of 16
ORIGINAL RESEARCH
published: 19 February 2018
doi: 10.3389/fmicb.2018.00260
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1
February 2018 | Volume 9 | Article 260
Edited by:
Martin G. Klotz,
Washington State University Tri-Cities,
United States
Reviewed by:
Donald A. Bryant,
Pennsylvania State University,
United States
Trinity L. Hamilton,
University of Minnesota Twin Cities,
United States
Wesley Douglas Swingley,
Northern Illinois University,
United States
*Correspondence:
Lewis M. Ward
LMWARD20111@gmail.com
Specialty section:
This article was submitted to
Evolutionary and Genomic
Microbiology,
a section of the journal
Frontiers in Microbiology
Received:
28 August 2017
Accepted:
01 February 2018
Published:
19 February 2018
Citation:
Ward LM, Hemp J, Shih PM,
McGlynn SE and Fischer WW (2018)
Evolution of Phototrophy in the
Chloroflexi Phylum Driven by
Horizontal Gene Transfer.
Front. Microbiol. 9:260.
doi: 10.3389/fmicb.2018.00260
Evolution of Phototrophy in the
Chloroflexi Phylum Driven by
Horizontal Gene Transfer
Lewis M. Ward
1
*
, James Hemp
2
, Patrick M. Shih
3,4
, Shawn E. McGlynn
5
and
Woodward W. Fischer
1
1
Division of Geological and Planetary Sciences, California
Institute of Technology, Pasadena, CA, United States,
2
Department of Gastroenterology, University of Utah School
of Medicine, Salt Lake City, UT, United States,
3
Department of
Energy, Joint BioEnergy Institute, Emeryville, CA, United S
tates,
4
Environmental Genomics and Systems Biology Division,
Lawrence Berkeley National Laboratory, Berkeley, CA, Unit
ed States,
5
Earth-Life Science Institute, Tokyo Institute of
Technology, Meguro, Japan
The evolutionary mechanisms behind the extant distributio
n of photosynthesis is a
point of substantial contention. Hypotheses range from the
presence of phototrophy
in the last universal common ancestor and massive gene loss i
n most lineages, to
a later origin in Cyanobacteria followed by extensive horiz
ontal gene transfer into the
extant phototrophic clades, with intermediate scenarios t
hat incorporate aspects of
both end-members. Here, we report draft genomes of 11 Chloro
flexi: the phototrophic
Chloroflexia isolate
Kouleothrix aurantiaca
as well as 10 genome bins recovered from
metagenomic sequencing of microbial mats found in Japanese
hot springs. Two of
these metagenome bins encode photrophic reaction centers a
nd several of these bins
form a metabolically diverse, monophyletic clade sister to
the Anaerolineae class that
we term
Candidatus
Thermofonsia. Comparisons of organismal (based on conserv
ed
ribosomal) and phototrophy (reaction center and bacterioc
hlorophyll synthesis) protein
phylogenies throughout the Chloroflexi demonstrate that tw
o new lineages acquired
phototrophy independently via horizontal gene transfer (H
GT) from different ancestral
donors within the classically phototrophic Chloroflexia cl
ass. These results illustrate a
complex history of phototrophy within this group, with meta
bolic innovation tied to HGT.
These observations do not support simple hypotheses for the
evolution of photosynthesis
that require massive character loss from many clades; rathe
r, HGT appears to be the
defining mechanic for the distribution of phototrophy in man
y of the extant clades in
which it appears.
Keywords: lateral gene transfer, comparative genomics, microb
ial metabolism, phylogenetics, microbial diversity
INTRODUCTION
Multiple hypotheses exist for the origin and subsequent evolut
ion of photosynthesis, but little is
known with certainty. It is widely held that anoxygenic prece
ded oxygenic photosynthesis, but
which of the extant taxa—if any—invented phototrophy and/or w
ere phototrophic progenitors on
the early Earth remains unclear (
Fischer et al., 2016
). While simple forms of photoheterotrophy
can be driven by proton-pumping rhodopsins, light-driven elect
ron transport—and therefore
the possibility of light-driven carbon fixation (i.e., photos
ynthesis)—is only known to be driven
by organisms utilizing phototrophic reaction centers. Here w
e focus on reaction center-based
Ward et al.
Evolution of Phototrophy in Chloroflexi
phototrophy, as it can drive electron transport and therefore
photosynthesis, and was responsible for major environmental
transitions through Earth history (
Fischer et al., 2016; Ward,
2017
). To date, reaction center-based phototrophy has been
identified in seven bacterial phyla—the Cyanobacteria, Chlo
robi,
Chloroflexi, Acidobacteria, Firmicutes, Gemmatimonadete
s, and
Proteobacteria. Of these, only one—the Cyanobacteria—con
tains
members that possess two photosystems, coupled in series
to perform oxygenic photosynthesis. The others perform
anoxygenic phototrophy, and possess only a single reaction
center, either of the Type 1 (Chlorobi, Heliobacteria, and
Acidobacteria) or Type 2 (Proteobacteria, Gemmatimonadete
s,
and Chloroflexi) variety. It was hypothesized that photosynth
esis
was present in the last common ancestor of all bacteria (
Woese
et al., 1985; Woese, 1987
) or a broad clade containing all
extant phototrophs (
Cardona, 2016
), followed by extensive loss
in most lineages; however, this idea remains controversial
. The
distribution of phototrophy across the bacterial tree is spars
e,
with phototrophic clades scattered across the domain rather
than forming a single clade of phototrophs. Even Type 1- and
Type 2-reaction center bearing phototrophs are mixed (e.g.,
the closest phototrophic relative of the phototrophic Chlorobi
are phototrophic Gemmatimonadetes; the former has a Type
1 reaction center and the latter a Type 2—a relationship
inconsistent with vertical inheritance;
Fischer et al., 2016
). This
pattern suggests instead an alternative scenario involving
a
later origin of phototrophy (sometime after the origin of the
bacterial domain), followed by multiple instances of horizo
ntal
gene transfer (HGT) that resulted in the modern distributio
n
of phototrophy (e.g.,
Igarashi et al., 2001; Raymond et al.,
2002; Hohmann-Marriott and Blankenship, 2011; Nagashima
and Nagashima, 2013; Zeng et al., 2014; Fischer et al., 2016
).
