Nutrient Acquisition and the Metabolic Potential of Photoferrotrophic Chlorobi

Abstract

Anoxygenic photosynthesis evolved prior to oxygenic photosynthesis and harnessed energy from sunlight to support biomass production on the early Earth. Models that consider the availability of electron donors predict that anoxygenic photosynthesis using Fe(II), known as photoferrotrophy, would have supported most global primary production before the proliferation of oxygenic phototrophs at approximately 2.3 billion years ago. These photoferrotrophs have also been implicated in the deposition of banded iron formations, the world’s largest sedimentary iron ore deposits that formed mostly in late Archean and early Proterozoic Eons. In this work we present new data and analyses that illuminate the metabolic capacity of photoferrotrophy in the phylum Chlorobi. Our laboratory growth experiments and biochemical analyses demonstrate that photoferrotrophic Chlorobi are capable of assimilatory sulfate reduction and nitrogen fixation under sulfate and nitrogen limiting conditions, respectively. Furthermore, the evolutionary histories of key enzymes in both sulfur (CysH and CysD) and nitrogen fixation (NifDKH) pathways are convoluted; protein phylogenies, however, suggest that early Chlorobi could have had the capacity to assimilate sulfur and fix nitrogen. We argue, then, that the capacity for photoferrotrophic Chlorobi to acquire these key nutrients enabled them to support primary production and underpin global biogeochemical cycles in the Precambrian.

Full text

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ORIGINAL RESEARCH
published: 06 July 2017
doi: 10.3389/fmicb.2017.01212
Edited by:
Trinity L. Hamilton,
University of Cincinnati, United States
Reviewed by:
Katja Laufer,
Aarhus University, Denmark
Erin Field,
East Carolina University, United States
*Correspondence:
Sean A. Crowe
sean.crowe@ubc.ca
Specialty section:
This article was submitted to
Microbiological Chemistry
and Geomicrobiology,
a section of the journal
Frontiers in Microbiology
Received: 01 April 2017
Accepted: 14 June 2017
Published: 06 July 2017
Citation:
Thompson KJ, Simister RL,
Hahn AS, Hallam SJ and Crowe SA
(2017) Nutrient Acquisition
and the Metabolic Potential
of Photoferrotrophic Chlorobi.
Front. Microbiol. 8:1212.
doi: 10.3389/fmicb.2017.01212
Nutrient Acquisition and the
Metabolic Potential of
Photoferrotrophic Chlorobi
Katharine J. Thompson1, Rachel L. Simister1, Aria S. Hahn1, Steven J. Hallam1and
Sean A. Crowe1,2*
1Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada, 2Departments of
Earth, Ocean and Atmospheric Sciences, University of British Columbia, Vancouver, BC, Canada
Anoxygenic photosynthesis evolved prior to oxygenic photosynthesis and harnessed
energy from sunlight to support biomass production on the early Earth. Models that
consider the availability of electron donors predict that anoxygenic photosynthesis
using Fe(II), known as photoferrotrophy, would have supported most global primary
production before the proliferation of oxygenic phototrophs at approximately 2.3 billion
years ago. These photoferrotrophs have also been implicated in the deposition of
banded iron formations, the world’s largest sedimentary iron ore deposits that formed
mostly in late Archean and early Proterozoic Eons. In this work we present new data
and analyses that illuminate the metabolic capacity of photoferrotrophy in the phylum
Chlorobi. Our laboratory growth experiments and biochemical analyses demonstrate
that photoferrotrophic Chlorobi are capable of assimilatory sulfate reduction and nitrogen
fixation under sulfate and nitrogen limiting conditions, respectively. Furthermore, the
evolutionary histories of key enzymes in both sulfur (CysH and CysD) and nitrogen
fixation (NifDKH) pathways are convoluted; protein phylogenies, however, suggest
that early Chlorobi could have had the capacity to assimilate sulfur and fix nitrogen.
We argue, then, that the capacity for photoferrotrophic Chlorobi to acquire these
key nutrients enabled them to support primary production and underpin global
biogeochemical cycles in the Precambrian.
Keywords: Chlorobi, Archean ocean, nitrogen, photoferrotrophy, sulfur
INTRODUCTION
Modern global primary production is supported through oxygenic photosynthesis, which converts
sunlight and CO2into biomass, fuelling the biosphere and driving fluxes of matter and energy
at global scales (Field et al., 1998). Primary production is limited by the availability of nutrients
that are essential for growth such as phosphorus, nitrogen, and sulfur (Howarth, 1988). Primary
producers thus expend valuable energy to meet their nutrient quotas. In the modern oceans, for
example, cyanobacteria can fix nitrogen in the photic zone to support their nitrogen requirements
(Karl et al., 1997). This in turn provides a competitive advantage that frequently allows nitrogen-
fixing cyanobacterial species like Trichodesmium to outcompete non-nitrogen fixing species and
can lead to cyanobacterial blooms (Capone and Carpenter, 1982;Capone et al., 2005). In addition
to their role as primary producers in the modern oceans, cyanobacteria play a key role in
the acquisition and redistribution of nutrients (Carpenter and Romans, 1991), driving global
biogeochemical cycles since their evolution and proliferation in the Precambrian Eons.
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Oxygenic photosynthesis and cyanobacteria emerged early in
the Archean Eon (Crowe et al., 2013; Planavsky et al., 2014),
evolving from anoxygenic phototrophs (Xiong et al., 2000), which
arose as early as 3.8 Ga (Czaja et al., 2013). Like oxygenic
phototrophs, anoxygenic phototrophs fix carbon dioxide into
biomass, but instead of water as the electron donor they use
a diverse set of inorganic species [e.g., H2, H2S, and Fe(II)]
to replace electrons transferred from the photosystem to CO2
(Blankenship et al., 2006). Most anoxygenic phototrophs that
grow in illuminated anoxic waters today use reduced sulfur
species as their electron donors. During much of Earth’s early
history, however, reduced sulfur species were likely scarce and
the chemistry of marine sediments suggests that the oceans
were overwhelmingly iron-rich (ferruginous) for long stretches
of both the Archean and Proterozoic Eons (Canfield et al.,
2008;Planavsky et al., 2011;Poulton and Canfield, 2011). Under
these ferruginous conditions, ferrous iron would have been the
most abundant and available inorganic electron donor (Canfield
et al., 2006). Models for primary production in these ferruginous
oceans suggest that anoxygenic phototrophs using Fe(II) as
their electron donor—photoferrotrophs—could have supported
up to 10% of modern day primary production before the
proliferation of cyanobacteria (Canfield et al., 2006;Jones et al.,
2015). Together, the evolutionary history of the photosystem and
current knowledge on the history of ocean redox states imply
that photoferrotrophs could have played a key role in driving
global fluxes of matter and energy throughout the Precambrian
Eons.
