of 32
1
Title: Comparative genomics on cultivated and uncultivated, freshwater and marine
Candidatus
1
Manganitrophaceae species implies their worldwide reach in manganese chemolithoautotrophy
2
3
Running title: Phylogenomics of manganese
-
oxidizing
Nitrospirota
4
5
Keywords: autotroph, lithotroph, chemolithoautotroph, manganese oxide, manganese carbonate,
6
Nitrospirae, Nitrospirota, Mn, Mn2+, Mn(II), Mn (II)
7
8
Authors: Hang Yu
a
, Grayson L. Chadwick
a
, Us
ha F. Lingappa
a
, Jared R. Leadbetter
a,b
9
10
a
Division of Geological & Planetary Sciences and
b
Division of Engineering & Applied Science,
11
California Institute of Technology, Pasadena, CA, USA 91125
12
13
Correspondence: Hang Yu (hyu@caltech.edu) and Jared R. Leadbe
tter (jleadbetter@caltech.edu)
14
15
.
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;
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2
Abstract
16
Chemolithoautotrophic manganese oxidation has long been theorized, but only recently
17
demonstrated in a bacterial co
-
culture. The majority member of the co
-
culture,
Candidatus
18
Manganitrophus noduliformans, is a distinct but not yet isolated lineage in the p
hylum
Nitrospirota
19
(
Nitrospirae
). Here, we established two additional MnCO
3
-
oxidizing cultures using inocula from
20
Santa Barbara (USA) and Boetsap (South Africa). Both cultures were dominated by strains of a
21
new species, designated
Candidatus
Manganitrophus
morganii. The next abundant members
22
differed in the available cultures, suggesting that while
Ca.
Manganitrophus species have not been
23
isolated in pure culture, they may not require a specific syntrophic relationship with another
24
species. Phylogeny of cul
tivated
Ca.
Manganitrophus and related metagenome
-
assembled
25
genomes revealed a coherent taxonomic family,
Candidatus
Manganitrophaceae, from both
26
freshwater and marine environments and distributed globally. Comparative genomic analyses
27
support this family
being Mn(II)
-
oxidizing chemolithoautotrophs. Among the 895 shared genes
28
were a subset of those hypothesized for Mn(II) oxidation (Cyc2 and PCC_1) and oxygen reduction
29
(TO_1 and TO_2) that could facilitate Mn(II) lithotrophy. An unusual, plausibly reverse C
omplex
30
1 containing 2 additional pumping subunits was also shared by the family, as were genes for the
31
reverse TCA carbon fixation cycle, which could enable Mn(II) autotrophy. All members of the
32
family lacked genes for nitrification found in
Nitrospira
spe
cies. The results suggest that
Ca.
33
Manganitrophaceae share a core set of candidate genes for the newly discovered manganese
34
dependent chemolithoautotrophic lifestyle, and likely have a broad, global distribution.
35
36
Importance
37
Manganese (Mn) is an abundant r
edox
-
active metal that cycled in many of Earth’s biomes. While
38
diverse bacteria and archaea have been demonstrated to respire Mn(III/IV), only recently have
39
bacteria been implicated in Mn(II) oxidation dependent growth. Here, two new Mn(II)
-
oxidizing
40
enric
hment cultures originated from two continents and hemispheres were examined. By
41
comparing the community composition of the enrichments and performing phylogenomic analysis
42
on the abundant
Nitrospirota
therein, new insights are gleaned on cell interactions,
taxonomy, and
43
machineries that may underlie Mn(II)
-
based lithotrophy and autotrophy.
44
45
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3
Introduction
46
Members of the bacterial phylum
Nitrospirota
(formerly
Nitrospirae
) are best known for having
47
physiologies that exploit the utilization of high potential electron donors or low potential electron
48
acceptors
(1, 2)
. Cultivated organisms representing this phylum cluster within 4 clades. Order
49
Nitrospirales
(formerly genus
Nitrospira
) plays an important role in the nitrogen cycle, carrying
50
o
ut nitrite oxidation
(3, 4)
and complete ammonium oxidation to nitrate
(5, 6)
. Class
Leptospirilla
51
(formerly genus
Leptospirillum
) thrive in low pH environments oxidizing iron
(7)
. Class
52
Thermodesulfovibria
(formerly genus
Thermodesulfovibrio
) includes high temperature
53
dissimila
tory sulfate
-
reducers
(8)
, some with the capacity of S disproportionation
(9)
, as well as
54
uncultivated magnetotactic bacteria
(10)
. Recently, a bacterial co
-
culture was demonstrated to
55
p
erform Mn(II) oxidation dependent chemolithoautotrophic growth
(11)
. This metabolism wa
s
56
attributed to a member of a previously uncultivated clade of
Nitrospirota
,
Candidatus
57
Manganitrophus noduliformans strain Mn1, given that the minority member in the co
-
culture,
58
Ramlibacter lithotrophicus
(
Comamonadaceae
; formerly within the
Betaproteobac
teria
, now
59
within
Gammaproteobacteria
) could be isolated yet would not oxidize Mn(II) alone
(11)
. Based
60
on 16S rRNA gene phylogeny, several relatives of strain Mn1 were identified
(11)
. However,
61
whether or not these relatives might share the same Mn(II) oxidation metabolism was not
62
something that could be gleaned from their rRNA genes.
