Enrichment
and
characterization
of
metal
oxidizing
photoautotrophs
MBL
Microbial
Diversity
2009
Benjamin
J
Tully
2
Abstract
Understanding the metabolic pathway of photoautotrophic iron oxidation, the oxidation of Fe2+
to Fe3+ coupled to the fixation of CO2, can provide detailed insight in to how microbial
communities shaped the early Earth in a period before oxygenic photosynthesis. The anaerobic,
high-light and high levels of free Fe(II) at School Street Marsh is an ideal environment to isolate
photoferroautotrophs. By developing new molecular probes it may be possible to track the
number of organisms possessing the genetic potential to perform phototrophic iron oxidation and
monitor the development of these organisms in enrichment cultures. This project potentially has
developed a successful set of primers capable of amplifying key phototrophic iron oxidation
genes from the environment and used them to monitor development inconjunction with qPCR
assays. Characterization of the organisms in three enrichments through 16S clone libraries was
performed and reveals a pattern of unique clades in all three enrichments, wholly distinct from
current 16S rRNA databases, including from an enrichment for Mn(II) oxdizing photoautotrophs.
Introduction
Understanding photoautotrophic oxidation of ferrous iron has a direct impact on our
understanding of how microbial communities have impacted the Earth over large geologic
timescales. Current theories suggest that photoferroautotrophs possess an ancient metabolic
pathway that proliferated in a period of time on the Earth when the atmosphere was anoxic and
most abundant reducing power was in the form of Fe2+(Walker 1983). Such organims are
capable of the following metabolic reaction:
4Fe2+ + CO2 + 11H2O + hv --> [CH2O] + 4Fe(OH)3 + 8H+
The ancient nature of this pathway is supported by the wide diversity of organisms that are
capable of utilizing it, including the green sulfur lineage of the Bacteriodetes and the purple
photosynthetic lineage in the Proteobacteria (Ehrenreich 1994). It has been suggested that the
intermediate redox potential of Fe(II) could have allowed it act as a form of transition
metabolism between anoxygenic and oxygenic photosynthesis during the course of evolution of
photosynthesis (Olson 2004). And in turn, the production of iron oxides in the absence of oxygen
through microbial actions may explain the presence of band iron formations (BIFs) in periods
throughout the Precambrian prior to the oxidation event that would follow the evolution of
oxygenic photosynthesis (Konhauser 2002). Continued study of this process and the organisms
that perform it will help to increase our understanding of the early Earth and the way
microorganisms shaped the early history of the planet.
Organisms capable of phototrophic Fe(II) oxidation possess a three gene operon known
as the pio operon (phototrophic iron oxidation). This operon has been shown to be required for
organisms that utilize Fe(II) as the source of electrons in anoxygenic photosynthesis. In brief,
pioA has been determined to be a putative c-cytochrome used to carry electrons donated from
Fe(II); pioB is a putative porin allowing Fe(II) to enter the periplasm; pioC contains an ironsulfur active site (Jiao 2007). Primers targeting conserved regions of these genes within the
operon could be used to search the environment for organisms capable of this unique metabolic
pathway.
3
Figure 1 Orginization of pio operon genes in R. palustris TIE-1, with arrows indicating the orientation of
transcription
In a similar vein to photoferroautotrophs, it should be metabolically feasible for
microorganisms to survive using a lifestyle that is photoautotrophic in nature and instead of
oxidizing Fe(II), oxidizes Mn(II) as the source of electrons. To date, there has been no successful
isolation or characterization of organisms capable of the process.
This project will attempt to increase our understanding of both these processes and the
organisms that perform them. Ease of access to an environment the possesses anaerobic
conditions and high levels of both Fe(II) and Mn(II), in the form of School Street Marsh, offers a
prime opportunity to design enrichments that would select for organisms capable of these
processes. The ability to detect organisms capable of phototrophic iron oxidation from
environmental samples would be a useful tool for tracking how prolific these organisms are and
allow for the monitoring of development of such organisms in an enriched community. The goal
of this experiment is to enrich for organisms capable of performing photosynthetic Fe(II)- and
Mn(II)-oxidation, while monitoring enrichment development using environmental primers
designed using the known sequence of the pio operon.
