40
A. Meyerdierks and F.O. Glöckner
bulk DNA. Selective lysis of cells, based for example on a difference in cell wall
composition, or biochemical properties may also be used, when an enrichment of a
subpopulation of a community is necessary.
Cell populations may also be tagged by in situ hybridisation using 16S rRNAtargeted nucleic acid probes. These probes can be labelled, for example using
biotin, and target cells captured with streptavidin coated paramagnetic beads and
a magnet (Stoffels et al. 1999). Another enrichment method using microplates has
also been described (Zwirglmaier et al. 2004). An improved protocol combining
catalysed-reporter deposition fluorescence in situ hybridisation (CARD-FISH) with
magnetic bead capture, called Magneto-FISH, has recently been used to enrich
methanotrophic microbial consortia from sediments (Pernthaler et al. 2008). Cell
populations stained by fluorescence in situ hybridisation (FISH), have also been
sorted by flow cytometry (Podar et al. 2007).
In addition to these cell enrichment methods based on physical or molecular
characteristics, cell populations can also be separated based on their metabolic capabilities. The genomic DNA of the active fraction of a microbial community can
be labelled by a short incubation with 5-bromo-2 -deoxyuridine (BrdU). BrdU is a
thymidine analogue, which is incorporated into newly synthesised DNA of replicating cells. Such labelled cells can be visualised by immunostaining and sorted
by flow cytometry. Alternatively, the labelled DNA can be captured with BrdUspecific monoclonal antibodies coupled to paramagnetic beads (Urbach et al. 1999,
Mou et al. 2008). This method can also be used to label cells growing in response
to a specific stimulus (Borneman 1999). Alternatively, microbial communities can
be incubated with substrates labelled with stable isotopes (Dumont and Murrell
2005, Dumont et al. 2006, Neufeld et al. 2008). In one study, marine surface water
was incubated with 13 C-labelled methanol. Microorganisms capable of metabolizing methanol incorporated 13 C. The resulting heavy DNA could be separated from
light DNA by caesium chloride density gradient centrifugation, and was used for
metagenome analysis of the genetic capability of the methanol utilizing fraction of
the microbial community (Neufeld et al. 2008).
Another way of increasing the fraction of target cells is to enrich using classical
microbiological techniques. The circular genome of the methanogenic, rice cluster I
bacterium (Erkel et al. 2006) and the almost complete genome of the uncultured
anammox bacterium Kuenenia stuttgartiensis (Strous et al. 2006) were obtained
from metagenomic approaches based on such enrichments.
A general trend in the analysis of uncultured prokaryotes is single cell genomics.
In one study, for example, single cells of marine bacterioplankton were sorted on 96well plates by high speed fluorescence activated cell sorting (FACS) (Stepanauskas
and Sieracki 2007). Moreover, microfluidic devices have been developed to allow
single cell isolation based on dilution (Ottesen et al. 2006), or targeted selection
of individual cells (Marcy et al. 2007). Micromanipulation has been applied to isolate FISH-stained cells (Ishoy et al. 2006, Kvist et al. 2007). Micromanipulation
using optical tweezers (Huber et al. 1995), and laser capture microdissection (Gloess
et al. 2008, Thornhill et al. 2008) are alternative tools to select single cells from
environmental samples.
A. Meyerdierks and F.O. Glöckner
bulk DNA. Selective lysis of cells, based for example on a difference in cell wall
composition, or biochemical properties may also be used, when an enrichment of a
subpopulation of a community is necessary.
Cell populations may also be tagged by in situ hybridisation using 16S rRNAtargeted nucleic acid probes. These probes can be labelled, for example using
biotin, and target cells captured with streptavidin coated paramagnetic beads and
a magnet (Stoffels et al. 1999). Another enrichment method using microplates has
also been described (Zwirglmaier et al. 2004). An improved protocol combining
catalysed-reporter deposition fluorescence in situ hybridisation (CARD-FISH) with
magnetic bead capture, called Magneto-FISH, has recently been used to enrich
methanotrophic microbial consortia from sediments (Pernthaler et al. 2008). Cell
populations stained by fluorescence in situ hybridisation (FISH), have also been
sorted by flow cytometry (Podar et al. 2007).
In addition to these cell enrichment methods based on physical or molecular
characteristics, cell populations can also be separated based on their metabolic capabilities. The genomic DNA of the active fraction of a microbial community can
be labelled by a short incubation with 5-bromo-2 -deoxyuridine (BrdU). BrdU is a
thymidine analogue, which is incorporated into newly synthesised DNA of replicating cells. Such labelled cells can be visualised by immunostaining and sorted
by flow cytometry. Alternatively, the labelled DNA can be captured with BrdUspecific monoclonal antibodies coupled to paramagnetic beads (Urbach et al. 1999,
Mou et al. 2008). This method can also be used to label cells growing in response
to a specific stimulus (Borneman 1999). Alternatively, microbial communities can
be incubated with substrates labelled with stable isotopes (Dumont and Murrell
2005, Dumont et al. 2006, Neufeld et al. 2008). In one study, marine surface water
was incubated with 13 C-labelled methanol. Microorganisms capable of metabolizing methanol incorporated 13 C. The resulting heavy DNA could be separated from
light DNA by caesium chloride density gradient centrifugation, and was used for
metagenome analysis of the genetic capability of the methanol utilizing fraction of
the microbial community (Neufeld et al. 2008).
Another way of increasing the fraction of target cells is to enrich using classical
microbiological techniques. The circular genome of the methanogenic, rice cluster I
bacterium (Erkel et al. 2006) and the almost complete genome of the uncultured
anammox bacterium Kuenenia stuttgartiensis (Strous et al. 2006) were obtained
from metagenomic approaches based on such enrichments.
A general trend in the analysis of uncultured prokaryotes is single cell genomics.
In one study, for example, single cells of marine bacterioplankton were sorted on 96well plates by high speed fluorescence activated cell sorting (FACS) (Stepanauskas
and Sieracki 2007). Moreover, microfluidic devices have been developed to allow
single cell isolation based on dilution (Ottesen et al. 2006), or targeted selection
of individual cells (Marcy et al. 2007). Micromanipulation has been applied to isolate FISH-stained cells (Ishoy et al. 2006, Kvist et al. 2007). Micromanipulation
using optical tweezers (Huber et al. 1995), and laser capture microdissection (Gloess
et al. 2008, Thornhill et al. 2008) are alternative tools to select single cells from
environmental samples.