The most straightforward tests of these hypotheses arise by
comparing the organismal phylogenies of phototrophic bacteria
to phylogenies of photosynthesis genes—concordance of the tr
ees
would be consistent with shared ancestry, while discrepanci
es
between them would indicate a history of horizontal gene
transfer (
Doolittle, 1986
). While the structure of the bacterial
tree of life is still debated (e.g.,
Woese, 1987; Williams et al.,
2013; McInerney et al., 2014; Hug et al., 2016; Schulz et al.,
2017
), intra-phylum organismal relationships tend to be robust
(e.g., reproduced via many different markers) despite enduring
uncertainty in relationships between phyla (
Pace, 2009
). As a
result, the history of metabolic characters like photosynth
esis
within a phylum is more straightforward to assess than it is fo
r
the bacteria as a whole. If a major role for horizontal gene tr
ansfer
can be demonstrated within a particular phylum, the HGT-
driven phototrophy hypothesis will be strengthened, whereas
a concordance of organismal and gene trees would be more
consistent with an ancient origin and vertical inheritance
of the
metabolism. While tests of this kind have been made previously
in the Proteobacteria, suggesting intra-phylum horizontal
gene
transfer (
Igarashi et al., 2001; Swingley et al., 2009; Nagashima
and Nagashima, 2013
), this has not previously been possible
in other phototrophic phyla due to the limited diversity of
phototrophic members within each. However, the discovery
of new phototrophic lineages via metagenomic sequencing
provides opportunities for querying the evolutionary history
of
phototrophs. In particular, the recent description of
Candidatus
Roseilinea gracile—a phototrophic Chloroflexi closely related
to the non-phototrophic Anaerolineae class and only distantly
related to known phototrophic Chloroflexi in the Chloroflexia
class (
Klatt et al., 2011; Thiel et al., 2016, 2017; Tank et al.,
2017
)—suggests that the diversity and evolutionary history of
phototrophy in the Chloroflexi is richer than previously though
t.
The Chloroflexi (e.g., Green Non-sulfur Bacteria) are a
phylum of primarily gliding, filamentous bacteria possessing
a wide diversity of metabolisms and ecological roles, but are
best known as photoheterotrophs (
Overmann, 2008
). Chloroflexi
are notably abundant in a range of environments (e.g., marin
e
sediments and groundwater,
Inagaki et al., 2003; Hug et al.,
2013
). Despite their environmental richness revealed by culture
-
independent surveys, most well-characterized Chloroflexi be
long
to a few subclades isolated from hot springs (
Yamada
and Sekiguchi, 2009
), including the anoxygenic phototrophic
Chloroflexus
(
Pierson and Castenholz, 1974; Hanada et al.,
1995
) and
Roseiflexus
(
Hanada et al., 2002
). Based on
phylogenetic analysis of chlorophyll and bacteriochlorophyll
synthesis genes, it was suggested that anoxygenic phototroph
y
in this group predates the evolution of oxygenic photosynthesi
s
in Cyanobacteria (
Xiong et al., 2000
); if correct it would
imply that this group is remarkably ancient, and therefore
might provide a window into phototrophy on the early Earth.
Recent genomic sequencing projects have expanded the known
taxonomic and metabolic diversity of the Chloroflexi phylum
(e.g., the Ardenticatenia class, capable of nitrate- and iron
oxide- reduction,
Kawaichi et al., 2013, 2015; Hemp et al.,
2015b
). Newly discovered Chloroflexi are diverse in terms of
morphology, metabolism, and other traits (
Table 1
), but continue
to be recovered as a monophyletic clade in phylogenetic trees
(
Figure 1
) and have sufficient sequence similarity to be classified
as a single phylum (
Hanada, 2014
).
Here, we report eleven draft Chloroflexi genomes: ten
recovered from hot spring microbial mats in Japan as well as
one previously cultured isolate. These genomes include two ne
w
phototrophs outside of the classically phototrophic Chloroflexi
a
class, as well as several members of a novel class-level clade s
ister
to the Anaerolinea. Distinct phylogenetic patterns of organi
smal
and phototrophic proteins demonstrate the role of horizontal
gene transfer during the evolution of phototrophy within this
phylum.
MATERIALS AND METHODS
Genomic Sequencing of
Kouleothrix
aurantiaca
The genome of
K. aurantiaca
COM-B (JCM 19913) was
sequenced as part of a project to expand the phylogenetic breadth
of Chloroflexi genomes (
Hemp et al., 2015a,b,c; Pace et al., 2015;
Ward et al., 2015a,b
).
K. aurantiaca
, a member of the group
formerly known as ‘Eikenboom morphotype 1851’ (
Seviour and
Blackall, 1999
), was isolated from activated sludge in an industrial
wastewater treatment facility (
Kohno et al., 2002
). It forms
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Evolution of Phototrophy in Chloroflexi
TABLE 1 |
Characteristics of Chloroflexi classes.
Chloroflexia
a
Thermomicrobia
b
Anaerolineae
c
Caldilineae
d
Ardenticatenia
e
Ktedenobacteria
f
Thermoflexia
g
Dehalococcoidetes
h
Ca.
Thermofonsia
i
Phototrophy
+
(reaction
center-based)
Some (reaction
center- or
rhodopsin-based)
Aerobic
respiration
+
+
Genes present
Genes present
+
+
Microaerophilic Genes rarely present Genes present
Complex III
bc, ACIII, or both ACIII or both
ACIII, bc, or neither bc
bc
bc
b
c
Neither
bc, also ACIII with
RCII
Morphology
Filamentous
Rod
Filamentous or rods Filamentou
s
Filamentous
Filamentous
(branched)
Filamentous
Coccoidal, discs
Unknown
Motility
Gliding
Flagellar
Flagellar or none
Unknown
—no
flagellar genes
Other
metabolic
traits
Nitrite oxidation
Iron and nitrogen
respiration
Dehalogenation
Nitrogen respiration
Temperature
range
10–67
43–80
20–73
37–65
30–75
17–74
67.5–75
15–35
32–59
% GC
48–62
56–63
48–58
59–65
51.5
54–60
69
49–54
46–63
a
(
Garrity and Holt, 2001; Gupta et al., 2013
).
b
(
Hugenholtz and Stackebrandt, 2004; Sorokin et al., 2012
).
c
(
Yamada et al., 2006; Hemp et al., 2015a,b,c; Pace et al., 2015; Ward et al., 2015a
,b
).
d
(
Yamada et al., 2006
).
e
(
Kawaichi et al., 2013
).
f
(
Cavaletti et al., 2006; Yabe et al., 2010; Chang et al., 2011
).
g
(
Dodsworth et al., 2014
).
h
(
Moe et al., 2009; Löffler et al., 2013
).
i
This study.
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February 2018 | Volume 9 | Article 260
Ward et al.
