acdS gene is used to infer the presence of ACC deaminase in a population,
nonspecific amplification of acdS homologs and mis-annotation of acdS genes in
genomes may also lead to false positives (Li et al. 2015). Hence, explicit detection of
the ACC deaminase structural gene (acdS) is important for predicting ACC deaminase activity and for identifying ACC deaminase producing bacteria and must be
complemented by ACC deaminase activity measurements as described above.
Recently, a rapid method to screen ACC deaminase-producing bacteria has been
reported (Patil et al. 2016). In this case, the bacteria are grown on minimal media
supplemented with ACC and two indicator dyes, namely bromothymol blue and
phenol red. Ammonia is produced as a consequence of the ACC deaminase catalyzed cleavage of ACC, which turns the media to a highly basic pH, a change that
can be readily observed due to the presence of the pH indicator dyes. Unfortunately,
this method, while rapid, is only semiquantitative and a detailed quantitative
enzyme-based assay should be performed to confirm the presence and amount of
active ACC deaminase.
Recently, the use of consensus-degenerate hybrid oligonucleotide primers for the
specific amplification of bona fide acdS genes has been described (Li et al. 2015).
More recently, a robust molecular tool was described for monitoring the size,
transcription levels, and diversity of acdS genes in a variety of environmental niches
(Bouffaud et al. 2018). This study is based on quantitative real-time PCR and is able
to detect highly diverse alleles of acdS. As mentioned above, acdS genes are extant
among Proteobacteria, Actinobacteria, Deinococcus-Thermus, and microeukaryotes. This method did not cover the thermophilic genus Meiothermus of the
Deinococcus, however, all other phylogenetic groups were well represented, and no
known overestimation has been found (Bouffaud et al. 2018).
The activity of purified ACC deaminase has also been measured using UV–Vis
spectrophotometry where enzyme activity was monitored in a coupled reaction
either with L-lactate dehydrogenase (Fedorov et al. 2013; Hontzeas et al. 2004),
which is used to measure the amount α-ketobutyrate produced, or with glutamate
dehydrogenase which measures the amount of ammonia produced (Fedorov et al.
2013) by monitoring the disappearance of the cofactor NADH at 340 nm
(ε ¼ 6220 M
–1 cm
–1 ). The K M values for all of the three enzyme-catalyzed reactions
were determined by monitoring the disappearance of ACC and plotting the reaction
rate versus the substrate concentration and then fitting the data to the Michaelis–
Menten equation (Fedorov et al. 2013; Hontzeas et al. 2004). The use of molecular
tools for the detection of acdS genes has been widely reported (Caballero-Mellado
et al. 2007; Govindasamy et al. 2008; Ma et al. 2003; Nikolic et al. 2011; OnofreLemus et al. 2009; Shah et al. 1998). However, due to the high similarity of the acdS
genes to its homologs, primer biased, mis-annotation of acdS genes within genomes
may occur, and most importantly, nonspecific amplification of acdS gene homologs
may also occur. Therefore, care must be taken in documenting such results
(Table 19.3).
19 Biochemistry and Molecular Biology of the Enzyme ACC Deaminase
375
nonspecific amplification of acdS homologs and mis-annotation of acdS genes in
genomes may also lead to false positives (Li et al. 2015). Hence, explicit detection of
the ACC deaminase structural gene (acdS) is important for predicting ACC deaminase activity and for identifying ACC deaminase producing bacteria and must be
complemented by ACC deaminase activity measurements as described above.
Recently, a rapid method to screen ACC deaminase-producing bacteria has been
reported (Patil et al. 2016). In this case, the bacteria are grown on minimal media
supplemented with ACC and two indicator dyes, namely bromothymol blue and
phenol red. Ammonia is produced as a consequence of the ACC deaminase catalyzed cleavage of ACC, which turns the media to a highly basic pH, a change that
can be readily observed due to the presence of the pH indicator dyes. Unfortunately,
this method, while rapid, is only semiquantitative and a detailed quantitative
enzyme-based assay should be performed to confirm the presence and amount of
active ACC deaminase.
Recently, the use of consensus-degenerate hybrid oligonucleotide primers for the
specific amplification of bona fide acdS genes has been described (Li et al. 2015).
More recently, a robust molecular tool was described for monitoring the size,
transcription levels, and diversity of acdS genes in a variety of environmental niches
(Bouffaud et al. 2018). This study is based on quantitative real-time PCR and is able
to detect highly diverse alleles of acdS. As mentioned above, acdS genes are extant
among Proteobacteria, Actinobacteria, Deinococcus-Thermus, and microeukaryotes. This method did not cover the thermophilic genus Meiothermus of the
Deinococcus, however, all other phylogenetic groups were well represented, and no
known overestimation has been found (Bouffaud et al. 2018).
The activity of purified ACC deaminase has also been measured using UV–Vis
spectrophotometry where enzyme activity was monitored in a coupled reaction
either with L-lactate dehydrogenase (Fedorov et al. 2013; Hontzeas et al. 2004),
which is used to measure the amount α-ketobutyrate produced, or with glutamate
dehydrogenase which measures the amount of ammonia produced (Fedorov et al.
2013) by monitoring the disappearance of the cofactor NADH at 340 nm
(ε ¼ 6220 M
–1 cm
–1 ). The K M values for all of the three enzyme-catalyzed reactions
were determined by monitoring the disappearance of ACC and plotting the reaction
rate versus the substrate concentration and then fitting the data to the Michaelis–
Menten equation (Fedorov et al. 2013; Hontzeas et al. 2004). The use of molecular
tools for the detection of acdS genes has been widely reported (Caballero-Mellado
et al. 2007; Govindasamy et al. 2008; Ma et al. 2003; Nikolic et al. 2011; OnofreLemus et al. 2009; Shah et al. 1998). However, due to the high similarity of the acdS
genes to its homologs, primer biased, mis-annotation of acdS genes within genomes
may occur, and most importantly, nonspecific amplification of acdS gene homologs
may also occur. Therefore, care must be taken in documenting such results
(Table 19.3).
19 Biochemistry and Molecular Biology of the Enzyme ACC Deaminase
375