76
reverse transcriptase (RT) step to copy single stranded RNA molecules to doublestranded DNA (Church et al. 2005b).
The PCR generates DNA that can be sequenced and contains copies of sequences
from different gene copies or microorganisms in the sample thus providing a measure of diversity, but not quantity. The development of real-time (sometimes also
called RT, which is distinct from “RT” also used to designate reverse-transcriptase,
see below) made it possible to quantitatively measure the abundance of specific
gene types, so that abundances and distributions of individual species or strains of
marine N 2 -fixing microorganisms could be determined (Church et al. 2005a;
Langlois et al. 2005). This method can also be used to target mRNA (Church et al.
2005b; Short and Zehr 2005) providing a measure of the relative activity of different
species or relative activity as a function of space and time. These methods for gene
and mRNA quantification are called qPCR and RT-qPCR (for reverse-transcriptase), respectively. The qPCR approach has been important for obtaining distribution and abundance data on diazotrophs, many of which are uncultivated. The
approach has limitations, however, since it is affected by gene copy number, and
polyploidy (Sargent et al. 2016) and extraction efficiency. Furthermore, qPCR primers are most reliable when specific for a single or closely related sequences, which
mean they target sequences that are known, not unidentified uncultivated genotypes.
The degenerate PCR approach, or metagenomics (Delmont et al. 2018; Salazar
et al. 2019) are important for retrieving unidentified sequence types, which then can
be targeted by qPCR.
A typical cultivation-independent approach for studying microorganisms in the
environment is FISH (Amann et al. 1995). This method uses hybridization of a
nucleic acid “probe” labeled by a variety of means with a fluorescent molecule to
intracellular nucleic acids, usually ribosomal RNA (rRNA). It has been very useful
for studies of organisms discovered by 16S rRNA gene surveys (Amann et al. 1995).
However, since the functional gene approach provides only a sequence of the nifH
gene and prior to metagenomics no possibility of linking to a 16S rRNA gene, it has
been impossible to use this approach for most uncultivated N 2 -fixing microbes. One
exception to this is the symbiont UCYN-A whose genome was sequenced prior to
cultivation, and the 16S rRNA gene of the genome was used to make rRNA HISH
(halogen in situ hybridization (Krupke et al. 2013; Thompson et al. 2012)) and
FISH probes (using CARD-FISH) to visualize the symbiosis (Cabello et al. 2016).
Stable Isotope Probing (SIP) covered in the next section is another means of linking
16S rRNA gene sequences to N 2 -fixers.
The accelerating development of next generation sequence technologies led to
the ability to characterize microbial communities by genomic DNA and mRNA
sequencing called metagenomics and metatranscriptomics (DeLong 2002; FriasLopez et al. 2008; Moran 2009; Tringe et al. 2005; Venter et al. 2004). These
approaches are not dependent on the prior knowledge of sequences that PCR
requires for primer design, and thus, have less potential bias. Metagenomic studies
have provided new information on marine microbial assemblages (Sunagawa et al.
2015). These approaches sequence all the microorganisms in a sample, and N 2 -
fixing microorganisms are typically a small fraction of the community (less than 1%).
5 Measurements of Organism Abundances and Activities
reverse transcriptase (RT) step to copy single stranded RNA molecules to doublestranded DNA (Church et al. 2005b).
The PCR generates DNA that can be sequenced and contains copies of sequences
from different gene copies or microorganisms in the sample thus providing a measure of diversity, but not quantity. The development of real-time (sometimes also
called RT, which is distinct from “RT” also used to designate reverse-transcriptase,
see below) made it possible to quantitatively measure the abundance of specific
gene types, so that abundances and distributions of individual species or strains of
marine N 2 -fixing microorganisms could be determined (Church et al. 2005a;
Langlois et al. 2005). This method can also be used to target mRNA (Church et al.
2005b; Short and Zehr 2005) providing a measure of the relative activity of different
species or relative activity as a function of space and time. These methods for gene
and mRNA quantification are called qPCR and RT-qPCR (for reverse-transcriptase), respectively. The qPCR approach has been important for obtaining distribution and abundance data on diazotrophs, many of which are uncultivated. The
approach has limitations, however, since it is affected by gene copy number, and
polyploidy (Sargent et al. 2016) and extraction efficiency. Furthermore, qPCR primers are most reliable when specific for a single or closely related sequences, which
mean they target sequences that are known, not unidentified uncultivated genotypes.
The degenerate PCR approach, or metagenomics (Delmont et al. 2018; Salazar
et al. 2019) are important for retrieving unidentified sequence types, which then can
be targeted by qPCR.
A typical cultivation-independent approach for studying microorganisms in the
environment is FISH (Amann et al. 1995). This method uses hybridization of a
nucleic acid “probe” labeled by a variety of means with a fluorescent molecule to
intracellular nucleic acids, usually ribosomal RNA (rRNA). It has been very useful
for studies of organisms discovered by 16S rRNA gene surveys (Amann et al. 1995).
However, since the functional gene approach provides only a sequence of the nifH
gene and prior to metagenomics no possibility of linking to a 16S rRNA gene, it has
been impossible to use this approach for most uncultivated N 2 -fixing microbes. One
exception to this is the symbiont UCYN-A whose genome was sequenced prior to
cultivation, and the 16S rRNA gene of the genome was used to make rRNA HISH
(halogen in situ hybridization (Krupke et al. 2013; Thompson et al. 2012)) and
FISH probes (using CARD-FISH) to visualize the symbiosis (Cabello et al. 2016).
Stable Isotope Probing (SIP) covered in the next section is another means of linking
16S rRNA gene sequences to N 2 -fixers.
The accelerating development of next generation sequence technologies led to
the ability to characterize microbial communities by genomic DNA and mRNA
sequencing called metagenomics and metatranscriptomics (DeLong 2002; FriasLopez et al. 2008; Moran 2009; Tringe et al. 2005; Venter et al. 2004). These
approaches are not dependent on the prior knowledge of sequences that PCR
requires for primer design, and thus, have less potential bias. Metagenomic studies
have provided new information on marine microbial assemblages (Sunagawa et al.
2015). These approaches sequence all the microorganisms in a sample, and N 2 -
fixing microorganisms are typically a small fraction of the community (less than 1%).
5 Measurements of Organism Abundances and Activities