Evolution of Phototrophy in Chloroflexi
FIGURE 1 |
Reference phylogeny of Chloroflexi based on RpoB protein sequ
ences, with our newly sequenced strains indicated with dagg
ers, phototrophic strains
highlighted (pink for fused
pufLM
, green for unfused), and
Candidatus
Thermofonsia noted. Most phototrophic Chloroflexi occur wit
hin a monophyletic clade in the
Chloroflexia class, yet two distinct lineages of phototrophs
occur outside of this class, separated by many non-phototro
phic lineages. This phylogeny is based on
RpoB—a single organismal marker protein which was recovere
d in all
Ca.
Thermofonsia genome bins—and is primarily intended as a ref
erence for the critical
phylogenetic relationships presented here (e.g., diverge
nce of
Ca.
Thermofonsia from Anaerolineae, separation of phototroph
ic Thermofonsia from phototrophic
Chloroflexia). Potentially more robust organismal phylogen
ies (e.g., 16S or larger concatenated protein datasets) wil
l be possible with higher completeness
Ca.
Thermofonsia genomes.
orange-pigmented cells organized into long mm-scale filament
s,
grows on pyruvate and by fermentation of certain sugars, and c
an
reduce nitrate to nitrite (
Kohno et al., 2002
). It is closely related
to members of the genus
Roseiflexus
(
Beer et al., 2002
), however
phototrophy has not been observed in these organisms in
culture.
Genome sequencing was performed at Seqmatic (Fremont,
CA) using the Illumina MiSeq sequencing platform. SPAdes 3.1.
1
(
Nurk et al., 2013
) was used to assemble the genome. The genome
was screened for contaminants based on sequence coverage, G
C
composition, and BLAST hits of conserved single copy genes.
Genome annotation was performed using the NCBI Prokaryotic
Genome Annotation Pipeline.
Metagenomic Sample Collection
Four metagenomic datasets were recovered from two
hot springs in Japan: Jinata Onsen and Nakabusa Onsen
(
Supplemental Table 1
). Genome bins labeled JP1 or JP3 were
derived from Jinata Onsen, while CP1 and CP2 were derived
from Nakabusa Onsen (
Table 2
).
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Ward et al.
Evolution of Phototrophy in Chloroflexi
TABLE 2 |
Genome statistics of sequenced strains.
Genome
size
% GC No. coding
sequences
No. Contigs Completeness Contamination
Strain
Heterogeneity
tRNAs Source
CP1_1M
1.39
59
1,182
138
42.28
1.81
50
14
Nakabusa Cone Pool 1
CP2_2F
1.99
59
1,734
20
49.46
0
0
23
Nakabusa Cone Pool 2
CP2_20G
3.09
48
2,678
852
78.54
3.55
33.33
32
Nakabusa Cone Po
ol 2
CP2_42A
3.3
59
2,897
2,024
79.44
10.42
16.13
31
Nakabusa Cone
Pool 2
JP1_8
2.21
51
1,973
601
58.13
0.13
0
17
Jinata Pool 1
JP1_16
4.06
44
3,238
1,764
95.15
17.31
0
45
Jinata Pool 1
JP1_20
3.36
46
2,878
1,139
79.09
4.78
20
34
Jinata Pool 1
JP1_191
0.417
47
334
883
10.63
1.8
0
7
Jinata Pool 1
JP3_7
3.62
63
3,078
1,331
87
12.85
7.32
46
Jinata Pool 3
JP3_13
3.67
60
3,116
1,259
96.17
10.87
75
46
Jinata Pool 3
Kouleothrix
aurantiaca
8.7
62
8,993
5,539
85
0
0
97
Isolate from
wastewater sludge
Jinata genome bins were assembled from two metagenomes
from Jinata Onsen, on Shikinejima Island, Tokyo Prefecture a
t
34.326111N, 139.216E. The geochemistry and microbial dive
rsity
of this spring were described in detail elsewhere (
Ward, 2017
).
Shikinejima is part of the Izu Islands, a chain of volcanic isl
ands
that formed in the past 2-3 million years along the northern
edge of the Izu-Bonin-Mariana Arc (
Kaneoka et al., 1970
). The
source water of Jinata Onsen emerges anoxic, iron-rich, and
gently bubbling from the spring source (
Supplemental Figure 1
).
Temperature at the source was
62
C. This spring water flows
into a series of pools that mix progressively more with seawate
r
during high tide, creating a range of geochemical condition
s
over short spatial and temporal scales as hot, iron-rich, oxyg
en-
poor spring water mixes with cold, oxygen-rich seawater. The
metagenomes from which JP1 bins were sequenced came from
samples of thin (
1 mm) microbial mats in an iron-oxide rich
pool near the spring source (Pool 1), while JP3 genomes were
recovered from a Cyanobacteria-rich microbial mat in Pool 3
, the
most downstream section of the hot spring before it flows into
the coastal ocean. Dissolved oxygen (DO), pH, and temperature
measurements were performed
in situ
using an Exetech DO700 8-
in-1 Portable Dissolved Oxygen Meter. Iron concentrations w
ere
measured using the ferrozine assay (
Stookey, 1970
) following
acidification with 40 mM sulfamic acid to inhibit iron oxidat
ion
by O
2
or oxidized nitrogen species (
Klueglein and Kappler, 2013
).
At the time of sampling, Pool 1 was 59
C, pH 5.8, contained
1.8 mg/L DO and 265
μ
M Fe
2
+
; Pool 3 was 46
C, pH 6.7, and
contained 5.6 mg/L DO and 100
μ
M Fe
2
+
.
Nakabusa genome bins were assembled from two
metagenome samples collected from microbial mats from
Nakabusa Onsen, located at 36.392429N, 137.748038E in the
Japanese Alps near Azumino, Nagano Prefecture. Geochemical
and microbiological characterization of the sampling site at
Nakabusa Onsen is described in detail elsewhere (
Kubo et al.,
2011; Everroad et al., 2012; Otaki et al., 2012; Ward, 2017
).
Nakabusa Onsen is a sulfidic, moderately alkaline hot spring
with source waters near 70
C (
Supplemental Figure 2
). The
samples from which the metagenomes were sequenced were of
cone-forming microbial mats at two points along the outflow
from the hot spring source; Cone Pool 1 (the source of CP1
genomes) was a Chloroflexi-dominated mat located near the
hot spring source, which at the time of sampling was 48
C and
pH 8.1, while Cone Pool 2 (the source of the CP2 genomes) was
collected from a cone-forming, Cyanobacteria-rich microb
ial
mat several meters downstream, which at the time of sampling
was 32
C and pH 8.3.