63
64
Mn is the third most abundant redox
-
active metal in the Earth’s crust and is actively cycled
(12
65
14)
. Microbial reduction of Mn oxides for growth has been demonstrated in numerous bacterial
66
and archaeal ph
yla
(14
18)
. The notion that microbial oxidation of Mn(II) with O
2
could serve as
67
the basis for chemolithoautotrophic growth was first theorized decades ago
(13, 14, 19, 20)
. This
68
metabolism, while energetically favorable (ΔG°′ = −68 kJ/mol Mn),
poses a biochemical challenge
69
to the cell because of the high average potential of the two Mn(II)
-
derived electrons
70
(Mn(II)/Mn(IV), E°′ = +466 mV
(11)
). These electrons would need their redox potential to be
71
lowered by nearly a full volt in order to
reduce the ferredoxin (E°′ = −320 to
-
398 mV
(21)
)
72
employed in their CO
2
fixation pathway
(11)
. This is a larger and more significant mismatch in
73
redox potential than similar chemolithotrophic metabolisms, such as nitrite or iron oxidation (NO
2
-
74
/NO
3
-
, E°′ = +433 mV
(21)
; Fe(II)/Fe(III), E°′ ~ 0 mV
(22)
). Based on deduced homology with
75
characterized proteins involved with Fe(II) oxidation or aerobic metabolism,
genes for 4 putative
76
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4
Mn
-
oxidizing complexes and 5 terminal oxidases were identified in strain Mn1 and proposed as
77
candidates for energy conservation via electron transport phosphorylation
(11)
. Remarkably, gene
78
clusters for 3 different Complex I exist in strain Mn1
and could facilitate the
otherwise
endergonic
79
coupling of Mn(II) oxidation to
CO
2
reduction, allowing for autotrophic growth via reverse
80
electron transport, i.e. expending motive force to drive down electron reduction potential
(11)
. The
81
apparent redundancy of diverse novel complexes
in several members of the family
remains
82
puzzling
.
It seems clear that the identification and analysis of additional strains and genome
s of
83
Mn(II)
-
oxidizing chemolithoautotrophs could likely shed light on the complexes essential for this
84
newfound mode of metabolism.
85
86
The ever increasing number of metagenome
-
assembled genomes (MAGs) available in the
87
databases provides for an unprecedented
opportunity to learn about the gene content and potential
88
functions of many uncultured microorganisms. Yet, cultivation remains critical to forming
89
interconnections between the genomes of both cultured and uncultivated microbes and their
90
metabolisms. Here
in, we successfully established new in vitro enrichment cultures performing
91
chemolithoautotrophic Mn oxidation from two disparate environmental inoculum sources. By
92
comparing the MAGs of the most abundant organisms present in these enrichments, also member
s
93
of the
Nitrospirota,
as well as 66 newly and publicly available MAGs in the databases belonging
94
to Nitrospirota clades with unexamined metabolisms, we gain insight into a core set of candidate
95
genes for facilitating chemolithoautotrophic Mn oxidation, as
well as the phylogenetic and
96
geographic distribution of known and putatively Mn
-
oxidizing
Nitrospirota
.
97
98
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5
Results
99
Reproducible cultivation of Mn
-
oxidizing chemolithoautotrophs.
Ca.
