Tatyana N. Chernikova

Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis

Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation–related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.

Chaperonins govern growth of Escherichia coli at low temperatures

Sabine Louët responds: In researching the news story, I had several interviews with Huub Schellekens, who explained to me the key findings of his laboratory’s research on Eprex. At no point during these interviews did he strongly underline the fact that there was such a level of uncertainty regarding the findings of his study. However, it is clear that the activity of a therapeutic protein is likely to depend on many factors; indeed, the news article pointed out this fact: “Not only could the immunogenic reaction be triggered by a change in formulation—as in the Eprex case—but also by variations in amino acid sequence, glycosylation or even by impurities cropping up during manufacturing or administration of the drug.” The adverse events associated with the manufacture, formulation and administration of Ortho Biotech’s (a Johnson & Johnson affiliate) erythropoietin alpha (Eprex) exemplify the difficulties faced by companies that seek to manufacture and formulate generic biopharmaceuticals. control the Plac promoter in E. coli strain XLOLR and examined the growth characteristics of the transgene after induction of expression with isopropyl-Dgalactopyranoside (IPTG; Fig. 1a). The strain bearing the construct grows much faster than the parental strain at low temperatures: 3-fold faster than the parental strain at 15 °C, 36-fold faster at 10 °C and 141-fold faster at 8 °C (growth rate of parental E. coli ∼ 0.002 h–1; that of the transgenic strain ∼ 0.282 h–1). No growth of the parental E. coli was detected below 8 °C, whereas the transgenic strain grew at temperatures below 4 °C As determined using the square-root growth model of Ratkowsky et al.6, the theoretical minimum temperatures for the parental and transgenic E. coli would be 7.5 °C and –13.7 °C, respectively (see Supplementary Methods online). To rule out the possibility that hyperexpression of chaperones per se lowers the growth limit of E. coli, we also expressed the GroEL and GroES chaperonins to similar cellular levels— 160 μg GroEL/ES per milligram of protein versus 120 μg Cpn60/10 per milligram of protein, using plasmids pBB528 and pBB541 (kindly provided by E. Betiku and U. Rinas (GBF)), in which the chaperonins are expressed from the same Plac promoter (for details, see Supplementary Fig. 1 online). The growth characteristics of E. coli at temperatures below 15 °C were not influenced by hyperexpression of the homologous chaperonins (data not shown). This demonstrates that the depression of the lower limit of growth of E. coli by Cpn60 and Cpn10 is due to a

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

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.

Expression of a Temperature-Sensitive Esterase in a Novel Chaperone-Based Escherichia coli Strain

ABSTRACT A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4°C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8T, that allow E. coli to grow at high rates at 4°C (maximum growth rate, 0.28 h−1) (M. Ferrer, T. N. Chernikova, M. Yakimov, P. N. Golyshin, and K. N. Timmis, Nat. Biotechnol. 21:1266-1267, 2003). The expression of a temperature-sensitive esterase in this host at 4 to 10°C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37°C (32,380 versus 190 μmol min−1 g−1). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5°C).

Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail

BackgroundA 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.ResultsIn 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 (p NPbetaG) and p NP-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 p NPbetaG 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 p NPbetaG). 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.ConclusionsThe 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.

Functional Metagenomics Unveils a Multifunctional Glycosyl Hydrolase from the Family 43 Catalysing the Breakdown of Plant Polymers in the Calf Rumen

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.

Unveiling microbial life in the new deep-sea hypersaline Lake Thetis. Part II: a metagenomic study.

So far only little is known about the microbial ecology of Mediterranean deep-sea hypersaline anoxic lakes (DHALs). These brine lakes were formed by evaporite dissolution/brine seeps and are important model environments to provide insights into possible metabolisms and distributions of microorganisms on the early Earth. Our study on the Lake Thetis, a new thalassohaline DHAL located South-East of the Medriff Corridor, has revealed microbial communities of contrasting compositions with a high number of novel prokaryotic candidate divisions. The major finding of our present work is co-occurrence of at least three autotrophic carbon dioxide fixation pathways in the brine-seawater interface that are likely fuelled by an active ramified sulphur cycle. Genes for the reductive acetyl-CoA and reductive TCA pathways were also found in the brine suggesting that these pathways are operational even at extremely elevated salinities and that autotrophy is more important in hypersaline environments than previously assumed. Surprisingly, genes coding for RuBisCo were found in the highly reduced brine. Three types of sulphide oxidation pathways were found in the interface. The first involves a multienzyme Sox complex catalysing the complete oxidation of reduced sulphur compounds to sulphate, the second type recruits SQR sulphide:quinone reductase for oxidation of sulphide to elemental sulphur, which, in the presence of sulphide, could further be reduced by polysulphide reductases in the third pathway. The presence of the latter two allows a maximal energy yield from the oxidation of sulphide and at the same time prevents the acidification and the accumulation of S(0) deposits. Amino acid composition analysis of deduced proteins revealed a significant overrepresentation of acidic residues in the brine compared with the interface. This trait is typical for halophilic organisms as an adaptation to the brines extreme hypersalinity. This work presents the first metagenomic survey of the microbial communities of the recently discovered Lake Thetis whose brine constitutes one of saltiest water bodies ever reported.

