Molecular identification of picoplankton populations in contrasting waters of the Arabian Sea

The composition of picoplankton in the southern oligotrophic, northern mesotrophic waters and deep oxygen minimum zone (OMZ) of the Arabian Sea was determined by 16S ribosomal RNA gene cloning and fluorescence in situ hybridisation (FISH). It was hypothesised that the composition of the heterotrophic picoplankton would be different in these contrasting waters. To reduce the total diversity, cells were sorted by flow cytometry according to their scatter and DNA content before PCR amplification. The 16S rRNA clone libraries resulting from flow-sorted populations were different and often dominated by a small number of clades. Libraries from the Prochlorococcus-dominated southerly waters were dominated by sequences related to uncultured clusters of SAR11, SAR86 and Actinobacteria (HGC I). From surface waters of the Synechococcus-dominated northern part of the Arabian Sea, mostly sequences related to the uncultured gammaproteobacterial group ‘Svalbard’ and HGC I were retrieved. The clone libraries from the OMZ were also dominated by sequences falling in the clades SAR11 and SAR406, but included sequences related to those of sulfate-reducing (Desulfosarcina, Desulfofrigus) and sulfide-oxidising bacteria (endosymbionts of Riftia and Calyptogena). With a recently developed more sensitive FISH protocol approximately 60% of all DAPI stained cells could be identified by general probes as Bacteria, Cren- or Euryarchaeota in both provinces of the Arabian Sea; 40% remained undetected. On this level and on that of the major phylogenetic groups like Alpha- and Gammaproteobacteria only minor differences were detected by FISH. However, the composition of heterotrophic picoplankton clearly differed for the proteobacterial subgroups SAR86, SAR11 and SAR116. These were more abundant in the oligotrophic waters throughout the water column than in the mesotrophic surface waters and the OMZ. This supports our original hypothesis that the contrasting waters in the Arabian Sea harbor different heterotrophic picoplankton communities. In the future, FISH with a larger set of probes for more narrow phylogenetic groups will enable us to quantify these differences in more detail.

[1]  R. Amann,et al.  Isolation of Novel Pelagic Bacteria from the German Bight and Their Seasonal Contributions to Surface Picoplankton , 2001, Applied and Environmental Microbiology.

[2]  F. Azam,et al.  Bacterial community composition during two consecutive NE Monsoon periods in the Arabian Sea studied by denaturing gradient gel electrophoresis (DGGE) of rRNA genes , 1999 .

[3]  R Amann,et al.  Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga-flavobacter-bacteroides in the natural environment. , 1996, Microbiology.

[4]  Thomas Huber,et al.  Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases. , 2003, International journal of systematic and evolutionary microbiology.

[5]  D. Halpern,et al.  Primary productivity and its regulation in the Arabian Sea during 1995 , 2001 .

[6]  R. Amann,et al.  Section 3 update: Sensitive multi-color fluorescence in situ hybridization for the identification of environmental microorganisms , 2004 .

[7]  E. Casamayor,et al.  Bacterial Community Structure Associated with a Dimethylsulfoniopropionate-Producing North Atlantic Algal Bloom , 2000, Applied and Environmental Microbiology.

[8]  V. Sarma.  An evaluation of physical and biogeochemical processes regulating perennial suboxic conditions in the water column of the Arabian Sea , 2002 .

[9]  D. Caron,et al.  Response of microbial community structure to environmental forcing in the Arabian Sea , 1998 .

[10]  E. Virginia Armbrust,et al.  Phylogenetic Analysis of Particle-Attached and Free-Living Bacterial Communities in the Columbia River, Its Estuary, and the Adjacent Coastal Ocean , 1999, Applied and Environmental Microbiology.

[11]  S. Giovannoni,et al.  Genetic diversity in Sargasso Sea bacterioplankton , 1990, Nature.

[12]  R. Olson,et al.  Arabian Sea phytoplankton during Southwest and Northeast Monsoons 1995: composition, size structure and biomass from individual cell properties measured by flow cytometry , 2001 .