Compelling, but indirect, evidence for photoferrotrophy
during Archean and Paleoproterozoic times comes from the
deposition of banded iron formations (BIFs) (Garrels et al.,
1973;Konhauser et al., 2002;Kappler et al., 2005). BIFs are
massive iron ore deposits that were mostly deposited toward the
end of the Neoarchean, though their deposition spans from the
Eoarchean through to the Neoproterozoic Eras (Klein, 2005).
Classical models for the deposition of iron from seawater to
form BIF invoke large-scale oxidation of seawater Fe(II) by
oxygen produced as a by-product of cyanobacterial growth
and the subsequent precipitation and sedimentation of ferric
iron minerals (Cloud, 1973;Garrels et al., 1973;Walker,
1987). Oxygen levels through the Archean, however, appear
too low to support oxidation of Fe(II) at rates sufficient to
sustain the rapid ferric Fe deposition needed to form even
some of the apparently small BIFs like the Isua Greenstone
belt in Greenland (Czaja et al., 2013). Instead, Fe(III) could
have come from abiotic photochemical iron oxidation through
UV photolysis (Garrels et al., 1973;Hartman, 1984), but this
also appears too slow to support ferric iron deposition at
rates recorded in BIFs (Konhauser et al., 2007). Alternatively,
direct photosynthetic iron oxidation through photoferrotrophy
could supply ferric Fe to form BIFs (Widdel et al., 1993;
Konhauser et al., 2002). Accepting that oxygen levels were too
low to drive Fe(II) oxidation and that UV photolysis appears
similarly ineffective, photoferrotrophy may be the only viable
mechanism to support appreciable ferric iron deposition and
BIF formation. Nevertheless, the role of photoferrotrophs in
BIF deposition remains controversial since direct evidence,
like lipid biomarkers in BIFs, to diagnose photoferrotrophy,
remain elusive. Extant cultures of photoferrotrophic bacteria
are thus employed in efforts to further test the possible role
of photoferrotrophs in BIF deposition and to identify signals
that might be used to diagnose photoferrotrophy in the rock
record.
A total of eight enrichments and isolates of photoferrotrophic
bacteria have been brought into laboratory collections over the
last 30 years. These cultures were largely obtained from a variety
of benthic environments, such as marine mud flats and freshwater
sediments (Widdel et al., 1993;Ehrenreich and Widdel, 1994;
Heising and Schink, 1998;Heising et al., 1999;Straub et al.,
1999), with a single isolate originating from a ferruginous
water column (Llirós et al., 2015). Laboratory cultures of
photoferrotrophs are distributed across the Alphaproteobacteria,
the Gammaproteobacteria, and the Chlorobi and experiments
conducted with these cultures reveal diverse physiological traits
that translate to differential growth rates across a wide range of
culture conditions (Kappler and Newman, 2004;Hegler et al.,
2008;Posth et al., 2010). Notably, under modest light availability,
many of these cultures grow sufficiently fast to oxidize Fe(II)
at rates that would support the deposition of some of the
largest BIFs (Konhauser et al., 2002;Kappler et al., 2005).
This gives confidence in the capacity of photoferrotrophs to
deposit BIFs, but laboratory culturing media are notoriously
nutrient rich. Natural settings, on the other hand, are typically
nutrient poor in comparison (Moore et al., 2013), and thus
the role of photoferrotrophs in both BIF deposition and
primary production would have depended on their capacity
to grow and acquire nutrients from Precambrian seawater at
concentrations almost certainly much lower than typical culture
media.
Many laboratory experiments have been conducted
with photoferrotrophs from the Alphaproteobacteria and
Gammaproteobacteria (Kappler and Newman, 2004;Hegler
et al., 2008;Posth et al., 2010;Bose and Newman, 2011;Pereira
et al., 2012), but the ecological relevance of these groups
in natural ferruginous settings is uncertain. In all modern
ferruginous environments supporting photoferrotrophy,
members of the Chlorobi appear to dominate (Crowe et al.,
2008;Walter et al., 2014;Llirós et al., 2015). Furthermore,
most or many extant photosynthetic communities dominated
by anoxygenic phototrophs are comprised mostly of Chlorobi
(Garrity et al., 2001). While anoxygenic photosynthesis by
the Proteobacteria likely evolved early (Xiong et al., 2000),
more recent phylogenomic analyses imply that the original
phototrophs belonged to the Chlorobi (Sadekar et al., 2006;
Bryant et al., 2012;Satoh et al., 2013). The reason for the
apparent prevalence of the Chlorobi in modern environments
is uncertain, but it is likely related to their ability to grow
under environmentally relevant conditions including low
nutrient availability and low light (Borrego and Garciagil,
1995;Manske et al., 2005;Gregersen et al., 2009;Romero-
Viana et al., 2010;Biderre-Petit et al., 2011;Crowe et al.,
2014a). Thus, despite the fact that photoferrotrophy by
Proteobacteria may be relevant to Precambrian ecosystems,
here, we focus our analyses on the Chlorobi because of
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their apparent ecological prominence in many modern
systems and their deeper ancestry compared to phototrophic
Proteobacteria.
Both phosphorus and nitrogen often limit photosynthetic
activities and primary production in the modern oceans and
in freshwater environments (Howarth, 1988). Phosphorus is
generally considered the ultimate limiting nutrient on geological
time scales as nitrogen can be fixed from the atmosphere
when phosphorus is available (Tyrrell, 1999). Phosphorus is
essential for life and is required in phospholipid, nucleic acid,
and adenosine tri-phosphate (ATP) biosynthesis. Phosphorus
throughout the Precambrian Eons was scarce with seawater
concentrations orders of magnitude lower than today (Jones et al.,
2015). This phosphorus scarcity would have led to low primary
production, influencing the ecology and elemental stoichiometry
of the photosynthetic primary producers (Reinhard et al., 2016).
While phosphorus scarcity likely played an outsized role in
shaping the Precambrian biosphere, nitrogen scarcity may have
developed locally and transiently throughout the Precambrian
Eons (Canfield et al., 2010;Michiels et al., 2017). Nitrogen is
required to build essential cellular components such as DNA
and amino acids. Biologically available nitrogen is supplied
to the oceans through rock weathering and volcanism, but
ammonium uptake and ultimate burial, however, would have
eventually depleted the oceanic bioavailable nitrogen reservoir
(Johnson and Goldblatt, 2015). In the modern ocean, biological
fixation of atmospheric nitrogen keeps pace with phosphate
supplies over geologic time scales (Moore et al., 2009). Nitrogen
fixation is one of the most energetically expensive processes
in the metabolic repertoire of life and yet it is distributed
across distantly related groups of microorganisms (Meyer et al.,
1978;Raymond et al., 2004). This underscores the importance
of nitrogen fixation to microbial growth and production, is
consistent with the early evolution and radiation of nitrogen
fixation (Boyd et al., 2015;Stüeken et al., 2015;Weiss et al.,
2016), and exemplifies how the distribution of core metabolic
machinery across diverse lineages and functional guilds ensures
survival of essential biogeochemical functions over geologic
time (Falkowski et al., 2008). While the genomic potential for
nitrogen fixation exists within the Chlorobi (Bryant et al., 2012),
the capacity of photoferrotrophic Chlorobi to conduct nitrogen
fixation and thus support Precambrian marine nitrogen quotas
remains untested and unsubstantiated. This leaves our knowledge
of the possible ecological role that photoferrotrophs may have
played in the acquisition and redistribution of nitrogen and
its attendant biogeochemical cycling in the Precambrian oceans
entirely unknown.
