Biology Reference
In-Depth Information
taking into account the top Blast hit, they identifi ed the species to which the sequence belongs and
performed the taxonomic assignment. Pyrosequencing is another technique that is dependent on
the characterization of PCR products of 16S rDNA. This is a cheap, labour saving method that does
not involve cloning of the gene. The original instrument known as GS20 Instrument that has been
fabricated by Margulies
et al
. (2005) sequences approximately 100 bases. The current GS FLX platform
can sequence 250 bases and the next generation GS XLR instrument is expected to sequence nearly
400 bases. Liu
et al
. (2008) performed accurate taxonomic assignments from the 16S rRNA gene
sequences produced by highly parallel pyrosequencers.
iii) 16S rRNA-23S rRNA operon copy number and the internally transcribed spacer (ITS)
sequences:
Bacterial rRNA genes are commonly present in a single operon in the order of 16S rRNA-
23S rRNA-5S rRNA each of which is separated by one ITS region (Srivastava and Schlessinger, 1990).
One or two tRNA genes (either tRNA
Glu
/tRNA
Ala
or both tRNA
Ala
and tRNA
Ile
) are often present in
the 16S rRNA-23S rRNA ITS region. However, in certain bacterial species like
Thermus thermophilus
(Srivastava and Schlessinger, 1990),
Leptospira interrogans
(Fukunaga and Mifuchi, 1989),
Borrelia
spp.
(Ojaimi
et al
., 1994) and
Wobachia pipientis
(Bensaadi-Merchermek
et al
., 1995) the rRNA genes are
separated into two distinct 16S rRNA and 23S rRNA operons. In case of bacteria, great variation in
between the number of ITS regions and alleles of the rRNA operon has been found. For example,
in
Escherichia
coli
there are 7 copies of operons coding for the three rRNAs (Morgan
et al
., 1977)
whereas in
Bacillus subtilis
(Loughney
et al
., 1982) and
Clostridium perfringens
(Garnier
et al
., 1991)
10 copies each of the operons coding for the three rRNAs are present. On the other hand, one copy
of the operon is present in
Mycobacterium
sp. (Bercovier
et al
., 1986) whereas one to two copies of
the operon may be present in
Mycoplasma
(Amikam
et al
., 1984). Moreover, greater heterogeneity
in these multiple copies has also been observed. In
E.
coli
where these copies are named as rrnA,
rrnB, rrnC, rrnD, rrnE, rrnG rrnH, the 16S rRNA-23S rRNA ITS region in the operons rrnB, rrnC,
rrnE and rrnG contains a gene coding for tRNA
glu-2
where as operons rrnA, rrnD and rrnH have
genes for tRNA
Ile-1
and tRNA
Ala-IB
(Antón
et al
., 1998). However, majority of gram-positive bacterial
species studied (15 out of 19) have no tRNA genes at all in the ITS region (Gürtler and Stanisich,
1996). This shows that there is a great variation in the number, length and composition of the 16S-
23S rDNA spacer regions of diverse range of eubacteria and archaebacteria. As a part of Human
Microbiome Project (accessible at http://nihroadmap.nih.gov/hmp), the diversity of 16S rRNA
genes in 883 prokaryotic genomes (including 13 from cyanobacteria) revealed 568 unique sequences.
Of these, 425 species showed 2 to 15 copies of rRNA genes per genome. There is every possibility
for taxonomic mis-classifi cation, based exclusively on 16S rRNA, of a number of species into more
than one species as it had happened with seven bacterial species associated with human disease
development (Pei
et al
., 2010). The diversity of 23S rRNA genes in 184 prokaryotic species has been
determined by Pei
et al
. (2009) who found multiple 23S rRNA genes in 113 genomes. Signifi cantly,
eight of the species exhibited intragenomic variation in the 23S rRNA gene sequences. Intervening
sequences ranging in length from 9 to 1471 nucleotides were found in seven species. In case of
Anabaena
sp. strain PCC 7120 there are four 23S rRNA genes of which rrnA 23S is 4299-nucleotides
long whereas the rest of the three each are 2828-nucleotides long, the difference in the lengths is
due to the presence of 1471-nucleotides long intervening sequence that encodes a transposase. The
second organism that has a transposase gene as an intervening sequence, within the 23S rRNA
gene, is
Deinococcus radiodurans
(Pei
et al
., 2009). Rastogi
et al
. (2009) constructed phylogenetic trees
based on rRNA copy numbers and genome sizes in which colour codes given with increasing rRNA
copy numbers and genome sizes can be correlated with the colour codes given on the phylogenetic
