In addition to its use for the definition of species in
prokaryotes, the 16S rDNA has also established itself as a
reference via molecular phylogeny for the delimitation of
higher taxonomic levels. However, the gene* for 16S ribosomal RNA is not the “yardstick” of bacterial taxonomy. It is
sometimes too conserved to be able to distinguish between
some close species such as in the case of the Bacillus cereus
species complex*. In addition, the genomes* of prokaryotes
can contain multiple copies of this gene, reaching 10 (Bacillus
subtilis) or even 15 copies (Rainey et al. 1996).
While in most cases, within a genome, the multiple copies
of genes coding for 16S rRNA are identical or very similar,
due to gene conversion (i.e., homologous recombination
events leading to a homogenization of copies of a same
gene in a genome), there are also taxa which are found to
have markedly different copies. For example, in Escherichia
coli, one of the seven copies present in the genome has up to
1 % difference (15 different bases/1,500 nt; Cilia et al.
1996), or in Thermomonospora, one of the six copies present
in the genome has 10 % difference (Yap et al. 1999),
showing thus the difficulty for phylogenetic studies
depending on the selected 16S rRNA gene copy.
Finally, the use of a single marker*, for the identification
of new microorganisms or to study their position in a phylogeny, can lead to errors when there are gene transfers (cf. Sect.
12.2; Daubin et al. 2001) or when mutations in different
lineages are convergent. This is why other genes are frequently used in addition to 16S rRNA. For example, for the
identification of bacterial strains, additional markers are
considered as the 23S ribosomal RNA gene, which is longer
and more variable than 16S, allowing comparisons at finer
scale. But a number of genes also known as housekeeping
genes* like gyrB, rpoB, etc., are more and more used through
approaches called multi-locus sequence typing or MLST*.
Despite the problems mentioned above, the use of the 16S
rRNA gene has many advantages, the first of these being
related to its abundance in public sequence databases. For
example, the Ribosomal Database Project II entirely dedicated to rRNA contains 2,765,278 16S rRNA sequences
aligned and annotated (release 10, update 32, May 14, 2013,
Fig. 6.1). This abundance of 16S rRNA sequences is due to
the fact that this molecule was used early as a reference
marker. Indeed, the 16S rRNA genes have a number of
advantages such as (1) ubiquity, that is, the presence in all
living beings without exception; (2) the stability of the function of the gene product; (3) a low rate of mutation (allowing
comparison across the living world) and making possible to
design primers called universal, which allow amplification of
almost all-known rRNA genes
1
; (4) a sufficient length; and
(5) a low frequency of horizontal transfer.
6.2
Obtaining a Prokaryotic Strain:
Strains Collection
Classical microbiology is based on obtaining prokaryotes
strains* or isolates, that is to say on prokaryote cultures
that may be kept for years, exchanged between laboratories,
and compared with other isolates. However, isolates evolve
over time so that subcultures can sometimes undergo
major changes such as loss of plasmids, genomic recombination events, invasion by insertion elements (Polzin and
McKay 1991), or have had point mutations with major
phenotypic consequences, etc. It is therefore important to
define the approach that will permit conservation of isolates
as stable as possible. After being characterized, an isolate
can become a reference strain which must be deposited in a
reference collection*, for example, ATCC in the USA
(www.atcc.org), the DSMZ in Germany (www.dsmz.de),
the NCIMB in England (http://www.ncimb.com/), the JCM
in Japan (http://www.jcm.riken.go.jp/), or the collection of
the Institute Pasteur in France (www.crbip.pasteur.fr). The
mission of these reference collections is to maintain the
different isolates and make them accessible to the entire
international community of researchers in microbiology.
Microorganisms are maintained as pure strains coded and
referenced, maintained under freezing conditions (at À80
C
or in liquid nitrogen), or freeze-dried.
Box 6.1 (continued)
Conclusion
The “genomic” definition of Eubacteria or Archaea
species is efficient and confers stability to the taxonomic nomenclature. This is, however, a fixed version
of the species concept that does not fit with the contemporary evolutionary concepts of speciation. Nevertheless, it may well be that it is within the “genomic
species” that the majority of genetic exchanges would
occur and thus that ecological innovations would be
shared. Genomic species could well be “ecological
species” adapted to specific ecological niches, at
least at the time of speciation. However, it is also
possible that genetic drift has played a major role in
the individualization of genomic species with the
result that their differentiations are purely contingent
to the vagaries of the history of each species. The
advantage of prokaryotes is that these alternatives are
testable via comparative analysis of their genomes. In
practice, it is possible to find the genes and functions
that determine the specific niche of each species.
1 This is an iterative process linked to the ongoing discovery of new
taxa in new environments or better explored. In 2002, for instance, a
bacteria was described in which 16S rRNA gene did not hybridize with
the “universal” primers commonly used.
