• shift from acetoclastic to H2-dependent methanogenesis in a west Siberian peat bog at low pH values and isolation of an acidophilic Methanobacterium strain.

      Kotsyurbenko, O R; Friedrich, M W; Simankova, M V; Nozhevnikova, A N; Golyshin, P N; Timmis, K N; Conrad, R; Institut für Mikrobiologie, Carolo-Wilhelmina Technische Universität zu Braunschweig, Biozentrum, Braunschweig, Germany. olk@helmholtz-hzi.de (2007-04)
      Methane production and archaeal community composition were studied in samples from an acidic peat bog incubated at different temperatures and pH values. H(2)-dependent methanogenesis increased strongly at the lowest pH, 3.8, and Methanobacteriaceae became important except for Methanomicrobiaceae and Methanosarcinaceae. An acidophilic and psychrotolerant Methanobacterium sp. was isolated using H(2)-plus-CO(2)-supplemented medium at pH 4.5.
    • Traditional cattle manure application determines abundance, diversity and activity of methanogenic Archaea in arable European soil.

      Gattinger, Andreas; Höfle, Manfred G; Schloter, Michael; Embacher, Arndt; Böhme, Frank; Munch, Jean Charles; Labrenz, Matthias; Institute of Soil Ecology, GSF-National Research Center for Environment and Health, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany. (2007-03)
      Based on lipid analyses, 16S rRNA/rRNA gene single-strand conformation polymorphism fingerprints and methane flux measurements, influences of the fertilization regime on abundance and diversity of archaeal communities were investigated in soil samples from the long-term (103 years) field trial in Bad Lauchstädt, Germany. The investigated plots followed a gradient of increasing fertilization beginning at no fertilization and ending at the 'cattle manure' itself. The archaeal phospholipid etherlipid (PLEL) concentration was used as an indicator for archaeal biomass and increased with the gradient of increasing fertilization, whereby the concentrations determined for organically fertilized soils were well above previously reported values. Methane emission, although at a low level, were occasionally only observed in organically fertilized soils, whereas the other treatments showed significant methane uptake. Euryarchaeotal organisms were abundant in all investigated samples but 16S rRNA analysis also demonstrated the presence of Crenarchaeota in fertilized soils. Lowest molecular archaeal diversity was found in highest fertilized treatments. Archaea phylogenetically most closely related to cultured methanogens were abundant in these fertilized soils, whereas Archaea with low relatedness to cultured microorganisms dominated in non-fertilized soils. Relatives of Methanoculleus spp. were found almost exclusively in organically fertilized soils or cattle manure. Methanosarcina-related microorganisms were detected in all soils as well as in the cattle manure, but soils with highest organic application rate were specifically dominated by a close phylogenetic relative of Methanosarcina thermophila. Our findings suggest that regular application of cattle manure increased archaeal biomass, but reduced archaeal diversity and selected for methanogenic Methanoculleus and Methanosarcina strains, leading to the circumstance that high organic fertilized soils did not function as a methane sink at the investigated site anymore.
    • Dynamics of reductive genome evolution in mitochondria and obligate intracellular microbes.

      Khachane, Amit N; Timmis, Kenneth N; Martins dos Santos, Vítor A P; Department of Environmental Microbiology, Helmholtz Center for Infection Research, Braunschweig, Germany. (2007-02)
      Reductive evolution in mitochondria and obligate intracellular microbes has led to a significant reduction in their genome size and guanine plus cytosine content (GC). We show that genome shrinkage during reductive evolution in prokaryotes follows an exponential decay pattern and provide a method to predict the extent of this decay on an evolutionary timescale. We validated predictions by comparison with estimated extents of genome reduction known to have occurred in mitochondria and Buchnera aphidicola, through comparative genomics and by drawing on available fossil evidences. The model shows how the mitochondrial ancestor would have quickly shed most of its genome, shortly after its incorporation into the protoeukaryotic cell and prior to codivergence subsequent to the split of eukaryotic lineages. It also predicts that the primary rickettsial parasitic event would have occurred between 180 and 425 million years ago (MYA), an event of relatively recent evolutionary origin considering the fact that Rickettsia and mitochondria evolved from a common alphaproteobacterial ancestor. This suggests that the symbiotic events of Rickettsia and mitochondria originated at different time points. Moreover, our model results predict that the ancestor of Wigglesworthia glossinidia brevipalpis, dated around the time of origin of its symbiotic association with the tsetse fly (50-100 MYA), was likely to have been an endosymbiont itself, thus supporting an earlier proposition that Wigglesworthia, which is currently a maternally inherited primary endosymbiont, evolved from a secondary endosymbiont.
    • Analysis of storage lipid accumulation in Alcanivorax borkumensis: Evidence for alternative triacylglycerol biosynthesis routes in bacteria.

