Peter N. Golyshin

Alcanivorax borkumensis gen. nov., sp. nov., a new, hydrocarbon-degrading and surfactant-producing marine bacterium

During screening for biosurfactant-producing, n-alkane-degrading marine bacteria, six heterotrophic bacterial strains were isolated from enriched mixed cultures, obtained from sea water/sediment samples collected near the isle of Borkum (North Sea), using Mihagol-S (C14,15-n-alkanes) as principal carbon source. These Gram-negative, aerobic, rod-shaped bacteria use a limited number of organic compounds, including aliphatic hydrocarbons, volatile fatty acids, and pyruvate and its methyl ether. During cultivation on n-alkanes as sole source of carbon and energy, all strains produced both extracellular and cell-bound surface-active glucose lipids which reduced the surface tension of water from 72 to 29 mN m-1 (16). This novel class of glycolipids was found to be produced only by these strains. The 16S rRNA gene sequence analysis showed that these strains are all members of the gamma-subclass of the Proteobacteria. Their phospholipids ester-linked fatty acid composition was shown to be similar to that of members of the genus Halmonas, although they did not demonstrate a close phylogenetic relationship to any previously described species. On the basis of the information summarized above, a new genus and species, Alcanivorax borkumensis, is described to include these bacteria. Strain SK2T is the type strain of A. borkumensis.

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.

Polychlorinated biphenyl-degrading microbial communities in soils and sediments

Recent advances in the degradation of polychlorinated biphenyls (PCBs) have focussed on the use of experimental enrichment cultures to obtain PCB-degrading communities, and the use of culture-independent approaches to characterize natural and experimental PCB-degrading communities and to identify the key members in this process. PCB-degrading communities can be surprisingly diverse. Novel types of composite bacteria-mineral biofilm communities have been described. Community metabolism of PCBs may lead to the formation of protoanemonin, a dead-end product in some instances but, in others, a seemingly productive intermediate. Analysis of isotope fractionation and preferred enantiomer degradation has provided new information on degradation of PCBs in anaerobic settings. The first defined community capable of dehalorespiration of PCBs has been described, and important community members identified. Here, we provide an overview of the current knowledge of aerobic and anaerobic degradation of PCBs in microbial consortia and in the environment, including novel approaches to determine in situ PCB degradation.

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

Novel Polyphenol Oxidase Mined from a Metagenome Expression Library of Bovine Rumen BIOCHEMICAL PROPERTIES, STRUCTURAL ANALYSIS, AND PHYLOGENETIC RELATIONSHIPS

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) >> phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μm and 18, 660, and 1175 s-1, respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.

Metagenomics for Mining New Genetic Resources of Microbial Communities

Recent progress has revealed that the capture of genetic resources of complex microbial communities in metagenome libraries allows the discovery of a richness of new enzymatic diversity that had not previously been imagined. Activity-based screening of such libraries has demonstrated that this new diversity is not simply variations on known sequence themes, but rather the existence of entirely new sequence classes and novel functionalities. This new diversity, the surface of which has thus far only been scratched, constitutes potential for a wealth of new and improved applications in industry, medicine, agriculture, etc., and promises to facilitate in a significant manner our transition to a sustainable society, by contributing to the transition to renewable sources of energy, chemicals and materials, the lowering of pollutant burdens, lower processes energies, etc. Current bottlenecks in metagenomics include insufficient functional characterization and amplifying non-validated annotations of proteins in databases.

Analysis of Storage Lipid Accumulation in Alcanivorax borkumensis: Evidence for Alternative Triacylglycerol Biosynthesis Routes in Bacteria

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.

