Biology Reference
In-Depth Information
populations of RIM2, RIM17, RIM24, RIM49/Syn9 and RIM50. These fi ve RIM strains co-occur in
the Rhode Island's coastal waters and have been frequently collected repeatedly over years. They
chose multilocus sequence analysis by selecting four core phage-specifi c genes (
g20
,
g23
,
g43
, DNA
polymerase gene; and cyanophage Syn9-g101 gene specifi c for tail fi ber protein) and four phage-
encoded host-derived genes (
psbA
,
psbD
,
cobS
, and
phoH
). The development of suitable primers for
the PCR amplifi cation of the selected genes and the sequence determinations for 60 concatenated
sequences led to the identifi cation of single nucleotide polymorphisms (SNPs) in these eight loci.
Intragenic recombination has been observed in two phage-encoded host genes (
psbA
and
psbD
)
and to a limited extent in the major capsid protein gene (
g23
). In most cases such SNPs are neutral
but in some others they resulted in amino acid substitutions. So they concluded that there is every
possibility for changes in the population dynamics of host-virus interactions. Metagenomic analysis
of the fi ve RIM strains revealed an average nucleotide identity of 99.3-99.8%. The GSC has developed
a checklist for annotation of Genome/Metagenome sequences. Following these guidelines, Duhaime
et al
. (2011) have enriched the public databases by annotating 30 genomes of marine bacteriophages
publicly available. Among these are included P-SS2,
Prochlorococcus
phages (PM-2, P-SSP7, P-SSM2
and P-SSM4) and
Synechococcus
phages (P60, S-PM2, S-RSM4, Syn5 and Syn9).
iii)
Enumeration techniques for marine cyanophages
:
(a) Transmission electron microscopy (TEM),
epifl uorescence microscopy and staining techniques
: The advantages of positive and negative staining of
aquatic viruses for the basic studies with electron microscopy have been highlighted by Ackermann
and Haldal (2010). The general stains employed for staining of aquatic viruses are phosphotungstate
and uranylacetate. The former confers only negative staining whereas the latter gives both positive
and negative staining. The probable artefacts encountered in staining, electron microscopy, counting
and size determinations of viruses have been discussed at length and refi nements in these techniques
suggested. TEM has been the best choice for viewing viruses directly because of the best resolution
of up to few nanometers and at the same time it permits the determination of shape of the virus.
Sieburth (1979) conducted fi rst studies on marine viruses by employing TEM. Torella and Morita
(1979) reported a titer of 10
4
viruses from coastal Oregon waters by using TEM. These studies were
followed by the investigations of a number of workers (Bergh
et al
., 1989; Børsheim
et al
., 1990;
Proctor and Fuhrman, 1990; Wommack
et al
., 1992; Bratbak and Heldal, 1993 Wilson
et al
., 1998).
Enumeration of marine viruses through TEM requires the transfer of viruses either directly or after
concentration by ultrafi ltration onto the electron microscopy grids. As it is a time consuming process
(and the equipment being bulky and expensive), it is not suited for fi eld studies (Paul
et al
., 1991;
Wommack
et al
., 1992; Bratbak and Heldal, 1993). The second approach is the use of epifl uorescence
microscopy combined with fl uorescent staining of viruses. In this technique, marine virus particles
are collected on 0.02 µm pore-size fi lters and then stained with a DNA-specifi c fl uorescent dye
such as DAPI (4', 6'-diamino-2-phenylindole) and observed under the epifl uorescnce microscopy
(Sieburth
et al
., 1988). DAPI was the fi rst fl uorochrome used for labelling and counting VLPs by
epifl uorescence microscopy (Proctor and Fuhrman, 1990; Hara
et al
., 1991; Fuhrman and Suttle, 1993;
Suttle, 1993). Other fl uorescent dyes used were YO-PRO-1 {4-[3-methyl-2,3-dihydro-(benzo-1,3-
oxazole)-2 methylmethyledene]-1-3, trimethylammoniumpropyl)-quinilinium diiodide}, YOYO-1,
POPO-1 or SBYR Green-1 that are reported to possess very high binding coeffi cients for nucleic
acids and such stained viruses have been viewed under epifl uorescence microscope (Hirons
et al
.,
1994; Hennes and Suttle, 1995). SBYR Green-stained samples of sea water when viewed through
epifl uorescence microscope revealed images of bacterial cells as bigger rounded structures and
viral particles as pin-head structures (Fig. 8). In most of the isolation protocols described, the use
