Biology Reference
In-Depth Information
iv) 16S and 23S rRNA ITS sequences
:
16S-23S rDNA ITS has helped in the recognition of three clusters
among 47 strains of the genus of
Microcystis
. In the fi rst cluster both toxic and non-toxic strains are
included while the second consisted of only toxic strains and the third cluster comprised of non-toxic
strains. Phylogenetic analysis based on the above studies enables us in understanding relationships
amongst closely related species and strains (Otsuka
et al
., 1999b).
Evolutionary relationships among toxic and non-toxic strains of the genus
Microcystis
were traced
with the help of 16S rRNA gene sequences. Strains of
Microcystis
with the ability to produce MCs
formed a distinct cluster consistent with cell morphology, gas vacuolation and low G + C content. This
cluster was shown to be quite distinct from the
Synechocystis
and
Nostoc
clusters.
Microcystis
-specifi c
16S rRNA sequence signatures were used to develop primers that enabled identifi cation of potentially
MC-producing strains of
Microcystis
via DNA amplifi cation. It was possible to selectively amplify
DNAs of all the strains belonging to
Microcystis
cluster by employing one of the primers located at 179
to 209 of the gene. The 209 F (forward) with a sequence of 5'-ATGTGCCGCGAGGTGAAACCTAAT-3'
was amplifi ed alongwith 409 R (reverse) primer with a sequence of 5'-TTACAA(C/T)CCAA(G/A)
(G/A)(G/A)CCTTCCTCCC-3'. Thus
M
.
aeruginosa
PCC 7806, PCC 7941, AWT 139, NIE 389 and PCC
7820,
M
.
viridis
NIES 102 and
M
.
wesenbergii
NIE 3107 were all producers of MCs as substantiated
by LD
50
in mouse bioassay and its associated histological studies.
v) DNA-dependent RNA Polymerase gene
:
DNA-dependent RNA polymerase gene (
rpoC1
) has been
recognized as a more discriminatory marker (Palenik and Haselkorn, 1992) than 16S rRNA that has
been used more extensively for the identifi cation of cyanobacteria (Lyra
et al
., 1997; Nuebel
et al
.,
1997; Rudi
et al
., 1998). A 609-bp region of
rpoC1
was amplifi ed by PCR with
C
.
raciborskii
-specifi c
primers. Molecular characterization of
C
.
raciborskii
isolates (19 of them) from different geographical
regions of Australia representing straight and coiled trichomes suggested that all isolates belonged to
the same species. The utility of RAPD and STRR sequence profi les to recognise
C
.
raciborskii
isolates
was of a limited value than the
rpoC1
gene analysis. The specifi c PCR targetting a specifi c region of
rpoC1
developed for
C
.
raciborskii
was found to be unique for this organism as it enabled identifi cation
of DNA from natural populations (Wilson
et al
., 2000).
A
.
circinalis
occurs in the blooms along with
morphologically similar coiled
Anabaena
sp. in Murray-Darling Basin of Australia. Because it produces
PSPs in this region of the world,
A
.
circinalis
-specifi c PCR assay of
rpoC1
gene has been developed
to detect this orgainsm from the environmental samples (Fergusson and Saint, 2000).
vi) mcyB and mcyA markers
:
The gene
mcyB
encodes a peptide synthetase of 242,334 Da protein
containing two modules each possessing adenylation, thiolation and condensation domains. The
mcyB
may be involved with the activation of “variable L-amino acids”. Thus genetic probes from
mcy
gene cluster, i.e.
mcyB
gene and an adenylation domain within this gene were used to detect
MC-producing cyanobacteria (Neilan
et al
., 1999; Nishizawa
et al
., 1999). Although these two probes
showed good correlation to toxin production, due to the sequence similarity of
mcyB
region with
other peptide synthetase loci these genetic loci did not fi nd favour for further application (Dittmann
et al
., 1997; Tillett
et al
., 1999). By using oligonucleotide primers for
mcyB
gene of the operon MC
synthetase gene, PCR amplifi cation of 60
Microcystis
spp. strains from 15 reservoirs distributed in
Brazil was performed. A correlation between the presence of
mcyB
gene and the presence of total
MC as determined by ELISA revealed that a unique amplifi ed product of approximately 780 bp
was present in 18 strains of
Microcystis
. The lake waters contained a mixture of toxic and non-toxic
strains of
Microcystis
(Bittencourt-Oliveira, 2003). A set of oligonucleotide primers was designed
according to the squence of the peptide synthetase gene
mapep1
(module 3 of
mcyB
). These are
forward primer mapep-2B with a sequence of 5'-GATTGAACGGATGGCAGCAC-3' and mapep