Samples of microbial mats were collected using sterile forceps
and spatulas (
0.25 cm
3
of material). Cells were lysed and DNA
preserved in the field using Zymo Terralyzer BashingBead Matri
x
and Xpedition Lysis Buffer (Zymo Research, Irvine, CA). Cells
were disrupted immediately by attaching tubes to the blade of a
cordless reciprocating saw (Black & Decker, Towson, MD) and
operating for 1 min.
Metagenomic Sequencing and Analysis
Following return to the laboratory, DNA was extracted and
purified with a Zymo Soil/Fecal DNA extraction kit (Zymo
Research, Irvine, CA). DNA was quantified with a Qubit 3.0
fluorimeter (Life Technologies, Carlsbad, CA) according to
manufacturer’s instructions following DNA extraction. Pu
rified
DNA was submitted to SeqMatic LLC (Fremont, CA) for
library preparation and 2
×
100 bp paired-end sequencing via
Illumina HiSeq 4,000 technology. Raw sequence reads were
assembled with MegaHit v. 1.02 (
Li et al., 2016
) and genome bins
constructed based on tetranucleotide frequency using MetaWa
tt
version 3.5.2 (
Strous et al., 2012
). Genomes were manually
screened for genes of interest and uploaded to RAST (
Aziz
et al., 2008
) for overall characterization. Genome bins were
assessed for completeness and contamination using CheckM
(
Parks et al., 2015
). Genes of interest (e.g., coding for ribosomal,
photosynthesis, and electron transport proteins) were screene
d
against outlier (e.g., likely contaminant) contigs as dete
rmined by
CheckM using tetranucleotide, GC, and coding density conten
t.
Phylogenetics
Sequences of ribosomal and phototrophy proteins used in
analyses (see below) were identified locally with the tblastn
function of BLAST
+
(
Camacho et al., 2008
), aligned with
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Ward et al.
Evolution of Phototrophy in Chloroflexi
MUSCLE (
Edgar, 2004
), and manually curated in Jalview
(
Waterhouse et al., 2009
). Positive BLAST hits were considered
to be full length (e.g.,
>
90% the shortest reference sequence
from an isolate genome) with e values greater than 1e-20.
Phylogenies were constructed using translated protein sequ
ences.
Phylogenetic trees were calculated using RAxML (
Stamatakis,
2014
) on the Cipres science gateway (
Miller et al., 2010
). Trees
were visualized with SeaView (
Gouy et al., 2010
) and the
Interactive Tree of Life viewer (
Letunic and Bork, 2016
).
Probability of Missing Genes
In order to estimate the probability that certain sets of gene
s
were missing from recovered genome bins, we calculated
the probability mass function of recovering zero genes of
a particular set from a genome of predicted size, given
estimated completeness and assuming random sampling without
replacement of individual genes. Though gene size varies
significantly and colocalization makes selection of relate
d genes
not entirely independent, we assumed here that all genes
have an equal probability of being selected. This simplifying
assumption is reasonable, as recovered phototrophy genes
largely reside on separate contigs (suggesting that colocal
ization
is limited—in contrast to phototrophic Proteobacteria and
Gemmatimonadetes, e.g.,
Igarashi et al., 2001; Nagashima
and Nagashima, 2013; Zeng et al., 2014
)—and the length
of phototrophy-related genes (e.g., coding for reaction cent
er
proteins, bacteriochlorophyll synthases, etc.) are within e
rror of
average gene length. The calculation took the form of
f
(
x
)
=
(
n
x
) (
T
n
r
x
)
/
(
T
r
)
, where
f
is the probability of recovering
x
genes of
set
r
from a genome containing
T
genes of which
n
are recovered.
In the case of our genome bins,
n
equaled the number of protein
coding sequences recovered in each bin,
T
equaled
n
divided
by the completeness of the genome as estimated by CheckM,
and
r
equaled 6 (representing
pufL
,
pufM
,
pufC
,
bchX
,
bchY
,
and
bchZ
). The probability that phototrophy genes existed in
Ca.
Thermofonsia genomes, but was not recovered in our bins,
ranged from
0.5 for JP1_191 (at only
10% completeness)
to
2
×
10
13
for JP3_13 (at over 96% completeness). The
probability of missing phototrophy genes was only
>
5% in
JP1_191, greatly improving confidence that the absence of
phototrophy from most strains of
Ca.
Thermofonsia is a real
signal and not due to incomplete genomic data.
A similar calculation can be made for the probability
that
bchL
,
bchN
,
bchB
,
bchM
, or
bchE
genes are present in
phototrophic Thermofonsia, but simply not recovered in the
genome bins. The probability of missing all five of these
genes is about 0.03% for CP2_42A and less than 0.005% for
JP3_7. It is therefore statistically likely that several (or
all)
of the missing bacteriochlorophyll synthesis genes are inde
ed
missing from the genomes of phototrophic Thermofonsia, where
the bacteriochlorophyll synthesis functions of these genes
are
potentially replaced by promiscuous homologs or other proteins
.
A complementary analysis of the probability of false positives
can be made to quantify the likelihood that all genes recover
ed
for a pathway were mistakenly recruited to the genome bin
(i.e., belong to the contaminant fraction). Given an estima
te of
contamination in a genome bin as assessed by CheckM, C, and
the number of contigs containing genes in a pathway of interes
t
recovered in the genome bin, k, the probability, P, that all of
these genes do not actually belong to the genome is given by P
=
C
k
. In the genome bins recovered here, phototrophy genes are
largely recovered on separate short contigs, and so k is typica
lly
equal to the number of phototrophy genes recovered. Following
the example above, the likelihood that
pufL
,
pufM
,
pufC
,
bchX
,
bchY
, and
bchZ
were all mistakenly assigned to bin CP2_42A is
P
=
0.1042
6
=
0.00000127998. This could also be considered a
conservative estimate, as it ignores the fraction of contam
inant
genes that are due to strain-level heterogeneity rather tha
n genes
from unrelated organisms (16.13% in the case of CP2_42A).
RESULTS AND DISCUSSION
Sequencing of both hot spring metagenomes and a cultured
isolate yielded draft genomes of three new reaction center-
containing phototrophic Chloroflexi lineages (
K. aurantiaca
,
JP3_7, and CP2_42A). In addition to these new phototrophs,
eight genome bins were recovered that are associated with a
new class-level clade, sister to the Anaerolineae (
Tables 1
3
,
Figure 1
,
Supplemental Figures 3
,
4
).
K. aurantiaca
represents
a thus-far monospecific genus within the class Chloroflexia,
basal to
Roseiflexus
; JP3_7 is a sister taxon to
Ca.
