In all three of these species, these two important pathways are
governed by the pleiotropic global regulator plcR, a transcriptional
activator that regulates expression of multiple extracellular virulence factors. However, in B. anthracis, the plcR gene contains a
single nucleotide mutation that introduces a stop codon, resulting
in a nonsense mutation. Because the resulting truncated protein is
nonfunctional and plcR is the major regulator of both these pathways, this renders both the hemolysis and motility pathways inactive [64, 65]. Functional complementation with active PlcR
reactivates hemolysis in B. anthracis [66], while deletion or inactivation of plcR in B. cereus and B. thuringiensis dramatically reduces
both their hemolysis [67] and motility [68]. Thus, a change in
literally a single nucleotide in B. anthracis is responsible for the
drastic difference in two major phenotypic characteristics. Phylogenic relationships inferred from these phenotypic differences
would erroneously suggest that these species are far more distantly
related than the biological reality.
While the example of the plcR mutation is extreme in its
functional inactivation of two major biological pathways, it serves
as a reminder that an organism’s phenotype is the observable
manifestation of its genotype, which consists of many complex
and interrelated pathways. Reliance on phenotypic observations
for species identification could be significantly problematic for
identification of members of the genus Mycobacterium, as species
often differ by only a few phenotypic characteristics. The functional
properties of strains are highly relevant for their general biological
characterization, but for the purposes of species identification,
more reliable genetic/genomic methods are advisable.
4.2 Individual
and Multiple Genes
As phenotypic identification of Mycobacterium species is potentially
unreliable, the next best alternative is sequencing-based identification using specific molecular markers.
For this type of molecular identification in bacteria, the most
commonly used target is the gene that encodes for the 16S rRNA
(colloquially referred to as “16S”). Because the 16S gene is present
in all bacteria and contains both highly conserved and hypervariable
regions, universal primers have been designed to bind to the conserved regions and sequence across the hypervariable regions. The
obtained 16S sequence can then be compared against gene databases to identify the species from which it was derived. This is a
commonly used technique in laboratories and provides a costeffective method for preliminary identification, in many cases to
the species level and in most cases to a genus level.
For mycobacteria, however, sequencing of the 16S gene alone
is typically not sufficient for determining an accurate species-level
identification. Many different species have 16S sequences that are
very similar [69–72]. Sequencing of different targets, primarily
hsp65 and rpoB, can yield better results. A study comparing the
Mycobacterial ID and Characterization Using WGS
421
governed by the pleiotropic global regulator plcR, a transcriptional
activator that regulates expression of multiple extracellular virulence factors. However, in B. anthracis, the plcR gene contains a
single nucleotide mutation that introduces a stop codon, resulting
in a nonsense mutation. Because the resulting truncated protein is
nonfunctional and plcR is the major regulator of both these pathways, this renders both the hemolysis and motility pathways inactive [64, 65]. Functional complementation with active PlcR
reactivates hemolysis in B. anthracis [66], while deletion or inactivation of plcR in B. cereus and B. thuringiensis dramatically reduces
both their hemolysis [67] and motility [68]. Thus, a change in
literally a single nucleotide in B. anthracis is responsible for the
drastic difference in two major phenotypic characteristics. Phylogenic relationships inferred from these phenotypic differences
would erroneously suggest that these species are far more distantly
related than the biological reality.
While the example of the plcR mutation is extreme in its
functional inactivation of two major biological pathways, it serves
as a reminder that an organism’s phenotype is the observable
manifestation of its genotype, which consists of many complex
and interrelated pathways. Reliance on phenotypic observations
for species identification could be significantly problematic for
identification of members of the genus Mycobacterium, as species
often differ by only a few phenotypic characteristics. The functional
properties of strains are highly relevant for their general biological
characterization, but for the purposes of species identification,
more reliable genetic/genomic methods are advisable.
4.2 Individual
and Multiple Genes
As phenotypic identification of Mycobacterium species is potentially
unreliable, the next best alternative is sequencing-based identification using specific molecular markers.
For this type of molecular identification in bacteria, the most
commonly used target is the gene that encodes for the 16S rRNA
(colloquially referred to as “16S”). Because the 16S gene is present
in all bacteria and contains both highly conserved and hypervariable
regions, universal primers have been designed to bind to the conserved regions and sequence across the hypervariable regions. The
obtained 16S sequence can then be compared against gene databases to identify the species from which it was derived. This is a
commonly used technique in laboratories and provides a costeffective method for preliminary identification, in many cases to
the species level and in most cases to a genus level.
For mycobacteria, however, sequencing of the 16S gene alone
is typically not sufficient for determining an accurate species-level
identification. Many different species have 16S sequences that are
very similar [69–72]. Sequencing of different targets, primarily
hsp65 and rpoB, can yield better results. A study comparing the
Mycobacterial ID and Characterization Using WGS
421