In addition to phosphorus and nitrogen, sulfur is also essential
for life and can limit biological production and growth when
scarce (Da Silva and Williams, 2001). Sulfur on the modern
Earth is abundantly available as the fully oxidized sulfate ion
due to high concentrations of oxygen in the atmosphere and
oceans, which promotes oxidative sulfur weathering and the
recycling of sulfur from anoxic marine sediments. During the
Precambrian Eons, however, marine sulfate concentrations were
much lower (Walker and Brimblecombe, 1985;Crowe et al.,
2014b) likely due to limited oxidative weathering and recycling
under low O2atmospheres (Walker and Brimblecombe, 1985;
Habicht et al., 2002;Crowe et al., 2014b;Paris et al., 2014;
Zhelezinskaia et al., 2014). Instead, sulfur was likely scarce
and biologically available as low concentrations of sulfate,
very low concentrations of sulfide, and possibly organic sulfur
(Crowe et al., 2014b). Assimilatory sulfate reduction (ASR),
therefore, would have been a key nutrient acquisition pathway,
supporting primary production under low sulfur conditions.
The genomic potential for ASR has been detected within
two members the Chlorobi [Chlorobium ferrooxidans and
Chlorobium luteolum (Frigaard and Bryant, 2008)], yet the
role of ASR in photoferrotrophic growth remains uncertain.
Photosynthetic growth of C. ferrooxidans on ferrous iron and
without reduced sulfur compounds implies that the genomic
potential for ASR translates into physiological capacity to convert
sulfate into biomass sulfur (Heising et al., 1999). Given the
likely low sulfate and extremely low sulfide concentrations
perceived for the Precambrian oceans, ASR may have been
absolutely critical for photoferrotrophs to operate as primary
producers and contribute to a reservoir of biologically available
reduced sulfur compounds in the ocean. The evolutionary
history of ASR in the photoferrotrophic Chlorobi has not been
explored, nor has sulfate uptake been quantitatively assessed.
The role of photoferrotrophs in driving sulfur cycling during the
Precambrian remains underappreciated and untested creating
another gap in our knowledge of nutrient acquisition and
redistribution in the Precambrian oceans.
To address the response of photoferrotrophy to nitrogen and
sulfur scarcity, and to create new knowledge relevant to nitrogen
and sulfur acquisition and redistribution in the Precambrian
oceans, we examined two extant demonstrably photoferrotrophic
Chlorobi: benthic C. ferrooxidans (grown in co-culture with
Geospirillum sp. KoFum) (Heising et al., 1999), and pelagic
Chlorobium phaeoferrooxidans (Llirós et al., 2015;Crowe et al.,
2017). We also examined putative benthic photoferrotroph
C. luteolum, postulated to grow through photoferrotrophy
because of its genomic potential for ASR (Frigaard and Bryant,
2008). We verified the capacity of photoferrotrophic Chlorobi to
fix inorganic nitrogen and sulfur, and constrained the antiquity
of this capacity in the Chlorobi through phylogenetic analyses.
RESULTS AND DISCUSSION
Nitrogen Fixation
The process of fixing dinitrogen is kinetically challenging and
energetically expensive as it involves overcoming the activation
energy required in breaking the triple bond between the two
nitrogen molecules. The enzyme necessary for nitrogen fixation,
nitrogenase, is a multi-subunit protein that is assembled and
regulated by a series of other related proteins. All nitrogenases
require a metal ion cofactor – molybdenum, iron, or vanadium –
with each cofactor being recruited and incorporated into the
nitrogenase by a different set of proteins, Nif, Anf, and Vnf,
respectively. Current studies indicate that the majority of
nitrogenases depend on the molybdenum ion cofactor for their
enzymatic activity (reviewed in Rubio and Ludden, 2008), while
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FIGURE 1 | Nitrogenase gene cassettes of the photoferrotrophic Chlorobi, detailing the position of each gene and the differences and similarities between the gene
cassettes.
the iron and vanadium dependant nitrogenases may play a role
in molybdenum limiting environments (Joerger et al., 1988).
Phylogenetic evidence suggests that the molybdenum-dependant
version of the enzyme evolved first (Boyd et al., 2011), which is
further supported by the observation that organisms identified as
having an iron or vanadium dependant nitrogenase all contain
a copy of the molybdenum-dependant nitrogenase (Raymond
et al., 2004;Soboh et al., 2010). There are up to 25 proteins,
depending on the species, required to assemble and regulate
the nitrogenase including three conserved structural proteins:
NifD, NifK, and NifH. NifH is often used as the marker gene
for nitrogen fixation in natural environments, due to its role
in the main enzyme structure and in cofactor recruitment.
Further phylogenetic information, however, can be obtained
when all three structural proteins (NifDKH) are concatenated
due to increased sequence information and the conserved nature
of all three proteins. Here we explored these key structural
proteins to test for the metabolic potential for nitrogen fixation
in the photoferrotrophic Chlorobi. We compare nitrogen fixation
in photoferrotrophic Chlorobi to the other members of the
phylum Chlorobi and to representatives from all phyla capable of
nitrogen fixation to assess the evolutionary history of nitrogenase
in relevant to photoferrotrophy in the Chlorobi and to place
constraints on the possible role of photoferrotrophs in supplying
fixed nitrogen to the Precambrian oceans.
Distribution of Nitrogen Fixation Pathways within
Chlorobi
Previous analyses of Chlorobi genomes identified that the
metabolic capacity for nitrogen fixation is distributed across the
phylum with the exception of the Ignavibacterium sp. (Bryant
et al., 2012;Liu et al., 2012;Hiras et al., 2016). Ignavibacterium
sp. is the deepest branching member of the Chlorobi and the only
class of non-photosynthetic organisms in the phylum. Here we
show that genes coding for the proteins required for nitrogen
fixation are present in the genomes of the photoferrotrophic
Chlorobi C. ferrooxidans and C. phaeoferrooxidans, putative
photoferrotroph C. luteolum (Figure 1), and in genomes of all
other members of the Chlorobi (data not shown). Specifically, we
identified one homolog of each of the molybdenum-dependant
nitrogenase proteins in all three photoferrotrophic Chlorobi. No
homologs of the alternative vanadium or iron-only nitrogenase
proteins were detected (PSI-Blast, expect threshold 10). These
results indicate that the photoferrotrophic Chlorobi have the
genomic capacity to fix nitrogen. Furthermore, nitrogen fixation
is wide spread among the Chlorobi, with all available Chlorobi
genome sequences coding the necessary proteins apart from
Ignavibacterium album.