Roseilinea
gracile; and CP2_42A and the other genome bins reported here
form a new clade sister to the Anaerolineae. Genome statistic
s
and summaries of the key metabolic proteins encoded by these
genomes are reported in
Tables 2
,
3
.
Organismal phylogenies of the Chloroflexi phylum, including
the novel phototrophs and other draft genome described
here, were constructed using conserved, single-copy protein
sequences including RpoB (
Figure 1
,
Supplemental Figure 4
)
and concatenated ribosomal proteins (
Supplemental Figure 3
).
RpoB is a core information processing protein, found as a
single copy, and offers a character set that is commonly
vertically inherited (
Hansmann and Martin, 2000
), and moreover
was recovered in even low-completion genome bins, allowing
the placement of additional low completion genomes into
Ca.
Thermofonsia (
Figure 1
,
Table 3
). Concatenated ribosomal
protein sequences provide a large, robust dataset for resolvin
g
organismal relationships, and were used following methods fr
om
Hug et al. (2016)
. Due to low genome completeness and the
recovery of only a partial RpoB sequence, bin JP1_191 was not
included in figures.
Phylogenetic trees of reaction center proteins (i.e., PufL a
nd
PufM) (
Figure 2
,
Supplemental Figure 5
) show
Kouleothrix
in
the same position relative to other Chloroflexia as in organis
mal
trees (i.e., basal to
Roseiflexus
), but these analyses place CP2_42A
and JP3_7 very differently—with CP2_42A as branching between
Kouleothrix
and
Roseiflexus
, and JP3_7 branching sister to the
Roseiflexus
+
CP2_42A
+
Kouleothrix
clade.
Kouleothrix aurantiaca
K. aurantiaca
encodes for all of the genes required for anoxygenic
phototrophy; a Type 2 reaction center (RC2) (including a fused
pufLM
and
pufC
), a complete bacteriochlorophyll biosynthesis
pathway, and a cytochrome
bc
complex, but no Alternative
Complex III.
K. aurantiaca
encodes a form 1 RuBisCO and
a phosphoribulokinase gene, suggesting that it is capable of
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Evolution of Phototrophy in Chloroflexi
TABLE 3 |
Presence/absence of selected organismal markers and metabo
lic genes in genomes reported here.
16S RpoB Type 2 reaction
center
bc
complex ACIII Rhodopsin Denitrification A-Family HCO B-Family
HCO 3HP Calvin Cycle
CP1_1M
+
+
CP2_2F
+
+
CP2_20G
+
+
nirK
+
CP2_42A
+
+
(fused)
+
+
+
+
(two)
+
JP1_8
+
JP1_16
+
+
+
JP1_20
+
+
nirK, NOR
+
(three)
JP1_191
+
-
JP3_7
+
+
(unfused)
+
+
+
JP3_13
+
+
+
+
(two)
Kouleothrix aurantiaca
+
+
+
(fused)
+
nirK
+
+
+
FIGURE 2 |
Phylogeny of Type 2 phototrophic reaction center proteins m
ade from concatenated sequences of PufL and PufM. Lineages w
ith a fused
pufLM
gene are
highlighted in pink while lineages with unfused reaction ce
nter genes are shown in green. The phylogeny of reaction cent
er proteins is incongruent with the organismal
tree (
Figure 1
), suggesting a history of horizontal gene transfer. Howeve
r, the monophyly of fused
pufLM
genes (pink) is consistent with a singular gene fusion event
.
carbon fixation via the Calvin Cycle. It does not, however,
encode key genes in the 3-hydroxypropionate bicycle (3HP)
used for carbon fixation in
Chloroflexus
and
Roseiflexus
(
Klatt
et al., 2007; Shih et al., 2017
).
K. aurantiaca
falls within the
phototrophic Chloroflexia, with a consistent position basal to
Roseiflexus
in both organismal and photosynthetic gene trees
(
Figures 1
,
2
). This suggests that phototrophy is a synapomorphy
of the Chloroflexales order (i.e., the members of the Chlorofl
exia
class after the divergence of the basal members
Herpetosiphon
and
Kallotenue
), with a single acquisition at the base of the clade,
before the divergence of the
Chloroflexus
and
Roseiflexus
lineages,
with no known instances of loss of phototrophy.
Candidatus
Thermofonsia—A
Metabolically Diverse Class of Chloroflexi,
Sister to Anaerolineae
Several of the draft genomes reported here (CP1_1M, CP2_2F,
CP2_20G, CP2_42A, JP1_8, JP1_16, JP1_20, JP1_191, and
JP3_13) cluster together in phylogenies based on organismal
marker genes (e.g., RpoB,
Figure 1
, and concatenated ribosomal
protein sequences,
Supplemental Figure 3
), forming a
monophyletic clade sister to the Anaerolineae class. Additi
onally,
JP3_7 and
Ca.
Roseilinea gracile (the “Anaerolineae-like”
phototroph recovered from a Yellowstone National Park
metagenome) (
Klatt et al., 2011; Thiel et al., 2016, 2017; Tank
et al., 2017
) are tentatively assigned to this class as they cluster
together under some analyses (e.g., RpoB,
Figure 1
), though in
concatenated ribosomal protein phylogenies these strains clu
ster
with
Thermoflexus hugenholtzii
in a lineage basal to Anaerolineae
and the new class described here (
Supplemental Figure 3
).
Genome analyses show that the members of the new class
described here encode diverse heterotrophic metabolic trait
s,
including photoheterotrophy and several pathways for both
aerobic and anaerobic respiration (
Table 3
).
For this new clade, we propose the name
Candidatus
Thermofonsia, from the Latin for hot spring, and the suffix -
ia
for a class level, with official classification pending isolatio
n
and characterization of at least one member. The members of
Ca.
Thermofonsia described here fall into three lower-order
clades in organismal trees, each composed of sequences from
members recovered from multiple hot spring metagenomes.
The monophyly of each of these clades was recovered in all
organismal phylogenies even if relationships between them
vary depending on analysis (e.g., placement of JP3_7 and
Ca
Roseilinea gracile,
Figure 1
and
Supplemental Figure 3
). These
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Evolution of Phototrophy in Chloroflexi
clades appear to vary in their metabolic characteristics base
d
on the genomes recovered so far, but are overall more similar
to each other than they are to the neighboring Anaerolineae
class. In particular, the Thermofonsia appear to have more
abundant and diverse pathways for high potential metabolism,
including aerobic respiration, reaction center-based photr
ophy,
and denitrification.
Shared Characters of
Ca.
