75
Wilson and colleagues (Wilson et al. 2010a, b, 2013) have also considered the
relationship between H 2 concentrations and production and N 2 fixation. They have
developed assays to estimate total nitrogenase activity with Argon Induced
Hydrogen Production (AIHP) which can provide a rapid field assay distinct from
C 2 H 2 reduction methods (Wilson et al. 2021). AIHP can also be developed for automated underway sampling similar but distinct from the CRDLAS method (Cassar
et al. 2012, 2018) earlier mentioned.
5.6 Functional Gene Analysis
While the infusion of molecular biological methods (e.g. analysis of 16S rRNA
genes in DNA) had begun to revolutionize our understanding of microbiological
phylogeny (Woese and Fox 1977) and diversity, including marine microbiology
(Giovannoni et al. 1990), the application of molecular biological approaches for
studies of functional genes began with the development of polymerase chain reaction (PCR) methods for nitrogenase in the environment (Zehr and McReynolds
1989; Zehr et al. 1998). The polymerase chain reaction uses enzymes to amplify a
short section of DNA, in this case a fragment of the nifH gene. This gene is usually
used since it is highly conserved, meaning the sequence is predictable at least in
certain regions, in all diazotrophic microorganisms. This approach has been used on
environmental DNA (and RNA) samples and provides information on the genetic
diversity of N 2 -fixing microorganisms in the environment. It was first used to show
that Trichodesmium had cyanobacterial nitrogenase genes, thereby along with other
studies (Bergman and Carpenter 1991) confirming that Trichodesmium and not
associated bacteria, was responsible for N 2 fixation.
More importantly PCR was applied to determine the diversity of potential N 2 -
fixing microorganisms in the oligotrophic ocean (Zehr et al. 1998), microbial mats
(Omoregie et al. 2004), marine sediments (Burns et al. 2002) and the deep sea
(Mehta et al. 2003). This led to the discovery of new organisms, for example, the
symbiont UCYN-A (Zehr et al. 2001), and has highlighted the large number of
bacteria that have nitrogenase genes and could potentially be N 2 -fixers. nifDK has
been used in some studies, but less so because it is less conserved and the databases
are smaller. The nifH sequence database now has tens of thousands of sequences
from diverse environments (Gaby and Buckley 2011). The sizes of databases has
required parallel development of bioinformatic approaches for analyzing sequences
(Heller et al. 2014).
Methods that target nucleic acid sequences make it possible to amplify messenger RNA (mRNA) instead of DNA (Zani et al. 2000). The advantage of targeting
mRNA is that mRNA has to be copied from genomic DNA, and has a short half-life,
thus showing not only that the organism is alive, but that it is expressing the gene
(Zehr and Paerl 2008). In the case of nifH, this is important, since nifH gene expression is highly regulated in response to multiple environmental factors (see Chap. 2).
mRNA, such as nifH gene transcripts, can be assayed by the PCR subsequent to a
5.6 Functional Gene Analysis
Wilson and colleagues (Wilson et al. 2010a, b, 2013) have also considered the
relationship between H 2 concentrations and production and N 2 fixation. They have
developed assays to estimate total nitrogenase activity with Argon Induced
Hydrogen Production (AIHP) which can provide a rapid field assay distinct from
C 2 H 2 reduction methods (Wilson et al. 2021). AIHP can also be developed for automated underway sampling similar but distinct from the CRDLAS method (Cassar
et al. 2012, 2018) earlier mentioned.
5.6 Functional Gene Analysis
While the infusion of molecular biological methods (e.g. analysis of 16S rRNA
genes in DNA) had begun to revolutionize our understanding of microbiological
phylogeny (Woese and Fox 1977) and diversity, including marine microbiology
(Giovannoni et al. 1990), the application of molecular biological approaches for
studies of functional genes began with the development of polymerase chain reaction (PCR) methods for nitrogenase in the environment (Zehr and McReynolds
1989; Zehr et al. 1998). The polymerase chain reaction uses enzymes to amplify a
short section of DNA, in this case a fragment of the nifH gene. This gene is usually
used since it is highly conserved, meaning the sequence is predictable at least in
certain regions, in all diazotrophic microorganisms. This approach has been used on
environmental DNA (and RNA) samples and provides information on the genetic
diversity of N 2 -fixing microorganisms in the environment. It was first used to show
that Trichodesmium had cyanobacterial nitrogenase genes, thereby along with other
studies (Bergman and Carpenter 1991) confirming that Trichodesmium and not
associated bacteria, was responsible for N 2 fixation.
More importantly PCR was applied to determine the diversity of potential N 2 -
fixing microorganisms in the oligotrophic ocean (Zehr et al. 1998), microbial mats
(Omoregie et al. 2004), marine sediments (Burns et al. 2002) and the deep sea
(Mehta et al. 2003). This led to the discovery of new organisms, for example, the
symbiont UCYN-A (Zehr et al. 2001), and has highlighted the large number of
bacteria that have nitrogenase genes and could potentially be N 2 -fixers. nifDK has
been used in some studies, but less so because it is less conserved and the databases
are smaller. The nifH sequence database now has tens of thousands of sequences
from diverse environments (Gaby and Buckley 2011). The sizes of databases has
required parallel development of bioinformatic approaches for analyzing sequences
(Heller et al. 2014).
Methods that target nucleic acid sequences make it possible to amplify messenger RNA (mRNA) instead of DNA (Zani et al. 2000). The advantage of targeting
mRNA is that mRNA has to be copied from genomic DNA, and has a short half-life,
thus showing not only that the organism is alive, but that it is expressing the gene
(Zehr and Paerl 2008). In the case of nifH, this is important, since nifH gene expression is highly regulated in response to multiple environmental factors (see Chap. 2).
mRNA, such as nifH gene transcripts, can be assayed by the PCR subsequent to a
5.6 Functional Gene Analysis