• Acidiplasma aeolicum gen. nov., sp. nov., a euryarchaeon of the family Ferroplasmaceae isolated from a hydrothermal pool, and transfer of Ferroplasma cupricumulans to Acidiplasma cupricumulans comb. nov.

      Golyshina, Olga V; Yakimov, Michail M; Lünsdorf, Heinrich; Ferrer, Manuel; Nimtz, Manfred; Timmis, Kenneth N; Wray, Victor; Tindall, Brian J; Golyshin, Peter N; Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany. p.golyshin@bangor.ac.uk (2009-11)
      A novel acidophilic, cell-wall-less archaeon, strain V(T), was isolated from a hydrothermal pool on Vulcano Island, Italy. The morphology of cells was observed to vary from pleomorphic to coccoid. The temperature range for growth of strain V(T) was 15-65 degrees C with an optimum at 45 degrees C. The pH for growth ranged from pH 0 to 4 with an optimal at pH 1.4-1.6. Strain V(T) was able to grow aerobically and anaerobically, oxidizing ferrous iron and reducing ferric iron, respectively. The isolate grew chemo-organotrophically with yeast extract and yeast extract with glucose as the sources of energy and carbon. The molar G+C content in the DNA was 36 mol%. 16S rRNA gene sequence analysis demonstrated that strain V(T) was a member of the family Ferroplasmaceae, order Thermoplasmatales, phylum Euryarchaeota, showing sequence identities of 100 % with Ferroplasma cupricumulans BH2(T), 95.4 % with Ferroplasma acidiphilum Y(T), 94 % with Picrophilus torridus DSM 9790(T) and 92 % with Picrophilus oshimae DSM 9789(T). 16S rRNA gene sequence-based phylogenetic analysis showed that strain V(T) formed a monophyletic cluster together with F. cupricumulans BH2(T) and all other thermophilic isolates with available 16S rRNA gene sequences, whereas F. acidiphilum Y(T) formed another cluster with mesophilic isolates within the family Ferroplasmaceae. DNA-DNA hybridization values between strain V(T) and F. cupricumulans BH2(T) were well below 70 %, indicating that the two strains belong to separate species. Principal membrane lipids of strain V(T) were dibiphytanyl-based tetraether lipids containing pentacyclic rings. The polar lipids were dominated by a single phosphoglycolipid derivative based on a galactosyl dibiphytanyl phosphoglycerol tetraether, together with smaller amounts of monoglycosyl and diglycosyl dibiphytanyl ether lipids and the corresponding phosphoglycerol derivatives. The major respiratory quinones present were naphthoquinone derivatives. Given the notable physiological and chemical differences as well as the distinct phylogenetic placement of the new isolate relative to the type species of the genus Ferroplasma, we propose strain V(T) as a member of a new genus and species, Acidiplasma aeolicum gen. nov., sp. nov. The type strain of Acidiplasma aeolicum is strain V(T) (=DSM 18409(T) =JCM 14615(T)). In addition, we propose to transfer Ferroplasma cupricumulans Hawkes et al. 2008 to the genus Acidiplasma as Acidiplasma cupricumulans comb. nov. (type strain BH2(T) =DSM 16551(T) =JCM 13668(T)).
    • 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.
    • Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

      Dammeyer, Thorben; Timmis, Kenneth N; Tinnefeld, Philip (2013-05-20)
      Abstract Background In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Results Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. Conclusion The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the new tools facilitate metabolic engineering and yield assessment for cytoplasmic or periplasmic protein production in a number of different expression hosts when yields in one initially selected are insufficient.
    • Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein.

      Dammeyer, Thorben; Timmis, Kenneth N; Tinnefeld, Philip; Institut für Physikalische und Theoretische Chemie, NanoBioSciences, Technische Universität Braunschweig, Hans Sommer Str, 10, Braunschweig 38106, Germany. T.Dammeyer@tu-braunschweig.de (2013)
      In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains.
    • Characterization and role of a metalloprotease induced by chitin in Serratia sp. KCK.

      Kim, Hyun-Soo; Golyshin, Peter N; Timmis, Kenneth N; Department of Environmental Microbiology, The Helmholtz Center for Infection Research, Braunschweig, Germany. hyun1006@korea.ac.kr (2007-11)
      A metalloprotease induced by chitin in a new chitinolytic bacterium Serratia sp. Strain KCK was purified and characterized. Compared with other Serratia enzymes, it exhibited a rather broad pH activity range (pH 5.0-8.0), and thermostability. The cognate ORF, mpr, was cloned and expressed. Its deduced amino acid sequence showed high similarity to those of bacterial zinc-binding metalloproteases and a well-conserved serralysin family motif. Pretreatment of chitin with the Mpr protein promoted chitin degradation by chitinase A, which suggests that Mpr participates in, and facilitates, chitin degradation by this microorganism.
    • Characterization of marine isoprene-degrading communities.