Manganitrophus
100
noduliformans strain Mn1 was accidentally enriched in tap water
(11)
. U
sing the defined Mn(II)
101
carbonate medium in this previous study
(11)
, new Mn
-
oxidizing
enrichment cultures were
102
successfully established from two distinct sample sources. One inoculum was material from a Mn
103
oxide containing rock surface near Boetsap, Northern Cape, South Africa (“South Africa
104
enrichment”), and the other inoculum was material
from an iron oxide microbial mat in Santa
105
Barbara, California, USA (“Santa Barbara enrichment”). While the new enrichments grew in the
106
same defined freshwater medium, they exhibited different temperature optima. The South Africa
107
enrichments initially grew
at 28.5°C, although they oxidized Mn(II) faster at 32°C, similar to the
108
previous enrichment from the Pasadena drinking water distribution system (“Pasadena
109
enrichment”)
(11)
. The Santa Barbara enrichments grew at 28.5°C, but not at 32°C. Otherwise, no
110
striking differences in appearance (e.g. formation of small Mn oxide nodular products) bet
ween
111
the three cultures was observed. These results indicate that the defined Mn(II) carbonate medium
112
can successfully be employed during intentional, directed attempts to cultivate Mn
-
oxidizing
113
chemolithoautotrophs from diverse terrestrial and aquatic fre
shwater environments.
114
115
Community analysis of Mn
-
oxidizing enrichment cultures from three origins.
As was the case
116
with cultures of
Ca.
M. noduliformans, repeated attempts to identify single colonies of the
117
lithotrophs responsible for Mn oxidation were not
successful on an agar
-
solidified, defined Mn(II)
118
carbonate medium. Sequencing of partial 16S rRNA genes amplified from the liquid cultures
119
revealed differences in community structures between the Mn
-
oxidizing enrichments. The most
120
abundant microorganism fr
om the South Africa and Santa Barbara enrichments belonged to the
121
same taxon as the previously described
Ca.
M. noduliformans (Figure 1). However, the identities
122
of the next most abundant members of the communities differed. The previously described
123
Pasade
na enrichment containing
Ca.
M. noduliformans had
Ramlibacter lithotrophicus
as the
124
second most abundant member throughout the enrichment refining process (Supplementary Table
125
1).
R. lithotrophicus
could be isolated from the enrichment using the same defined medium but
126
with other electron donors such as succinate and hydrogen, but
could not oxidize Mn(II) as an
127
isolate
(11)
. Organisms belonging to the same taxon as
R. lithotrophicus
were present in the South
128
Africa enrichments, varying from 2
-
28 in rank abundance, but were not abundant in Santa Barbara
129
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enrichments (<0.5% relative abundance) (Figure 1 and Supplementary Table 1). In the South
130
Africa enrichments, the se
cond most abundant member varied between a
Pseudomonas
species
131
(
Gammaproteobacteria
), a member of the
Zavarziniales
(
Alphaproteobacteria
),
R. lithotrophicus
,
132
and
Hydrogenophaga
(a
Comamonadaceae
closely related to
R. lithotrophicus
) (Figure 1). In the
133
Sant
a Barbara enrichments, the second most abundant member was a member of the
134
Anaerolineaceae
(phylum
Chloroflexi
or
Chloroflexota
; Figure 1). Changing the incubation
135
temperature did not affect the identities of the 3 most abundant taxa in the South Africa
136
en
richments (Figure 1). However, the choice of nitrogen source in the medium resulted in a shift
137
in community member relative abundances (Figure 1). Notably, the only other shared organism
138
between South Africa, Santa Barbara and Pasadena enrichments with >1%
relative abundance was
139
a member of the
Zavarziniales
(Figure 1 and Supplementary Table 1). Its relative abundance
140
markedly increased when the South Africa enrichments were grown in medium with nitrate instead
141
of ammonia as the nitrogen source. Overall, wh
ile the community composition varied between the
142
Mn
-
oxidizing enrichments, strains of
Ca.
Manganitrophus were consistently the most abundant
143
species in all such cultures.
144
145
Expansion of metagenome
-
assembled genomes of cultivated and environmental Mn
-
146
oxidizi
ng
Nitrospirota
.
We performed shotgun metagenomic sequencing on two of the new Mn
-
147
oxidizing enrichments in order to gain phylogenetic and functional insights into the newly
148
cultivated
Ca.
Manganitrophus strains. We reconstructed high
-
quality MAGs (>97%
149
com
pleteness, <5% contamination)
(23)
of the most abundant organism from each metagenome
150
(Supplementary Table 1). We refer to these MAGs as strain SA1 and SB1 to indicate that they
151
o
riginated from South Africa and Santa Barbara, respectively. Both genome and 16S rRNA gene
152
phylogenies confirmed that strain SA1 and strain SB1 were related to the previously characterized
153
Ca.