150
P. Caumette et al.
prokaryotes, the 16S rDNA has also established itself as a
reference via molecular phylogeny for the delimitation of
higher taxonomic levels. However, the gene* for 16S ribosomal RNA is not the “yardstick” of bacterial taxonomy. It is
sometimes too conserved to be able to distinguish between
some close species such as in the case of the Bacillus cereus
species complex*. In addition, the genomes* of prokaryotes
can contain multiple copies of this gene, reaching 10 (Bacillus
subtilis) or even 15 copies (Rainey et al. 1996).
While in most cases, within a genome, the multiple copies
of genes coding for 16S rRNA are identical or very similar,
due to gene conversion (i.e., homologous recombination
events leading to a homogenization of copies of a same
gene in a genome), there are also taxa which are found to
have markedly different copies. For example, in Escherichia
coli, one of the seven copies present in the genome has up to
1 % difference (15 different bases/1,500 nt; Cilia et al.
1996), or in Thermomonospora, one of the six copies present
in the genome has 10 % difference (Yap et al. 1999),
showing thus the difficulty for phylogenetic studies
depending on the selected 16S rRNA gene copy.
Finally, the use of a single marker*, for the identification
of new microorganisms or to study their position in a phylogeny, can lead to errors when there are gene transfers (cf. Sect.
12.2; Daubin et al. 2001) or when mutations in different
lineages are convergent. This is why other genes are frequently used in addition to 16S rRNA. For example, for the
identification of bacterial strains, additional markers are
considered as the 23S ribosomal RNA gene, which is longer
and more variable than 16S, allowing comparisons at finer
scale. But a number of genes also known as housekeeping
genes* like gyrB, rpoB, etc., are more and more used through
approaches called multi-locus sequence typing or MLST*.
Despite the problems mentioned above, the use of the 16S
rRNA gene has many advantages, the first of these being
related to its abundance in public sequence databases. For
example, the Ribosomal Database Project II entirely dedicated to rRNA contains 2,765,278 16S rRNA sequences
aligned and annotated (release 10, update 32, May 14, 2013,
Fig. 6.1). This abundance of 16S rRNA sequences is due to
the fact that this molecule was used early as a reference
marker. Indeed, the 16S rRNA genes have a number of
advantages such as (1) ubiquity, that is, the presence in all
living beings without exception; (2) the stability of the function of the gene product; (3) a low rate of mutation (allowing
comparison across the living world) and making possible to
design primers called universal, which allow amplification of
almost all-known rRNA genes
1
; (4) a sufficient length; and
(5) a low frequency of horizontal transfer.
6.2
Obtaining a Prokaryotic Strain:
Strains Collection
Classical microbiology is based on obtaining prokaryotes
strains* or isolates, that is to say on prokaryote cultures
that may be kept for years, exchanged between laboratories,
and compared with other isolates. However, isolates evolve
over time so that subcultures can sometimes undergo
major changes such as loss of plasmids, genomic recombination events, invasion by insertion elements (Polzin and
McKay 1991), or have had point mutations with major
phenotypic consequences, etc. It is therefore important to
define the approach that will permit conservation of isolates
as stable as possible. After being characterized, an isolate
can become a reference strain which must be deposited in a
reference collection*, for example, ATCC in the USA
(www.atcc.org), the DSMZ in Germany (www.dsmz.de),
the NCIMB in England (http://www.ncimb.com/), the JCM
in Japan (http://www.jcm.riken.go.jp/), or the collection of
the Institute Pasteur in France (www.crbip.pasteur.fr). The
mission of these reference collections is to maintain the
different isolates and make them accessible to the entire
international community of researchers in microbiology.
Microorganisms are maintained as pure strains coded and
referenced, maintained under freezing conditions (at À80
C
or in liquid nitrogen), or freeze-dried.
Box 6.1 (continued)
Conclusion
The “genomic” definition of Eubacteria or Archaea
species is efficient and confers stability to the taxonomic nomenclature. This is, however, a fixed version
of the species concept that does not fit with the contemporary evolutionary concepts of speciation. Nevertheless, it may well be that it is within the “genomic
species” that the majority of genetic exchanges would
occur and thus that ecological innovations would be
shared. Genomic species could well be “ecological
species” adapted to specific ecological niches, at
least at the time of speciation. However, it is also
possible that genetic drift has played a major role in
the individualization of genomic species with the
result that their differentiations are purely contingent
to the vagaries of the history of each species. The
advantage of prokaryotes is that these alternatives are
testable via comparative analysis of their genomes. In
practice, it is possible to find the genes and functions
that determine the specific niche of each species.
1 This is an iterative process linked to the ongoing discovery of new
taxa in new environments or better explored. In 2002, for instance, a
bacteria was described in which 16S rRNA gene did not hybridize with
the “universal” primers commonly used.
150
P. Caumette et al.