Methodology
Media construction. Media for enrichment of Fe(II)- and Mn(II)-oxidizing photoautoptrophs
was adapted from Croal 2004. To 1L deionized H2O, the following was added:
Compound Mass (g)
NH4Cl
0.3
KH2PO4
0.5
MgCl2
0.4
MgSO4
0.2
CaCl2
0.1
This solution was autoclaved at 15 psi and 121°C for 40 minutes with a stir bar present. After
initial cooling on ice (~5-10 min), the media was allowed to cool under bubbling of 20%
CO2/80% N2, with constant stirring. Once cooled, 22 mL of 1M NaHCO3 (sterile, autoclaved
under CO2 headspace) was added. The Microbial Diversity 2009 stock vitamin, trace elements
and vitamin B12 solutions were added to media at volumes of approximately 0.5, 0.5 and 0.7
mL, respectively. The media was then moved to the anaerobic glove box and electron donors
were added. Stock solutions of FeSO4, FeCl2, MnCl2 and MnSO4 were prepared using sterile,
anaerobic H2O:
4
mL to bring
enrichment to 25 mL
total
Electron donor
mols/25 mL
enrichment
FeSO4
0.0002
FeCl2
0.000375
1.0
1.187
MnCl2
0.0001
0.93
0.532
MnSO4
0.0001
0.794
0.532
g/25 mL stock
1.307
1.063
Stocks for both Fe(III) chelators were prepared as such: 1.606g of NTA added to 25 mL sterile,
anaerobic H2O (final concentration, 25 mM) and 0.13g of EDTA to 25 mL sterile, anaerobic H2O
(final concentration, 12.5 mM). One mL was added to each desired 25 mL enrichment (total
volume, 26 mL).
Addition of the metal ions results in the precipitation of a white, fluffy material. According to
Emerson 2005, the precipitate is most likely vivianite (Fe[PO4]2·8H2O) or siderite (FeCO3) and
"[t]he precipitate should not affect cell growth."
Sample collection, enrichments & extractions. For the enrichments, all media containing only
the electron donor were inoculated in duplicate, while only single enrichments were created for
Fe(II) containing enrichments with NTA and EDTA chelators. The following negative controls
were also prepared: sterile media with sample and no electron donor; incubations in the dark
with sample and all electron donors; incubation in the dark with no sample and FeCl2.
Two enrichments were inoculated from different core samples. Enrichment 1 consisted of
~0.25g of sample placed in 25mL of media in screw cap Pfennig bottles, which were removed
from the anaerobic glove box and incubated under aerobic atmospheric conditions. This core was
collected from School Street Marsh (July 8th, 2009) after a severe rainstorm, which removed
much of the visible evidence of mat structure. The vertical profiles of O2, H2S and pH of Core 1
was obtained using Unisense voltammetric probes. Enrichment 2 consisted of ~0.25g of sample
placed in 25mL of media in capped-and-sealed serum bottles under anaerobic conditions. This
core was collected from School Street Marsh (July 13th, 2009) after the water level had subsided
and mat structure appeared to reforming.
Incubations in the light were placed ~30 cm from a 40-W incandescent bulb and kept at
~24°C, while incubations in the dark ~23°C.
Secondary Enrichments. After 7 days, turbidity was noted in many of the Fe(II) containing
aerobic Pfennig enrichments. 0.5 mL of the 1° enrichment was added to sterile media containing
respective electron donor sources and placed in the light and dark, in the same fashion described
above.
DNA extractions. Environmental DNA extractions were performed using the Mobio Powersoil
kit, while enrichment DNA extractions were performed using the UltraClean Microbial DNA
Isolation kit, following standard kit protocols.