M. noduliformans strain Mn1 (Figure 2). Based on their average nucleotide identities (ANI)
154
and using 95% ANI as a possible metric for species delineation
(24
26)
, strains SA1 and SB1 were
155
provisionally considered to represent dis
tinct strains of the same species (96% ANI). Both could
156
be considered a different species than strain Mn1 (94% ANI) (Supplementary Table 3
)
.
The
157
genome sizes of these 2 new strains were smaller (4.3 Mb) than that of strain Mn1 (5.2 Mb)
158
(Supplementary Table
2). The arrangement of homologous regions in strains SA1 and SB1 were
159
similar (Supplementary Figure 1a), but were different from strain Mn1 (Supplementary Figure 1b).
160
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These differences were also observed at the deduced protein level, with strains SA1 and
SB1 more
161
closely related to each other than to strain Mn1 (Supplementary Table 4). These variations in the
162
proteins were not concentrated in one genomic region, but instead scattered throughout the genome
163
(Supplementary Figure 1c). Further, de novo gene cl
ustering showed that strains SA1 and SB1
164
shared more genes with each other than with strain Mn1 (Supplementary Figure 1d). All together,
165
our results support strains SA1 and SB1
as
a distinct species, which we designate as
Candidatus
166
Manganitrophus morganii
(Supplementary Text). T
hese 3 cultivated
Ca.
Manganitrophus strains
167
in
two different species
provide
a basis to examine the phylogenetic and genomic diversity of their
168
shared metabolism, namely Mn
-
oxidizing chemolithoautotrophy.
169
170
In addition to reconstructing MAGs from Mn
-
oxidizing enrichments, we also analyzed publicly
171
available MAGs in the phylum
Nitrospirota
. We screened for MAGs that did not belong in the
172
three characterized clades, namely
Nitrospirales
,
Leptospirilla
and
Therm
odesulfovibria
. As of 26
173
March 2019, only 3 MAGs had met this taxonomic criteria with completeness >50% and
174
contamination <5%
(11)
. However, as of March 30 2021, 64 new public high
-
quality (>90%
175
completeness, <5% contamination) and 2 medium
-
quality (>50% completeness, <10%
176
contamination) MAGs meeting this taxonomic criteria had become availa
ble (Supplementary
177
Table 5). These 66 MAGs allowed for a much more detailed phylogenomic view into the
178
uncultivated
Nitrospirota
and their potential ability to oxidize Mn.
179
180
16S rRNA gene and multilocus protein phylogeny reveal robust taxonomic groups.
The
181
available MAGs provide a phylogenetic resolution that matches the traditionally employed 16S
182
rRNA genes (Figure 2). The MAGs were spread out across different phylogenetic clusters within
183
the phylum (Figure 2a). Using the 14 MAGs that also contained 16S rRN
A genes, we were able
184
to link the genome phylogeny to the 16S rRNA gene phylogeny, and observed similar clusterings
185
between the two phylogenetic approaches (Figure 2). The 3 cultivated strains all resided within
186
the genus
Ca.
Manganitrophus. Other members
of
Ca.
Manganitrophus, based on either their
187
genomes or 16S rRNA genes, were from terrestrial, aquatic and engineered environments, and all
188
freshwater in origin (Figure 2). Our phylogeny revealed a sister genus of marine origin (Figure 2).
189
Together, these
two genera form a coherent and well supported phylogenetic clade, hereafter
190
termed family
Candidatus
Manganitrophaceae (Figure 2).
191
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192
Previously, the class
Candidatus
Troglogloea was proposed to encompass strain Mn1 and
193
Candidatus
Troglogloea absoloni (an u
ncultivated species from Vjetrenica cave in the Dinaric
194
Karst), based on their 16S rRNA gene phylogeny
(11)
. Based on our new phylogenomic analysis,
195
we propose that the order
Ca.
Troglogloeales includes the family
Ca.
Manganitrophaceae,
Ca.
T.
196
absoloni, and its relatives (Figure 2), together constituting a sister group distinct from the orde
r
197
Nitrospirales
(which includes the cultivated nitrite and ammonia
-
oxidizing
Nitrospirota
). These
198
genera, family, and order proposals are consistent with the latest taxonomic classification in the
199
Genome Taxonomy Database (GTDB) release 06
-
RS202 April 2021
(27, 28)
, even though GTDB
200
currently contains fewer genomes. Based on the current GTDB taxonomy, both orders
Ca.
