Development of molecular tools to detect and quantify atmospheric
CO-oxidizing bacteria and identification of environmental factors
regulating their distribution and activity
Liliana Quiza, Isabelle Lalonde, Philippe Constant
INRS-Institut Armand Frappier, Laval (Québec).
Abstract
Methodology
Soil bacteria scavenging carbon monoxide (CO) are responsible for
the biological sink of atmospheric CO. These bacteria exert a
significant influence on atmospheric photochemical processes,
because the soil uptake of CO mitigates an important fraction of the
global emissions of CO from natural and anthropogenic sources.
Mitigation of these emissions is of critical importance since CO
indirectly regulates the atmospheric lifetime of methane (CH4) - the
second most powerful greenhouse gas. Whether the soil uptake of
atmospheric CO is vulnerable or not to global change remains to be
elucidated.
1) Sampling
• Three neighbouring sampling sites - different land-use types:
Results
A.
- Undisturbed deciduous forest (Site F)
Table 1. Soil physicochemical properties
- Larch monoculture (Site M)
*
• Triplicate samples (0-10 cm)
Fig 4 . Composite soil samples
for each triplicate (3 sampling
stations per sampling site).
**
5) Soil biological activity, physicochemical properties (Table 1; centered
explanatory variables) and coxL gene sequences (Hellinger-transformed
OTU frequency data) were integrated into redundancy analysis (RDA) in
order to identify clone sequences linked to elevated CO uptake activity and
the environmental factors influencing their distribution. The most
parsimonious model to explain coxL gene sequence distribution was
obtained by stepwise forward selection of environmental variables. Soil
water content and CO uptake activity explained >40% of coxL gene
distribution in soil (Fig. 9).
Introduction
Fig 5. Localisation (St-Amable, Qc) and picture of the sampling sites.
- “Universal” (OMP + BMS) coxL degenerated primers have
been developed from gene sequences available in public
database (NCBI) in order to target the as much diversity of
coxL as possibly.
- Generation of 9 coxL clone library (one per sampling
station; 344 clones in total).
Studies of microbial CO metabolism unveiled the occurrence of two
functional groups of CO-oxidizing bacteria:
•
“carboxydovore” bacteria unable to grow chemoautotrophically
with CO.
The CO-dehydrogenase (CODH) is the enzyme catalyzing the CO
oxidation reaction in bacteria:
Fig 9. Parsimonious
RDA
triplot
of
Hellinger-transformed
coxL
OTUs
data
explained by soil water
content and CO uptake
activity. OTU whose
distribution
was
correlated to elevated
soil CO uptake activity
are highlighted in the
triplot as well as the
phylogenetic
trees
(Fig. 6). Most of them
belong to the OMP
group.
2) Development of molecular tools to detect and quantify coxL in
the three environments:
Soil bacteria are a significant alternative to sink and oxidize the
atmospheric CO which keep it below toxic levels as well as minimize
its release to the atmosphere.
“carboxydotroph”, bacteria capable of using CO as the sole
source of carbon and energy (Fig. 1).
**
Fig 8. CO oxidation rate in pmol h-1 g(dw)-1.
Work is currently in progress to isolate high affinity CO-oxidizing
bacteria and relate coxL gene expression profile to CO uptake
activity in soil.
•
4) A gas chromatographic assay allowed to measure the potential soil CO
uptake activity of the three sampling sites (Fig. 8). CO uptake activity was
significantly higher in deciduous forest (Mann-Whitney, P<0.05) and was
positively correlated to soil carbon and nitrogen content (P<0.01).
- Maize field (Site A)
The objective of this investigation was to relate the diversity of coxL
gene sequences with CO soil uptake activity, land-use and soil
physicochemical properties. Diversity of coxL gene sequence was
more important than anticipated and land-use change was shown to
influence CO-oxidizing bacterial community structure and activity.
CO is an atmospheric trace gas released as a result of terrestrial and
aquatic organic mater oxidation, volcanic activity and incomplete
combustion. It indirectly contributes to global warming as it strongly
competes with greenhouse gases such as methane to react with the
hydroxyl radical (OH-) of the atmosphere - the cleansing radical able to
destroy CO, CH4 and other reduced trace gases.(1)
Results and Discussion
- PCR (universal) and qPCR (OMP-specific) assays were
developed.
