Biology Reference
In-Depth Information
4) Diversity of cyanobacteria determined on the basis of molecular markers
: During recent years,
diversity studies of cyanobacteria based on sequencing of 16S rRNA, 16S rRNA-23S rRNA ITS region,
ARDRA, T-RFLP and PCR with DGGE techniques have been made circumventing the collection-and
culture-based studies (i.e. metagenomic analysis). In some of the cases, the molecular taxonomic
data have been correlated with the cultures of cyanobacteria raised from the same habitats. Some
of these are summarized here.
The molecular techniques described above play a meaningful role in assessing the diversity of
cyanobacteria from a wide variety of ecosystems (Kumari
et al
., 2009). These require the extraction
of nucleic acids and do not allow the identifi cation of individual cells from the environmental
samples. However, the identifi cation of whole fi xed cells by
in situ
hybridization with rRNA targeted
oligonucleotide probes has been advocated (Amann
et al.
, 1995). This technique is so powerful that
cyanobacteria in deeper layers of microbial mats can be identifi ed. One disadvantage is that the
autofl uorescence exhibited by cyanobacterial cells due to the presence of chlorophyll disrupts the
fl uorescence label associated with oligonucleotide probes upon hybridization within the target cells.
To overcome this problem, Schönhuber
et al
. (1999) developed a non-fl uorescent assay method, based
on horseradish peroxidase (HRP)-labelled oligonucleotides, for identifi cation of cyanobacterial
cultures as well as cyanobacteria existing in microbial mats. With the help of cyanobacteria-specifi c
primers designed by them (CYA 361, CYA 664, CYA 762, CIV/V 1342 together with EUB 338 that
targets most bacteria) and dot blot hybridization, the cells can be detected by the formation of a
coloured precipitate by oxidative polymerization of the non-fl uorescent substrate diaminobenzidine.
Additionally, they also evaluated the automatic fl uorescence detection system based on enzymatic
signal amplifi cation (Schönhuber
et al
., 1997; Van Gijlswijk
et al
., 1996). This is known as tyramide
signal amplifi cation (TSA) system that involves covalent binding of fl uorochrome tyramide substrate
molecules to electron rich moieties such as tyrosine and tryptophan in the HRP-containing cells.
It was possible to identify
in situ
cyanobacterial diversity from microbial mats from alpine lake in
Switzerland. According to these workers, this method complements the traditional methods based
on phenotype and the identifi cation of cyanobacteria will be more reliable. Another approach,
suggested to overcome the problem of autofl uorescence of the cells, is to hybridize with biotin-
labelled probes and allow reaction with a streptavidin-HRP conjugate, prior to detection employing
TSA. This has successfully been done in case of bacteria (Lebaron
et al
., 1997). With the help of probe
CYA664 (5'-GGAATTCCCTCTGCCCC-3') that targets all known marine species of
Synechococcus
and
Prochlorococcus
and in combination with TSA, closely related
Prochlorococcus
genotypes belonging
to HL-adapted and LL- adapted were shown to be distributed at different depths of North Atlantic
Ocean and the Red Sea. While LL-adapted strains are distributed in deeper layers in both these
waters, the HL-adapted
Prochlorococcus
populations not only exhibited distinct distribution patterns
for each region but also are distributed at different depths (West
et al
., 2001). However, the cell fi xation
step and permeabilization of cells using alcohol and/or detergents used in these studies prior to
hybridization with labelled 16S rRNA-targeted probe sometimes damages the cells. This is especially
true for
Prochlorococcus
or
Synechococcus
cells from oceanic samples. In light of this, another method
promising in the analysis of individual cells is whole-cell hybridization with fl uorescently labelled
rRNA-targeted oligonucleotide probes combined with fl ow cytometry (Amann
et al
., 1990). This has
been successfully achieved to quantify individual cells from cultures of
Synechococcus
(Chisholm,
1992). A relationship between the rRNA content of
Synechococcus
cells and growth rate has been
demonstrated by Binder and Liu (1998). At slow growth rates, the rRNA content almost remained
constant but at intermediate growth rates the rRNA content per cell increased proportionally with
growth rate and a corresponding decrease in its content was noted at saturating light intensities