201
Troglogloeales and
Nitrospirales
are placed within the class
Nitrospiria
, but this is incongruent
202
with analyses of their 16S rRNA phylogeny (Figure 2b). Numerous
Nitrospirota
MAGs fall
203
outside of the three known groups of
Nitrospirota
(
Nitrospirales
,
Leptosprillia
and
204
Thermodesulfovibriona
) and are over
-
represented in subsurfa
ce and aquatic environments.
205
However, 16S rRNA gene surveys indicate that members of many of the uncultivated clades exist
206
from marine, soil and sediment environments, but are not as of yet represented by genomes (Figure
207
2b). Overall, while the taxonomic r
elationship between orders
Ca.
Troglogloeales and
208
Nitrospirales
and the assignment of classes in
Nitrospirota
remains to be resolved, our proposals
209
of the genus
Ca.
Manganitrophus, family
Ca.
Manganitrophaceae, and order
Ca.
Troglogloeales
210
are supported by
both 16S rRNA gene and genome phylogenetic approaches, and additionally
211
reveal members of a novel marine genus that possibly oxidize Mn lithotrophically.
212
213
Genome comparison streamlines the hypothesized genes for Mn
-
oxidizing lithotrophy.
We
214
next compared
the MAGs of members of the family
Ca.
Manganitrophaceae to understand which
215
genes might be candidates as essential for Mn oxidation, and whether these are found in
216
representatives of the marine genus or other members in the phylum. Four routes for Mn oxida
tion
217
and electron uptake had been previously hypothesized in strain Mn1, including a Cyc2 and three
218
different porin
-
dodecaheme cytochrome
c
(PCC) complexes
(11)
. Cyc2 homologs are not only
219
identified in the majority of
Ca.
Troglogloeales (Figure 3a), but also in other members of the
220
phylum, including characterized clades such as acidophilic,
iron
-
oxidizing
Leptospirilla
and nitrite
221
or ammonia
-
oxidizing
Nitrospirales
(29, 30)
. Of the 3 PCCs in strain Mn1, only PC
C_1 was found
222
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in the strains SA1 and SB1 (Figure 3a). PCC_1 was also identified in other MAGs in both marine
223
and freshwater genera of
Ca.
Manganitrophaceae, but not in the extant MAGs and genomes of
224
Nitrospirota
species falling out outside of this family. These results point to PCC_1, possibly
225
together with Cyc2, as being central to chemolithotrophic Mn oxidation by
Ca.
226
Manganitrophaceae.
227
228
We identified five possible routes in strain Mn1 to reduce oxygen and conserve energy using
229
electrons from Mn(II). The canonical Complex IV (
cbb3
-
type cytochrome
c
oxidase) was
230
identified in the cultivated and uncultivated members of the freshwater genus,
but not in the
231
uncultivated members of the marine genus (Figure 3a). However, the expression of this Complex
232
IV had been observed to be low (24th percentile) in strain Mn1, especially so for a catabolic
233
process, and therefore may not be the primary route f
or oxygen respiration
(11)
. Genes for a
234
canonical cytochrome
bd
oxidase, which has been
hypothesized to reduce oxygen in
Leptospirilla
235
(31)
, were not found in strain Mn1 or other members in the order
Ca.
Troglogloeales (Figure 3a).
236
However, genes for a numbe
r of cytochrome
bd
oxidase
-
like (
bd
-
like) proteins that were
237
phylogenetically distinct and predicted to have many more transmembrane helices than
238
cytochrome
bd
oxidase
(32)
, were identified in strain Mn1
(11)
. These
bd
-
like oxidases are
239
clustered w
ith other genes potentially involved in electron transfer and energy conservation; we
240
refer to these
bd
-
like oxidase containing gene clusters as terminal oxidase (TO) complexes. While
241
all 4 TO complexes were found in other members of
Ca.
Troglogloeales, th
eir taxonomic
242
distributions differed (Figure 3a). TO_1 was found in the majority of
Ca.
Troglogloeales (Figure
243
3a), and have been well discussed in other
Nitrospirota
including
Nitrospirales
(32, 33)
. TO_1 is
244
composed of a
bd
-
like oxidase clade I protein, two cytochrome
c
and a periplasmic cytochrome
b
,
245
and was the highest expressed TO complex (98th percentile) in strain Mn1
(11)
. Contrasting with
246
the more widespread distribution of the TO_1 complex across the phylum, complexes TO_2, TO_3
247
and TO_4 were restricted to
Ca.
