Cleaning of oil-polluted bottom sediments of the boreal lake, Samotlor oil field, North Russia: case report.

Small lakes in areas of intensive crude oil production may be susceptible to oil pollution arising from accidental spills and leaks, eventually leading to the pollution of bottom sediments. Effective cleaning of aquatic bottom sediments remains a challenge. Flotation is a potentially simple and reliable approach for the cleanup of bottom sediments without their excavation from the water body. Full-scale testing of flotation-based technology using the specially designed airlift plant allowed the cleaning of bottom sediments of an unnamed boreal lake ('the lake') within the Samotlor oil field, North Russia, heavily polluted with crude oil several decades ago. The lake bottom sediments are dominated by peat and unevenly polluted with oil. The average oil content in the lake bottom sediments was 111 g kg-1. During the 1.5 months' field test in July-August 2018, the average total oil concentration in the bottom sediments of the lake was reduced to 1.99 g kg-1. Secondary water contamination was minimal; the content of oil hydrocarbons in the water after completion of work did not exceed 0.09 ± 0.04 mg L-1. This study demonstrates that flotation-based technology can be applied for in situ cleaning of oil-contaminated lake bottom sediments including those in boreal climates.

Identification, Heterologous Expression, and Functional Characterization of Bacillus subtilis YutF, a HAD Superfamily 5'-Nucleotidase with Broad Substrate Specificity.

5'-nucleotidases (EC 3.1.3.5) catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides as well as complex nucleotides, such as uridine 5'-diphosphoglucose (UDP-glucose), nicotinamide adenine dinucleotide and flavin adenine dinucleotide, to their corresponding nucleosides plus phosphate. These enzymes have been found in diverse species in intracellular and membrane-bound, surface-localized forms. Soluble forms of 5'-nucleotidases belong to the ubiquitous haloacid dehalogenase superfamily (HADSF) and have been shown to be involved in the regulation of nucleotide, nucleoside and nicotinamide adenine dinucleotide (NAD+) pools. Despite the important role of 5'-nucleotidases in cellular metabolism, only a few of these enzymes have been characterized in the Gram-positive bacterium Bacillus subtilis, the workhorse industrial microorganism included in the Food and Drug Administration's GRAS (generally regarded as safe) list. In the present study, we report the identification of a novel 5'-nucleotidase gene from B. subtilis, yutF, which comprises 771 bp encoding a 256-amino-acid protein belonging to the IIA subfamily of the HADSF. The gene product is responsible for the major p-nitrophenyl phosphatase activity in B. subtilis. The yutF gene was overexpressed in Escherichia coli, and its product fused to a polyhistidine tag was purified and biochemically characterized as a soluble 5'-nucleotidase with broad substrate specificity. The recombinant YutF protein was found to hydrolyze various purine and pyrimidine 5'-nucleotides, showing preference for 5'-nucleoside monophosphates and, specifically, 5'-XMP. Recombinant YutF also exhibited phosphohydrolase activity toward nucleotide precursors, ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate. Determination of the kinetic parameters of the enzyme revealed a low substrate specificity (Km values in the mM concentration range) and modest catalytic efficiencies with respect to substrates. An initial study of the regulation of yutF expression showed that the yutF gene is a component of the yutDEF transcription unit and that YutF overproduction positively influences yutDEF expression.

Enhancement of extracellular purine nucleoside accumulation by Bacillus strains through genetic modifications of genes involved in nucleoside export.

Using a simple method to introduce genetic modifications into the chromosome of naturally nontransformable Bacillus, a set of marker-free inosine-producing and 5-aminoimidazole-4-carboxamide (AICA) ribonucleoside-producing Bacillus amyloliquefaciens strains has been constructed. These strains differ in expression levels of the genes responsible for nucleoside export. Overexpression of B. amyloliquefaciens pbuE and heterologous expression of Escherichia coli nepI, which encode nucleoside efflux transporters, each notably enhanced inosine production by a B. amyloliquefaciens nucleoside-producing strain. pbuE overexpression was found to increase AICA ribonucleoside accumulation, indicating that the substrate specificity of the PbuE pump extends to this nucleoside. These results demonstrate that identifying genes whose products facilitate transport of a desired nucleoside out of cells and enhancing their expression can improve the performance of strains used for industrial production.

A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains.

A simple method to introduce marker-free deletions, insertions, and point mutations into the chromosomes of naturally nontransformable Bacillus amyloliquefaciens strains has been developed. The method is efficient and fast, and it allows for the generation of genetic modifications without the use of a counter-selectable marker or a special prerequisite strain. This method uses the combination of the following: the effective introduction of a delivery plasmid into cells for gene replacement; a two-step replacement recombination procedure, which occurs at a very high frequency due to the use of a thermosensitive rolling-circle replication plasmid; and colony polymerase chain reaction (PCR) analysis for screening. Using PCR primers with mismatches at the 3' end enables the selection of strains that contain a single nucleotide substitution in the target gene. This approach can be used as a routine method for the investigation of complex physiological pathways and for the metabolic engineering of food-grade industrial B. amyloliquefaciens and other Bacillus strains.

A new function for the Bacillus PbuE purine base efflux pump: efflux of purine nucleosides.

The pbuE (ydhL) gene from Bacillus subtilis is known to encode the purine base efflux pump, and its expression is controlled by an adenine-dependent riboswitch. We cloned the pbuE gene from Bacillus amyloliquefaciens and examined gene expression by its own cis-acting regulatory elements in Escherichia coli. Regulation of pbuE expression, previously found in B. subtilis, was retained in this heterologous expression: it was induced by adenine and activated by a mutation in the 5' untranslated region, which disrupted transcription termination. This observation supports the model that the adenine-dependent riboswitch directly regulates pbuE expression, without requiring additional factors. Overexpression of the PbuE pump conferred upon the E. coli strain resistance to higher concentrations of inosine, adenosine and guanosine, and increased exogenous inosine accumulation by E. coli cells deficient in purine nucleoside phosphorylase. Overexpression of the PbuE pump also enhanced hypoxanthine excretion by the E. coli hypoxanthine-producing strain and inosine excretion both by the E. coli and B. amyloliquefaciens nucleoside-producing strains. Thus, for the first time, we obtained direct evidence for the involvement of PbuE in efflux of not only purine bases, but also purine ribonucleosides. A possible new role for the pump in cell physiology is discussed.

The yicM (nepI) gene of Escherichia coli encodes a major facilitator superfamily protein involved in efflux of purine ribonucleosides.

The yicM gene of Escherichia coli was found by selection for resistance to 6-mercaptopurine. Translation and transcription initiation sites of yicM were determined. Overexpression of yicM increased resistance of sensitive cells to inosine and guanosine, decreased E. coli growth rate in medium containing these ribonucleosides as the sole carbon source, led to inosine accumulation by the E. coli strain deficient in purine nucleoside phosphorylase and enhanced the rate of inosine excretion by an inosine-producing strain. These results suggest that yicM encodes a purine ribonucleoside exporter and we have accordingly renamed it nepI (for 'nucleoside efflux permease-inosine').