• 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.
    • 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)
    • Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen.

      Ferrer, Manuel; Ghazi, Azam; Beloqui, Ana; Vieites, José María; López-Cortés, Nieves; Marín-Navarro, Julia; Nechitaylo, Taras Y; Guazzaroni, María-Eugenia; Polaina, Julio; Waliczek, Agnes; et al. (2012)
      Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional β-xylosidase, α-xylanase, α-L-arabinase and α-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit β-1,4 xylosidase, α-1,5 arabinofur(pyr)anosidase, β-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, β-galactosidase and α-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.
    • Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

      Kube, Michael; Chernikova, Tatyana N; Al-Ramahi, Yamal; Beloqui, Ana; Lopez-Cortez, Nieves; Guazzaroni, María-Eugenia; Heipieper, Hermann J; Klages, Sven; Kotsyurbenko, Oleg R; Langer, Ines; et al. (2013-07-23)
      Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis-the paradigm of mesophilic hydrocarbonoclastic bacteria-O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.
    • Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

      Del Pozo, Mercedes V; Fernández-Arrojo, Lucía; Gil-Martínez, Jorge; Montesinos, Alejandro; Chernikova, Tatyana N; Nechitaylo, Taras Y; Waliszek, Agnes; Tortajada, Marta; Rojas, Antonia; Huws, Sharon A; et al. (2012-09-21)
      Abstract Background A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases.
    • Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail.

      Del Pozo, Mercedes V; Fernández-Arrojo, Lucía; Gil-Martínez, Jorge; Montesinos, Alejandro; Chernikova, Tatyana N; Nechitaylo, Taras Y; Waliszek, Agnes; Tortajada, Marta; Rojas, Antonia; Huws, Sharon A; et al. (2012)
      A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product.
    • 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.
    • 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. (American Society for Microbiology, 2006-06)
    • 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.