      Alvarez, Laura Acuña; Exton, Daniel A; Timmis, Kenneth N; Suggett, David J; McGenity, Terry J; Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UK. (2009-12)
      Isoprene is a volatile and climate-altering hydrocarbon with an atmospheric concentration similar to that of methane. It is well established that marine algae produce isoprene; however, until now there was no specific information about marine isoprene sinks. Here we demonstrate isoprene consumption in samples from temperate and tropical marine and coastal environments, and furthermore show that the most rapid degradation of isoprene coincides with the highest rates of isoprene production in estuarine sediments. Isoprene-degrading enrichment cultures, analysed by denaturing gradient gel electrophoresis and 454 pyrosequencing of the 16S rRNA gene and by culturing, were generally dominated by Actinobacteria, but included other groups such as Alphaproteobacteria and Bacteroidetes, previously not known to degrade isoprene. In contrast to specialist methane-oxidizing bacteria, cultivated isoprene degraders were nutritionally versatile, and nearly all of them were able to use n-alkanes as a source of carbon and energy. We therefore tested and showed that the ubiquitous marine hydrocarbon-degrader, Alcanivorax borkumensis, could also degrade isoprene. A mixture of the isolates consumed isoprene emitted from algal cultures, confirming that isoprene can be metabolized at low, environmentally relevant concentrations, and suggesting that, in the absence of spilled petroleum hydrocarbons, algal production of isoprene could maintain viable populations of hydrocarbon-degrading microbes. This discovery of a missing marine sink for isoprene is the first step in obtaining more robust predictions of its flux, and suggests that algal-derived isoprene provides an additional source of carbon for diverse microbes in the oceans.
    • Diversity of Bacillus-like organisms isolated from deep-sea hypersaline anoxic sediments.

      Sass, Andrea M; McKew, Boyd A; Sass, Henrik; Fichtel, Jörg; Timmis, Kenneth N; McGenity, Terry J; Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, UK. sassam@Cardiff.ac.uk (2008)
      The deep-sea, hypersaline anoxic brine lakes in the Mediterranean are among the most extreme environments on earth, and in one of them, the MgCl2-rich Discovery basin, the presence of active microbes is equivocal. However, thriving microbial communities have been detected especially in the chemocline between deep seawater and three NaCl-rich brine lakes, l'Atalante, Bannock and Urania. By contrast, the microbiota of these brine-lake sediments remains largely unexplored.
    • 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.
    • Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory.

      Dammeyer, Thorben; Steinwand, Miriam; Krüger, Sarah-C; Dübel, Stefan; Hust, Michael; Timmis, Kenneth N; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2011)
      Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs.
    • 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.
    • Obligate oil-degrading marine bacteria.

      Yakimov, Michail M; Timmis, Kenneth N; Golyshin, Peter N; Istituto per l'Ambiente Marino Costiero, CNR, Messina 98122, Italy. (2007-06)
      Over the past few years, a new and ecophysiologically unusual group of marine hydrocarbon-degrading bacteria - the obligate hydrocarbonoclastic bacteria (OHCB) - has been recognized and shown to play a significant role in the biological removal of petroleum hydrocarbons from polluted marine waters. The introduction of oil or oil constituents into seawater leads to successive blooms of a relatively limited number of indigenous marine bacterial genera--Alcanivorax, Marinobacter, Thallassolituus, Cycloclasticus, Oleispira and a few others (the OHCB)--which are present at low or undetectable levels before the polluting event. The types of OHCB that bloom depend on the latitude/temperature, salinity, redox and other prevailing physical-chemical factors. These blooms result in the rapid degradation of many oil constituents, a process that can be accelerated further by supplementation with limiting nutrients. Genome sequencing and functional genomic analysis of Alcanivorax borkumensis, the paradigm of OHCB, has provided significant insights into the genomic basis of the efficiency and versatility of its hydrocarbon utilization, the metabolic routes underlying its special hydrocarbon diet, and its ecological success. These and other studies have revealed the potential of OHCB for multiple biotechnological applications that include not only oil pollution mitigation, but also biopolymer production and biocatalysis.
    • 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.
    • 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.
    • Transcriptional profiling of the marine oil-degrading bacterium Alcanivorax borkumensis during growth on n-alkanes.

      Sabirova, Julia S; Becker, Anke; Lünsdorf, Heinrich; Nicaud, Jean-Marc; Timmis, Kenneth N; Golyshin, Peter N; Department of Bioscience and Bioengineering, Ghent University, Ghent, Belgium. julia.sabirova@ugent.be (2011-06)
      The marine oil-degrading bacterium Alcanivorax borkumensis SK2 has attracted significant interest due to its hydrocarbonoclastic lifestyle, its alkane-centered metabolism, and for playing an important ecological role in cleaning up marine oil spills. In this study, we used microarray technology to characterize the transcriptional responses of A. borkumensis to n-hexadecane exposure as opposed to pyruvate, which led to the identification of a total of 220 differentially expressed genes, with 109 genes being upregulated and 111 genes being downregulated. Among the genes upregulated on alkanes are systems predicted to be involved in the terminal oxidation of alkanes, biofilm formation, signal transduction, and regulation.