194
1. Giuliano et al.
(mean, 24%) of them were detected by the
group-specific G V probe (Figs.1a,bi 2a,b).
Discussion
The high percentage of coverage of the groupspecific probes indicated the suitability of the
previously described method to obtain enrichments of numerically representative autochthonous marine bacteria. Moreover, because the two
different group-specific probes were targeting
enriched bacterial populations from the same
dilution of the original sample, the equivalent
size of detected populations agreed with the predicted occurrence of populations within the natural community. The presence of bacterial populations representative of Vibrio group in pelagic
waters is well documented (Pinhassi et al. 1997).
According to previous works in different oceans
(Gonzalez and Moran 1997; Mullins et al. 1995),
the Rhodobacter group, affiliated to the ccProteobacteria, is best adapted to the conditions
of the euphotic zone when the water column is
highly stratified (e.g. sampling conditions), and
some representatives of this group have been isolated from the surfaces of unicellular algae (Lafay
et al. 1995). Because of the bloom event detected
at the sampling time in the DYFAMED station,
we could speculate about the possible existence
of some spatial relations between SRF- targeted
populations and some dominant algae.
Monitoring by the clone-specific probes did not
show any detectable signal under epifluorescence microscopy analysis. One of possible reasons for the absence in the natural sample of
populations hybridizing with the obtained clonespecific probes could be explained by the high
frequency of mutational events usually involving
the most variable regions of 16S rRNA operons
(Le. minor nucleotide substitutions). Such events
could cause the loss of the original sequences of
some hypervariable regions during succession of
bacterial generations and, therefore, the design
of probes complementary to mutated regions.
Acknowledgements. We would like to thank Dr. A. Bianchi
(CNRS, Marseilles, France) for generous hospitality, providing
us the possibility to collect the probes during the DYFABAC
cruise of rlv'Tethys II' (Programme DYFAMED, CNRS-INSU)
and Prof. K.N. Timmis (GBF, Braunschweig, Germany) for
helpful discussions. The assistance of Prof. E. De Domenico is
also gratefully acknowledged. This research work was supported in part by grants from University of Messina.
References
Amann R, Glockner OF, Neef A (1997) Modern methods in subsurface microbiology: in situ identification of microorganisms with nucleic acid probes. FEMS Microbiol Rev
20: 191-200
Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic indentificadon and in situ detection of individual microbial cells
without cultivation. Microbiol Rev 59: 143-169
Bianchi A, Giuliano L (1996). Enumeration of viable bacteria in
the marine pelagic environment. Appl Environ Microbiol62:
174-177
Boyle M, Ahle T. Molin 5 (1995) Application of a strain-specific
rRNA oligonucleotide probe targeting Pseudomonas fluorescens Agl in a mesocosm study of bacterial release into the
environment. Appl Environ Microbio161: 1384-1390
Fuhrman JA, Comeau DE, Hagstrom A. Chan AM (1988)
Extraction from natural planktonic microorganisms of DNA
suitable for molecular biological studies. Appl Environ
Microbiol 54: 1426-1429
Giuliano L, De Domenico M. De Domenico E, Hofle MG, Yakimov
MM (1998) Identification of culturable oligotrophic bacteria
within naturally occurring bacterioplankton communities
of the Ligurian Sea by 165 rRNA Sequencing and Probing.
Microb Eco137: 77-85
Glockner FO, Amann R, Alfreider A, Pernthaler T, Psenner R,
Trebesius K, Schleifer KH (1996} An in situ hybridization
protocol fur detection and identification of planktonic bacteria. Appl Microbiol19: 403-406
Gonzalez JM, Moran MA (1997) Numerical dominance of a group
of marine bacteria in the "-subclass of the class
Proteobacteria in coastal seawater. Appl Environ Microbiol
63: 4237-4242
Lafoy B, Ruimy R, Rausch de Traubenberg C, Breittmayer V,
Gauthier MI, Christen R (1995) Roseobacter algicola sp. nov.,
a new marine bacterium isolated from the phycosphere of
the toxin-producing dinoflagellate Prorocentrum lima. Int J
Syst Bacterio145: 290-296
Lane DJ (1991) 16/23 rRNA sequencing. In: Stackebrandt E.
Goodfellow M (eds) Nucleic acids techniques in bacterial
systematics. John Wiley and Sons, Chichester. UK, pp 115-175
Larsen N, Overbeck R, McCaughey MI, Woese CR (1994) The ribosomal RNA database project (RDP). Nucleic Acid Res 24: 82-85
Ludwig W, Schleifer KH (1994) Bacterial phylogeny based on 165
and 235 rRNA sequence analysis. FEMS Microbiol Rev 15:
155-173
Minas HI. Minas M (1990) New production considerations in the
Mediterranean Sea. EOS 71: 1395-1396
Morel A, Andre JM (1991) Pigment distribution and primary production in the western Mediterranean as derived and modelled from Coastal Zone Colour scanner observations. J
Geophys Res 96: 12685-12698
Mullins TD, Britschgi TB, Krest RL, Giovannoni SJ (1995) Genetic
comparisons reveal the same unknown bacterial lineage in
Atlantic and Pacific bacterioplankton communities. Limnol
Oceanogr 40: 148-158
Pernthaler J. Sattler B, Simek K. Schwarzenbacher A, Psenner R
(1996) Top-down effects on the size-biomass distribution of
a freshwater bacterioplankton community. Aquat Microb
Ecol 10: 255-263
Pinhassi T, ZWeifel UL, Hagstrom A (1997) Dominant marine bacterioplankton species found among colony-forming bacteria. Appl Environ Microbio163: 3359-3366
1. Giuliano et al.
(mean, 24%) of them were detected by the
group-specific G V probe (Figs.1a,bi 2a,b).