3) Measurement of the potential CO oxidation activity using a
gas chromatography assay.
4) Determination of the soil physicochemical properties (i.e. pH,
C, N, P, K and water content).
B.
.
Results
The aerobic CODH (Fig. 2) belongs to the molybdenum hydroxylases
Fe-S family, the enzyme is a dimer of heterotrimers encoded by the
genes coxS, coxM and coxL (Fig. 3).
CoxL is the large subunit of the CODH. Phylogenetic analysis revealed
that coxL gene sequences encompass two main clusters: BMS and
OMP but the version conferring a high affinity for CO and the ability to
scavenge atmospheric CO is unknown.
Conclusion
1) New “Universal” primers targeting coxL (BMS + OMP) were
tested by generating coxL clone libraries (800 bp, including
CODH active site signature; see Fig. 2). The sequences were
aligned, classified into 199 OTU (10% cutoff level) and
integrated into a phylogenetic analysis (Fig. 6). Most of the
bacteria possessing coxL are unknown.
• Land-use change exerts a significant impact on coxL diversity as well as
CO oxidation activity (loss of the potential CO soil uptake activity
following the conversion of native forest to maize or larch plantation)
• Most of the coxL gene sequences retrieved from the soil samples were
not affiliated to sequences derived from microbial genome databases,
impairing a taxonomic identification of the potential CO-oxidizing bacteria
detected in soil.
2) UniFrac analysis demonstrated the correlation between
phylogeny and the origin of coxL sequences (unique sequences
found in deciduous forest, while sites A and M cannot be discriminated).
• Canonical ordination analysis allowed us to identify coxL sequences
belonging to potential high affinity CO-oxidizing bacteria.
3) The analysis suggests that conversion of deciduous forest
(site F) to larch or maize monocultures have exerted a
selective pressure to CO-oxidizing bacteria (Fig. 7).
Fig 1. Carboxydotroph
metabolic pathway.
• Work is currently in progress to isolate, assess the abundance and CO
uptake activity of these microorganisms in soil. Taken together, the
results of this work will be implemented into molecular models aimed at
predicting CO uptake activity in soil. These models will be utilized to
predict the response of the biological sink of CO to global change, while
determining how land management practices could protect this important
ecosystem service.
Fig 2. Classification of anaerobic (NiFe)
and aerobic (molybdenum) CODH.
Bibliography
• Weber & King, 2007. Nature Reviews in Microbiology (5): 107-118.
• Oelgeschläger & Rother, 2008. Archives of Microbiology (190): 257-269.
• Mörsdorf et al., 1992. Biodegradation (3): 61-82.
• Borcard et al., 2011, Numerical Ecology with R, Springer, 306 pp.
Fig 2. Crystal structure of CODH in Oligotropha
carboxidovorans (carboxydotroph bacteria).
Hypothesis: Conversion of deciduous forest to monoculture will exert a
significant effect on the CO soil uptake activity induced by change in
CO-oxidizing bacteria community structure (BMS and OMP groups).
Fig 7. UniFrac dendrogram based on coxL gene sequences detected in
different sampled ecosystems. Significance of the nodes is shown by the
circles (Jackknife resampling, 1000 permutations). Bar = Unifrac units.
Fig 6. Maximum likelihood phylogenetic tree based on in silico translated coxL
sequences derived from clones libraries and NCBI genome sequence database.
(A) Three main clusters were identified (Type I-OMP, Type II-BMS and the
novel Type III). (B) Visualization of the OMP group. Color code identify coxL
gene sequences for which the occurrence was shown to be correlated to
elevated CO uptake activity (see RDA analysis, Fig. 9)
• Dobbek et al., 1999. Proceedings of the National Academy of Sciences
(96): 8884-8889.
Acknowledgements
Fonds de recherche du Québec - Nature et technologies (FQRNT) for funding
and Prof. Claude Guertin for guidance in the field.