Manganitrophaceae, with the latter two limited to the freshwater
248
genus (Figure 3a). While
both TO_3 and TO_4 contain two
bd
-
like oxidase clade V proteins, their
249
predicted interactions with the quinol pool differ: TO_3 encodes for an Alternative Complex III,
250
whereas TO_4 encodes for a more canonical Complex III
(11)
. TO_3 and TO_4 were observed to
251
be moderately expressed at 55th and 67th percentile in strain Mn1, respectively
(11)
. Importantly,
252
TO_2 stands out as it was found in the majority of
Ca.
Manganitrophacea
e, but as yet to be
253
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identified in any genomes outside of this family (Figure 3a). The TO_2 gene arrangement differed
254
slightly between the two genera of
Ca.
Manganitrophaceae, but gene content was similar (Figure
255
3c). The TO_2 complex is composed of a membr
ane cytochrome
b
(similar to the petB/D or
256
cytochrome
bf
complex) and potentially interacts with the quinone pool, a periplasmic cytochrome
257
b
to receive electrons in the periplasm,
bd
-
like oxidase to reduce oxygen, multiple cytochrome
c
258
to transfer electro
ns, and two ion
-
pumping mrpD
-
like subunits that might be coupled to the
259
generation or dissipation of a motive force (Figure 3c). Genes for the TO_2 complex had also been
260
observed to be highly expressed in strain Mn1 (79th percentile)
(11)
. Taken together, our
261
comparative genomic analyses point to TO_2, possibly together with TO_1, as being
central to
262
Mn(II)
-
oxidation
-
dependent oxygen respiration by
Ca.
Manganitrophaceae.
263
264
Autotrophic pathway predicted in Mn
-
oxidizing
Nitrospirota
.
In addition to coupling the
265
oxidation of Mn(II) to oxygen reduction, strain Mn1 was also shown to be capable of
CO
2
fixation
266
and autotrophic growth using Mn(II) as its electron donor
(11)
. Carbon fixation pathways such as
267
the reverse tricarboxylic acid (rTCA) cycle, imp
licated in autotrophy by strain Mn1, require low
268
potential electrons in the form of both NAD(P)H and ferredoxin (E°′ = −320 to
-
398 mV
(21)
)
269
(34)
. Yet, electrons derived from Mn(II) are likely high pot
ential (E°′ = +466 mV)
(11, 12)
. Run
270
in reverse, Complex I has been shown or postulated to couple the dissipation of motive force to
271
the generation of low potential electrons and production of
NAD(P)H or possibly ferredoxin
(11,
272
35, 36)
.
273
274
Remar
kably, in strain Mn1, 3 different Complex I gene clusters were previously identified
(11
)
.
275
Complex_I_1 and Complex_I_2 are similar to canonical Complex I with gene clusters containing
276
nuoA
-
N
genes in order (Supplementary Figure 2). Here, phylogenomic analyses revealed that
277
Complex_I_1 was shared by all members of both genera of
Ca.
Mangani
trophaceae, whereas
278
Complex_I_2 was restricted to members of the freshwater genus (Figure 3a). Of note,
279
Complex_I_3 appears unique in the known biological world, having two additional ion
-
pumping
280
subunits (Figure 4). This highly unusual gene cluster was fo
und in nearly all of
Ca.
281
Manganitrophaceae (Figure 3a) and is not apparent in any other member of the phylum. Unusual
282
Complex I with one additional ion
-
pumping subunit have been previously observed in various
283
bacterial groups including
Nitrospirales
, terme
d 2M Complex I given the extra
nuoM
in the gene
284
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11
cluster
(35)
, and rhizobia, termed Green Complex I in which 2
mrpD
-
like subunits have replaced
285
the standard
nuoL
(37)
. Sequence comparison of Complex_I_3 subunits showed that the two
286
MrpD
-
like subunits were most cl
osely related to those in rhizobia Green Complex I, while the
287
other subunits in the gene cluster were most closely related to those in
Nitrospira
2M Complex I
288
(Figure 4a). Sequence alignment of MrpD2 revealed a 26 amino acid insertion in the C
-
terminal
289
amp
hipathic helix (HL) in all of
Ca.