Molecular probe design. Created an alignment using ClustalW of the 4 known sequences of
pioB in 4 Rhodopseudomonas palustris strains. The corresponding locus tags in each genome are
as follows:
5
Locus tag
Strain R. palustris
Rpal_0816
Length of amplicon (bp)
TIE-1
203
RPA0745
CGA009
203
RPC_2959
BisB18
196
RPE_0832
BisA53
176
The following primers were developed from the alignment. The names of the primer correspond
to the base pair the primer begin on in strain CGA009. Forward primer: pioBF_RPA477 5' GAG
CAT CTG GAA CGG CGT 3'. Reverse primer: pioBR_RPA679 5' CGG GHG TCC AGC GAT
AGG 3'. Self-dimer and hetero-dimer melt temperatures were determined using the
oligonucleotide analyzer on the Integrated DNA Technologies website.
PCR. A stock culture of R. palustris TIE-1 was obtained from the Newman laboratory at MIT,
grown up overnight in sterile YP media (0.3% yeast extract and 0.3% Bacto Peptone) (Jiao 2007)
and genomic DNA was extracted using the UltraClean Microbial DNA Isolation kit. Using this
kit, 24.7 ng/µL of DNA was extracted and diluted to a working stock concentration of 0.5 ng/µL.
Amplification was optimized for a 25µL reaction, with the corresponding volumes:
Volume (µL)
Taq Mix
12.5
pioBF (10 µM) 0.75
pioBR (10 µM) 0.875
H2 O
4.875
Template
6
The cycle protocol was also optimized, such that the initial melt time was 5 minutes at 95°C,
followed by 35 cycles of 95°C melting for 30 seconds, 60°C annealing for 30 seconds and 72°C
extension for 40 seconds, with a 5 minute final extension at 95°C.
For environmental samples. To inactivate the large amounts of humics present in
environmentally extracted DNA, 0.4 µL of H20 was substituted with 0.4 µL of 10 mg/mL bovine
albumen serum (BSA).
Quantitative PCR. Standards. DNA standards were designed by applying the TOPO TA
cloning kit (Invitrogen) to standard PCR pioB products amplified from R. palustris TIE-1.
Plasmids containing the pioB PCR products were used to transform chemically competent E.
coli, and purified using the QIAprep spin Miniprep kit (Qiagen). A 16S standard plasmid was
obtained in a similar manner from previously constructed 16S rDNA clone libraries. For the
standard curve, an initial dilution of the plasmid 1:100, was followed 7 times by a 1:10 serial
6
dilution. For initial runs, 6 of the 8 dilutions were used in the standard curve, to cover the
potential range of DNA concentrations in samples.
Real-Time PCR. All reactions were performed in 25 µL (12.5 µL Sybr Green, 0.4 µL BSA, x µL
forward primer, y µL reverse primer, z µL H2O, 1 µL DNA) reactions using iQ Syber Green
Supermix (BioRad) and the Applied BioSystems StepOnePlus Real-Time PCR system.
Reactions amplifying pioB used the primers described above. Reactions amplifying 16S rDNA
used previously determined primers Bac_331F and Bac_797R. The cycle protocol used consisted
of an initial 10 minute, 95°C melt step, followed by 40 cycles which consisted of 95°C melting
for 15 seconds and 60°C annealing and extension for 1 minute. Following amplification, a melt
curve was determined for all samples using the Applied BioSystems, standard 2 hour melt
protocol. As described above, BSA was added to inactivate humic substances. Extracted DNA
was diluted 5X in order to further dilute the humic substances present.
