Imaging Marine Bacteria with Unique 16S rRNA V6 Sequences by Fluorescence in situ Hybridization and Spectral Analysis

Fluorescence in situ hybridization (FISH) combined with spectral analysis was performed to image specific bacteria from seawater using probes targeting the V6 hypervariable region of small subunit ribosomal RNA (SSU rRNA), corresponding to positions 984 to 1047 of E. coli 16S rRNA gene. For each target, we designed two probes, each with a distinct fluorescent reporter and a unique hybridization site. Spectral imaging analysis of bacteria incubated with a pair of specific probes and a general bacterial probe enabled the detection of three probe-conferred spectra in each target cell, increasing the reliability of target identification in a diverse bacterial community.

[1]  M. Crowell,et al.  Human gut microbiota in obesity and after gastric bypass , 2009, Proceedings of the National Academy of Sciences.

[2]  Susan M. Huse,et al.  Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing , 2008, PLoS genetics.

[3]  Susan M. Huse,et al.  Pyrosequencing analysis of the Oral Microflora of healthy adults , 2008, Journal of dental research.

[4]  R. Amann,et al.  Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques , 2008, Nature Reviews Microbiology.

[5]  W. Ludwig,et al.  SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB , 2007, Nucleic acids research.

[6]  Susan M. Huse,et al.  Microbial Population Structures in the Deep Marine Biosphere , 2007, Science.

[7]  Susan M. Huse,et al.  Accuracy and quality of massively parallel DNA pyrosequencing , 2007, Genome Biology.

[8]  J. R. Price,et al.  A novel fluorescence imaging technique combining deconvolution microscopy and spectral analysis for quantitative detection of opportunistic pathogens. , 2006, Journal of microbiological methods.

[9]  Susan M. Huse,et al.  Microbial diversity in the deep sea and the underexplored “rare biosphere” , 2006, Proceedings of the National Academy of Sciences.

[10]  Yuval Garini,et al.  Spectral imaging: Principles and applications , 2006, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[11]  Hideki Harada,et al.  Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes , 2006, Applied and Environmental Microbiology.

[12]  D. White,et al.  New methodology for viability testing in environmental samples. , 2006, Molecular and cellular probes.

[13]  O. Hoegh‐Guldberg,et al.  Fluorescence In Situ Hybridization and Spectral Imaging of Coral-Associated Bacterial Communities , 2006, Applied and Environmental Microbiology.

[14]  Michael Wagner,et al.  daime, a novel image analysis program for microbial ecology and biofilm research. , 2006, Environmental microbiology.

[15]  J. Handelsman,et al.  Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness , 2005, Applied and Environmental Microbiology.

[16]  James R. Cole,et al.  The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis , 2004, Nucleic Acids Res..

[17]  D. Noguera,et al.  Mechanistic Approach to the Problem of Hybridization Efficiency in Fluorescent In Situ Hybridization , 2004, Applied and Environmental Microbiology.

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

[19]  Yasushi Hiraoka,et al.  Multispectral imaging fluorescence microscopy for living cells. , 2002, Cell structure and function.

[20]  M E Dickinson,et al.  Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. , 2001, BioTechniques.

[21]  S. Chisholm,et al.  In Situ Hybridization of Prochlorococcusand Synechococcus (Marine Cyanobacteria) spp. with rRNA-Targeted Peptide Nucleic Acid Probes , 2000, Applied and Environmental Microbiology.

[22]  Poul Nielsen,et al.  LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition , 1998 .

[23]  N. Sugimoto,et al.  Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. , 1995, Biochemistry.

[24]  Peter E. Nielsen,et al.  PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules , 1993, Nature.

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

[26]  D. Lipman,et al.  Improved tools for biological sequence comparison. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[27]  P.K Sahoo,et al.  A survey of thresholding techniques , 1988, Comput. Vis. Graph. Image Process..

[28]  B L Maidak,et al.  The RDP-II (Ribosomal Database Project) , 2001, Nucleic Acids Res..

[29]  S. Chisholm,et al.  In Situ Hybridization of Prochlorococcus and Synechococcus (Marine Cyanobacteria) spp. with rRNA-Targeted Peptide Nucleic Acid Probes , 1999 .

[30]  N. Pace,et al.  The Analysis of Natural Microbial Populations by Ribosomal RNA Sequences , 1986 .