Biochemical Verification of Nitrogen Fixation
To test for the biochemical capacity to fix nitrogen during
photosynthetic growth on Fe(II), nitrogen free (below limit
of detection ammonium, ammonia, nitrate, or nitrite) media
was inoculated with C. phaeoferrooxidans or C. ferrooxidans.
Both species were also grown in the standard growth medium
containing 5.6 mM ammonium (Hegler et al., 2008), for
comparison. Both species were able to fix nitrogen while
growing through photosynthetic Fe(II) oxidation with
doubling times of 45 and 36 h for C. phaeoferrooxidans and
C. ferrooxidans, respectively (Figures 2A,B). Fe(II) oxidation
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FIGURE 2 | Fe(II) concentrations and cell counts over time for both C. phaeoferrooxidans (A,C) and C. ferrooxidans (B,D) under two sets of media: no bioavailable
nitrogen – N2as sole nitrogen source (A,B) and ammonium rich (C,D). Data points used to calculate growth rates and Fe(II) oxidation rates are highlighted in each
panel.
rates, during exponential growth phase, were 4.8 ±0.33 µM/hour
(C. phaeoferrooxidans) and 16 ±0.56 µM/hour (C. ferrooxidans)
(Figures 2A,B). Growth under ammonium-rich conditions
supported shorter doubling times (15 and 27 h) and higher rates
of Fe(II) oxidation (50 ±2.4 µM/hour and 23 ±0.7 µM/hour)
for C. phaeoferrooxidans and C. ferrooxidans, respectively
(Figures 2C,D). These results indicate that both pelagic
C. phaeoferrooxidans and benthic C. ferrooxidans are capable
of using dinitrogen gas as their sole source of nitrogen during
growth, but that the need to fix N decreases growth rates.
To further explore the metabolic capacity of photoferrotrophic
Chlorobi under both sets of conditions, cell specific Fe(II)
oxidation rates were calculated for each species. C. phaeoferro
oxidans oxidized Fe(II) at 21.2 ±1.4 fmol/cell while fixing
nitrogen and 47.8 ±2.3 fmol/cell under ammonium-rich
conditions. Conversely, C. ferrooxidans oxidized Fe(II) at
30.0 ±0.9 fmol/cell and 29.4 ±1.0 fmol/cell in ammonium free
and ammonium-rich media, respectively, with no appreciable
difference during N-fixation. The apparent insensitivity of
C. ferrooxidans to N-availability may be related to the presence
of its co-culture partner, Geosprillum sp. KoFum. Further
experiments with KoFum could help constrain its possible
role in N metabolism within the co-culture The observation
that C. phaeoferrooxidans has lower cell specific growth rates
under N scarcity, however, implies lower growth yields during
N fixation. Both species are ultimately capable of growth
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TABLE 1 | Gene length (bp), codon adaptation index (CAI), and GC content (%) for each of the genes in the nitrogenase cassette.
Gene C. phaeoferrooxidans C. ferrooxidans C. luteolum
Length (bp) CAI GC content (%) Length (bp) CAI GC content (%) Length (bp) CAI GC content (%)
NifB 1275 0.80 52.63 1275 0.76 53.18 1263 0.75 60.89
NifN 1353 0.79 53.22 1350 0.75 53.48 1353 0.74 59.42
NifE 1362 0.79 50.07 1362 0.75 49.63 1362 0.74 57.34
NifK 1383 0.81 53.51 1383 0.73 52.78 1380 0.76 60.14
NifD 1635 0.79 49.54 1635 0.76 49.66 1641 0.77 57.22
PII regulator 378 0.81 50.00 378 0.72 50.00 378 0.72 59.26
PII regulator 357 0.76 49.30 357 0.73 48.74 357 0.70 56.30
NifH 825 0.83 49.58 825 0.79 49.21 825 0.81 59.03
Each parameter was calculated for all three photoferrotrophic Chlorobi, whose whole genome GC contents are: 49.72% for Chlorobium phaeoferrooxidans, 49.9% for
C. ferrooxidans, and 58.1% for C. luteolum.
FIGURE 3 | Phylogenies of the Chlorobi and Bacteroidetes using (A) the concatenated NifDKH proteins and (B) 16S rRNA with bootstrap values shown at each
node (maximum likelihood/maximum parsimony). The blue colors delineate the organisms of the Phylum Chlorobi, with each shade representing a different genus,
while the purple color delineates the Phylum Bacteroidetes. The orange lines indicate the position of the photoferrotrophic Chlorobi. The trees were rooted with four
cyanobacterial organisms. Note: Azobacteroides pseudotrichonymphae CFP2 is abbreviated from Candidatus Azobacteroides pseudotrichonymphae genomovar.
CFP2.
and Fe(II) oxidation while fixing nitrogen but the differential
response of cell specific iron oxidation rates to N-scarcity
implies that nutrient availability can influence the ecology of
photoferrotrophs in the environment.
Evolutionary History of Nitrogen Fixation in the
Chlorobi
To assess the evolutionary history of nitrogen fixation in the
Chlorobi we tested for horizontal gene transfer (HGT) within
the photoferrotrophic Chlorobi and conducted phylogenetic
analyses of Nif proteins, which we compared to small subunit
16S ribosomal RNA (SSU rRNA) genes. Deviations in the
branching orders between these phylogenies would indicate non-
vertical inheritance and HGT. To test for horizontal transfer
of Nif genes in the photoferrotrophic Chlorobi, we looked
for characteristic signatures of HGT within nif gene cassettes.
Codon adaptation index (CAI) values, a metric used to describe
differences in codon usage between specific genes and the
genomic background, were calculated for all individual nif
genes belonging to C. ferrooxidans,C. phaeoferrooxidans, and
C. luteolum. All CAI values were greater than the threshold
value, 0.70, below which HGT is indicated (Table 1). In addition,
GC contents of nif genes were very similar to GC contents of
genomic backgrounds providing no evidence for HGT (Table 1).
Our analyses also failed to identify tRNAs, transposases, or
other genetic elements commonly associated with gene mobility
in close proximity (within 5000 bp) to the nitrogenase gene
cassette in any of the photoferrotrophic Chlorobi. The general
lack of tRNAs or transposases near the nif cassettes in the
photoferrotrophic Chlorobi, combined with super threshold CAI
values and nif gene GC contents that are homogenous against
genomic backgrounds, imply nif gene acquisition through
vertical decent.
To test the evolutionary history of the nif genes in the
Chlorobi, we conducted phylogenetic analyses of concatenated
NifDKH proteins of all cultured and sequenced Chlorobi that
have the genomic potential to fix dinitrogen. The Chlorobi
sequences were aligned with selected sequences from the
next closest phylum – Bacteroidetes – and the tree was
rooted using four Cyanobacterial species as an out-group
(Figure 3A). The genus Chlorobium, which includes all three
photoferrotrophic Chlorobi, the genus Chlorobaculum, and
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FIGURE 4 | Phylogenies of (A) the concatenated NifDKH proteins and (B) 16S rRNA for two to four representatives of several nitrogen-fixing phyla with bootstrap
values shown at each node (maximum likelihood/maximum parsimony). The green color delineates the Chlorobi/Bacteroidetes monophyletic grouping, while the
orange line indicates the position of the photoferrotrophic Chlorobi. Note: Azobacteroides pseudotrichonymphae CFP2 is abbreviated from Candidatus
Azobacteroides pseudotrichonymphae genomovar. CFP2.
the genus Prosthecochloris all form monophyletic groups that
are collectively part of the phylum Chlorobi clade. Likewise,
the Bacteroidetes form a monophyletic group and share a
common ancestor with the members of the phylum Chlorobi.