Analysis. Number of gene copies present in the standard curve was determined using the
following formula:
# gene copies = (i0 ng/µL) x (1/109) x (1/660) x 6.02x1023 x (1/vector length)
In order to relate Ct with gene copy, a linear regression of the relationship between Ct and
log(gene copy) was calculated, such that:
Ct = m x (log[gene copy]) + b
Using the calculated slope and x-intercept, Ct values of DNA samples were used to calculate the
log(gene copies) amplified in a given sample. Furthermore, the number of gene copies per a
given unit of sample was calculated using:
per unit sample = 10(log gene copy) x dilution factor in qPCR x (volume of extracted DNA/total
amount sample)
Ferrozine assay. Ferrozine. Ferrozine was prepared as such: 0.1% (w/v) Ferrozine in 50% (w/v)
ammonium acetate, stored in the dark at 4°C.
Standards. A calibration curve was created using a several concentrations (0.25µM - 20µM) of
ammonium ferrous sulfate ((NH4)2Fe(SO4)2·6H2O) dissolved in 1M HCl and diluted further in
1M HCl 1,000-fold. This dilution was performed serially using 1:10 dilutions, due the nature of
iron chemistry. An equal volume of ferrozine was added, allowed to react for 10 minutes and
then absorbance was measured spectrophotometrically at 562 nm.
Sampling dissolved Fe(II). Under anaerobic conditions, 0.1 mL of sample was added to 0.9 mL
1M HCl. Such additions stabilize Fe(II) in solution and prevents oxidation under aerobic
conditions. Note: samples can be left in this state several days (< 4 days) at 4°C. Samples were
further diluted 100X using serial dilutions. An equal volume of ferrozine was added, allowed to
react for 10 minutes and then absorbance was measured spectrophotometrically at 562 nm.
Analysis. Using a linear regression of the standard curve, absorbance at 562 nm can be directly
related to initial concentration of Fe(II) in sample:
absorbance = m x (Fe(II) concentration) + b
Clone libraries, sequence and analysis. The following clone libraries were constructed using
the TOPO TA cloning kit (Invitrogen), following product protocols.
pioB functional gene library. The above pioB primers were used to amplify the target gene from
environmental DNA extracts. Successful amplification through PCR was monitored using 1.0%
agarose gel electrophoresis. Successfully amplified samples obtained from Core 1 and Core 2
7
were inserted into the TOPO-4 plasmid and cloned in to chemically competent E. coli. For each
sample, 10 E. coli colonies containing the plasmid were picked and sequenced at the Josephine
Bay Paul Center. NCBI BLAST (basic local alignment search tool) against the nr database was
used to identify the best hit for sequences.
16S rDNA library. The general bacterial 16S rDNA primers 8F and 1492R were used to amplify
16S sequences from all enrichments showing evidence of growth 8 days after inoculation of
aerobic Pfennig bottle enrichments and 5 days after inoculation of anaerobic enrichments.
Successful amplification through PCR was monitored using 1.0% agarose gel electrophoresis. Of
the successfully amplified enrichments, three were chosen that appeared to exhibit evidence of
Fe(II) oxidation, determined based on turbidity and color of sample: 2° enrichment of aerobic
FeSO4 + NTA, 1° enrichment of anaerobic FeSO4 + NTA, and 1° enrichment of anaerobic
MnSO4. After insertion in to the TOPO-4 plasmid and cloning in to E. coli, 20 colonies were
picked for the 2° enrichment of aerobic FeSO4 + NTA, while 29 colonies were picked for the 1°
enrichment of anaerobic FeSO4 + NTA and 1° enrichment of anaerobic MnSO4 and sequenced at
the Josephine Bay Paul Center.
16S rDNA library analysis. After preliminary analysis of the sequence data using the Ribosomal
Database Project, sequences were aligned using the Silva database Aligner, and uploaded into
ARB. Sequences were added to the ARB tree using a quick parsimony addition. The position in
the ARB tree of the sequences was used to select "nearest neighbors". All sequences and select
nearest neighbors were then used to create a maximum likelihood tree using Phyml parameters
and the Bacteria_94 filter.
