Culturability and In situ abundance of pelagic bacteria from the North Sea

Abstract

The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.

FIG. 1

Rarefaction curves for the different ARDRA patterns of all of the isolates used in this study. The expected number of ARDRA patterns is plotted versus the number of isolates (○). Rarefaction curves were also calculated for the fraction of gamma Proteobacteria (●). The dotted lines represent 95% confidence intervals.

FIG. 2

Phylogenetic tree based on comparative analysis of 16S rDNA from selected clones and isolates of the alpha and gamma subclasses of Proteobacteria. Brackets indicate probe specificity. Selected sequences from the beta subclass of Proteobacteria were used to root the tree. The bar indicates 10% sequence divergence.

FIG. 3

Epifluorescence micrographs of bacteria in bacterioplankton from the North Sea station Helgoland Roads. Hybridization with CY3-labeled probes (right) and the same microscopic field with UV excitation (DAPI staining, left). Panels: A, probe ALT1413 (18.08.98); B, probe G V (12.09.97); C, probe SAR86-1249 (20.08.98); D, probe G Rb (12.09.97). Scale bars, 5 μm.