77
For example, no nif genes were recovered from over 12,000 single-cell genome
sequence libraries from seawater samples (Pachiadaki et al. 2019). Thus, detection
and characterization of diazotrophs in some habitats by ‘omics approaches can be
limited. Nonetheless sequencing and assemblies have provided genomic level information on a number of uncultivated N 2 -fixing microorganisms (Delmont et al.
2018), yielding information on their metabolism and identifying new microorganisms that can be targeted for quantification and cultivation by other methods.
Meetagenomics and metatranscriptomics will continue to be important methods for
identifying N 2 -fixing microorganisms in microbial communities.
5.7 Stable Isotope Probing
A very powerful method which couples stable enriched tracer uptake of an element
(e.g.
13
C or
15
N) with molecular analysis is Stable Isotope Probing (SIP) (Buckley
2011; Dumont and Murrell 2005). While the method has recently been revived for
biogeochemical studies of carbon and nitrogen, it was the approach which originally and elegantly resolved that DNA was replicated semi-conservatively (Meselson
and Stahl 1958). In principal, SIP allows phylogenetic identification using the 16S
rRNA gene of specific organisms involved in biochemical anapleurotic and biogeochemical pathways.
Briefly, SIP is based on the uptake of the
15
N during growth of an organism after
addition and incubation with enriched
15
N 2 and its subsequent incorporation into
nucleic acids, either DNA or RNA. Protocols for SIP methods focus on one or the
other. Nucleic acids which have incorporated the stable isotope are literally “heavier”
than those that are biosynthesized in the presence of the natural low levels of the
heavy isotope in the environment. For N, this is approximately 0.37% naturally.
Artificially enriched N can approach 99%
15
N.
Once sufficient enriched isotope is assimilated into the nucleic acids (typically
over at least one doubling of the cell), the nucleic acids which have taken up the
heavy isotope can be physically separated from the unlabeled nucleic acids by
cesium gradient ultracentrifugation, which can take 1–2 days of spinning. The products can then be subjected to molecular analysis by a variety of methods for identifying active organisms with respect to the large 16S (prokaryotic) or 18S (eukaryotic)
rRNA sequence databases of microbial phylogenies. Sophisticated methods have
been developed to avoid the timely and tedious centrifugation step (Mayali et al.
2010), but because of the need to use highly complex nanoSIMS analyzers which
are only of limited availability and not yet broadly applied.
The identification of N 2 -fixers in soils has been one application of SIP (Buckley
et al. 2007) including arid soil crusts (Pepe-Ranney et al. 2016). Recently SIP has
been used to identify N 2 -fixers in a deep marine sediment (Kapili et al. 2019).
Applications are developing rapidly in the SIP field including applications that
allow quantitative interpretation (Hungate et al. 2015). Linking High Throughput
Sequencing with SIP yields enormous data sets and software specific to SIP has
5.7 Stable Isotope Probing
For example, no nif genes were recovered from over 12,000 single-cell genome
sequence libraries from seawater samples (Pachiadaki et al. 2019). Thus, detection
and characterization of diazotrophs in some habitats by ‘omics approaches can be
limited. Nonetheless sequencing and assemblies have provided genomic level information on a number of uncultivated N 2 -fixing microorganisms (Delmont et al.
2018), yielding information on their metabolism and identifying new microorganisms that can be targeted for quantification and cultivation by other methods.
Meetagenomics and metatranscriptomics will continue to be important methods for
identifying N 2 -fixing microorganisms in microbial communities.
5.7 Stable Isotope Probing
A very powerful method which couples stable enriched tracer uptake of an element
(e.g.
13
C or
15
N) with molecular analysis is Stable Isotope Probing (SIP) (Buckley
2011; Dumont and Murrell 2005). While the method has recently been revived for
biogeochemical studies of carbon and nitrogen, it was the approach which originally and elegantly resolved that DNA was replicated semi-conservatively (Meselson
and Stahl 1958). In principal, SIP allows phylogenetic identification using the 16S
rRNA gene of specific organisms involved in biochemical anapleurotic and biogeochemical pathways.
Briefly, SIP is based on the uptake of the
15
N during growth of an organism after
addition and incubation with enriched
15
N 2 and its subsequent incorporation into
nucleic acids, either DNA or RNA. Protocols for SIP methods focus on one or the
other. Nucleic acids which have incorporated the stable isotope are literally “heavier”
than those that are biosynthesized in the presence of the natural low levels of the
heavy isotope in the environment. For N, this is approximately 0.37% naturally.
Artificially enriched N can approach 99%
15
N.
Once sufficient enriched isotope is assimilated into the nucleic acids (typically
over at least one doubling of the cell), the nucleic acids which have taken up the
heavy isotope can be physically separated from the unlabeled nucleic acids by
cesium gradient ultracentrifugation, which can take 1–2 days of spinning. The products can then be subjected to molecular analysis by a variety of methods for identifying active organisms with respect to the large 16S (prokaryotic) or 18S (eukaryotic)
rRNA sequence databases of microbial phylogenies. Sophisticated methods have
been developed to avoid the timely and tedious centrifugation step (Mayali et al.
2010), but because of the need to use highly complex nanoSIMS analyzers which
are only of limited availability and not yet broadly applied.
The identification of N 2 -fixers in soils has been one application of SIP (Buckley
et al. 2007) including arid soil crusts (Pepe-Ranney et al. 2016). Recently SIP has
been used to identify N 2 -fixers in a deep marine sediment (Kapili et al. 2019).
Applications are developing rapidly in the SIP field including applications that
allow quantitative interpretation (Hungate et al. 2015). Linking High Throughput
Sequencing with SIP yields enormous data sets and software specific to SIP has
5.7 Stable Isotope Probing