Bacterial population and biodegradation potential in chronically crude oil-contaminated marine sediments are strongly linked to temperature

Two of the largest crude oil-polluted areas in the world are the semi-enclosed Mediterranean and Red Seas, but the effect of chronic pollution remains incompletely understood on a large scale. We compared the influence of environmental and geographical constraints and anthropogenic forces (hydrocarbon input) on bacterial communities in eight geographically separated oil-polluted sites along the coastlines of the Mediterranean and Red Seas. The differences in community compositions and their biodegradation potential were primarily associated (P < 0.05) with both temperature and chemical diversity. Furthermore, we observed a link between temperature and chemical and biological diversity that was stronger in chronically polluted sites than in pristine ones where accidental oil spills occurred. We propose that low temperature increases bacterial richness while decreasing catabolic diversity and that chronic pollution promotes catabolic diversification. Our results further suggest that the bacterial populations in chronically polluted sites may respond more promptly in degrading petroleum after accidental oil spills.

Functional consequences of single:double ring transitions in chaperonins: life in the cold

The cpn60 and cpn10 genes from psychrophilic bacterium, Oleispira antarctica RB8, showed a positive effect in Escherichia coli growth at low temperature, shifting its theoretical minimal growth temperature from +7.5°C to −13.7°C [Ferrer, M., Chernikova, T.N., Yakimov, M., Golyshin, P.N., and Timmis, K.N. (2003) Nature Biotechnol 21: 1266–1267]. To provide experimental support for this finding, Cpn60 and 10 were overproduced in E. coli and purified to apparent homogeneity. Recombinant O.Cpn60 was identical to the native protein based on tetradecameric structure, and it dissociates during native PAGE. Gel filtration and native PAGE revealed that, in vivo and in vitro, (O.Cpn60)7 was the active oligomer at 4–10°C, whereas at > 10°C, this complex was converted to (O.Cpn60)14. The dissociation reduces the ATP consumption (energy‐saving mechanism) and increases the refolding capacity at low temperatures. In order for this transition to occur, we demonstrated that K468 and S471 may play a key role in conforming the more advantageous oligomeric state in O.Cpn60. We have proved this hypothesis by showing that single and double mutations in K468 and S471 for T and G, as in E.GroEL, produced a more stable double‐ring oligomer. The optimum temperature for ATPase and chaperone activity for the wild‐type chaperonin was 24–28°C and 4–18°C, whereas that for the mutants was 45–55°C and 14–36°C respectively. The temperature inducing unfolding (TM) increased from 45°C to more than 65°C. In contrast, a single ring mutant, O.Cpn60SR, with three amino acid substitutions (E461A, S463A and V464A) was as stable as the wild type but possessed refolding activity below 10°C. Above 10°C, this complex lost refolding capacity to the detriment of the double ring, which was not an efficient chaperone at 4°C as the single ring variant. We demonstrated that expression of O.Cpn60WT and O.Cpn60SR leads to a higher growth of E. coli at 4°C (µmax, 0.22 and 0.36 h−1 respectively), whereas at 10–15°C, only E. coli cells expressing O.Cpn60 or O.Cpn60DR grew better than parental cells (–cpn). These results clearly indicate that the single‐to‐double ring transition in Oleispira chaperonin is a wild‐type mechanism for its thermal acclimation. Although previous studies have also reported single‐to‐double ring transitions under many circumstances, this is the first clear indication that single‐ring chaperonins are necessary to support growth when the temperature falls from 37°C to 4°C.

Reactome array: forging a link between metabolome and genome.

Metabolite Arrays Methods suitable for the biochemical analysis of multiple metabolic pathways in mixed samples are in short supply. Beloqui et al. (p. 252) report a method to sample the global metabolic state of an organism or mixture of organisms using an array of more than 1500 metabolites linked to a glass slide. The substrates are linked to the plate so that the reaction of an enzyme with one of the metabolites releases a fluorescent dye, which allows sensitive detection of the enzymatic activity. From a sample with small numbers of a mixture of bacteria, the authors were able to collect DNA, amplify it in a host bacterium, and measure its encoded metabolic activity with the array. Furthermore, by coating the substrates on nanoparticles with a specially designed linker, the authors could trap and purify enzymes that reacted with the immobilized substrate. The metabolite array may be useful in the characterization of environmental samples, in diagnostic procedures, and in enzyme discovery. A microarray technique uses trapped, dye-associated metabolites to allow rapid global characterization of metabolic activity. We describe a sensitive metabolite array for genome sequence–independent functional analysis of metabolic phenotypes and networks, the reactomes, of cell populations and communities. The array includes 1676 dye-linked substrate compounds collectively representing central metabolic pathways of all forms of life. Application of cell extracts to the array leads to specific binding of enzymes to cognate substrates, transformation to products, and concomitant activation of the dye signals. Proof of principle was shown by reconstruction of the metabolic maps of model bacteria. Utility of the array for unsequenced organisms was demonstrated by reconstruction of the global metabolisms of three microbial communities derived from acidic volcanic pool, deep-sea brine lake, and hydrocarbon-polluted seawater. Enzymes of interest are captured on nanoparticles coated with cognate metabolites, sequenced, and their functions unequivocally established.