Thermofonsia
and Divergence from Anaerolineae
Based on analysis of fairly complete Thermofonsia genomes
(
>
75% completeness, i.e., CP2_20G, CP2_42A, JP1_16, JP1_20,
JP3_7, and JP3_13), several traits appear to be common
characteristics of these organisms, likely inherited from
the last
common ancestor of the class (i.e., synapomorphies). Some of
these traits (e.g., carotenoid synthesis) are shared with o
ther
Chloroflexi, while others (such as O
2
metabolism) distinguish the
Thermofonsia from their closest relatives.
The Thermofonsia described here possess many genes
for metabolizing O
2
that distinguish them from their sister
class, Anaerolineae. The Anaerolineae are typically descri
bed
as obligate anaerobes (e.g.,
Yamada and Sekiguchi, 2009
),
though genes for aerobic respiration have been recovered in
a number of Anaerolineae genomes (e.g.,
Hemp et al., 2015a;
Ward et al., 2015a
). Phylogenetic analysis of electron transport
and respiration genes in the Thermofonsia and Anaerolineae
reveal metabolic protein trees that are incongruent with
organismal relationships, implying independent acquisition
s of
respiration in these two clades (
Supplemental Figures 6
9
).
The Thermofonsia identified thus far utilize a
bc
complex
for respiration, while the Anaerolineae commonly use an
Alternative Complex III (ACIII). Furthermore, the Heme
Copper Oxidoreductases (HCOs) in these organisms are not
closely related (
Supplemental Figures 6
,
7
). Thermofonsia use
a low-O
2
affinity A-family HCO closely related to those of
Cyanobacteria, while those in Anaerolineae are closely rel
ated
to those found in the Chloroflexi class Caldilineae. The A-
family HCOs found in Thermofonsia are closely related to
each other, potentially reflecting vertical inheritance fro
m their
last common ancestor. These genes are not closely related to
those of other members of the Chloroflexi, potentially reflect
ing
acquisition of aerobic respiration at the base of the class,
rather than at the origin of the phylum. This suggests that
stem group lineages of these classes diverged prior to the
acquisition of aerobic respiration, followed by diversifica
tion
after receiving this metabolism through horizontal gene tr
ansfer,
or alternatively loss and replacement (from a different source
)
in at least one lineage. Similarly, phylogenies of the
bc
complex
in Thermofonsia (
Supplemental Figure 8
) largely recapitulate
organismal relationships. In this case, however, the closes
t
relatives of Thermofonsia sequences are those from other
Chloroflexi, potentially reflecting an earlier acquisition of
Complex III or intra-phylum HGT. Few Thermofonsia (only
CP2_20G and JP3_13) encode
bd
oxidase enzymes used for
respiration or O
2
detoxification at low O
2
concentrations
(
Borisov et al., 2011
), whereas this enzyme is common in the
Anaerolineae (
Hemp et al., 2015c; Pace et al., 2015; Ward
et al., 2015a
). Moreover, the members of Thermofonsia reported
here tend to encode fewer oxidative stress response genes
than is typical for the Anaerolineae as annotated in RAST
(mean of 10 vs. 19 among fairly complete Thermofonsia and
Anaerolineae, respectively). Together these lines of eviden
ce
support interpretations of
Ca.
Thermofonsia being adapted to
a more aerobic lifestyle than the Anaerolineae. It is theref
ore
possible that the acquisition of aerobic respiration via HGT by
early diverging ancestors of the Thermofonsia may have trigge
red
diversification and radiation of this clade associated with i
nvasion
of newly accessible metabolic niches.
While some members of the Thermofonsia encode genes
for nitrogen respiration (discussed below), other anaerobi
c
respiration pathways—such as sulfate reduction—were not
observed, nor were genes for bioenergetic nitrogen or sulfu
r
oxidation. No Thermofonsia genomes described here contain
genes for nitrogenase. Overall, the gene content of the
Thermofonsia described here are characteristic with a life
style as
aerobic heterotrophs.
Like other Chloroflexi, most members of
Ca.
Thermofonsia
encode genes associated with carotenoid synthesis, such as
phytoene synthase, phytoene desaturase, and lycopene cyclase
,
though these appear to be absent in JP1_16, despite the
relative completeness of this genome, suggesting that carote
noid
synthesis may be a common but not universal trait within this
class. No genes for flagellar synthesis were identified in mem
bers
of the Thermofonsia, but it is possible that they are capable of
gliding motility like other members of the Chloroflexi. Mark
er
genes for this trait have not yet been identified, but genes fo
r
chemotaxis regulation (e.g., CheA, CheR, CheY) are common.
Ca.
Thermofonsia Clade 1: JP3_13,
CP1_1M, and CP2_2F
The first clade within
Ca
. Therofonsia is represented here by
genome bins JP3_13, CP1_1M, and CP2_2F, of which JP3_13 is
the most complete (
96% as estimated by CheckM). Members
of this clade characterized thus far have GC content
60%
and predicted average estimated full genome size of
3.7 Mb.
While the CP1_1M and CP2_2F genomes are too incomplete
for thorough metabolic characterization, JP3_13 was used her
e
as representative of the clade. JP3_13 encodes a
bc
complex
and two A-family Heme-Copper Oxidoreductases for aerobic
respiration. CP2_20G and JP3_13 also contain genes for a
bd
oxidase, an O
2
reductase adapted to low O
2
concentrations.
Two members of Clade 1 contain rhodopsin genes (CP2_2F and
JP3_13). These rhodopsin genes have highly similar sequences
,
and were likely inherited from the last common ancestor of th
ese
strains. These rhodopsins are related to the “Actinorhodopsin
s”
found in
Roseiflexus
sp. RS-1, which are thought to be functional
as light-driven proton pumps (
Sharma et al., 2008
). Despite the
presence of rhodopsins in diverse members of the Chloroflexi,
including the Thermofonsia described here as well as
Roseiflexus
,
Ktedonobacter racemifer
, and
Bellilinea caldifistulae
(members of
the Chloroflexia, Ktedonobacteraceae, and Anaerolineae cla
sses
of the Chloroflexi, respectively), the rhodopsins in each of the
se
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Evolution of Phototrophy in Chloroflexi
Chloroflexi lineages are not closely related, and likely refl
ect
independent acquisitions via horizontal gene transfer from
other
phyla and not a shared history of rhodopsins in the Chloroflexi
phylum.
Ca.
