Genetic diversity of total, active and culturable marine bacteria in coastal seawater

Abstract

The genetic diversity of marine bacteria from coastal Mediterranean water was analyzed using denaturing gradient gel electrophoresis (DGGE) and comparative sequence analysis of PCR-amplified 16S rRNA genes. The diversity of the whole bacterial assemblage was compared to the diversity of the fraction of actively respiring bacterial cells and of culturable bacteria. Culturable bacteria were isolated on agar plates using 4 different culture media, as well as in filtered autoclaved seawater following dilution to extinction. The cell fractions exhibited varied genetic diversity. High similarity between DGGE patterns obtained from the whole bacterial assemblage and those obtained from the active cell fraction (representing only 3% of total cells) indicated the simultaneous presence of both active and inactive cells within populations corresponding to numerous bacterial phylotypes defined as DGGE bands. Furthermore, an important source of genetic diversity corresponding to viable organisms, detected by culturability on agar media and in dilution culture with unamended seawater, was not detectable by DGGE patterns obtained from total cells. Most of the strains isolated by dilution cultures were different from those isolated on solid agar media. These results suggest that studies on the structure of complex marine bacterial communities do not necessarily reflect the physiological heterogeneity of ecologically important populations and may ignore populations present at low relative abundance that can play a key ecological role.KEY WORDS: Activity · Cell sorting · Culturability · Genetic diversity · Marine bacteria · Oligotrophy Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 23: [1][2][3][4][5][6][7][8][9][10][11] 2000 not yet been cultured. Several media with different compositions have been proposed for isolating new species (Martin & MacLeod 1984, Gonzalez & Moran 1997) and a dilution culture technique has been developed to isolate oligotrophic species, which do not grow on nutrient-rich medium . However, culturability is also a function of the physiological state of a cell, and cells of cultured species may become unculturable under adverse environmental conditions (Schut et al. 1997). Recent studies have demonstrated that, even if the CFU proportion remains very low, cultured species sometimes represent a significant part of the community DNA (Pinhassi et al. 1997). This suggests that non-culturable cells of cultured species may represent a numerically important fraction of natural communities.Different culture-independent methods have been developed to quantify metabolically active bacteria in natural communities (Joux & Lebaron 2000). Among these, the detection of actively respiring cells via cellspecific reduction of the fluorogenic dye 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC; Polyscience Europe) is often considered as one of the most universal methods applicable for complex natural communities. Although this method remains controversial (Thom et ...

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