Furthermore, when the NifDKH tree is compared to a 16S
rRNA tree of the same organisms (Figure 3B), all of the
genera within the phylum Chlorobi branch in an identical
order to those in the 16S rRNA phylogeny. The phylogenetic
relationship between the Chlorobi and Bacteroidetes is the same
for both NifDKH and 16S rRNA sequences, indicating that
the common ancestor to the phyla Chlorobi and Bacteroidetes
likely contained a nitrogenase and therefore the ability to fix
dinitrogen. Ignavibacterium sp., the sole members of the phylum
Chlorobi who do not posses a nitrogenase, likely lost the
capability to fix nitrogen as the remainder of the Chlorobi and
the phylum Bacteroidetes bracket the phylogenetic position of the
Ignavibacterium sp. Taken together, available data imply vertical
decent.
Accepting largely vertical descent of NifDKH from the
common ancestor of the Chlorobi and Bacteroidetes, NifDKH
must have emerged within this line of descent before the
divergence of the Chlorobi and Bacteroidetes. The timing of
this divergence has been estimated using a whole genome
molecular clock (David and Alm, 2011) to between 3 and
1.6 Gya, which implies the capacity to fix N in the ancestors
of the Chlorobi before this time. Independent N isotope data
from metasedimentary kerogen implies N fixation by at least
3.2 Gy (Stüeken et al., 2015). Combined, the evidence for vertical
inheritance of NifDKH in the Chlorobi on the taxonomic levels
of genus and phylum, the timing of divergence between the
Chlorobi and the Bacteroidetes, and the N isotope record, imply
that ancestors of modern Chlorobi likely had capacity to fix
nitrogen in the iron-rich oceans of the paleoproterozoic and
perhaps as early as the mesoarchean eras.
To place N fixation in the Chlorobi, and Bacteroidetes, within
the broader context of nitrogenase evolution in general, we
conducted further phylogenetic analyses using a greater diversity
of organisms. We analyzed the NifDKH phylogeny using two
to four representatives from every phylum that had a cultured
and sequenced species with previously documented genomic
potential for nitrogen fixation (Figure 4A). This phylogeny places
the Nif proteins found in the Chlorobi and Bacteroidetes in
a single clade, supporting their emergence from a common
ancestor and the vertical inheritance of NifDKH from this
ancestor. The phylogeny of the NifDKH protein is, however,
incongruent with that of the 16S rRNA gene from the same
organisms (Figure 4B). While the Chlorobi and Bacteroidetes
group together in both phylogenies, the Spirochetes, Chloroflexi,
and Firmicutes also group with the Chlorobi in the NifDKH
phylogeny, but belong to distinct clades in the 16S rRNA gene
phylogeny. The differences between these phylogenies confound
further constraints on the evolutionary history of nitrogenase
within the Chlorobi based on phylogeny and add to the
overwhelming evidence for horizontal transfer of NifDKH genes
(Raymond et al., 2004;Falkowski et al., 2008;Boyd and Peters,
2013).
Ecology of Nitrogen Fixation in Chlorobi, Past and
Present
Members of the phylum Chlorobi underpin biological
production in many modern anoxic environments, both
sulfidic (Overmann, 1997;Tonolla et al., 2004;Gregersen et al.,
2009;Kondo et al., 2009;Meyer et al., 2011) and ferruginous
(Walter et al., 2014;Llirós et al., 2015), through their ability to
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harness light energy and fix inorganic carbon into biomass, even
at low light intensities (Manske et al., 2005). Chlorobi further
contribute to biogeochemical cycling in these systems through
the acquisition and redistribution of essential nutrients, such
as nitrogen. This ecological role would have extended to global
scales in the low oxygen Precambrian oceans. Our analyses
confirm the genomic potential to fix N in all but one of the
Chlorobi lineages and directly demonstrate the capacity of the
photoferrotrophic Chlorobi to fix dinitrogen as their sole source
of nitrogen while oxidizing Fe(II). Rates of Fe(II) oxidation
are, however, slower when photosynthetic growth is supported
through N-fixation rather than ammonium assimilation. To test
the impact of slower rates of Fe(II) oxidation, and therefore
growth, on the deposition of BIFs, we ran our cell counts
and Fe(II) oxidation rates through the calculation outlined
by Konhauser et al. (2002). Our data indicates that both
photoferrotrophic strains would be capable of generating even
the largest BIFs (i.e., the Hamersley BIF) with maximum of
2.44 ×103photoferrotrophic cells/mL required in the basin.
Thus, photoferrotrophic growth coupled to N-fixation could
support BIF deposition, even in the face of nitrogen scarcity.
Chlorobium phaeoferrooxidans, and C. ferrooxidans exhibit
differential responses to N scarcity that manifest in different
cell specific Fe(II) oxidation rates and different ratio’s between
microbial growth (cell doubling times) and Fe(II) oxidation.
C. phaeoferrooxidans has a cell doubling time to Fe(II)
oxidation ratio of 9.4 under N-fixing conditions compared to
0.3 when there is ample ammonium, whereas C. ferrooxidans
has comparable ratio’s of 2.3 and 1.2 for N-fixing and
ammonium-rich conditions comparatively. This differential
response indicates that under ammonium-rich conditions
C. phaeoferrooxidans grows more efficiently (i.e., with a
higher growth yield) whereas when N-fixation is required
C. ferrooxidans grows more efficiently. This creates niches for
each microorganism defined by N availability. The differential
response also implies that the stoichiometry of Fe-oxidation to
biomass production and cell growth is partly decoupled and
depends on N availability. Essentially, this decoupling means
that more Fe(II) is oxidized to produce an individual cell during
growth supported by N-fixation than by ammonium assimilation.
Such a decoupling thus requires either the diversion of reducing
equivalents (NADH) produced during photosynthesis into
compounds not used directly in cell growth, or that cell growth
and division requires more fixed carbon during N-fixation. The
former could include conversion of N2to ammines and the
biosynthesis of cell exudates, and the latter might include the
biosynthesis of cellular proteins needed to conduct N-fixation.
Such a decoupling would influence the overall biogeochemical
functioning and ecology of ecosystems supported through
primary production by photoferrotrophy. The overall activity
of the marine biosphere through the Precambrian Eons may
thus have been influenced by the availability of fixed N to
photoferrotrophs.
