CYANOPHAGES - Cyanobacteria - page 455

kb. The cyanophages infecting Synechococcus (represented to 7.3 x 10 4 ml -1 ) have been detected at a
concentration of 7.2 x 10 3 particles ml -1 . The genetic diversity of cyanophages has been determined
by the amplifi cation of g20 gene sequence (by CPS1 and CPS2 primers) and the homology of all
viruses belonged to cyanomyoviruses. Seasonal shifts in the populations of cyanophages were
noted with one community dominantly represented in spring and summer and a different one in
fall (Sandaa and Larsen, 2006). Isothermal nucleic acid amplifi cation assay, i.e. signal-mediated
amplifi cation of RNA technology (SMART) has been used for the fi rst time by Hall et al . (2002) to
detect DNA extracted from marine cyanophages. This technique is based on the target-dependent
production of multiple copies of an RNA signal that is measured by an enzyme linked oligosorbent
assay. Cyanophage S-PM2 and S-BnM1 specifi c signals for g20 gene sequences could be identifi ed
very easily. Extensive purifi cation of phage DNA is not required as signals could be obtained from
crude phage lysates as well. So this technique does not appear to be best suited for identifying
phage DNA sequences but helps in detecting RNA transcripts. By using this technique it has been
possible to differentiate the S-PM2 phage-infected and non-infected cells of Synechococcus sp. strain
WH7803 and the expression of g20 mRNA peaked at 10 h after infection coinciding with the onset
of lysis (Fig. 11; Wharam et al ., 2007).
From the above studies, it has been possible to (i) determine diversity of marine cyanophages
as measured by variability in the sequence of g20 across gradients in time and space from different
environments, (ii) the g20 diversity was as great within the sample as between Oceans, (iii) the phage
diversity increased as Synechococcus abundance increased, and (iv) temporal studies correlated
cyanophage diversity with Synechococcus diversity. The marine cyanophage diversity described by
various workers on the basis of g20 sequences could be grouped into nine phylogenetic clusters and
with the g20 sequences originated from freshwater environments constituted the tenth cluster. Of the
nine marine clusters, three are those from the cultured representatives of marine cyanophages whereas
the remaining six marine clusters (Clusters A to F) and the freshwater cluster represent environmental
samples. Sullivan et al . (2008) are of the opinion that the g20 marker falls short of explaining the
diversity of marine cyanophages. This conclusion was based on a comprehensive phylogenetic study
of (i) all the marine cyanophages isolated by other workers to date, (ii) their own cyanophage isolates
obtained after enlarging the range of host strains during isolation, (iii) the assemblages identifi ed after
metagenomic analysis of marine waters from different oceans and (iv) also the data from Global Ocean
Sampling expedition (GOS). Comeau et al . (2010) conducted a comprehensive metagenomic analysis
of more than 300 scaffolds of T4-type cyanophages from GOS project. They designated the T4-type
cyanophages as Cyano-T4s and compared the gene order with those of the cultured representatives
of Cyano-T4s. There is a closer resemblance in the synteny between the gene sequences of uncultured
Cyano-T4s with the core genome of known and cultured Cyano-T4s. According to them the cultured
Cyano-T4s represent the great diversity of such phages from marine surface waters.
In order to fi nd out a diagnostic marker for detecting freshwater cyanophages, Baker et al .
(2006) have tried to amplify the g20 sequence by the PCR primers developed earlier (CPS1 and CPS2
primers developed by Fuller et al ., 1998; CPS1 and CPS8 primers described by Zhong et al ., 2002 and
g20 fD2 and CPS2 primers developed by Hambly, 2002). But as the g20 gene sequences could not
be amplifi ed from freshwater cyanophages, they developed special primers for the amplifi cation
of phage AN-15 gene that encodes major capsid protein (MCP). First they identifi ed the MCP by
subjecting the purifi ed viral particles to SDS-PAGE analysis. A dominant protein at approximately
37 kDa was supposed to represent the MCP. N-terminal amino acid analysis of this protein revealed
the presence of amino acids Ala-Leu-Thr-Thr-Leu-Thr-Pro. A degenerate primer PAN 15 F1
(5'-ACN ACN YTN CAN CC-3') was designed based on the amino acid sequence that helped in the