Thermofonsia Clade 2: CP2_20G,
CP2_42A, JP1_8, JP1_16, JP1_20, and
JP1_191
The second clade of Thermofonsia described here contains
the genomes CP2_20G, JP1_20, CP2_42A, JP1_8, JP1_16, and
JP1_191. Of these, JP1_16 is the most complete (
95%) and
JP1_191 the least (
11%), while the others are of
80%
completeness. Due to its incompleteness and the recovery of on
ly
a partial RpoB sequence, JP1_191 was excluded from most figures
and the following discussion. GC content of this clade appears
to
be typically lower than for Clade 1, ranging between 44 and 51%
for most genomes with the single outlier of CP2_42A at 59%.
The average predicted genome size (recovered genome divided
by estimated completeness) is slightly larger than for Clade 1
(4.0
vs. 3.7 Mb).
This clade encompasses members with the potential for
rhodopsin- and reaction center-based phototrophy (CP2_42A),
partial denitrification (CP2_20G and JP1_20), and aerobic
respiration (all genomes
>
50% completeness). Aerobic
respiration in this clade is largely associated with A-famil
y
HCOs and
bc
complexes, consistent with Thermofonsia
Clade 1 with whom these genes share a phylogenetic affinity
(
Supplemental Figures 6
,
8
). CP2_42A is the only member of
this clade that contains genes for Alternative Complex III an
d a
B-family HCO, potentially related to its capacity for phototro
phy
(see below). While CP2_42A appears to be capable of reaction
center-based phototrophy (see below), no other members of thi
s
clade encode the necessary genes for phototrophy. JP1_8, the
sister taxon to CP2_42A in organismal phylogenies, contains
no marker genes for phototrophy; given the completeness of
this genome, and the calculations for statistical confidenc
e of
the absence of genes from a metagenome bin, there is less than
a 0.5% chance that this organism is a reaction center-based
phototroph but the genes simply failed to be recovered in the
genome bin (
Supplemental Figure 12
). While the presence of
phototrophy in CP2_42A and absence in JP1_8 may be a result
of presence in their last common ancestor followed by loss in
JP1_8, it is equally parsimonious to assume HGT into CP2_42A
from another source, a scenario that is discussed in greater
detail
below. CP2_42A encodes a rhodopsin gene most closely related
to xanthorhodopsin, a proton-pumping rhodopsin shown to
use light-harvesting antenna carotenoids (
Balashov et al., 2005
).
CP2_42A also encodes a NiFe hydrogenase, a feature that was
not recovered in any other Thermofonsia genomes.
Ca.
Thermofonsia Clade 3: JP3_7 and
Ca.
Roseilinea Gracile
The third clade of
Ca.
Thermofonsia consists of
Ca.
Roseilinea
gracile, described elsewhere (e.g.,
Klatt et al., 2011; Tank et al.,
2017
), and JP3_7, described here. The phylogenetic placement
of this clade is tentative, as it varies somewhat between
marker sets (e.g., RpoB,
Figure 1
, and concatenated ribosomal
proteins,
Supplemental Figure 3
). Both members of this clade
described so far encode a Type 2 reaction center and genes
for bacteriochlorophyll synthesis (discussed in detail bel
ow) but
not genes for carbon fixation, suggesting a photoheterotrophi
c
lifestyle. These organisms contain genes for aerobic respir
ation
via both an A- and B-family HCO as well as a
bc
complex.
Unlike other phototrophic Chloroflexi, Alternative Complex III
was recovered not in these genomes.
Anoxygenic Phototrophy in Chloroflexi
Outside the Chloroflexia Class
The draft genomes reported here include two organisms from
outside the Chloroflexia class (JP3_7 and CP2_42A) that conta
in
genes for anoxygenic phototrophy via Type 2 reaction centers
(though some genes for bacteriochlorophyll synthesis were n
ot
recovered, see below).
JP3_7 is most closely related to
Ca.
Roseilinea gracile—the
“Anaerolineae-like” phototrophic Chloroflexi assembled fro
m a
metagenome from Yellowstone National Park (
Klatt et al., 2011;
Thiel et al., 2016, 2017; Tank et al., 2017
), though it is genetically
distinct at the species and possibly the genus level (
78% average
nucleotide identity across the genome,
Goris et al., 2007
). JP3_7
encodes genes for anoxygenic phototrophy: a Type 2 reaction
center (including
pufL
,
pufM
, and
pufC
), bacteriochlorophyll
a
synthesis, and a cytochrome
bc
complex, but no Alternative
Complex III. Interestingly, JP3_7 (as well as
Ca
. Roseilinea
gracile) possesses fused
pufL
and
pufM
genes, a rare feature
previously only observed in
Roseiflexus
(
Youvan et al., 1984;
Yamada et al., 2005
). JP3_7 and
Ca.
Roseilinea gracile branch with
Ca.
Thermofonsia in RpoB phylogenies (
Figure 1
), albeit with
weak bootstrap support (
Supplemental Figure 4
). However, in
concatenated ribosomal protein trees, JP3_7 and
Ca.
Roseilinea
gracile cluster with
T. hugenholtzii
as a lineage branching basal
to Anaeolineae
+
Thermofonsia (
Supplemental Figure 3
). Note
that the uncertainty in the exact placement of this lineage
does not affect interpretations of evolutionary relationships
of these organisms (e.g., HGT of phototrophy genes, see
below).
CP2_42A encodes genes for anoxygenic phototrophy;
a Type 2 reaction center (including a fused
pufLM
and
pufC
), bacteriochlorophyll
a
biosynthesis, a cytochrome
bc
complex, and Alternative Complex III. CP2_42A falls
within
Ca.
Thermofonsia, and is separated from its closest
phototrophic relatives (JP3_7 and
Ca.
Roseilinea gracile) by
several nonphototrophic lineages (
Figure 1
).
While the draft genomes reported here are largely too
fragmented to recover informational genes on the same conti
gs
as phototrophy related genes, the
rpoB
and
bchP
genes of JP3_7
were collocated on the same contig, providing strong support fo
r
the inference of phototrophy in this lineage from these genome
bins. Moreover, given the relatively low contamination in th
ese
genome bins as estimated by CheckM (
<
13% in both JP3_7 and
CP2_42A, much of which is due to strain-level heterogeneity
rather than contamination from unrelated organisms,
Table 2
),
the likelihood of multiple contigs bearing phototrophy-relat
ed
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Evolution of Phototrophy in Chloroflexi
genes being mistakenly assigned to these genome bins is low (
e.g.,
10
6
for
pufL
,
pufM
,
pufC
,
bchX
,
bchY
, and
bchZ
in CP2_42A),
providing statistical confidence that phototrophy genes belon
g to
these genome bins.