Discussion
The high percentage of coverage of the groupspecific probes indicated the suitability of the
previously described method to obtain enrichments of numerically representative autochthonous marine bacteria. Moreover, because the two
different group-specific probes were targeting
enriched bacterial populations from the same
dilution of the original sample, the equivalent
size of detected populations agreed with the predicted occurrence of populations within the natural community. The presence of bacterial populations representative of Vibrio group in pelagic
waters is well documented (Pinhassi et al. 1997).
According to previous works in different oceans
(Gonzalez and Moran 1997; Mullins et al. 1995),
the Rhodobacter group, affiliated to the ccProteobacteria, is best adapted to the conditions
of the euphotic zone when the water column is
highly stratified (e.g. sampling conditions), and
some representatives of this group have been isolated from the surfaces of unicellular algae (Lafay
et al. 1995). Because of the bloom event detected
at the sampling time in the DYFAMED station,
we could speculate about the possible existence
of some spatial relations between SRF- targeted
populations and some dominant algae.
Monitoring by the clone-specific probes did not
show any detectable signal under epifluorescence microscopy analysis. One of possible reasons for the absence in the natural sample of
populations hybridizing with the obtained clonespecific probes could be explained by the high
frequency of mutational events usually involving
the most variable regions of 16S rRNA operons
(Le. minor nucleotide substitutions). Such events
could cause the loss of the original sequences of
some hypervariable regions during succession of
bacterial generations and, therefore, the design
of probes complementary to mutated regions.
Acknowledgements. We would like to thank Dr. A. Bianchi
(CNRS, Marseilles, France) for generous hospitality, providing
us the possibility to collect the probes during the DYFABAC
cruise of rlv'Tethys II' (Programme DYFAMED, CNRS-INSU)
and Prof. K.N. Timmis (GBF, Braunschweig, Germany) for
helpful discussions. The assistance of Prof. E. De Domenico is
also gratefully acknowledged. This research work was supported in part by grants from University of Messina.
References
Amann R, Glockner OF, Neef A (1997) Modern methods in subsurface microbiology: in situ identification of microorganisms with nucleic acid probes. FEMS Microbiol Rev
20: 191-200
Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic indentificadon and in situ detection of individual microbial cells
without cultivation. Microbiol Rev 59: 143-169
Bianchi A, Giuliano L (1996). Enumeration of viable bacteria in
the marine pelagic environment. Appl Environ Microbiol62:
174-177
Boyle M, Ahle T. Molin 5 (1995) Application of a strain-specific
rRNA oligonucleotide probe targeting Pseudomonas fluorescens Agl in a mesocosm study of bacterial release into the
environment. Appl Environ Microbio161: 1384-1390
Fuhrman JA, Comeau DE, Hagstrom A. Chan AM (1988)
Extraction from natural planktonic microorganisms of DNA
suitable for molecular biological studies. Appl Environ
Microbiol 54: 1426-1429
Giuliano L, De Domenico M. De Domenico E, Hofle MG, Yakimov
MM (1998) Identification of culturable oligotrophic bacteria
within naturally occurring bacterioplankton communities
of the Ligurian Sea by 165 rRNA Sequencing and Probing.
Microb Eco137: 77-85
Glockner FO, Amann R, Alfreider A, Pernthaler T, Psenner R,
Trebesius K, Schleifer KH (1996} An in situ hybridization
protocol fur detection and identification of planktonic bacteria. Appl Microbiol19: 403-406
Gonzalez JM, Moran MA (1997) Numerical dominance of a group
of marine bacteria in the "-subclass of the class
Proteobacteria in coastal seawater. Appl Environ Microbiol
63: 4237-4242
Lafoy B, Ruimy R, Rausch de Traubenberg C, Breittmayer V,
Gauthier MI, Christen R (1995) Roseobacter algicola sp. nov.,
a new marine bacterium isolated from the phycosphere of
the toxin-producing dinoflagellate Prorocentrum lima. Int J
Syst Bacterio145: 290-296
Lane DJ (1991) 16/23 rRNA sequencing. In: Stackebrandt E.
Goodfellow M (eds) Nucleic acids techniques in bacterial
systematics. John Wiley and Sons, Chichester. UK, pp 115-175
Larsen N, Overbeck R, McCaughey MI, Woese CR (1994) The ribosomal RNA database project (RDP). Nucleic Acid Res 24: 82-85
Ludwig W, Schleifer KH (1994) Bacterial phylogeny based on 165
and 235 rRNA sequence analysis. FEMS Microbiol Rev 15:
155-173
Minas HI. Minas M (1990) New production considerations in the
Mediterranean Sea. EOS 71: 1395-1396
Morel A, Andre JM (1991) Pigment distribution and primary production in the western Mediterranean as derived and modelled from Coastal Zone Colour scanner observations. J
Geophys Res 96: 12685-12698
Mullins TD, Britschgi TB, Krest RL, Giovannoni SJ (1995) Genetic
comparisons reveal the same unknown bacterial lineage in
Atlantic and Pacific bacterioplankton communities. Limnol
Oceanogr 40: 148-158
Pernthaler J. Sattler B, Simek K. Schwarzenbacher A, Psenner R
(1996) Top-down effects on the size-biomass distribution of
a freshwater bacterioplankton community. Aquat Microb
Ecol 10: 255-263
Pinhassi T, ZWeifel UL, Hagstrom A (1997) Dominant marine bacterioplankton species found among colony-forming bacteria. Appl Environ Microbio163: 3359-3366