      Kalscheuer, Rainer; Stöveken, Tim; Malkus, Ursula; Reichelt, Rudolf; Golyshin, Peter N; Sabirova, Julia S; Ferrer, Manuel; Timmis, Kenneth N; Steinbüchel, Alexander; Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Corrensstrasse 3, D-48149 Münster, Germany. (2007-02)
      Marine hydrocarbonoclastic bacteria, like Alcanivorax borkumensis, play a globally important role in bioremediation of petroleum oil contamination in marine ecosystems. Accumulation of storage lipids, serving as endogenous carbon and energy sources during starvation periods, might be a potential adaptation mechanism for coping with nutrient limitation, which is a frequent stress factor challenging those bacteria in their natural marine habitats. Here we report on the analysis of storage lipid biosynthesis in A. borkumensis strain SK2. Triacylglycerols (TAGs) and wax esters (WEs), but not poly(hydroxyalkanoic acids), are the principal storage lipids present in this and other hydrocarbonoclastic bacterial species. Although so far assumed to be a characteristic restricted to gram-positive actinomycetes, substantial accumulation of TAGs corresponding to a fatty acid content of more than 23% of the cellular dry weight is the first characteristic of large-scale de novo TAG biosynthesis in a gram-negative bacterium. The acyltransferase AtfA1 (ABO_2742) exhibiting wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) activity plays a key role in both TAG and WE biosynthesis, whereas AtfA2 (ABO_1804) was dispensable for storage lipid formation. However, reduced but still substantial residual TAG levels in atfA1 and atfA2 knockout mutants compellingly indicate the existence of a yet unknown WS/DGAT-independent alternative TAG biosynthesis route. Storage lipids of A. borkumensis were enriched in saturated fatty acids and accumulated as insoluble intracytoplasmic inclusions exhibiting great structural variety. Storage lipid accumulation provided only a slight growth advantage during short-term starvation periods but was not required for maintaining viability and long-term persistence during extended starvation phases.
    • Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2.

      Sabirova, Julia S; Ferrer, Manuel; Lünsdorf, Heinrich; Wray, Victor; Kalscheuer, Rainer; Steinbüchel, Alexander; Timmis, Kenneth N; Golyshin, Peter N; Department of Environmental Microbiology, HZI-Helmholtz Center fro Infection Research, Braunschweig, Germany. jsa05@helmholtz-hzi.de (2006-12)
      A novel mutant of the marine oil-degrading bacterium Alcanivorax borkumensis SK2, containing a mini-Tn5 transposon disrupting a "tesB-like" acyl-coenzyme A (CoA) thioesterase gene, was found to hyperproduce polyhydroxyalkanoates (PHA), resulting in the extracellular deposition of this biotechnologically important polymer when grown on alkanes. The tesB-like gene encodes a distinct novel enzyme activity, which acts exclusively on hydroxylated acyl-CoAs and thus represents a hydroxyacyl-CoA-specific thioesterase. Inactivation of this enzyme results in the rechanneling of CoA-activated hydroxylated fatty acids, the cellular intermediates of alkane degradation, towards PHA production. These findings may open up new avenues for the development of simplified biotechnological processes for the production of PHA as a raw material for the production of bioplastics.
    • Proteomic insights into metabolic adaptations in Alcanivorax borkumensis induced by alkane utilization.

      Sabirova, Julia S; Ferrer, Manuel; Regenhardt, Daniela; Timmis, Kenneth N; Golyshin, Peter N; Institute of Microbiology, Technical University of Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany. jsa05@gbf.de (2006-06)
      Alcanivorax borkumensis is a ubiquitous marine petroleum oil-degrading bacterium with an unusual physiology specialized for alkane metabolism. This "hydrocarbonoclastic" bacterium degrades an exceptionally broad range of alkane hydrocarbons but few other substrates. The proteomic analysis presented here reveals metabolic features of the hydrocarbonoclastic lifestyle. Specifically, hexadecane-grown and pyruvate-grown cells differed in the expression of 97 cytoplasmic and membrane-associated proteins whose genes appeared to be components of 46 putative operon structures. Membrane proteins up-regulated in alkane-grown cells included three enzyme systems able to convert alkanes via terminal oxidation to fatty acids, namely, enzymes encoded by the well-known alkB1 gene cluster and two new alkane hydroxylating systems, a P450 cytochrome monooxygenase and a putative flavin-binding monooxygenase, and enzymes mediating beta-oxidation of fatty acids. Cytoplasmic proteins up-regulated in hexadecane-grown cells reflect a central metabolism based on a fatty acid diet, namely, enzymes of the glyoxylate bypass and of the gluconeogenesis pathway, able to provide key metabolic intermediates, like phosphoenolpyruvate, from fatty acids. They also include enzymes for synthesis of riboflavin and of unsaturated fatty acids and cardiolipin, which presumably reflect membrane restructuring required for membranes to adapt to perturbations induced by the massive influx of alkane oxidation enzymes. Ancillary functions up-regulated included the lipoprotein releasing system (Lol), presumably associated with biosurfactant release, and polyhydroxyalkanoate synthesis enzymes associated with carbon storage under conditions of carbon surfeit. The existence of three different alkane-oxidizing systems is consistent with the broad range of oil hydrocarbons degraded by A. borkumensis and its ecological success in oil-contaminated marine habitats.
    • New lineage of filamentous, spore-forming, gram-positive bacteria from soil.

