Biology Reference
In-Depth Information
tree constructed with the 31 housekeeping genes from 578 bacterial genomes. This will be useful in
determining microbial diversity and reduces chances for mis-identifi cation due to the existence of
variable rRNA copy numbers.
A number of cyanobacteria have been studied for the sequences of the 16S rRNA-23S rRNA ITS
domain. Three types of ITS regions have been identifi ed. The fi rst type contains both tRNA
Ile
and
tRNA
Ala
sequences. These are found in
Anabaena
sp. (Lu, 1999),
Arthrospira
PCC 7345 (Nelissen
et
al
., 1994),
Nostoc
sp.
(Lu, 1999),
Synechococcus
PCC 6301 (“
Anacystis
”, Tomioka and Sugiura, 1984),
and
Trichodesmium
NIBB 1067 (Wilmotte
et al
., 1994). The second type of ITS regions contain only
tRNA
Ile
found in 47 strains of
Microcystis
(Otsuka
et al
., 1999),
Spirulina
PCC 6313 (Nelissen
et al
.,
1994) and the unicellular
Synechocystis
sp. strain PCC 6803 (Kaneko
et al
., 1996). Kaneko
et al
. (1996)
identifi ed two rRNA operons both consisting of tRNA
Ile
present in inverted repeats in
Synechocystis
sp. strain PCC 6803. The third type of ITS regions do not contain any tRNA as noted in
Nodularia
BCNO D9427 (Hayes and Barker, 1997). The 16S rRNA-23S rRNA ITS sequences of two other
cyanobacteria
Mastigocladus
HTF strain PCC 7518 of uncertain identity and that of the cyanelle
Cyanophora paradoxa
have also been determined (Iteman
et al
., 2000). There is a great size variation in
ITS regions of cyanobacterial origin. They vary from 354 to 545 nucleotides with the exception of 287
nucleotides in the cyanelle
C
.
paradoxa.
The existence of multiple rRNA operons was for the fi rst time
reported in three species of
Anabaena
and one species of
Nostoc
(Nichols
et al
., 1982). The existence
of four rrn operons in
Anabaena
sp. strain
PCC 7120 was reported by Ligon
et al
. (1991). Three PCR
products have been generated from the 16S rRNA-23S rRNA ITS region of
Anabaena
sp. strain PCC
7120. Of these, two of them represented the true ITS regions while the third fragment was longer
(with 512 nucleotides) and possessed tRNA
Ile
and tRNA
Ala
genes separated by a large stem-loop
structure (Iteman
et al
., 2000). Further, these workers identifi ed highly conserved motifs important
for folding and maturation of rRNA transcripts (homologous to bacterial antiterminators boxB-
boxA) that can serve as potential targets of PCR primers and oligonucleotide probes for detection
and identifi cation of cyanobacteria. The 16S rRNA-23S rRNA ITS regions of
Anabaena
sp. strain
PCC 7120,
Mastigocladus
HTF strain PCC 7518 and
C
.
paradoxa
contain tRNA
Ile
and tRNA
Ala
. Boyer
et al
. (2001) examined variability in the ITS regions among multiple rRNA operons in fi ve species of
cyanobacteria (
Scytonema hyalinum
,
Toplypothrix distorta
,
Calothrix parietina
,
Coelodesmium wrangelii
and a new genus designated by isolates SRS 6 and SRS 70). The ITS regions from these organisms
transcribe two tRNA molecules tRNA
Ile
and tRNA
Ala
. So in this respect, these species belong to the fi rst
type of ITS region described above. However,
C
.
parietina
and
S
.
hyalinum
also contained ITS regions
without tRNA. In addition,
S
.
hyalinum
contained two non-coding ITS regions that are identical in
length but differed in their sequences. The species included in their study has wider representation
taxonomically because three of the species belonged to three of the four families of Nostocales. Boyer
et al
. (2001) cautioned that although the potential of ITS region as a tool for studying molecular
systematic and population genetics is great but the presence of multiple non-identical rRNA operons
poses a problem, especially in sequence comparison and secondly when restriction digests of PCR
products are analyzed. So as to enable investigators to align and compare the highly variable ITS
region, D'Auria
et al
. (2006) created a software known as Word Count based (IWoCS) system with
32,061,819 entries of which 482 sequences pertain to cyanobacterial ITS sequences. Of these, 204
sequences pertain to Chroococcales, 56 of Nostocales, 60 of Oscillatoriales, 67 of Prochlorales, 2 of
Stigonematales and 93 unidentifi ed/unknown sequences. This provides a novel tool for the use of
ITS sequences in typing and identifi cation of bacteria and cyanobacteria.
iv)
Amplifi ed rDNA restriction analysis (ARDRA):
Ever since this method was discovered by
Grimont and Grimont (1986) more than 2000 research papers have been published on 16S rRNA
