Specific detection, isolation, and characterization of selected, previously uncultured members of the freshwater bacterioplankton community

Abstract

High-throughput cultivation was combined with rapid and group-specific phylogenetic fingerprinting in order to recover representatives of three freshwater bacterioplankton communities. A total of 570 bacterial cultures were obtained by employing the most probable number and MicroDrop techniques. The majority of the cultured bacteria were closely related to previously uncultured bacteria and grouped with the alpha-Proteobacteria, beta-Proteobacteria, Actinobacteria, Firmicutes, or Flavobacteria-Cytophaga lineage. Correspondingly, the natural bacterioplankton community was analyzed by high-resolution phylogenetic fingerprinting of these five bacterial lineages. 16S rRNA gene fragments were generated for each lineage and subsequently separated by denaturing gradient gel electrophoresis. By the combination of five group-specific PCR protocols, the total number of 16S rRNA gene fingerprints generated from the natural communities was increased sixfold compared to conventional (eubacterial) fingerprinting. Four of the environmental alpha-Proteobacteria 16S rRNA gene sequences obtained from the natural community were found to be identical to those of bacterial isolates. One of these phylotypes was detected in 14 different cultures and hence represented the most frequently cultured bacterium. Three of these 14 strains were characterized in detail. Their complete 16S rRNA gene sequences showed only 93% similarity to that of Sandaracinobacter sibiricus, the closest relative described so far. The novel phylotype of bacterium is a strict aerobe capable of using numerous organic carbon substrates and contains bacteriochlorophyll a bound to two different photosynthetic light-harvesting complexes. Dot blot hybridization revealed that the strains occur in lakes of different trophic status and constitute up to 2% of the microbial community.

FIG. 1.

16S rRNA gene fragments of five different phylogenetic groups of planktonic bacteria separated by DGGE. A negative image of a SybrGold-stained gel is shown. The letters s, w, and z denote samples from Starnberger See, Walchensee, and Zwischenahner Meer, respectively. The letters j and o denote samples obtained in July and October 2001, respectively. ST, standard. Numbers indicate DNA bands that were excised and sequenced. The same numbers mark positions of identical sequences. The percentages on the left margin give the concentrations of denaturant.

FIG. 2.

Maximum likelihood phylogenetic trees calculated for 16S rRNA gene sequences of α-Proteobacteria obtained in this study (given in boldface) and those of their closest relatives. Sequences were obtained from Starnberger See (s), Walchensee (w), and Zwischenahner Meer (z) and in July (j) or October (o) 2001. Sequences of cultured bacteria are marked “CULTURE”; sequences of the three strains investigated in detail are marked “STRAIN.” Sequences from bacteria cultured in MPN series are marked M. “ENVSEQ” denotes 16S rRNA gene sequences recovered by culture-independent DGGE fingerprinting; the numbers of these environmental sequences refer to those in Fig. 1. Environmental sequences and sequences of cultured bacteria that are identical are shaded in gray.

FIG. 3.

Quantification of genomic DNAs of strains so36, so42, and wo26 in the natural bacterioplankton communities by dot blot hybridization. For nomenclature of strains, see the legend to Fig. 1. Different concentrations of genomic DNA of strain wo26 served as standards. DNA of Sphingomonas cloacae was used as a control for specificity. The values below the dots indicate the concentrations of genomic DNA employed (in ng μl⁻¹).

FIG. 4.

(A) Phase-contrast photomicrograph of cells of isolate so42. Bar, 5 μm. (B) Absorption spectra of whole cells (solid line) and acetone extracts (dashed line) of isolate so42. The inset depicts long-wavelength absorption maxima of photosynthetic complexes at 800 and 865 nm and shoulder at 837 nm.

FIG. 5.

Effects of the peptone and the yeast extract on growth rates and lag phases of three α-Proteobacteria bacterioplankton isolates, so36, so42, and wo26, grown in synthetic freshwater mineral media. The error bars represent 1 standard deviation.