• Composition and dynamics of bacterial communities of a drinking water supply system as assessed by RNA- and DNA-based 16S rRNA gene fingerprinting.

      Eichler, Stefan; Christen, Richard; Höltje, Claudia; Westphal, Petra; Bötel, Julia; Brettar, Ingrid; Mehling, Arndt; Höfle, Manfred G; Department of Environmental Microbiology, GBF-German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. (2006-03)
      Bacterial community dynamics of a whole drinking water supply system (DWSS) were studied from source to tap. Raw water for this DWSS is provided by two reservoirs with different water characteristics in the Harz mountains of Northern Germany. Samples were taken after different steps of treatment of raw water (i.e., flocculation, sand filtration, and chlorination) and at different points along the supply system to the tap. RNA and DNA were extracted from the sampled water. The 16S rRNA or its genes were partially amplified by reverse transcription-PCR or PCR and analyzed by single-strand conformation polymorphism community fingerprints. The bacterial community structures of the raw water samples from the two reservoirs were very different, but no major changes of these structures occurred after flocculation and sand filtration. Chlorination of the processed raw water strongly affected bacterial community structure, as reflected by the RNA-based fingerprints. This effect was less pronounced for the DNA-based fingerprints. After chlorination, the bacterial community remained rather constant from the storage containers to the tap. Furthermore, the community structure of the tap water did not change substantially for several months. Community composition was assessed by sequencing of abundant bands and phylogenetic analysis of the sequences obtained. The taxonomic compositions of the bacterial communities from both reservoirs were very different at the species level due to their different limnologies. On the other hand, major taxonomic groups, well known to occur in freshwater, such as Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes, were found in both reservoirs. Significant differences in the detection of the major groups were observed between DNA-based and RNA-based fingerprints irrespective of the reservoir. Chlorination of the drinking water seemed to promote growth of nitrifying bacteria. Detailed analysis of the community dynamics of the whole DWSS revealed a significant influence of both source waters on the overall composition of the drinking water microflora and demonstrated the relevance of the raw water microflora for the drinking water microflora provided to the end user.
    • Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea.

      Labrenz, Matthias; Brettar, Ingrid; Christen, Richard; Flavier, Sebastien; Bötel, Julia; Höfle, Manfred G; Department of Environmental Microbiology, GBF-German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany. (2004-08)
      We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 x 10(3) to 4.4 x 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 x 10(1) to 2.2 x10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.
    • Evaluation of a microarray-hybridization based method applicable for discovery of single nucleotide polymorphisms (SNPs) in the Pseudomonas aeruginosa genome.

      Dötsch, Andreas; Pommerenke, Claudia; Bredenbruch, Florian; Geffers, Robert; Häussler, Susanne; Helmholtz Centre for Infection Research, Braunschweig, Germany. andreas.doetsch@helmholtz-hzi.de (2009)
      BACKGROUND: Whole genome sequencing techniques have added a new dimension to studies on bacterial adaptation, evolution and diversity in chronic infections. By using this powerful approach it was demonstrated that Pseudomonas aeruginosa undergoes intense genetic adaptation processes, crucial in the development of persistent disease. The challenge ahead is to identify universal infection relevant adaptive bacterial traits as potential targets for the development of alternative treatment strategies. RESULTS: We developed a microarray-based method applicable for discovery of single nucleotide polymorphisms (SNPs) in P. aeruginosa as an easy and economical alternative to whole genome sequencing. About 50% of all SNPs theoretically covered by the array could be detected in a comparative hybridization of PAO1 and PA14 genomes at high specificity (> 0.996). Variations larger than SNPs were detected at much higher sensitivities, reaching nearly 100% for genetic differences affecting multiple consecutive probe oligonucleotides. The detailed comparison of the in silico alignment with experimental hybridization data lead to the identification of various factors influencing sensitivity and specificity in SNP detection and to the identification of strain specific features such as a large deletion within the PA4684 and PA4685 genes in the Washington Genome Center PAO1. CONCLUSION: The application of the genome array as a tool to identify adaptive mutations, to depict genome organizations, and to identify global regulons by the "ChIP-on-chip" technique will expand our knowledge on P. aeruginosa adaptation, evolution and regulatory mechanisms of persistence on a global scale and thus advance the development of effective therapies to overcome persistent disease.
    • Ultrastructural and electron energy-loss spectroscopic analysis of an extracellular filamentous matrix of an environmental bacterial isolate.

      Böckelmann, Uta; Lünsdorf, Heinrich; Szewzyk, Ulrich; Department of Environmental Microbiology, Technical University Berlin, Franklin Str. 29, 10587 Berlin, Germany. uta.boeckelmann@tu-berlin.de (2007-09)
      Strain F8, a bacterial isolate from 'river snow', was found to produce extracellular fibres in the form of a filamentous network. These extracellular filaments, which were previously shown to be composed of DNA, have been studied for the first time by ultrastructural and electron energy-loss spectroscopy in the present work. 'Whole mount' preparations of strain F8 indicate these polymers are ultrastructurally homogeneous and form a network of elemental filaments, which have a width of 1.8-2.0 nm. When incubated at pH 3.5 with colloidal cationic ThO(2) tracers they become intensely stained (electron dense), affording direct evidence that the fibres are negatively charged and thus acidic chemically. Elemental analysis of the extracellular filaments by Energy-filtered Transmission Electron Microscopy revealed phosphorus to be the main element present and, because pretreatment of F8 cells with DNase prevented thorium labelling, the fibres must be composed of extracellular DNA (eDNA). Neither ultrathin sections nor 'whole mount negative stain' caused DNA release by general cell lysis. Additionally, cells infected with phages were never observed in ultrathin sections and phage particles were never detected in whole mount samples, which rules out the possibility of phages being directly involved in eDNA release.