Results & Discussion
Sample collection, characterization and enrichments. As shown in Figure 2, the vertical
profile of the core (Core 1) used to inoculate the aerobic Pfennig enrichments was gathered for
O2 and H2S concentraions and pH. The O2 profile clearly shows the removal of oxygen from the
core at depth of ~2 mm. The curvature of this line demonstrates the biological impact on oxygen
consumption within the core. The H2S profile is more varied in the anaerobic portion of the core,
but is quickly consumed below 10 mm, which appears abiotic in nature due to the linear trend of
the line.
8
Figure 2 Vertical profiles of Core 1, used as the source of inoculum for aerobic Pfennig enrichments. Profiles
of (a) O2, (b) H2S, (c) pH.
The varying amount of sulfide present is likely the result of both biolgical sulfur reduction and
consumption of free sulfide through the abiotic reaction with Fe(II), which forms FeS (pyrite).
The pH curve demonstrates the near neutral nature of the core through its depth. Variations in pH
may be a result of sulfide concentration, but no obviosu trend seems to be visible. The neutral
condition of this core indicates that under aerobic conditions, the available Fe(II) would
spontaneously oxidize and form iron oxides in the sediment. Because photoferroautotrophs under
neutral conditions need the surrounding environment to be anoxic in order to have access to
Fe(II), sampling for a source of inoculum was collected below 2 mm, but as near the surface as
possible to potentially capture organisms using light as an energy source. Care was also taken to
remain above the highest levels of free sulfide to maximize the amount of available Fe(II) in the
environment.
For the aerobic Pfennig bottle enrichments, growth, in the form of solution turbidity, was
visible after 4-5 days in many of the enrichments. Anerobic enrichments required about 7 days
before turbidity was visible. As Figures 3 & 4 demostrates, the appearance of the enrichments
varied greatly depending on both the initial Fe(II) salt used and the time observed after
inoculation. Such variation in enrichment color and turbidity was the basis for which
enrichments would be further characterized through 16S rDNA clone libraries. Based on this
9
Figure 3 Various anaerobic enrichments after 10 days of incubation. Conditions of each enrichment are: (a)
No electron donor, (b) FeSO4+NTA, (c) FeCl2 and (d) MnSO4.
Figure 4 Anaerobic enrichments at various time points, (a) lacks electron donor, while (b-d) contains
FeSO4+NTA at different time points. (b) 7 days, (c) 9 days and (d) 11 days after inoculation
criteria, DNA as extracted from the aerobic 2° enrichment of FeSO4+NTA (not pictured) and the
anaerobic 1° enrichments containing FeSO4+NTA (Figure 3b) and MnSO4 (Figure 3d). The light
pink color in Figure 3d, suggests the growth of an organism containing pigments, potentially
capable of Mn(II)-oxidizing phototrophy. As is evident from Figure 4, development of visible
iron oxide precipitate occurred in the absence of oxygen suggesting such activity was performed
by Fe(II) oxidizers. The bands of iron oxide visible in Figure 4d occurred on the side facing the
light source.
Ferrozine assay. Results from the ferrozine assay showed several trends. As can be seen in the
aerobic Pfennig enrichments the general trend shows the consumption of Fe(II) compared the
initial amounts of Fe(II) added. However, the enrichments with the most drastic consumption of
Fe(II) appears to be the dark controls, which cannot be explained using the consumption of by
phototrophic iron oxidizers. It is possible that due to the aerobic nature of the Pfennig
enrichments that oxidation was the result of abiotic oxidation, but increased precipitate was not
noted.
