16S rRNA gene-based phylogenetic microarray for simultaneous identification of members of the genus Burkholderia.

For cultivation-independent and highly parallel analysis of members of the genus Burkholderia, an oligonucleotide microarray (phylochip) consisting of 131 hierarchically nested 16S rRNA gene-targeted oligonucleotide probes was developed. A novel primer pair was designed for selective amplification of a 1.3 kb 16S rRNA gene fragment of Burkholderia species prior to microarray analysis. The diagnostic performance of the microarray for identification and differentiation of Burkholderia species was tested with 44 reference strains of the genera Burkholderia, Pandoraea, Ralstonia and Limnobacter. Hybridization patterns based on presence/absence of probe signals were interpreted semi-automatically using the novel likelihood-based strategy of the web-tool Phylo- Detect. Eighty-eight per cent of the reference strains were correctly identified at the species level. The evaluated microarray was applied to investigate shifts in the Burkholderia community structure in acidic forest soil upon addition of cadmium, a condition that selected for Burkholderia species. The microarray results were in agreement with those obtained from phylogenetic analysis of Burkholderia 16S rRNA gene sequences recovered from the same cadmiumcontaminated soil, demonstrating the value of the Burkholderia phylochip for determinative and environmental studies.

PhyloDetect: a likelihood-based strategy for detecting microorganisms with diagnostic microarrays.

MOTIVATION: Detection and identification of microbes using diagnostic arrays is still subject of ongoing research. Existing significance-based algorithms consider an organism detected even if a significant number of the microarray probes that match the organism are called absent in a hybridization. Further, they do generate redundant results if the target organisms show high sequence similarity and the microarray probes cannot discriminate all of them. RESULTS: We propose a new analysis strategy that considers organism similarities and calls organisms only present if the probes that match the organism but are absent in a hybridization can be explained by random events. In our strategy, we.rst identify the groups of target organisms that are actually distinguishable by the array. Subsequently, these organism groups are placed in a hierarchical tree such that groups matching only less specific probes are closer to the tree root, and groups that are discriminated only by few probes are close to each other. Finally, we compute for each group a likelihood score that is based on a hypothesis test with the null hypothesis that the group was actually present in the hybridized sample. We have validated our strategy using datasets from two different array types and implemented it as an easy-to-use web application. AVAILABILITY: http://www.fgcz.ethz.ch/PhyloDetect. SUPPLEMENTARY INFORMATION: Example data is available at http://www.fgcz.ethz.ch/PhyloDetect.

High specificity but contrasting biodiversity of Sphagnum-associated bacterial and plant communities in bog ecosystems independent of the geographical region.

Mosses represent ecological niches that harbor a hitherto largely uncharacterized microbial diversity. To investigate which factors affect the biodiversity of bryophyte-associated bacteria, we analyzed the bacterial communities associated with two moss species, which exhibit different ecological behaviors and importance in bog ecosystems, Sphagnum magellanicum and Sphagnum fallax, from six temperate and boreal bogs in Germany and Norway. Furthermore, their surrounding plant communities were studied. Molecular analysis of bacterial communities was determined by single-strand conformation polymorphism (SSCP) analysis using eubacterial and genus-specific primers for the dominant genera Burkholderia and Serratia as well as by sequence analysis of a Burkholderia 16S rRNA gene clone library. Plant communities were analyzed by monitoring the abundance and composition of bryophyte and vascular plant species, and by determining ecological indicator values. Interestingly, we found a high degree of host specificity for associated bacterial and plant communities of both Sphagnum species independent of the geographical region. Calculation of diversity indices on the basis of SSCP gels showed that the S. fallax-associated communities displayed a statistically significant higher degree of diversity than those associated with S. magellanicum. In contrast, analyses of plant communities of Sphagnum-specific habitats resulted in a higher diversity of S. magellanicum-specific habitats for all six sites. The higher content of nutrients in the S. fallax-associated ecosystems can explain higher diversity of microorganisms.

Investigations of the structure and function of bacterial communities associated with Sphagnum mosses.

High acidity, low temperature and extremely low concentration of nutrients form Sphagnum bogs into extreme habitats for organisms. Little is known about the bacteria associated with living Sphagnum plantlets, especially about their function for the host. Therefore, we analysed the endo- and ectophytic bacterial populations associated with two widely distributed Sphagnum species, Sphagnum magellanicum and Sphagnum fallax, by a multiphasic approach. The screening of 1222 isolates for antagonistic activity resulted in 326 active isolates. The bacterial communities harboured a high proportion of antifungal (26%) but a low proportion of antibacterial isolates (0.4%). Members of the genus Burkholderia (38%) were found to be the most dominant group of antagonistic bacteria. The finding that a large proportion (89%) of the antagonistic bacteria produced antifungal compounds may provide an explanation for the well-known antimicrobial activity of certain Sphagnum species. The secondary metabolites of the Sphagnum species themselves were analysed by HPLC-PDA. The different spectra of detected compounds may not only explain the antifungal activity but also the species specificity of the microbial communities. The latter was analysed using cultivation-independent single-stranded conformation polymorphism (SSCP) analysis. Using Burkholderia-specific primers we found a high diversity of Burkholderia isolates in the endophytic and ectophytic habitats of Sphagnum. Furthermore, a high diversity of nitrogen-fixing bacteria was detected by using nifH-specific primers, especially inside Sphagnum mosses. In conclusion, this study provides evidence that both Sphagnum species were colonized by characteristic bacterial populations, which appear to be important for pathogen defence and nitrogen fixation.

Burkholderia bryophila sp. nov. and Burkholderia megapolitana sp. nov., moss-associated species with antifungal and plant-growth-promoting properties.

A polyphasic taxonomic study including DNA-DNA reassociation experiments and an extensive biochemical characterization was performed on 14 Burkholderia isolates from moss gametophytes of nutrient-poor plant communities on the southern Baltic Sea coast in northern Germany. The strains were classified within two novel species, for which the names Burkholderia bryophila sp. nov. and Burkholderia megapolitana sp. nov. are proposed. The former species also includes isolates from grassland and agricultural soil collected in previous studies. Strains Burkholderia bryophila 1S18(T) (=LMG 23644(T) =CCUG 52993(T)) and Burkholderia megapolitana A3(T) (=LMG 23650(T) =CCUG 53006(T)) are the proposed type strains. They were isolated from Sphagnum rubellum and Aulacomnium palustre, respectively, growing in the 'Ribnitzer Grosses Moor' nature reserve (Mecklenburg-Pommern, Germany). All moss isolates of both novel species showed antifungal activity against phytopathogens as well as plant-growth-promoting properties.