Diversity and phylogenetic affiliations of morphologically conspicuous large filamentous bacteria occurring in the pelagic zones of a broad spectrum of freshwater habitats

Abstract

Filamentous bacteria with a conspicuous morphology were found in the majority of the bacterioplankton samples from a variety of freshwater habitats that were studied. These heterotrophic filaments typically account for < 1 to 11% of the total number of bacteria. The biovolume of this morphotype can exceed 40% of the biovolume for all bacteria. Surprisingly, we found hardly any data on these morphologically conspicuous filaments in the literature. Mixed cultures containing these filamentous bacteria were established by cultivation and isolation experiments with samples from different freshwater lakes. Nearly full-length 16S rRNA gene sequences were obtained from several mixed cultures and environmental samples from habitats in Europe, Africa, China, Australia, and New Zealand. Phylogenetic analysis of the sequences showed that three groups form a single monophyletic cluster, the SOL cluster, in the family Saprospiraceae. We developed a set of six nested probes for fluorescence in situ hybridization. Of the six probes, one probe was specific for Haliscomenobacter hydrossis, three probes were specific for the three subclusters (each probe was specific for one subcluster), one probe was specific for the entire SOL cluster, and another probe targeted almost the entire Saprospiraceae family. Specific hybridization of environmental samples and enrichments showed that the members of the three subclusters exhibited the same filamentous morphology. So far, using the subcluster-specific probes, we have not been able to detect any bacteria with a differing morphology. We conclude that the SOL cluster bacteria are an integral part of bacterioplankton in many freshwater habitats. They potentially account for a large fraction of the total bacterial biomass but have been underrepresented in molecular diversity studies so far.

FIG. 1.

Microscopic image of a DAPI-stained bacterioplankton sample from Lake Victoria (31 May 2001) presenting a filament of the SOL morphotype. These filaments always possess blunt ends, and flagella were never observed. These characteristics together with the unbowed rigid habit of the filaments allows for a reliable discrimination of members of the SOL morphotype against other filamentous bacteria by standard microscopic techniques. The upper arrow marks the filament with yellowish granule. The lower arrow marks a sheath covering the filament. A similar sheath is also known to cover the wild-type cells of H. hydrossis. Magnification, ×1,250.

FIG. 2.

Contribution of SOL morphotype bacteria to the number and biovolume of all bacteria in 17 samples from 16 different freshwater habitats. Selected samples represent a broad range of ecologically different habitat types.

FIG. 3.

Phylogenetic tree showing the affiliation of new bacteria of the Saprospiraceae family (in bold type) from mixed cultures and environmental samples. The neighbor-joining tree was calculated with almost full-length 16S rRNA gene sequences. Seven shorter sequences (765 to 1,165 nucleotides) (indicated by a small solid circle) were added by the ARB parsimony method without changing the tree topology. H. hydrossis sequence AJ784892 is the resequenced sequence. Bootstrap values (1,000 iterations) of >50% are given. Brackets indicate the specificity of FISH probes (probe names in italic type). Probe SAP-309 targets all SOL cluster bacteria and >80% of the remaining sequences within the Saprospiraceae family.

FIG. 4.

Pairs of microscopic images of the same microscopic field with UV excitation (DAPI stained; left column) and green excitation (hybridized with Cy3-labeled oligonucleotide probes; right column). Probes SAP-309 (A), SOL-852 (B), HAL-844 (C), and LD2-1261 (D) were used. All coccoid signals on the right-hand images arise from phototrophic organisms (autofluorescent signals). All photomicrographs were taken at a magnification of ×1,250. The scale bar is valid for all microscopic images.