10
As for the results of the ferrozine assay for the anaerobic enrichments, a more plausible
explanation for the observed trends is possible. Following inoculation, it appears as if the free
Fe(II) concentration increases in the enrichments. It is possible that phototrophic iron oxidizers
are capable of remineralizing the Fe(II) that precipitated upon addition to the media in the form
Figure 5 Results of the ferrozine assay for the aerobic Pfennig enrichments , 12 days after inoculation.
vivianite or siderite. This seems to be a plausible explanation because in an environment like
School Street Marsh, much of the free Fe(II) is bound up with the humic substances present. The
physiological ability to remineralize bound Fe(II) would be an evolutionary benefit to organisms
performing such a metabolic pathway in this type of environment. It should be noted that the
extent of white percipitate did decrease over the course of the enrichment process. And the
decrease of Fe(II) apparent 11 days after inoculation may indicate the continued consumption of
released Fe(II). These results suggest there is active utilization of iron species in these
enrichments by the organisms inoculated from School Street Marsh.
11
Figure 6 Results of the ferrozine assay for the anaerobic enrichments inoculated on July 13th at various time
points.
Quantitative PCR. Quantitative PCR was used to determine the copy number of certain genes
from environmental samples and enrichment experiments. The results from the 16S rDNA qPCR
can be used to approximate the number of organisms in a selected sample, assuming that each
organisms present has only one copy of the 16S rDNA present in its genome. The low copy
number from the Core 1 Sample 1 is likely the result of an overloading the Mobio Powersoil kit
with almost 1.0 g of a humic rich environmental sample. Figure 7 suggests that all the
environments sampled have approximately the same number of organisms present, if the results
are added. These gene copy numbers are low compared to normal bacterial concentration in
organic rich environments, and may be the result of suboptimal qPCR conditions.
Figure 8 indicates that there is a decrease in the number of cells present in the 2° aerobic
Pfennig enrichments, which may be the result of enriching for particular organisms within this
enrichment. It should be noted that the turbidity in these samples was low, indicating a low
bacterial concentration. Figure 9 seems to suggest that the enrichments with the highest bacterial
concentration were those containing electron donors and that cell concentrations decrease in
accordance with the decrease of available Fe(II) seen in the ferrozine assays above, possibly
suggesting a dependence on cellular abundance and available Fe(II).
12
Figure 7 Results of quantative PCR for DNA samples from various environmental conditions
Figure 8 Results of quantitative PCR for DNA extracts from various aerobic Pfennig enrichments, including
from the 1° and 2° enrichments
An attempt was made to perform qPCR on both environmental samples and enrichments
using the pioB primers. The level of detection was extremely limited with the lower standard
concentrations, the negative control and all samples passing the Ct threshold at the same cycle
value. This did not occur in the standards containing the highest concentration of target gene.
These results were checked via gel electrophoresis (data not shown). This data suggests that all
samples and controls with similar Ct values as the negative are in fact negative for pioB at a
detectable level for the this qPCR assay. Further analysis revealed that one enrichment possessed
a Ct value significantly higher than the negative control and other samples.
13
Figure 9 Results of quantitative PCR for the anaerobic 1° enrichments at two time points
Figure 10 Quantitative PCR results for the anaerobic FeSO4+EDTA enrichment for both pioB and 16S
The results displayed in Figure 10 correspond to the image in Figure 4d, specifically after
the appearance of iron oxide bands within the enrichment. The qPCR data suggests that pioB is
present in the anaerobic FeSO4+EDTA enrichment, and in accordance with the 16S qPCR
results, assuming a 1:1 ratio of pioB:16S, the organisms containing pioB are the dominant
bacterial cell type in the enrichment. This data seems to suggest the pioB primers are capable of
amplifying pioB from conditions in which it is present. Further optimization of the qPCR
protocol is needed to increase the sensitivity for samples with lower gene copy number.
14
Clone libraries. A functional gene library was developed using the results of the PCR products
displayed in Figure 11. The PCR products of Core 1 Sample 1 at a 60°C annealing temperature
and of Core 2 Sample 2 at a 64°C annealing temperature were cloned and sequenced, dispite the
presence of bands in the negative control. It was reasoned that if the reagents had been
completely contaminated PCR products should be visible in all lanes, which fails to occur. It is
possible that positive DNA was added to the negative controls or that placement with another
sample was switched, but due to time constraints and similar results in a previous PCR with a
60°C annealing temperature, these 2 PCR products were selected for further analysis. Each PCR
product had 10 sequences performed from the plasmids of transformed E. coli. Of the 20
possible sequences only 3 returned positive hits to pioB using NCBI BLAST. For 1 of the
sequences, there was 99% identity to pioB in two strains, TIE-1 and CGA009, or R. palustris.
