Michail M. Yakimov

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.

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.

Stratified prokaryote network in the oxic-anoxic transition of a deep-sea halocline

The chemical composition of the Bannock basin has been studied in some detail. We recently showed that unusual microbial populations, including a new division of Archaea (MSBL1), inhabit the NaCl-rich hypersaline brine. High salinities tend to reduce biodiversity, but when brines come into contact with fresher water the natural haloclines formed frequently contain gradients of other chemicals, including permutations of electron donors and acceptors, that may enhance microbial diversity, activity and biogeochemical cycling. Here we report a 2.5-m-thick chemocline with a steep NaCl gradient at 3.3 km within the water column betweeen Bannock anoxic hypersaline brine and overlying sea water. The chemocline supports some of the most biomass-rich and active microbial communities in the deep sea, dominated by Bacteria rather than Archaea, and including four major new divisions of Bacteria. Significantly higher metabolic activities were measured in the chemocline than in the overlying sea water and underlying brine; functional analyses indicate that a range of biological processes is likely to occur in the chemocline. Many prokaryotic taxa, including the phylogenetically new groups, were confined to defined salinities, and collectively formed a diverse, sharply stratified, deep-sea ecosystem with sufficient biomass to potentially contribute to organic geological deposits.

Microbial community dynamics during assays of harbour oil spill bioremediation: a microscale simulation study

Aims:  Microcosm experiments simulating an oil spill event were performed to evaluate the response of the natural microbial community structure of Messina harbour seawater following the accidental load of petroleum.

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.

The potential of Bacillus licheniformis strains for in situ enhanced oil recovery

Abstract The ability of microorganisms isolated from oil reservoirs to increase oil recovery by in situ growth and metabolism following the injection of laboratory grown microbial cells and nutrients were studied. Four strains isolated from Northern German oil reservoirs at depths of 866 to 1520 m, and identified as Bacillus licheniformis, were characterized taxonomically and physiologically. All strains grew on a variety of substrates at temperatures of up to 55°C and at salinities of up to 12% NaCl. Extracellular polymer production occurred both aerobically and anaerobically over a wide range of temperatures, pressures and salinities, though it was optimal at temperatures around 50°C and at salinities between 5 and 10% NaCl. Strain BNP29 was able to produce significant amounts of biomass, polymer, fermentation alcohols and acids in batch culture experiments under simulated reservoir conditions. Oil recovery (core flooding) experiments with strain BNP29 and a sucrose-based nutrient were performed with lime-free and lime-containing, oil-bearing sandstone cores. Oil recovery efficiencies varied from 9.3 to 22.1% of the water flood residual oil saturation. Biogenic acid production that accompanied oil production, along with selective plugging, are important mechanisms leading to increased oil recovery, presumably through resulting changes in rock porosity and alteration of wettability. These data show that strain BNP29 exhibits potential for the development of enhanced oil recovery processes.

Crude oil‐induced structural shift of coastal bacterial communities of rod bay (Terra Nova Bay, Ross Sea, Antarctica) and characterization of cultured cold‐adapted hydrocarbonoclastic bacteria

For preliminary screening of human impact on Antarctic coasts, the compositions of microbial communities were analyzed in seawater at two sites located in the Terra Nova Bay of Antarctica (Ross Sea) by a combination of 16S rRNA gene sequencing and culture techniques. The bacterial community in the sample from the Rod Bay site, located at the proximity to the Italian Station, was characterized by a high abundance of 16S rRNA gene sequences belonging to the microflora typically found in soil and freshwater environments. In contrast, the seawater sample from the Adelie Cove station, a pristine reference site, contained 16S rRNA gene sequences typically found in marine areas affected by algal blooms and sea ice decay. The addition of crude oil to the Rod Bay seawater sample rapidly induced a shift in the composition of the bacterial community with appearance of novel taxonomic groups and a dramatic increase in the relative abundance of gamma-Proteobacteria sequences, whereas no significant changes were detected in the bacterial community of the Adelie Cove sample under the same conditions. Bacteria-exhibiting features with potential interest for industrial and environmental applications were isolated from the Rod Bay oil-enriched sample. In particular, hydrocarbon-degrading, cold-adapted bacteria were selectively enriched, isolated and screened for their ability to synthesize polyunsaturated fatty acids. Twenty two bacterial strains were isolated from the oil enrichment culture and identified. Eighteen isolates were found to be members of gamma-Proteobacteria, while the remainder were representatives of alpha-Proteobacteria, CFB and high G + C divisions.

Contribution of crenarchaeal autotrophic ammonia oxidizers to the dark primary production in Tyrrhenian deep waters (Central Mediterranean Sea)

Mesophilic Crenarchaeota have recently been thought to be significant contributors to nitrogen (N) and carbon (C) cycling. In this study, we examined the vertical distribution of ammonia-oxidizing Crenarchaeota at offshore site in Southern Tyrrhenian Sea. The median value of the crenachaeal cell to amoA gene ratio was close to one suggesting that virtually all deep-sea Crenarchaeota possess the capacity to oxidize ammonia. Crenarchaea-specific genes, nirK and ureC, for nitrite reductase and urease were identified and their affiliation demonstrated the presence of ‘deep-sea’ clades distinct from ‘shallow’ representatives. Measured deep-sea dark CO2 fixation estimates were comparable to the median value of photosynthetic biomass production calculated for this area of Tyrrhenian Sea, pointing to the significance of this process in the C cycle of aphotic marine ecosystems. To elucidate the pivotal organisms in this process, we targeted known marine crenarchaeal autotrophy-related genes, coding for acetyl-CoA carboxylase (accA) and 4-hydroxybutyryl-CoA dehydratase (4-hbd). As in case of nirK and ureC, these genes are grouped with deep-sea sequences being distantly related to those retrieved from the epipelagic zone. To pair the molecular data with specific functional attributes we performed [14C]HCO3 incorporation experiments followed by analyses of radiolabeled proteins using shotgun proteomics approach. More than 100 oligopeptides were attributed to 40 marine crenarchaeal-specific proteins that are involved in 10 different metabolic processes, including autotrophy. Obtained results provided a clear proof of chemolithoautotrophic physiology of bathypelagic crenarchaeota and indicated that this numerically predominant group of microorganisms facilitate a hitherto unrecognized sink for inorganic C of a global importance.