Manganitrophaceae as compared to the MrpD subunits found
290
in the rhizobia Green Complex I (Figure 4b). This type of insertion was previously identified in
291
all gene clusters containing a second ion
-
pumping subunit (35). Such
insertions were not unique
292
to Complex_I_3, as they were also found in the NuoL of Complex_I_1 and Complex_I_2 in
Ca.
293
Manganitrophaceae (Supplementary Figure 2) and could represent evolutionary intermediates en
294
route to being able to support additional ion
-
pumping subunits in the protein complex (Figure 4c).
295
296
The majority of
Ca.
Troglogloeales and all of
Ca.
Manganitrophaceae analyzed had complete sets
297
of genes for the rTCA cycle (Figure 3a). Only a minor difference was observed in the rTCA cycle
298
gene cont
ent: members of the freshwater genus contained class II fumarate hydratase, whereas
299
those of the marine genus contained class I fumarate hydratase. To further assimilate pyruvate,
300
nearly all genes of the gluconeogenic pathway (Embden
-
Meyerhof
-
Parnas pathwa
y) were
301
observed in
Ca.
Troglogloeales. However, one key gluconeogenic pathway gene, namely fructose
-
302
biphosphate aldolase, was absent in strain Mn1
(11)
and also appears absent from the majority of
303
Ca.
Troglogloeales, save for except two of the MAGs (NCBI assembly accession:
304
GCA_004297235 and GCA_013151935). Moreover, our comparative analysi
s revealed that only
305
1 of the 5 pyruvate dehydrogenases encoded by the genome of strain Mn1 (IMG gene ID:
306
Ga0306812_1021045
-
Ga0306812_1021047, Ga0306812_102629) was common to the other
307
members of the
Ca.
Manganitrophaceae. Overall, despite apparently minor
differences between
308
their MAGs, the majority of
Ca.
Manganitrophaceae shared the same unique Complex_I_3,
309
pathways for CO
2
fixation and pathways for central metabolism as had been previously identified
310
in strain Mn1.
311
312
Core genome of
Ca.
Manganitrophaceae in marine and freshwater environments.
De novo
313
gene clustering revealed that 8 analyzed members of
Ca.
Manganitrophaceae shared a total of 895
314
gene clusters, which included the above
-
mentioned Cyc2, PCC_1, TO_1, TO_2, Complex_I_1
315
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12
and Com
plex_I_3 (Supplementary Table 6). Several other shared genes and pathways appear
316
noteworthy: assimilatory sulfate reduction (
sat
,
aprA/B
,
aSir
), cytochrome c biogenesis, heme
317
exporters, 2 multicopper oxidases, and type IV pilus assembly. These confirm the
basis for the
318
ability of the cultivated strains to use sulfate as an anabolic sulfur source, make cytochrome c for
319
anabolism and catabolism, and suggest the potential for surface twitching motility. Notably
320
missing among the shared genes were those for the
carbon
-
monoxide dehydrogenase complex that
321
had been observed to be highly expressed (95th percentile) during Mn(II) dependent growth by
322
strain Mn1
(11)
. Together, our comparative genomic analyses shed light on common gene sets of
323
Mn
-
oxidizing chemolithoautotrophs in both marine and freshwater environments.
324
325
326
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13
Discussion
327
Cultivation of novel microorganisms with previously undemonstrated physiologies remains a key
328
cornerstone to our expanding understanding of the metabolic potential of the as yet largely
329
uncultured microbial diversity in nature
(38, 39)
. Aerobic, Mn(II)
-
oxidizing chemolithoautotrop
hs
330
were long theorized, but only recently demonstrated to exist in vitro in a bacterial co
-
culture
(11)
.
331
The majority member was a distinct member of the phylum
Nitrospirota
,
Ca.
Manganitrophus
332
noduliformans strain Mn1, and only distantly related to any other cultivated biota
(11)
. Curiously,
333
the initial enrichment of Mn(II)
-
oxidizing chemolithoautotrophs from Caltech’s campus tap was
334
unintentional
(11)
. Here cultivation attempts were intentionally initiated with the specific goal of
335
successfully establishing ne
w Mn
-
oxidizing enrichment cultures. These attempts were successful
336
using a media formulation refined during the course of the earlier study using inocula obtained
337
from two different continents and hemispheres. Community analyses on these two new enrichment
338
cultures revealed that the most abundant microorganisms in each were closely related to, but of a
339
different species than
Ca.
M. noduliformans strain Mn1. The enrichment cultures also harbored a
340
diversity of taxa varying in their relative abundances and id
entities (Figure 1). The results support
341
the notion that members of the genus
Ca.
