CYANOBACTERIAL TOXINS - Cyanobacteria - page 553

2A reverse primer with a sequence of 5'GGCCGCTCTAGAAACAGGTA-3' and for module 1 of
mcyA gene M1F2 forward primer with a sequence of 5'-CAGTGACGAGTAATCCCGAT-3' and
M1R2 reverse primer with a sequence of 5'-GCCATTCTCGCATCTGAGGA-3'. PCR amplifi cation
of 20 toxic strains and 18 non-toxic strains of Microcystis revealed that PCR amplifi ed products
could be seen only in case of toxic strains. It was possible to enumerate the number of toxic cells
(10 3 -10 5 cells ml -1 ) on the basis number of amplifi ed products. A positive correlation existed in the
toxin concentration and density of toxic cells in the environmental samples from Lakes of Mikata
and Suigetsu (Yoshida et al ., 2003).
N-methyltransferase (NMT) domain encoded by mcyA gene has been chosen as a molecular
probe to identify toxic Microcystis . NMT region was specifi cally present in 18 toxigenic strains of
Microcystis but was not detected in non-toxic strains (17) of this genus. NMT-specifi c DNA was
detected in natural populations and correlated well with PP-inhibition assays. Further, these results
have been compared with those of 16S rRNA gene sequences and PC-IGS sequences. The strains of
Microcystis formed a monophyletic group based on 16S rRNA gene sequence analysis while PC-IGS
enabled the recognition of two groups, i.e. both toxic and non-toxic strains of the genus were randomly
distributed. Interestingly, mcyA gene marker helped in the recognition of two coherent groups. In
all NMT-positive strains a gene of unknown function ( umaI ) existed at a distance from mcyC gene.
Though gene umaI also was detected in non-toxic strains it was not transcribed along with mcyABC
(Tillett et al ., 2001). Analysis of naturally occurring strains of Anabaena sp. revealed a 1236 bp in-frame
deletion that corresponded to the loss of the entire NMT domain but the synthesis of MCs continued
in nine of these strains as they incorporated dehydroalanine in place of N-methyldehydroalanine
and also elevated levels of L-Ser (Fewer et al ., 2008). However, majority of the non-toxic strains of
Planktothrix sp. from nine European countries have lost the large mcy gene cluster but still retained
mcyT gene along with fl anking regions of mcy gene cluster. The involvement of mcyT in the synthesis
of MCs has been confi rmed by the isolation of mcyT inactivation mutant of a MC-producing strain
that showed a reduction of 94 ± 2% in MC synthesis (Christiansen et al ., 2008).
The distribution of MC-producing and non-MC-producing genotypes of M . aeruginosa and
M . botrys in European freshwater bodies was studied by PCR amplifi cation of gene regions mcyA
and mcyB . Individual colonies of the two species were estimated for the presence of the two MC-
synthesizing genes, the type of MC-variants and total MC content. More than 75% of the colonies of
the two species contained mcy genes whereas only 20% of the colonies identifi ed as M . ichthyoblabe
and M . viridis were positive for the amplifi cation of mcy genes. However, colonies of M . wesenbergi
did not give a PCR product for either of the mcy genes. A relationship between 'morphospecies'
and MC genotype as well as 'morphospecies' and MC production have also been established (Via-
Ordorika et al ., 2004).
The amplifi cation of six characteristic segments of mcy gene cluster, three corresponding to
NRPS genes ( mcyA , mcyB , and mcyC ) and three to PKS genes ( mcyD , mcyE and mcyG ), helped in
the distinction of MC-producing and non-producing strains of Microcystis . These studies revealed
that (i) all the MC-producing strains could be identifi ed with help of specifi c amplifi cons and (ii)
simultaneous amplifi cation of several specifi c gene regions is feasible with cultured cells and fi eld
samples. The authors further suggested that the potential toxicity of a fi eld sample could easily be
determined on the basis of amplifi cation of these two sets of genes from fi eld samples (Ouahid et al .,
2005). The detection of MC-producing strains of cyanobacteria by the amplifi cation of mcyF gene by
PCR enabled Rantala et al . (2006) to identify Microcystis sp. (70%), Planktothrix sp. (63%) and Anabaena
sp. (37%) with variable population densities in 70 Finnish Lakes The proportion of the three genera
in the lakes very much depended on the trophic status of the lakes and eutrophic lakes supported