate
“
Candidatus
Manganitrophus morganii
”
(Text S1). These 3 cultivated
“
Ca.
Manganitrophus
”
strains in two different species provide a basis to examine the phylo-
genetic and genomic diversity of their shared metabolism, namely, Mn-oxidizing
chemolithoautotrophy.
In addition to reconstructing MAGs from Mn-oxidizing enrichments, we also ana-
lyzed publicly available MAGs in the phylum
Nitrospirota
. We screened for MAGs that
did not belong in the three characterized clades, namely,
Nitrospirales
,
Leptospirilla
,
and
Thermodesulfovibria
. As of 26 March 2019, only 3 MAGs had met this taxonomic cri-
teria with completeness of
.
50% and contamination of
,
5% (11). However, as of 30
March 2021, 64 new public high-quality (
.
90% completeness,
,
5% contamination)
and 2 medium-quality (
.
50% completeness,
,
10% contamination) MAGs meeting
these taxonomic criteria had become available (Table S5). These 66 MAGs allowed for a
much more detailed phylogenomic view into the uncultivated
Nitrospirota
and their
potential ability to oxidize Mn.
16S rRNA gene and multilocus protein phylogeny reveal robust taxonomic
groups.
The available MAGs provide a phylogenetic resolution that matches the tradi-
tionally employed 16S rRNA genes (Fig. 2). The MAGs were spread out across different
phylogenetic clusters within the phylum (Fig. 2A). Using the 14 MAGs that also contained
16S rRNA genes, we were able to link the genome phylogeny to the 16S rRNA gene phy-
logeny and observed similar clusterings between the two phylogenetic approaches
(Fig. 2). The 3 cultivated strains all resided within the genus
“
Ca.
Manganitrophus.
”
Other
members of
“
Ca.
Manganitrophus,
”
based on either their genomes or 16S rRNA genes,
were from terrestrial, aquatic, and engineered environments and all freshwater in origin
(Fig. 2). Our phylogeny revealed a sister genus of marine origin (Fig. 2). Together, these
two genera form a coherent and well-supported phylogenetic clade, here termed family
“
Candidatus
Manganitrophaceae
”
(Fig. 2).
Previously, the class
“
Candidatus
Troglogloea
”
was proposed to encompass strain Mn1
and
“
Candidatus
Troglogloea absoloni
”
(an uncultivated species from Vjetrenica cave in
the Dinaric Karst) based on their 16S rRNA gene phylogeny (11). Based on our new phylo-
genomic analysis, we propose that the order
“
Ca.
Troglogloeales
”
includes the family
“
Ca.
Manganitrophaceae,
”“
Ca.
T. absoloni,
”
and its relatives (Fig. 2), together constituting a sis-
ter group distinct from the order
Nitrospirales
(which includes the cultivated nitrite and
ammonia-oxidizing
Nitrospirota
). These genera, family, and order proposals are consistent
with the latest taxonomic classi
fi
cation in the Genome Taxonomy Database (GTDB) release
06-RS202 April 2021 (27, 28), even though GTDB currently contains fewer genomes. Based
on the current GTDB taxonomy, both orders
“
Ca.
Troglogloeales
”
and
Nitrospirales
are
placed within the class
Nitrospiria
, but this is incongruent with analyses of their 16S rRNA
phylogeny (Fig. 2B). Numerous
Nitrospirota
MAGs fall outside the three known groups of
Nitrospirota
(
Nitrospirales
,
Leptosprillia
,and
Thermodesulfovibriona
) and are overrepre-
sented in subsurface and aquatic environments. However, 16S rRNA gene surveys indicate
that members of many of the uncultivated clades exist from marine, soil, and sediment
environments but are not represented by genomes (Fig. 2B). Overall, while the taxonomic
relationship between orders
“
Ca.
Troglogloeales
”
and
Nitrospirales
and the assignment of
classes in
Nitrospirota
remains to be resolved, our proposals of the genus
“
Ca.
Manganitrophus,
”
family
“
Ca.
Manganitrophaceae,
”
and order
“
Ca.
Troglogloeales
”
are
supportedbyboth16SrRNAgeneandgenomephylogeneticapproachesandreveal
members of a novel marine genus that possibly oxidize Mn lithotrophically.
Genome comparison streamlines the hypothesized genes for Mn-oxidizing
lithotrophy.
We next compared the MAGs of members of the family
“
Ca.
Manganitrophaceae
”
to understand which genes might be candidates essential for Mn
oxidation and whether these are found in representatives of the marine genus or other
members in the phylum. Four routes for Mn oxidation and electron uptake had been
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previously hypothesized in strain Mn1, including a fused cytochrome-porin protein with a
single heme
c
(Cyc2) and three different porin-dodecaheme cytochrome
c
(PCC) com-
plexes (11). Cyc2 homologs are not only identi
fi
ed in the majority of
“
Ca.
Troglogloeales
”
(Fig. 3A) but also in other members of the phylum, including characterized clades such as
acidophilic, iron-oxidizing
Leptospirilla
and nitrite- or ammonia-oxidizing
Nitrospirales
(29,
30). Of the 3 PCCs in strain Mn1, only PCC_1 was found in the strains SA1 and SB1
(Fig. 3A). PCC_1 was also identi
fi
ed in other MAGs in both marine and freshwater genera
of
“
Ca.
Manganitrophaceae
”
but not in the extant MAGs and genomes of
Nitrospirota
spe-
cies falling outside this family. These results point to PCC_1, possibly together with Cyc2,
as being central to chemolithotrophic Mn oxidation by
“
Ca.
Manganitrophaceae.
”
Not all previously proposed membrane complexes by which strain Mn1 might
reduce oxygen and conserve energy were shared by all
“
Ca.
Manganitrophaceae
”
members. A canonical complex IV (
cbb3
-type cytochrome
c
oxidase) was identi
fi
ed for
all members of the freshwater genus but not for any in the marine genus (Fig. 3A).
FIG 3
Metabolic genes and gene clusters of interest in metagenome-assembled genomes representing the order
“
Candidatus
Troglogloeales.
”
(A) The
multilocus protein phylogram and the presence (yellow
fi
lled square) or absence (empty square) of genes and gene clusters of interest in the
corresponding genomes. Putative functional assignments are proposed above the gene and gene cluster names. The phylogram (left) is extracted from
Fig. 2. (B and C) Comparison of gene clusters of porin cytochrome
c
1 (PCC_1) (B) and terminal oxidase 2 (TO_2) (C), both restricted to the family
“
Candidatus
Manganitrophaceae.
”
Members of the freshwater genus
“
Candidatus
Manganitrophus
”
share similar gene arrangements, which differ from
those representing the
Candidatus
marine genus (in brown).
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