CHAPTER 24
Distribution of Representative Oligotrophic Bacteria in a Pelagic
Marine Environment (Ligurian Sea) by in situ Hybridization with
rRNA-targeted, Fluorescently Labelled Oligonucleotides
L. Giuliano!, M. De Domenico l , and M.M. Yakimovl
ABSTRACT
The distribution of specific autochthonous oligotrophic bacteria in a surface layer (30 m depth)
of a pelagic seawater station (DYFAMED,28 miles off-shore, in the northwestern Mediterranean
Sea) was studied by in situ hybridization with rRNA-targeted fluorescently labelled oligonucleotide probes (FISH). The data reported here extend a previous analysis of 16S rONA done
libraries obtained by peR from enriched oligotrophic bacterial populations in order to relate the
previous enrichment results to population abundance and diversity in the original sample. For
such a purpose, the frequency of matching of six previously designed group- and clone-specific probes with the naturally occurring 16S rRNA sequences was analysed. The results of in situ
hybridization with the rRNA-targeted fluorescently labelled oligonucleotide probes support
reports of Vibrio and Rhodobacter groups in the surface layer. The high percentage of coverage
of Vibrio- and Rhodobacter- specific probes (24 and 19%, respectively) resulting from in situ
experiments indicates the suitability of the previously described method to obtain enrichment
of numerically representative autochthonous marine bacteria.
Introduction
Knowledge of microbial diversity has increased
dramatically in recent years, in part as a result of
sequencing of rRNA genes from DNA obtained
directly from uncultured microbiota, often by
the use of peR and rRNA-specific primers.
Fluorescence in situ hybridization (FISH) with
rRNA-targeted oligonucleotide probes selectively visualizes bacterial cells with defined phylogenetic affiliations (Amann et al. 1995; 1997). Based
on a rapidly growing set of 16S rRNA sequence
data, it is probably the phylogenetically most
sophisticated approach of a whole-cell in situ
identification (Ludwig and Schleifer 1994). In
contrast to other identification approaches, FISH
largely maintains the original features of the targeted microorganisms, i.e. their morphologies,
cell sizes (Pernthaler et al. 1996; Ramsing et al.
1996), and cellular rRNA content (Boyle et al.
1995; Poulsen et al. 1993). Although these
approaches have produced a diverse collection of
sequences and expanded our view of microbial
diversity, analysis of microbial 168 rDNA
sequences has limitations in relating specific
rDNA sequences to other aspects of organisms in
the environment under study. We have already
described a method to obtain seawater enrichments of numerically representative indigenous
oligotrophic marine bacteria within the naturally occurring microbial populations {Giuliano et
al.1998}. Such a method provided the possibility
to test physiologically the taxonomically characterized targeted populations representative of
the original communities. In the present study,
the density of specific populations from seawater
enrichments in the natural pelagic marine environment was determined by in situ hybridizations with the previously obtained rRNA-targeted oligonucleotide probes. Based on the
described hybridization protocol, the total density of the checked populations accounted for a
large fraction of the bacterioplankton, thus suggesting the suitability of the method for the proposed aim. By relating the previous enrichment
results to the population abundance and diversi1 Dipartimento di Biologia Animale ed Ecologia Marina, Universita ill Messina, Salita Sperone 31, 98166 Messina, Italy
2 Centro Siciliano per Ia Ricerca Atmosferica e la Fisica Applicata (CSRAFA), C. da Papardo 31, 98166 Messina, Italy
EM. Faranda, L. Gug1ielmo, G. Spezie (eds)
Mediterranean Ecosystems: Structures and Processes
© Springer-Verlag ltalia 2001
Distribution of Representative Oligotrophic Bacteria in a Pelagic
Marine Environment (Ligurian Sea) by in situ Hybridization with
rRNA-targeted, Fluorescently Labelled Oligonucleotides
L. Giuliano!, M. De Domenico l , and M.M. Yakimovl
ABSTRACT
The distribution of specific autochthonous oligotrophic bacteria in a surface layer (30 m depth)
of a pelagic seawater station (DYFAMED,28 miles off-shore, in the northwestern Mediterranean
Sea) was studied by in situ hybridization with rRNA-targeted fluorescently labelled oligonucleotide probes (FISH). The data reported here extend a previous analysis of 16S rONA done
libraries obtained by peR from enriched oligotrophic bacterial populations in order to relate the
previous enrichment results to population abundance and diversity in the original sample. For
such a purpose, the frequency of matching of six previously designed group- and clone-specific probes with the naturally occurring 16S rRNA sequences was analysed. The results of in situ
hybridization with the rRNA-targeted fluorescently labelled oligonucleotide probes support
reports of Vibrio and Rhodobacter groups in the surface layer. The high percentage of coverage
of Vibrio- and Rhodobacter- specific probes (24 and 19%, respectively) resulting from in situ
experiments indicates the suitability of the previously described method to obtain enrichment
of numerically representative autochthonous marine bacteria.
Introduction
Knowledge of microbial diversity has increased
dramatically in recent years, in part as a result of
sequencing of rRNA genes from DNA obtained
directly from uncultured microbiota, often by
the use of peR and rRNA-specific primers.
Fluorescence in situ hybridization (FISH) with
rRNA-targeted oligonucleotide probes selectively visualizes bacterial cells with defined phylogenetic affiliations (Amann et al. 1995; 1997). Based
on a rapidly growing set of 16S rRNA sequence
data, it is probably the phylogenetically most
sophisticated approach of a whole-cell in situ
identification (Ludwig and Schleifer 1994). In
contrast to other identification approaches, FISH
largely maintains the original features of the targeted microorganisms, i.e. their morphologies,
cell sizes (Pernthaler et al. 1996; Ramsing et al.
1996), and cellular rRNA content (Boyle et al.
1995; Poulsen et al. 1993). Although these
approaches have produced a diverse collection of
sequences and expanded our view of microbial
diversity, analysis of microbial 168 rDNA
sequences has limitations in relating specific
rDNA sequences to other aspects of organisms in
the environment under study. We have already
described a method to obtain seawater enrichments of numerically representative indigenous
oligotrophic marine bacteria within the naturally occurring microbial populations {Giuliano et
al.1998}. Such a method provided the possibility
to test physiologically the taxonomically characterized targeted populations representative of
the original communities. In the present study,
the density of specific populations from seawater
enrichments in the natural pelagic marine environment was determined by in situ hybridizations with the previously obtained rRNA-targeted oligonucleotide probes. Based on the
described hybridization protocol, the total density of the checked populations accounted for a
large fraction of the bacterioplankton, thus suggesting the suitability of the method for the proposed aim. By relating the previous enrichment
results to the population abundance and diversi1 Dipartimento di Biologia Animale ed Ecologia Marina, Universita ill Messina, Salita Sperone 31, 98166 Messina, Italy
2 Centro Siciliano per Ia Ricerca Atmosferica e la Fisica Applicata (CSRAFA), C. da Papardo 31, 98166 Messina, Italy
EM. Faranda, L. Gug1ielmo, G. Spezie (eds)
Mediterranean Ecosystems: Structures and Processes
© Springer-Verlag ltalia 2001