Assimilatory Sulfate Reduction (ASR)
Sulfate ions are biologically inert and organisms expend
tremendous energy ‘activating’ sulfate for three main functions:
(1) reduction and incorporation into amino acids; (2)
condensation and incorporation into sulfolipids and other
small molecules; and (3) for dissimilatory sulfate respiration.
In addition to the reduction of sulfate, organisms can acquire
organic sulfur compounds like amino acids, and hydrogen sulfide
directly from the environment. Acquisition of these reduced
sulfur compounds can considerably reduce the expenditure of
energy on sulfur acquisition. Here we focus on the first two
assimilatory pathways and the capacity for reductive sulfur
assimilation in the photoferrotrophic Chlorobi. The proteins
required to complete an entire ASR pathway include: CysD, the
sulfateadenyl transferase that activates sulfate to form APS; CysN
which catalyzes GTP hydrolysis providing the energy needed to
adenylate imported sulfate; CysC (a domain of CysN), the APS
kinase that phosphorylates APS to PAPS; and CysH, the APS
reductase which reduces the sulfur in APS to sulfite. We have
explored the metabolic potential for sulfate assimilation in the
genomes of the photoferrotrophic Chlorobi and directly tested
sulfate incorporation into biomass.
Distribution of ASR Pathways within Chlorobi
Previous analyses of Chlorobi genomes identified the metabolic
capacity for ASR in C. ferrooxidans and C. luteolum (Frigaard
and Bryant, 2008). Using all currently available genomic
information we identified components of the ASR pathways
distributed throughout the Chlorobi (Table 2). We find
that the photoferrotrophic Chlorobi, C. ferrooxidans and
C. phaeoferrooxidans, as well as putative photoferrotroph
C. luteolum, all possess the necessary proteins for ASR – CysD,
CysN/C, CysH – and therefore have the potential capacity
to synthesize amino acids from exogenous sulfate (Figure 5).
Notably, the presence of both CysD and CysN indicate that
sulfate activation to APS in these Chlorobi is coupled to
GTP hydrolysis. Sulfate assimilation in the Chlorobi, therefore,
offsets the energetic expense associated with sulfate activation.
The presence of CysN/C indicates the metabolic potential to
phosphorylate APS to PAPS implying that these strains might
have capacity to synthesize sulfate-containing compounds like
sulfolipids. Finally, while components of assimilatory sulfate
metabolisms are more broadly distributed throughout the
Chlorobi, genes coding for key components of the pathway are
mostly missing implying a lack of capacity for sulfate assimilation
outside the photoferrotrophic Chlorobi (Table 2). Given the
metabolic potential for ASR in the photoferrotrophic Chlorobi,
we sought to biochemically verify this process.
Biochemical Verification of ASR
Chlorobium phaeoferrooxidans and C. ferrooxidans are both
known to grow in media where sulfur is supplied exclusively
in the form of sulfate, which directly demonstrates the
physiological capacity for sulfate assimilation. We quantitatively
tested this capacity by measuring the uptake of 35S labeled
sulfate in low sulfate growth media. C. phaeoferrooxidans,
indeed took up 35S labeled sulfate into TCA extractable
biomass, demonstrating assimilatory reduction of sulfate and its
incorporation into amino acids. Over the course of these sulfate
uptake experiments, C. phaeoferrooxidans oxidized 3070 µM
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TABLE 2 | Assimilatory (orange) and dissimilatory (blue) sulfur proteins present in the genomes of green sulfur bacteria.
Organism CysD Sat CysNC CysH AprAb DsrAB
Chlorobium phaeoferrooxidans KB01 + − + + − −
Chlorobium ferrooxidans DSM 13101 + − + + − −
Chlorobium luteolum DSM 273 + − + + − +
Chlorobium phaeovibrioides DSM 265 + − + − − +
Prosthecochloris aestuarii DSM 271 + − + − − +
Chlorobium phaeobacteroides BS1 − + + − + +
Chlorobium chlorochromatii CaD3 − + + − + +
Pelodictyon phaeoclathratiforme − + − − + +
Chlorobium tepidum TLS − + − − + +
Chlorobium phaeobacteroides DSM 266 − − − − − +
Chlorobium limicola DSM 245 − − − − − +
Chlorobaculum parvum NCIB 8327 − − − − − +
Chloroherpeton thalassium − − − − − −
Ignavibacterium album − − − − − −
FIGURE 5 | Assimilatory sulfate reduction (ASR) gene cassettes for the photoferrotrophic Chlorobi, detailing the position of each gene and the differences and
similarities between the gene cassettes.
Fe(II). This implies the fixation of 770 µM C, based on
the 4:1 stoichiometry between Fe(II) oxidation and C fixation
observed for C. phaeoferrooxidans during growth on Fe(II),
and for photoferrotrophic organisms, more generally (Widdel
et al., 1993). A corresponding total of 3 µM S was fixed
demonstrating a ratio of 260:1 C to S, which we take as
approximately indicative of the S content of C. phaeoferrooxidans.
There are few data to compare with, but our results suggest
that C. phaeoferrooxidans has relatively low S quotas compared
to aquatic and cultured bacteria (C:S from 10–60) (Fagerbakke
et al., 1996) and particulate organic matter from the North
Pacific (C:S of 50) (Chen et al., 1996). By analogy to
C. phaeoferrooxidans, photoferrotrophic Chlorobi likely have
capacity to fix sulfate into biomass under low sulfate conditions,
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which they appear well adapted to do based on minimal cellular
sulfur quotas in comparison to other bacteria and marine organic
material.
Evolutionary History of ASR in the Chlorobi
To test for horizontal transfer of ASR genes to the
photoferrotrophic Chlorobi, we searched for characteristic
signatures of HGT within the ASR cassettes and conducted
phylogenetic analyses of ASR genes, which we compared to 16S
rRNA gene phylogenies. The CAI value for each of the ASR
genes belonging to C. ferrooxidans and C. phaeoferrooxidans
were all greater than the threshold value, 0.70, below which HGT
is indicated (Table 3). ASR genes in C. luteolum, however, had
sub-threshold CAI values, as low as 0.53, indicating possible
ASR gene acquisition through horizontal transfer. The GC
contents of ASR genes for all three species were very similar
to GC contents of their respective genomic backgrounds,
providing no evidence for HGT (Table 3). Collectively, these
data provide little evidence for the lateral acquisition of ASR
gene cassettes in the photoferrotrophic Chlorobi, although the
evidence for vertical descent is greater in C. phaeoferrooxidans
and C. ferrooxidans than in C. luteolum. A single transposase
(Figure 5) was found on a contig adjacent to that hosting the ASR
gene cassette in C. phaeoferrooxidans. The general lack of tRNAs
or transposases near the ASR cassettes in the photoferrotrophic
Chlorobi combined with super threshold CAI values and ASR
gene GC contents that are homogenous against the genomic
backgrounds, implies ASR gene acquisition through vertical
decent.
