Open AccessA new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems
Author(s) -
Bernard Laetitia,
Courties Claude,
Duperray Christophe,
Schäfer Hendrik,
Muyzer Gerard,
Lebaron Philippe
Publication year - 2001
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/1097-0320(20010401)43:4<314::aid-cyto1064>3.0.co;2-h
Subject(s) - biology , temperature gradient gel electrophoresis , phylotype , bacteria , flow cytometry , polymerase chain reaction , 16s ribosomal rna , microbiology and biotechnology , genetic diversity , gene , genetics , population , demography , sociology
Background Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. Methods Total bacteria were determined by SYBR‐II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate‐responsive cells (i.e., DVC + cells) were distinguished from nonresponsive cells (i.e., DVC ‐ cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)‐amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. Results The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell‐sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. Conclusion This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible. Cytometry 43:314–321, 2001. © 2001 Wiley‐Liss, Inc.