[13]  R. Amann,et al.  Linking the composition of bacterioplankton to rapid turnover of dissolved dimethylsulphoniopropionate in an algal bloom in the North Sea. , 2001, Environmental microbiology.

[14]  D. Stahl,et al.  Unexpected Population Distribution in a Microbial Mat Community: Sulfate-Reducing Bacteria Localized to the Highly Oxic Chemocline in Contrast to a Eukaryotic Preference for Anoxia , 1999, Applied and Environmental Microbiology.

[15]  S. Giovannoni,et al.  Genetic comparisons reveal the same unknown bacterial lineages in Atlantic and Pacific bacterioplankton communities , 1995 .

[16]  B. Fuchs,et al.  Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries. , 2002, Environmental microbiology.

[17]  Sharon L. Smith.  Understanding the Arabian Sea: Reflections on the 1994-1996 Arabian Sea Expedition , 2001 .

[18]  K. Schleifer,et al.  The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. , 1999, Systematic and applied microbiology.

[19]  Rudolf Amann,et al.  Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria , 2002, Applied and Environmental Microbiology.

[20]  T. Ferdelman,et al.  Structural and Functional Dynamics of Sulfate-Reducing Populations in Bacterial Biofilms , 1998, Applied and Environmental Microbiology.

[21]  P. Burkill,et al.  Synechococcus and its importance to the microbial foodweb of the northwestern Indian Ocean , 1993 .

[22]  T. Reinthaler,et al.  Combining Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization and Microautoradiography To Detect Substrate Utilization by Bacteria and Archaea in the Deep Ocean , 2004, Applied and Environmental Microbiology.

[23]  E. Delong,et al.  Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton. , 2001, Environmental microbiology.

[24]  G. Tarran,et al.  Phytoplankton community structure in the Arabian Sea during and after the SW monsoon, 1994 , 1999 .

[25]  R Amann,et al.  Flow sorting of microorganisms for molecular analysis , 1997, Applied and environmental microbiology.

[26]  R. Malmstrom,et al.  Contribution of SAR11 Bacteria to Dissolved Dimethylsulfoniopropionate and Amino Acid Uptake in the North Atlantic Ocean , 2004, Applied and Environmental Microbiology.

[27]  Thomas P. Curtis,et al.  Estimating prokaryotic diversity and its limits , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[28]  I. Joint,et al.  Bacterioplankton activity in the surface waters of the Arabian Sea during and after the 1994 SW monsoon , 1999 .

[29]  R. Amann.  In situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes , 1995 .

[30]  Rudolf Amann,et al.  Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization , 2004, Applied and Environmental Microbiology.

[31]  A. Uitterlinden,et al.  Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA , 1993, Applied and environmental microbiology.

[32]  R. Amann,et al.  Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations , 1990, Applied and environmental microbiology.

[33]  David C. Smith,et al.  Heterotrophic bacterioplankton in the Arabian Sea:: Basinwide response to year-round high primary productivity , 2001 .

[34]  R. Amann,et al.  Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. , 1993, Cytometry.

[35]  A. Chistoserdov,et al.  Phylogenetic analysis of the succession of bacterial communities in the Great South Bay (Long Island). , 2001, FEMS microbiology ecology.

[36]  Stefan Schouten,et al.  Distribution of Membrane Lipids of Planktonic Crenarchaeota in the Arabian Sea , 2002, Applied and Environmental Microbiology.

[37]  William A. Siebold,et al.  SAR11 clade dominates ocean surface bacterioplankton communities , 2002, Nature.

[38]  R. Amann,et al.  Comparison of Cellular and Biomass Specific Activities of Dominant Bacterioplankton Groups in Stratified Waters of the Celtic Sea , 2001, Applied and Environmental Microbiology.

[39]  Alessandra Ghiani,et al.  Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining , 2001, Applied and Environmental Microbiology.

[40]  J. Messing.  New M13 vectors for cloning. , 1983, Methods in enzymology.

[41]  S. Giovannoni,et al.  Detection of stratified microbial populations related to Chlorobium and Fibrobacter species in the Atlantic and Pacific oceans , 1996, Applied and environmental microbiology.

