Isolation of novel ultramicrobacteria classified as actinobacteria from five freshwater habitats in Europe and Asia

Abstract

We describe the first freshwater members of the class Actinobacteria that have been isolated. Nine ultramicro-size (<0.1 microm(3)) strains were isolated from five freshwater habitats in Europe and Asia. These habitats represent a broad spectrum of ecosystems, ranging from deep oligotrophic lakes to shallow hypertrophic lakes. Even when the isolated strains were grown in very rich media, the cell size was <0.1 microm(3) and was indistinguishable from the cell sizes of bacteria belonging to the smaller size classes of natural lake bacterioplankton. Hybridization of the isolates with oligonucleotide probes and phylogenetic analysis of the 16S rRNA gene sequences of the isolated strains revealed that they are affiliated with the class Actinobacteria and the family Microbacteriaceae. The previously described species with the highest levels of sequence similarity are Clavibacter michiganensis and Rathayibacter tritici, two phytopathogens of terrestrial plants. The 16S rRNA gene sequences of the nine isolates examined are more closely related to cloned sequences from uncultured freshwater bacteria than to the sequences of any previously isolated bacteria. The nine ultramicrobacteria isolated form, together with several uncultured bacteria, a diverse phylogenetic cluster (Luna cluster) consisting exclusively of freshwater bacteria. Isolates obtained from lakes that are ecologically different and geographically separated by great distances possess identical 16S rRNA gene sequences but have clearly different ecophysiological and phenotypic traits. Predator-prey experiments demonstrated that at least one of the ultramicro-size isolates is protected against predation by the bacterivorous nanoflagellate Ochromonas sp. strain DS.

FIG. 1.

(a and b) Survey views of ultrathin-sectioned (a) and shadow-cast (b) cells of strain MWH-Ta1. The small arrowheads indicate sites of binary fission. The large arrowhead in panel b indicates the direction of shadow casting. (c) Detailed view of a cross section of the cell wall. The cytoplasm (cp), the cytoplasmic membrane (cm), and the outer surface-like layer (sl) are indicated. The area enclosed by a box represents the measured area, and an averaged line scan density profile of this area is shown in panel d. The density features of the cell wall are shown in the area enclosed by a box in panel d. pg, peptidoglycan.

FIG. 2.

Bacterial size distribution of bacterioplankton in Lake Mondsee (including selenoid cells) (A), of ultramicro-size selenoid bacteria in Lake Mondsee (B), of isolate MWH-Mo1 grown in 9-g liter⁻¹ NSY medium (C), and of isolate MWH-Ta3 grown in 9-g liter⁻¹ NSY medium (D).

FIG. 3.

Neighbor-joining tree based on homologous 1,439-nucleotide sequence stretches of the 16S rRNA gene. Shorter partial sequences which were added by parsimony to the existing neighbor-joining tree are indicated by asterisks. Only the bootstrap values (percentage, 1,000 replicates) relevant for the Luna cluster are shown. The tree was rooted by using the sequence of Cellulomonas cellasea (Cellulomonadaceae). Except for the C. cellasea, clone GCP1, and “Brevibacterium helvolum” sequences, all the reference sequences are sequences of members of the family Microbacteriaceae. The positions of the Luna cluster and its two subclusters (Luna-1 and Luna-2) are indicated by brackets. The Luna cluster was named after the ecosystem from which the first isolate was obtained (Lake Mondsee [German Mond = English moon = Latin luna]). Bar = 1 nucleotide substitution per 10 nucleotides. DGGE, denaturing gradient gel electrophoresis.

FIG. 4.

Predation of strain MWH-Mo1 by Ochromonas sp. in experiment 2. See Table 3. The development of the flagellate and the MWH-Mo1 population in experiment 2 (based on triplicate preparations) and the development of Ochromonas sp. strain DS and heat-killed Pseudomonas sp. in control 2a− are shown. Data from the other three controls (controls 2a+, 2b−, and 2b+) are not shown. In experiment 2 the three preparations were initially inoculated with isolate MWH-Mo1, and 20 days after the start of the experiment they were inoculated with heat-killed Pseudomonas sp. cells. For experiment 2 the average cell numbers and standard deviations (error bars) for the three parallel preparations are shown.