High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates

Abstract

Microbial diversity studies based on the cloning and sequencing of DNA from nature support the conclusion that only a fraction of the microbial diversity is currently represented in culture collections. Out of over 40 known prokaryotic phyla, only half have cultured representatives. In an effort to culture the uncultured phylotypes from oligotrophic marine ecosystems, we developed high-throughput culturing procedures that utilize the concept of extinction culturing to isolate cultures in small volumes of low-nutrient media. In these experiments, marine bacteria were isolated and cultivated at in situ substrate concentrations-typically 3 orders of magnitude less than common laboratory media. Microtiter plates and a newly developed procedure for making cell arrays were employed to raise the throughput rate and lower detection sensitivity, permitting cell enumeration from 200-microl aliquots of cultures with densities as low as 10(3) cells/ml. Approximately 2,500 extinction cultures from 11 separate samplings of marine bacterioplankton were screened over the course of 3 years. Up to 14% of the cells collected from coastal seawater were cultured by this method, which was 14- to 1,400-fold higher than the numbers obtained by traditional microbiological culturing techniques. Among the microorganisms cultured were four unique cell lineages that belong to previously uncultured or undescribed marine Proteobacteria clades known from environmental gene cloning studies. These cultures are related to the clades SAR11 (alpha subclass), OM43 (beta subclass), SAR92 (gamma subclass), and OM60/OM241 (gamma subclass). This method proved successful for the cultivation of previously uncultured marine bacterioplankton that have consistently been found in marine clone libraries.

FIG. 1.

Flow chart of HTC procedures. DMSO, dimethyl sulfoxide.

FIG. 2.

Fluorescence microscopy images of several of the novel isolates. The cells were stained with DAPI. Size bars, 1 μm.

FIG.3.

Neighbor-joining trees showing phylogenetic relationships among the 16S rRNA genes of HTCC isolates compared to those of representative species and environmental clones. Scale bars indicate 0.1 change per nucleotide. Bootstrap values below 50 are not shown. Short sequences (approximately 600 bp) of HTCC isolates were added to the trees by using the parsimony insertion tool in ARB. HTCC230 and HTCC234 are close to full length and were put in the original tree. In parentheses next to HTCC isolates is the number of total cultures from the subset of 47 identified cultures that are included in that clade. However, not all of the HTCC sequences used in the tree are part of the subset of 47 identified cultures. (A) α-Proteobacteria phylogenetic tree. β- and γ-Proteobacteria were used to root the tree; 1,051 characters were used to infer the tree. (B) β-Proteobacteria phylogenetic tree. γ-Proteobacteria isolates were used to root the tree; 789 characters were used to infer the tree. (C) γ-Proteobacteria phylogenetic tree. β-Proteobacteria isolates were used to root the tree; 1,042 characters were used to infer the tree.