[42]  J. Fuhrman,et al.  Significance of Size and Nucleic Acid Content Heterogeneity as Measured by Flow Cytometry in Natural Planktonic Bacteria , 1999, Applied and Environmental Microbiology.

[43]  L. Codispoti,et al.  The oxygen minimum zone in the Arabian Sea during 1995 , 1999 .

[44]  K. Schleifer,et al.  ARB: a software environment for sequence data. , 2004, Nucleic acids research.

[45]  J. Brock,et al.  Interannual variability in phytoplankton blooms observed in the northwestern Arabian Sea during the southwest monsoon , 1992 .

[46]  K. Schleifer,et al.  Phylogenetic Oligodeoxynucleotide Probes for the Major Subclasses of Proteobacteria: Problems and Solutions , 1992 .

[47]  D. Vaulot,et al.  Enumeration and Cell Cycle Analysis of Natural Populations of Marine Picoplankton by Flow Cytometry Using the Nucleic Acid Stain SYBR Green I , 1997, Applied and environmental microbiology.

[48]  G. Tarran,et al.  Picoplanktonic community structure on an Atlantic transect from 50°N to 50°S , 1998 .

[49]  O. White,et al.  Environmental Genome Shotgun Sequencing of the Sargasso Sea , 2004, Science.

[50]  F. Rohwer,et al.  Use of 16S Ribosomal DNA for Delineation of Marine Bacterioplankton Species , 2002, Applied and Environmental Microbiology.

[51]  E. Delong,et al.  Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel , 1997, Applied and environmental microbiology.

[52]  N. Pace,et al.  Microbial ecology and evolution: a ribosomal RNA approach. , 1986, Annual review of microbiology.

[53]  R Amann,et al.  In situ probing of gram-positive bacteria with high DNA G + C content using 23S rRNA-targeted oligonucleotides. , 1994, Microbiology.

[54]  U. Göbel,et al.  Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. , 1997, FEMS microbiology reviews.

[55]  Rappé,et al.  Phylogenetic comparisons of a coastal bacterioplankton community with its counterparts in open ocean and freshwater systems. , 2000, FEMS microbiology ecology.

[56]  S. Giovannoni,et al.  Phylogenetic diversity of marine coastal picoplankton 16S rRNA genes cloned from the continental shelf off Cape Hatteras, North Carolina , 1997 .

[57]  J. Fuhrman,et al.  Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Pacific Oceans , 1993, Applied and environmental microbiology.

[58]  D. Stahl,et al.  Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences , 1993, Applied and environmental microbiology.

[59]  Frank Oliver Glöckner,et al.  An in situ hybridization protocol for detection and identification of planktonic bacteria , 1996 .

[60]  Philippe Lebaron,et al.  Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems? , 2001, Applied and Environmental Microbiology.

[61]  Harald Huber,et al.  In Situ Accessibility of Small-Subunit rRNA of Members of the Domains Bacteria, Archaea, and Eucarya to Cy3-Labeled Oligonucleotide Probes , 2003, Applied and Environmental Microbiology.

[62]  R. Amann,et al.  High Rate of Uptake of Organic Nitrogen Compounds by Prochlorococcus Cyanobacteria as a Key to Their Dominance in Oligotrophic Oceanic Waters , 2003, Applied and Environmental Microbiology.

[63]  H. Noller,et al.  Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. , 1981, Journal of molecular biology.

[64]  Rudolf Amann,et al.  Automated Enumeration of Groups of Marine Picoplankton after Fluorescence In Situ Hybridization , 2003, Applied and Environmental Microbiology.

[65]  S. Giovannoni,et al.  Cultivation of the ubiquitous SAR11 marine bacterioplankton clade , 2002, Nature.

[66]  J. Messing.  [2] New M13 vectors for cloning , 1983 .

[67]  G. Muyzer,et al.  Phylogenetic relationships ofThiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments , 1995, Archives of Microbiology.

[68]  R. Amann,et al.  Changes in community composition during dilution cultures of marine bacterioplankton as assessed by flow cytometric and molecular biological techniques. , 2000, Environmental microbiology.