To further test the evolutionary history of ASR, the CysH
protein was analyzed to examine the phylogenetic relationship
between the proteins used in the photoferrotrophic Chlorobi
and ASR in other organisms. The photoferrotrophic Chlorobi
grouped together forming a monophyletic clade within the
CysH phylogeny (Figure 6A). The photoferrotrophic Chlorobi
exhibit congruent phylogenies between the CysH protein and
16S rRNA gene (Figure 6B), providing further evidence in
support of vertical inheritance of the ASR pathway in the
photoferrotrophic Chlorobi. The more general evolutionary
history of the CysH protein, however, is convoluted given
abundant incongruences between the CysH protein and 16S
rRNA gene phylogenies. Accepting vertical inheritance of the
ASR pathway in the photoferrotrophic Chlorobi and the early
divergence of the Chlorobi from other organisms, we hypothesize
that gene loss explains the lack of a complete ASR pathways in
other photosynthetic Chlorobi and this hypothesis is supported
by the partial presence of ASR pathway components across the
phylum Chlorobi (Table 2).
Ecology of ASR in Chlorobi, Past and Present
The presence of an ASR pathway in all known photoferrotrophic
Chlorobi implies that ASR is advantageous to growth under
ferruginous conditions. The lack of the ASR pathway in the
canonically sulfur oxidizing Chlorobi makes sense in light of
the availability of reduced sulfur compounds in their preferred
habitats. The energetic expense of ASR would tend to favor
assimilation of reduced compounds when available. Conversely,
ferruginous environments are by definition sulfur poor and the
availability of reduced sulfur compounds can be limited by the
solubility of FeS. Sulfate, therefore, is likely the most abundant
and available sulfur source in modern ferruginous environments.
The rock record also demonstrates that ferruginous marine
conditions persisted throughout much of the Precambrian
Eons and reduced sulfur species were likely scarce with the
exception of in the apparently ephemeral developments of costal
euxinia. ASR may thus have supported sulfur requirements
of photoferrotrophic primary producers over long stretches of
Earth’s history.
The apparent role of ASR in supporting primary production
through photoferrotrophy implies that sulfate availability could
have been an important control on global productivity. At
28 mM, sulfate is the principle anion in modern seawater, but
sulfate concentrations could have been as low as a few µM
in the Archean oceans (Crowe et al., 2014b). Nutrients like
phosphorus and nitrogen are known to become limiting at such
low concentrations. The apparently low sulfur quotas of the
photoferrotrophic Chlorobi (260:1, C:S) thus seem well adapted
to growth in the low sulfate oceans of the Archean, which would
have enhanced productivity in the face of sulfur scarcity.
Under low sulfate conditions dissimilatory sulfate reduction
(DSR) would have played a comparatively small role in the
remineralization of organic matter in Archean oceans (Crowe
et al., 2014b). Qualitatively then ASR would have played an
outsized role in the reduction of sulfur and the global sulfur
cycle in the Archean oceans, relative to today. We therefore
hypothesize that primary production through photoferrotrophy
was a key pathway in the production of an organic reduced
sulfur pool, which would have provided an important vector
for sulfur to Archean sediments. We further hypothesize that
ASR may have predated DSR. Earliest evidence for DSR comes
from S-isotope fractionation recorded in 3.47Gya barites (Shen
et al., 2001), whereas photoferrotrophy likely operated as early
as 3.8Gya (Czaja et al., 2013) and presumably required ASR. The
idea that ASR predates DSR could be tested if homology could
be established in enzymes involved in both pathways. Although
ASR and DSR serve different functions – sulfate acquisition
versus energy transduction, respectively – both pathways actively
transport sulfate into the cell and the first enzymes in the
pathways are thus analogous. Comparison of amino acid
sequences of the first enzymes (CysD and Sat, respectively) in the
two pathways indicates a strong degree of homology implying
evolutionary relationships between components of ASR and
DSR.
To examine the phylogenetic relationships between enzymes
that transport sulfate for use in ASR and DSR, we aligned
CysD and Sat proteins from the majority of the Chlorobi and a
selection of representative microorganisms from diverse phyla.
The resulting phylogeny clearly separated amino acid sequences
annotated as CysD from those annotated as Sat (Figure 7). Both
CysD and Sat appear to support sulfate transport in relation to
multiple sulfur metabolisms, but the phylogenetic relationships
appear complicated and likely require more detailed analyses.
Nevertheless, homology between the two proteins implies a
possible evolutionary relationship between ASR and DSR that
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TABLE 3 | Gene length (bp), CAI, and GC content (%) for each of the genes in the ASR cassette.
Gene C. phaeoferrooxidans C. ferrooxidans C. luteolum
Length (bp) CAI GC content (%) Length (bp) CAI GC content (%) Length (bp) CAI GC content (%)
CysH 714 0.78 54.34 714 0.78 54.62 753 0.59 56.97
CsyD 882 0.80 56.12 882 0.78 55.56 915 0.61 57.38
CsyN/C 1800 0.81 54.28 1800 0.77 53.67 1800 0.69 58.06
Siroheme synthase 453 0.81 54.08 453 0.71 52.98 453 0.53 57.17
Uroporphyrin-III
C-methyltransferase
1287 0.73 57.96 1287 0.73 55.40 258 0.62 57.36
CysA 1074 0.77 53.26 1074 0.80 52.42 1074 0.67 59.22
CysW 870 0.79 53.22 870 0.79 52.76 870 0.64 58.74
CysT 834 0.79 52.64 834 0.81 53.36 834 0.66 58.03
Sulfate transporter 1020 0.82 53.14 1020 0.79 52.65 1008 0.75 59.52
Each parameter was calculated for all three photoferrotrophic Chlorobi, whose whole genome GC contents are: 49.72% for C. phaeoferrooxidans, 49.9% for
C. ferrooxidans, and 58.1% for C. luteolum.
FIGURE 6 | Phylogenies of (A) the CysH protein and (B) 16S rRNA with bootstrap values shown at each node (maximum likelihood/maximum parsimony). The
orange line indicates the position of the photoferrotrophic Chlorobi. The trees are rooted with two Archaeal species.
may inform evolutionary histories and should be tested in the
future.
OUTLOOK
Photoferrotrophy links the C and Fe biogeochemical cycles
through coupled CO2fixation and Fe(II) oxidation and has
likely done so since the early Archean Eon. Models for
photoferrotrophic growth in the Archean oceans remain poorly
constrained as they are extrapolated from growth rates in
nutrient rich laboratory culture media. Here we demonstrate
that photoferrotrophic Chlorobi have the physiological capacity
to fix inorganic N and S into biomass when availability of
these nutrients is low and have likely had this capacity since
the Archean Eon. Thus, under N and S limited ferruginous
conditions, photoferrotrophy underpins biogeochemical cycling
of C, N, S, and Fe. Nutrient availability, however, influences
growth and Fe(II) oxidation rates and has consequences for
the stoichiometric relationships between C, N, S, and Fe
transformations. Undoubtedly, these relationships should be
assessed and studied in more detail with additional physiological
experimentation and should be applied to further constrain
models of photoferrotrophy, biological production, and global
biogeochemical cycling in the Archean Eon.