      Cavaletti, Linda; Monciardini, Paolo; Bamonte, Ruggiero; Schumann, Peter; Rohde, Manfred; Sosio, Margherita; Donadio, Stefano; Vicuron Pharmaceuticals, 21040 Gerenzano, Italy. (2006-06)
      A novel bacterial strain that was isolated from an Italian soil and was designated SOSP1-21T forms branched mycelia in solid and liquid media and has a filamentous morphology similar to that of some genera belonging to the Actinobacteria. Electron microscopy showed that this organism has a grape-like appearance, resulting from interlacing of spores originating from sporophoric hyphae. Ten strains that are morphologically related to SOSP1-21T were recovered from soil. Phylogenetic analyses of 16S rRNA gene segments confirmed the relatedness of these strains to SOSP1-21T and indicated that the newly isolated strains form separate clades in a deeply branching lineage. The closest matches for the 16S rRNA sequences of all the strains (around 79% identity) were matches with representatives of the Chloroflexi, although the affiliation with this division was not supported by high bootstrap values. The strains are mesophilic aerobic heterotrophs and are also capable of growing under microaerophilic conditions. They all stain gram positive. Strain SOSP1-21T contains ornithine, alanine, glutamic acid, serine, and glycine as the peptidoglycan amino acids. In addition, an unusual level of C16:1 2OH (30%) was found in the cellular fatty acids. The G+C content of SOSP1-21T genomic DNA is 53.9%, and MK-9(H2) was the only menaquinone detected. All these data suggest that SOSP1-21T and the related strains may constitute a new division of filamentous, spore-forming, gram-positive bacteria. We propose the name Ktedobacter racemifer gen. nov., sp. nov. for strain SOSP1-21T.
    • Proteomic Insights into Metabolic Adaptations in Alcanivorax borkumensis Induced by Alkane Utilization

      Sabirova, Julia S; Ferrer, Manuel; Regenhardt, Daniela; Timmis, Kenneth N.; Golyshin, Peter N. (American Society for Microbiology, 2006-06)
    • The 'pH optimum anomaly' of intracellular enzymes of Ferroplasma acidiphilum.

      Golyshina, Olga V; Golyshin, Peter N; Timmis, Kenneth N; Ferrer, Manuel; Division of Microbiology, GBF--German Research Centre for Biotechnology, Braunschweig, Germany. (2006-03)
      A wide range of microorganisms, the so-called acidophiles, inhabit acidic environments and grow optimally at pH values between 0 and 3. The intracellular pH of these organisms is, however, close to neutrality or slightly acidic. It is to be expected that enzymatic activities dedicated to extracellular functions would be adapted to the prevailing low pH of the environment (0-3), whereas intracellular enzymes would be optimally active at the near-neutral pH of the cytoplasm (4.6-7.0). The genes of several intracellular or cell-bound enzymes, a carboxylesterase and three alpha-glucosidases, from Ferroplasma acidiphilum, a cell wall-lacking acidophilic archaeon with a growth optimum at pH 1.7, were cloned and expressed in Escherichia coli, and their products purified and characterized. The Ferroplasmaalpha-glucosidases exhibited no sequence similarity to known glycosyl hydrolases. All enzymes functioned and were stable in vitro in the pH range 1.7-4.0, and had pH optima much lower than the mean intracellular pH of 5.6. This 'pH optimum anomaly' suggests the existence of yet-undetected cellular compartmentalization providing cytoplasmic pH patchiness and low pH environments for the enzymes we have analysed.
    • 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.
    • Expression of a Temperature-Sensitive Esterase in a Novel Chaperone-Based Escherichia coli Strain

      Ferrer, Manuel; Chernikova, Tatyana N.; Timmis, Kenneth N.; Golyshin, Peter N. (American Society for Microbiology, 2004-08)
    • 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.
    • Metagenome Survey of Biofilms in Drinking-Water Networks

      Schmeisser, C.; Stöckigt, C.; Raasch, C.; Wingender, J.; Timmis, K. N.; Wenderoth, D. F.; Flemming, H.-C.; Liesegang, H.; Schmitz, R. A.; Jaeger, K.-E.; et al. (American Society for Microbiology, 2003-12)
    • Involvement of IHF protein in expression of the Ps promoter of the Pseudomonas putida TOL plasmid.

      Holtel, A; Goldenberg, D; Giladi, H; Oppenheim, A B; Timmis, K N (1995-06)
    • Carbon source-dependent inhibition of xyl operon expression of the Pseudomonas putida TOL plasmid.

      Holtel, A; Marqués, S; Möhler, I; Jakubzik, U; Timmis, K N (1994-03)
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