Isolation of strains belonging to the cosmopolitan Polynucleobacter necessarius cluster from freshwater habitats located in three climatic zones

Abstract

More than 40 bacterial strains belonging to the cosmopolitan Polynucleobacter necessarius cluster (Betaproteobacteria) were isolated from a broad spectrum of freshwater habitats located in three climatic zones. Sequences affiliated with the freshwater P. necessarius cluster are among the most frequently detected in studies on bacterial diversity in freshwater ecosystems. Despite this frequent detection with culture-independent techniques and the cosmopolitan occurrence of members affiliated with this cluster, no isolates have been reported thus far. The isolated strains have been obtained from lakes, ponds, and rivers in central Europe, the People's Republic of China, and East Africa by use of the filtration-acclimatization method. The 16S rRNA gene sequences of the isolates are 98.8 to 100% identical to reference sequences obtained by various authors by use of culture-independent methods. The isolates, aerobic heterotrophs, grew on a wide range of standard complex media and formed visible colonies on agar plates. Thus, the previous lack of isolates cannot be explained by a lack of appropriate media. Most of the isolates possess, under a wide range of culture conditions, very small cells (<0.1 micro m(3)), even when grown in medium containing high concentrations of organic substances. Thus, these strains are obligate ultramicrobacteria. The obtained strains have a C-shaped cell morphology which is very similar to that of recently isolated ultramicrobacterial Luna cluster strains (Actinobacteria) and the SAR11 cluster strains (Alphaproteobacteria).

FIG. 1.

Neighbor-joining tree showing the phylogenetic relationships of isolated and uncultured strains affiliated with the P. necessarius cluster. The tree was calculated with 1,240 nucleotide sequence stretches of the 16S rRNA genes. The tree is based on all publicly available sequences affiliated with the P. necessarius cluster which have sequence lengths of ≥1,292 nucleotides. The length limit has been defined by the maximum sequence length available for representatives of the various subclusters. Four proposed subclusters (A to D) are indicated by brackets. Alcaligenes faecalis (M22508) served as the outgroup (data not shown). R. basilensis is the closest known relative of the P. necessarius cluster bacteria. Bootstrap (1,000 replicates) values of ≥60% are shown. The scale bar indicates 1% estimated sequence divergence.

FIG. 2.

Photomicrograph of DAPI-stained cells of isolate MWH-VicM1 grown in 3-g liter⁻¹ NSY medium.

FIG. 3.

Growth of the isolate MWH-VicM1 in liquid 3-g liter⁻¹ NSY medium at 20°C and cell volumes observed during exponential and stationary stages. Data represent means and standard deviations of results from three replicates. Open circles, optical density (OD); closed squares, cell volume.