The other 2 sequences had 99% identity to pioB in TIE-1 and 96% identity to pioB in CGA009.
These results seem to support the notion that these samples were not contaminated, as only a
small fraction of the sequences belonged to pioB and the amplified sequences varied in their
nucleotide sequence, suggesting the presence of multiple alleles in the environment. These
results continue to support the idea that the desgined primers are capable of amplifiying pioB
from the environment.
Figure 11 Gel electrophoresis of standard PCR products used to create the pioB functional gene clone library.
Various annealing temperatures were used to increase specific binding of the primers at 40 cycles.
In total 76 16S rDNA sequences were sent to be sequenced, of which 71 returned actual
sequence. After initial classification using the Ribosomal Database Project, aligned sequences
were added to ARB and phylogenetic analysis was performed using a maximum likelihood tree
constructed using Phyml. Sequences fell into distinct clades based on the enrichment condition.
As would be expected, some sequences in the 2° aerobic FeSO4+NTA enrichment clustered with
sequences from the anaerobic FeSO4+NTA enrichment, suggesting that these enrichments have
15
some overlap in the types of organisms selected for, potentially belonging to phototrophic iron
oxidizers. Unlike both Fe(II) containing enrichments, the Mn(II) enrichment seems to be far
Figure 12 (a & b) are sequences that orginated from the anaerobic FeSO4+NTA enrichment, (c) contains
sequences predominantly from the 2° aerobic FeSO4+NTA enrichment, and (d) has sequences from the
MnSO4 enrichment.
more diverse, with several sequences branching elsewhere on the tree.
16
Comparison with the nearest neighbor of a sequence can sometimes offer insight into the
metabolic potential of unknown organism. The clade denoted in Figure 12 as (a) comes from the
FeSO4+NTA anaerobic enrichment. The nearest neighbor shown, Prosthecochloris vibrioformis,
is a member of the green sulfur bacteria, that possesses bacterial chlorophyll d (Bchl d) and is
photoautotrophic using sulfide and sulfur as an electron donor. In context of this enrichment, the
media used selects for green and purple bacteria, and photoferroautotrophs would contain
pigments, possibly suggesting the organisms in these enrichments should also have Bchl d, and
be capable of autotrophic growth. Figure 12b also comes from the FeSO4+NTA anaerobic
enrichment and the nearest neighbor is Geovibrio ferrireducens. G. ferrireducens is capable of
Fe(III) reduction coupled with the oxidation of amino acids. The presence of organisms related
to these strains may suggest that there exists some type of commensualism between these two
types of organisms predominant in the enrichment. Such cycling would be beneficial to both
organisms. Figure 12c from the FeSO4+NTA aerobic enrichment had no nearest neighbor for any
of the sequences. Figure 12d from the MnSO4 anaerobic enrichment clusters with an group of
unclassified Rhizobiales, which could exemplify the current lack of knowledge of such
organisms.
Closing Remarks
The pioB primers designed for this project seem capable of amplifying the target gene from
environmental and enrichment samples when above a certain threshold. Further optimization of
both the standard PCR and qPCR protocols would be required before confidence in these
outcomes could be possible. However, such a tool will be useful in tracking the presence and
quantity of these organisms in the environment. Furthermore, the enrichment protocol, based on
16S rDNA clone libraries, seems to be capable of enriching for Fe(II) utilizing organisms from
the environment. Added to this is a possiblity of a first glimpse at a group of organisms capable
of Mn(II) oxidative photoautotrophy that are of yet undescribed.
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