Manganitrophus are playing a key if not the central role
342
in chemolithoautotrophic Mn(II) oxidation in the laboratory cultures examined. The results also
343
suggest that
Ca.
Mang
anitrophus may not require an obligate partnership with
R. lithotrophicus
344
(the second species present in the previously described co
-
culture
(11)
), leaving open the
345
possibility that its eventual clonal isolation may be possible. The phylogenomic analyses here also
346
predict an assemblage of a marine genus within the family
Ca.
Manganitrophaceae that may also
347
carry out this mode of chemolithoautotroph
y (Figure 2
, 3, and 4
). However, our analyses do not
348
exclude other members in
Nitrospirota
carrying out Mn(II) lithotrophy using a different
349
mechanism than that we hypothesized for
Ca.
Manganitrophacae. With the increasing evidence
350
that the
Ca.
Manganitrop
haceae are distributed globally across marine and freshwater biomes
351
(Figure 5a), taken together the reported prevalence of Mn and Mn
-
reducing microorganisms in the
352
environment
(14, 40)
, chemolithoautotrophic Mn oxidation becomes particularly important to
353
reaching a better understanding of the redox biogeochemical cycle for manganese.
354
355
By comparing metagenome
-
assembled genomes of the 3 cultivated
Ca.
Manganitrophus strains
356
and related but uncultivated organisms available in public genome databases, our results narrow
357
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14
down the list of genes in
Ca.
Manganitrophaceae that may underlie Mn(II
) oxidation driven
358
chemolithoautotrophy. Unique to
Ca.
Manganitrophaceae among all
Nitrospirota
, and perhaps
359
across all of the biological world that has yet been analyzed, were PCC_1, as a candidate for being
360
the initial electron acceptor during Mn oxidati
on; TO_2, as a candidate respiratory complex for
361
productively coupling the electrons from Mn(II) oxidation to oxygen reduction and energy
362
conservation (Figure 3 and Figure 5b); and Complex_I_3, as a candidate complex catalyzing
363
reverse electron transport t
o generate low
-
potential reducing power from quinones during carbon
364
fixation (Figure 4, Figure 5b).
365
366
While not unique to
Ca.
Manganitrophaceae, the identification of Cyc2 and TO_1 in the majority
367
of the family members (Figure 3a and 4b), together with their comparable or even higher
368
expression than that of PCC_1 and TO_2, respectively, in strain Mn1
(11)
, suggests that these two
369
complexes may also be likely involved in Mn lithotrophy. Cyc2 is a fused cytochrome
-
p
orin
370
protein with a single heme
c
, whereas porin cytochrome
c
(PCC) are larger complexes composed
371
of a beta
-
barrel outer membrane protein and at least one multiheme cytochrome
c
(41
43)
. Best
372
understood in acidophilic and circum neutral pH Fe(II) oxidation, predicted structural differences
373
between Cyc2 and PCC homologs, specifically the smaller porin size of Cyc2 and the inner
374
placement of heme
c
within the porin
, have been suggested as meaning that Cyc2 may only react
375
with dissolved Fe
2+
species
(29)
, whereas PCC homologs might react wi
th both soluble and
376
insoluble forms of Fe(II). In the case of Mn(II) oxidation, the reaction is thought most likely to
377
proceed via two sequential one
-
electron oxidation steps
(44)
. In t
hat case, Cyc2 and PCC_1 might
378
react with different forms or oxidation states of Mn (e.g. Mn(H
2
O)
6
2+
vs MnCO
3
, Mn(II) vs Mn(III)
379
complexes) that have different solubilities. In comparison, known heterotrophs that catalyze
380
Mn(II) oxidation often employ multicopper oxidase (MCO) or heme peroxidase homologs to
381
oxidise this metal
(45
47)
. However,
the nature of these enzymes is to couple the oxidation of Mn
382
to the direct reduction of oxygen, without a clear path for conserving any of the potential free
383
energy energy for use by the cell. Members of
Ca.
Manganitrophaceae encodes two MCOs each
384
(Supplem
entary Table 6). It is possible that these too could be involved in the lithotrophic
385
oxidation of Mn(II); if so, it seems likely that the MCO would transfer Mn(II)
-
derived electrons
386
to a periplasmic electron carriers such as cytochrome
c
, rather than direc
tly to molecular oxygen,
387
to be able to conserve energy for the cell
(48)
.
388
.
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