The design of 16S ribosomal RNA-targeted oligonucleotide probes of detection of natural populations of autotrophic ammonia-oxidising bacteria

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Nearly identical 16S rRNA sequences recovered from lakes in North America and Europe indicate the existence of clades of globally distributed freshwater bacteria

We compared bacterial 16S ribosomal RNA gene sequences recovered from Lake Loosdrecht, the Netherlands, to reported sequences from lakes in Alaska and New York State. In each of the three lake systems, which differ in pH and trophic state, some sequence types were found without related sequences (sequence identity <90%) in the data sets from the other two systems. Two sequences in the Actinomycetes and Verrucomicrobia radiations were more closely related to sequences from the New York: lakes data set than to any other sequence in the global databases. However, the most striking similarities were found in the subdivisions alpha and beta of the Proteobacteria. In these subdivisions three different clusters of highly related bacteria were identified (97-100% sequence identity) that were represented in all three lake regions. The clusters contained no members other than freshwater bacteria. One cluster falls within a monophyletic aquatic supergroup that apparently diverged early in evolution into an exclusive freshwater cluster and an exclusive marine cluster, the so-called SAR11 cluster. The detection of these three bacterial clades in lakes distinguished by geographic distance as well as physical and chemical diversity suggests that these organisms are dispersed globally and that they possess unique functional capabilities enabling successful competition in a wide range of freshwater environments. [KEYWORDS: global distribution; freshwater bacteria; ribosomal RNA; SAR11 cluster; Proteobacteria; dispersal; bacterioplankton; community analysis; lakes Gradient gel-electrophoresis; ribosomal-rna genes; phylogenetic diversity; euplotes-aediculatus; sar11 cluster; arctic lake; endosymbiont; populations; communities; alignment]

13C incorporation into DNA as a means of identifying the active components of ammonia-oxidizer populations

Aims: To identify active CO2-assimilating species of ammonia-oxidizing bacteria in fresh water sediment. Methods and Results: Enrichment cultures were incubated in the presence of 13C labelled CO2, and 13C-DNA successfully resolved from 12C-DNA by caesium chloride density gradient ultracentrifugation of DNA extracts. Ammonia-oxidizer DNA recovered from these gradients was amplified and characterised by Temporal Temperature Gradient Gel Electrophoresis (TTGE), with confirmatory sequence analysis to identify the metabolically active components of the population. Conclusions: The 12C-DNA fraction was dominated by nitrosospiras, in contrast to the 13C-DNA fraction which was largely nitrosomonad DNA, in support of the hypothesis that nitrosomonads out-compete nitrosospiras in laboratory culture. Significance and Impact of the Study: The use of stable isotype incorporation into ammonia-oxidizer DNA could therefore circumvent the problems associated with RNA detection to identify metabolically active species in situ

Endosymbiosis In Statu Nascendi: Close Phylogenetic Relationship Between Obligately Endosymbiotic and Obligately Free-Living Polynucleobacter Strains (Betaproteobacteria)

Bacterial strains affiliated to the phylogenetically shallow subcluster C (PnecC) of the 28 Polynucleobacter cluster, which is characterized by a minimal 16S rRNA gene sequence similarity of approx. 98.5 percent, have been reported to occur as obligate endosymbionts of 30 ciliates (Euplotes spp.), as well as to occur as free-living cells in the pelagic zone of freshwater habitats. We investigated if these two groups of closely related bacteria represent 32 strains fundamentally differing in lifestyle, or if they simply represent different stages of a facultative endosymbiotic lifestyle. The phylogenetic analysis of 16S rRNA gene and 16S34 23S ITS sequences of five endosymbiont strains from two different Euplotes species and 40 pure culture strains demonstrated host-species-specific clustering of the endosymbiont 36 sequences within the PnecC subcluster. The sequences of the endosymbionts showed characteristics indicating an obligate endosymbiotic lifestyle. Cultivation experiments 38 revealed fundamental differences in physiological adaptations, and determination of the genome sizes indicated a slight size reduction in endosymbiotic strains. We conclude that the 40 two groups of PnecC bacteria represent obligately free-living and obligately endosymbiotic strains, respectively, and do not represent different stages of the same complex lifecycle. 42 These closely related strains occupy completely separated ecological niches. To our best knowledge, this is the closest phylogenetic relationship between obligate endosymbionts and 44 obligately free-living bacteria ever revealed