While other phototrophic Chloroflexi encode carbon fixation
via the 3-hydroxypropionate pathway (e.g.,
Chloroflexus)
or
the Calvin Cycle (e.g.,
Oscillochloris
) (
Shih et al., 2017
), these
pathways are absent from the draft genomes of CP2_42A, JP3_7,
and
Ca.
Roseilinea gracile, potentially reflecting a lifestyle as
photoheterotrophs.
Horizontal Gene Transfer of Phototrophy
within the Chloroflexi
The position of
Kouleothrix
in both organismal and gene trees
is consistent with a vertical inheritance of phototrophy from
the
last common ancestor of the
Roseiflexus
+
Chloroflexus
clade after
its divergence from the nonphototrophic
Herpetosiphon
and
Kallotenue
. However, the other two phototrophic Chloroflexi
reported here, along with
Ca.
Roseilinea gracile reported
previously, reveal a more complex history. In organismal
trees based on conserved vertically inherited proteins
(e.g., RpoB,
Figure 1
, or concatenated ribosomal proteins,
Supplemental Figure 3
), these two strains (CP2_42A and JP3_7)
sit well outside the Chloroflexia class where other phototroph
ic
Chloroflexi are found, separated by many nonphototrophic
lineages (
Figure 1
). However, phylogenetic relationships of
phototrophy-related genes (such as reaction centers and
bacteriochlorophyll synthesis genes) place these strains wi
thin
clades comprised of other phototrophic Chloroflexi (
Figure 2
,
Supplemental Figures 10
,
11
). In reaction center protein trees,
CP2_42A branches within the Chloroflexia, basal to the clade
of
Roseiflexus
and
Kouleothrix
. JP3_7, however, branches more
deeply, sister to the
Roseiflexus
+
Kouleothrix
+
CP2_42A clade
(
Figure 2
). Furthermore,
Kouleothrix
and CP2_42A have fused
pufL
and
pufM
genes, a feature which appears in reaction centers
of
Roseiflexus
(
Youvan et al., 1984; Yamada et al., 2005
), and so
appears to be a synapomorphy of this lineage of phototrophs,
supporting their inclusion at this point in the phototrophy tree
to the exclusion of JP3_7 (which has unfused
pufL
and
pufM
genes). This also suggests that the
pufLM
fusion is a rare event,
and therefore the presence of the fused form of these genes is
a useful trait for assessing the relatedness of reaction cen
ters
independent of overall sequence similarity.
The discordance of the topologies between organismal (e.g.,
concatenated ribosomal protein) and phototrophy (e.g., react
ion
center protein) trees for the novel phototrophic Chloroflexi
described here suggests that phototrophy genes were not
vertically inherited from the last common ancestor of the
phototrophic Chloroflexi. Instead, the differing branching ord
er
of JP3_7 and CP2_42A between organismal (e.g., concatenated
ribosomal protein) and phototrophy (e.g., reaction center
protein) trees, along with the presence of a conserved gene fus
ion
within the
Roseiflexus
+
Kouleothrix
+
CP2_42A clade, strongly
suggests that horizontal gene transfer has played a role in th
e
current distribution of phototrophy in the Chloroflexi phylum.
In light of these data, the simplest scenario for the evolutio
n
of phototrophy within the Chloroflexi requires at least two
instances of horizontal gene transfer to have occurred (
Figure 3
).
In this scenario, the acquisition of an unfused Type 2 reaction
center (and other phototrophy-related genes, such as those
for bacteriochlorophyll synthesis) occurred in an ancestor
of the phototrophic Chloroflexia after their divergence from
Herpetosiphon
and
Kallotenue
. Then, horizontal gene transfer
of phototrophy, including an unfused ancestral Type 2 reaction
center, from the branch leading to
Roseiflexus
into the JP3_7
lineage occurred, followed by a single
pufL
+
pufM
fusion event
in the lineage leading to
Roseiflexus
and
Kouleothrix
. Finally,
there was a second horizontal gene transfer event of phototro
phy,
including the now fused
pufLM
reaction center gene, into an
ancestor of CP2_42A from the
Roseiflexus
lineage; phylogenetic
relationships of the reaction centers show that this must hav
e
occurred after their divergence from
Kouleothrix
. It is important
to note that this is the most parsimonious interpretation that
honors all of the phylogenetic data. More complex scenarios
involving more than two instances of HGT, or extensive HGT in
addition to multiple losses, can also be envisioned.
Presence and HGT of Other Physiologically
Relevant Genes
The genome bins for CP2_42A and JP3_7 recover most, but not
all, of the bacteriochlorophyll synthesis pathway expected fo
r
phototrophic Chloroflexi. These genomes contain
bchX, bchY,
bchZ, bchP, bchF, bchG, bchI, bchD
, and a
bchH
-like gene, but not
bchL, bchN, bchB, bchM
, or
bchE
. While this may be a result of the
incompleteness of these genomes, the same bacteriochlorophy
ll
synthesis gene complement has been described in
Ca.
Roseilinea
gracile (
Klatt et al., 2011
). Microscopic analysis of organisms
tentatively identified as
Ca.
Roseilinea gracile has confirmed
that it exhibits fluorescence characteristic of bacterioch
lorophyll
a
(but not bacteriochlorophyll
c
), consistent with predictions
based on genome content (
Tank et al., 2017
), suggesting that
this organism is capable of bacteriochlorophyll synthesis de
spite
its reduced gene complement. It is possible that some or all
of these genes may actually be absent from these genomes,
functionally replaced by promiscuous homologs (e.g.,
bchL,
bchN
, and
bchB
are homologous to
bchX, bchY
, and
bchZ
,
and chimeras of other homologs of these genes have been
demonstrated to be functionally exchangeable, e.g.,
Cheng et al.,
2005; Wätzlich et al., 2009
). While
bchE
can be functionally
replaced by
acsF
, and
bchL, bchN,
and
bchB
can be functionally
replaced by the light-dependent POR enzyme (
Chew and Bryant,
2007
), these genes were also not recovered in
Ca
. Roseilinea
gracile, JP3_7, or CP2_42A genomes. Our estimates of the
probabilities of missing the same set of genes from multiple
genomes of relatively high (
>
50%) completeness are incredibly
low (
<<
1%); this supports the hypothesis that these genes
truly are absent from JP3_7, CP2_42A, and
Ca.
Roseilinea
gracile (
Supplemental Figure 12
). Ultimately, isolation and
biochemical characterization of the bacteriochlorophyll s
ynthesis
pathway in these organisms will be necessary to test this
notion.
Frontiers in Microbiology | www.frontiersin.org
10
February 2018 | Volume 9 | Article 260