MATERIALS AND METHODS
Strains and Growth Medium
Media was prepared after Hegler et al. (2008), and allocated into
serum bottles (100 mL media and 160 mL total volume), with
0.3 g/L NH4Cl, 0.5g/L MgSO4·7H2O, 0.1g/L CaCl2·2H2O, and
0.6g/L KH2PO4. After autoclaving, 22 mmol L−1bicarbonate,
trace elements, mixed vitamin solution, selenate-tungstate,
vitamin B12, and FeCl2were added and the pH was adjusted
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FIGURE 7 | Phylogeny of the Sat/CysD protein with bootstrap values shown at each node (maximum likelihood/maximum parsimony), to compare the ASR and
DSR pathway among a diverse set of organisms. The dashed line delineates the organisms with CysD versus those with Sat. The orange line indicates the position
of the photoferrotrophic Chlorobi.
to 6.8–6.9 under an N2/CO2atmosphere (80:20). 10 mmol L−1
FeCl2was added to all media (regular, NH4+deplete, and SO4−
poor) – Fe(II) concentrations from 200 µmol L−1to 10 mmol
L−1have been shown to produce the same growth rates under
nutrient rich conditions. The 10 mmol L−1media was filtered
after being made to remove any precipitates, which resulted in a
final Fe(II) concentration of 2 mmol L−1for the standard media
and 4mmol L−1for the NH4+deplete media. The low SO4−
media was left unfiltered with an Fe(II) concentration of 10 mmol
L−1. In the ammonium free media, NH4Cl was replaced with
0.3 g/L KCl and an additional 10 mL of N2gas was injected into
the headspace. In the low sulfate media, 0.0025 g/L MgSO4and
0.4 g/L MgCl2were added instead of the usual 0.5 g/L MgSO4.
Furthermore, approximately 10 kBq of carrier-free 35S was added
to all of the low sulfate cultures. The cultures for the N-fixation
experiments were grown in ammonium free conditions once and
then transferred into the final experimental bottles. The culture
for the S35 experiment was grown up in standard media, spun
down and decanted to avoid adding extra sulfate, before the cells
were inoculated into the final experimental bottles. All cultures
were grown under a constant light intensity of 14 µE m−2s−1.
Analytical Techniques
Spectrophotometric analysis of Fe(II) and Fe(III) concentrations
were performed using the ferrozine method; samples were
measured directly as well as after being fixed in 1 N
HCl – after Viollier et al. (2000). Pigments were measured
spectrophotometrically after 24 h extractions of 1 mL of pelleted
cells in acetone:methanol (7:2 v/v) (Frigaard et al., 1996).
Cells numbers were then obtained using a pigment to cell
count conversion factor of 6.3 ×10−10 pigment/cell/mL for
C. phaeoferrooxidans and 5.8 ×10−10 pigment/cell/mL for
C. ferrooxidans. The cells from the 35S experiment were collected
via filtration along with a liquid sample as a background
measurement. The filtered samples were subsequently washed
with 5% Trichloroacetic acid (TCA) in order to kill, wash, and
dissolve cellular material. TCA precipitates DNA and proteins,
leaving only these cellular components on the filter and therefore
any counts associated with the filtered samples would indicate 35S
that had been incorporated into this cellular biomass (Cuhel et al.,
1981). Five milliliter of scintillation fluid were added to the 35S
samples (1 mL of liquid or the filter) and all samples were counted
using a scintillation counter.
Bioinformatics
Genomes of Chlorobi stains used in this paper were retrieved
from NCBI under the following accession numbers with
the completion percentage of each genome in brackets after
the number: NC_008639.1 (99.45%), NZ_AASE00000000.1
(90.71%), NC_007514.1 (97.8%), NC_009337.1 (98.91%),
NC_010803.1 (99.98%), NC_002932.3 (97.8%), NC_011027.1
(98.89%), and NC_007512.1(98.91%). Genomes were
analyzed using MetaPathways V2.5.1, an open source
pipeline for predicting reactions and pathways using default
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settings1(Konwar et al., 2013, 2015) and using the following
databases: MetaCyc-v4-11-07-03 (Caspi et al., 2012), Kyoto
Encyclopedia of Genes and Genomes (KEGG-11-06-18)
(Kanehisa et al., 2008), SEED-14-01-302, Clusters of Orthologous
Groups (COG-13-12-27) (Kaufmann, 2006), Carbohydrate-
Active enZYmes (CAZY-14-09-04) (Cantarel et al., 2009), and
RefSeq-nr-14-01-18 (Agarwala et al., 2015) databases. Initially,
we identified all sequences with a functional assignment affiliated
with nitrogen fixation and assimilatory sulfur reduction using the
MetaPathways functional annotation table output.
Phylogenetic Trees for Nitrogen Fixation
Individual NifDKH gene sequences from all organisms outside
of the phylum Chlorobi were retrieved from NCBI searches
from described strains, concatenated, and then aligned using
the package software ClustalX2.1 (Larkin et al., 2007). To
rigorously test the evolutionary history of nitrogen fixation
multiple tree construction methods [Maximum likelihood (ML)
and Maximum parsimony (MP)] were employed. ML and MP
trees were constructed in MEGA version 7 (Tamura et al.,
2013;Kumar et al., 2016) and all trees bootstrapped 500 times.
Bootstrap values are indicated at the nodes.
Phylogenetic Trees for Assimilatory
Sulfate reduction
CysH and CysD/Sat gene sequences from all organisms outside
of the phylum Chlorobi were retrieved from NCBI searches
from described strains, and then aligned using the package
software ClustalX2.1 (Larkin et al., 2007). To rigorously test the
evolutionary history of ASR multiple tree construction methods
1https://github.com/hallamlab/metapathways2/wiki
2http://www.theseed.org/
(ML and MP) were employed. ML and MP trees were constructed
in MEGA version 7 (Tamura et al., 2013;Kumar et al., 2016)
and bootstrapped 500 times. Bootstrap values are indicated at the
nodes.
Phylogenetic Trees for 16S rRNA
16S rRNA sequences were retrieved from strains used in Nif
and ASR gene trees from the Silva online database – version
128 (Pruesse et al., 2007;Quast et al., 2012). Only full-length
(>1400 bp) sequences were selected, and these were aligned,
using the package software ClustalX2.1 (Larkin et al., 2007). To
rigorously test the evolutionary history of nitrogen fixation and
ASR multiple tree construction methods (ML and MP) were
employed. ML and MP trees were constructed in MEGA version
7 (Tamura et al., 2013) and all trees bootstrapped 500 times.
Bootstrap values are indicated at the nodes.
AUTHOR CONTRIBUTIONS
KT performed all laboratory work, except for biochemical
verification of assimilatory sulfate reduction, which was
performed by SC; KT, RS, and SC interpreted and analyzed the
data with bioinformatic data analysis conducted by AH; KT and
SC wrote the paper with input from RS. SH contributed to data
interpretation and insights, SC supervised the group.
FUNDING
This work was supported by Agouron Institute and NSERC
discovery grants to SC.
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