Novel Polyphenol Oxidase Mined from a Metagenome Expression Library of Bovine Rumen

RL5, a gene coding for a novel polyphenol oxidase, was identified through activity screening of a metagenome expression library from bovine rumen microflora. Characterization of the recombinant protein produced in Escherichia coli revealed a multipotent capacity to oxidize a wide range of substrates (syringaldazine > 2,6-dimethoxyphenol > veratryl alcohol > guaiacol > tetramethylbenzidine > 4-methoxybenzyl alcohol > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) >> phenol red) over an unusually broad range of pH from 3.5 to 9.0. Apparent Km and kcat values for ABTS, syringaldazine, and 2,6-dimetoxyphenol obtained from steady-state kinetic measurements performed at 40 °C, pH 4.5, yielded values of 26, 0.43, and 0.45 μm and 18, 660, and 1175 s-1, respectively. The Km values for syringaldazine and 2,6-dimetoxyphenol are up to 5 times lower, and the kcat values up to 40 times higher, than values previously reported for this class of enzyme. RL5 is a 4-copper oxidase with oxidation potential values of 745, 400, and 500 mV versus normal hydrogen electrode for the T1, T2, and T3 copper sites. A three-dimensional model of RL5 and site-directed mutants were generated to identify the copper ligands. Bioinformatic analysis of the gene sequence and the sequences and contexts of neighboring genes suggested a tentative phylogenetic assignment to the genus Bacteroides. Kinetic, electrochemical, and EPR analyses provide unequivocal evidence that the hypothetical proteins from Bacteroides thetaiotaomicron and from E. coli, which are closely related to the deduced protein encoded by the RL5 gene, are also multicopper proteins with polyphenol oxidase activity. The present study shows that these three newly characterized enzymes form a new family of functional multicopper oxidases with laccase activity related to conserved hypothetical proteins harboring the domain of unknown function DUF152 and suggests that some other of these proteins may also be laccases.

Oleiphilaceae fam. nov., to include Oleiphilus messinensis gen. nov., sp nov., a novel marine bacterium that obligately utilizes hydrocarbons.

A bacterial isolate, ME102T, was obtained from an n-hexadecane enrichment culture of seawater/sediment samples collected in the harbour of Messina (Italy). This gram-negative, aerobic, motile, rod-shaped bacterium used a narrow spectrum of organic compounds, including aliphatic hydrocarbons, alkanoates and alkanoles, as carbon and energy sources. None of the sugars, organic acids or amino acids tested was used. During cultivation on n-alkanes as the sole source of carbon and energy, the cells formed a biofilm on the surface of the alkane droplets. Large-scale (sometimes >50% of the cell mass) intracellular accumulation of alkanoates occurred in cells adsorbed on the alkane surface and under nitrogen-limiting conditions. 16S rRNA gene sequence analysis showed that this isolate represents a distinct lineage in the gamma-Proteobacteria and has about 91% sequence identity to members of Marinobacter and Alcanivorax, the closest genera. Four different types of polar lipid could be detected, phosphatidyl glycerol, phosphatidyl ethylamine, phosphatidyl dimethylethylamine and lipids belonging to an unknown type of phospholipid (m/z between 861 and 879). The principal fatty acids in the polar lipid fatty acid profile were 16:0 and 16:1. The putative gene encoding the key enzyme of alkane catabolism, alkane hydroxylase (AlkB), has been cloned. The protein sequence of the putative AlkB of the isolate ME102T was related to the AlkB of Pseudomonas oleovorans and Alcanivorax borkumensis, showing about 60% sequence identity. On the basis of physiological studies and taking into account the distant phylogenetic position of isolate ME102T relative to previously described organisms, a novel genus and species is proposed, Oleiphilus messinensis gen. nov., sp. nov., within a new family, Oleiphilaceae fam. nov. Strain ME102T (= DSM 13489T = LMG 20357T) is the type and only strain of O. messinensis.

Metagenomics approaches in systems microbiology.

The world of microorganisms comprises a vast diversity of live organisms, each with its individual set of genes, cellular components and metabolic reactions that interact within the cell and communicate with the environment in many different ways. There is a strong imperative to gain a broader view of the wired and interconnected cellular and environmental processes as a whole via the systems microbiology approach in order to understand and predict ecosystem functioning. On the other hand, currently we experience a rise of metagenomics as an emerging tool to study communities of uncultured microorganisms. In this review, we conducted a survey of important methodologies in metagenomics and describe systems microbiology-like approaches for gaining a mechanistic understanding of complex microbial systems to interrogate compositional, evolutionary and metabolic properties. The review also discusses how metagenomics can be used as a holistic indicator for ecosystem response in terms of matter, nutrient and energy sources and functional networking.