Vol 54, No 2 (2018)

The defense mechanisms of bacterial cells against antibiotics, which induce specific complexes of adaptive reactions at the levels of replication, transcription, translation, and enzymatic activity, are reviewed. These adaptive reactions are conventionally considered to be stress responses. Specific stress responses are integrated in an adaptive network that is flexible in its reaction to environmental signals and capable of increasing antibiotic tolerance and maintaining the viability of bacterial cells in order to restart their growth once environmental conditions are again optimal. This facilitates the selection of mutations with high resistance to antibiotics. A prerequisite of efficient tools for the inhibition of such resistance is a profound knowledge of the mechanisms responsible for the development of physiological tolerance.

The effect of anaplerotic pathways activation on CO₂-dependent anaerobic glucose utilization by Escherichia coli strains deficient in the main fermentation pathways and possessing a modified system of glucose transport and phosphorylation was studied. Intracellular CO₂ generation in the strains was ensured resulting from oxidative decarboxylation of pyruvic acid by pyruvate dehydrogenase. Sodium bicarbonate dissolved in the medium was used as an external source of CO₂. The genes of heterologous pyruvate carboxylase and native NADH-dependent malic enzyme were overexpressed in the strains to allow anaplerotic carboxylation of pyruvic acid to oxaloacetic or malic acid. The ability of the strains to reoxidize NADH utilizing carboxylation products was additionally increased due to enhanced expression of malate dehydrogenase gene. In the case of endogenous CO₂ formation, the activation of anaplerotic pathways did not cause a notable increase in the anaerobic glucose consumption by the constructed strains. At the same time, the expression of pyruvate carboxylase led to a pronounced decrease in the secretion of pyruvic acid with the concomitant increase in the yield of four-carbon metabolites. Further enhancement of NADH-dependent malic enzyme expression provoked activation of a pyruvate–oxaloacetate–malate–pyruvate futile cycle in the strains. The availability in the medium of the external CO₂ source sharply increased the anaerobic utilization of glucose by strains expressing pyruvate carboxylase. The activity of the futile cycle has raised with the increased malic enzyme expression and dropped upon enhancement of malate dehydrogenase expression. As a result, the efficiency of CO₂-dependent anaerobic glucose utilization coupled to the formation of four-carbon carboxylation products increased in the studied strains resulting from the primary anaplerotic conversion of pyruvic acid into oxaloacetic acid followed by the involvement of the precursor formed in NADH-consuming biosynthetic reactions dominating over the reactions of the revealed futile cycle.

An efficient biosurfactant-producing strain was isolated and cultured from Dagang oil field (China) using crude oil as sole source of carbon. Based on partial sequenced 16S rDNA analysis, the isolated strain was identified as Pseudomonas aeruginosa SNP0614. The bacterium P. aeruginosa SNP0614 produced a type of biosurfactant with excessive foam-forming properties. After microbial cultivation at 37°C and 150 rpm for 12 h, the produced biosurfactant was found to reduce the surface tension to 25.4 mN/m with critical micelle concentration (CMC) of 45.0 mg/L. After 20 days of incubation, the biosurfactant exhibited 90% emulsification activity (E₂₄) on crude oil. FTIR spectroscopy of extracted biosurfactant indicated the biosurfactant as lipopeptide. The significant synergistic effect between P. aeruginosa SNP0614 and the mixed oildegrading bacteria resulted in increasing n-alkanes degradation rate by 30%. The strain P. aeruginosa SNP0614 represented as a promising biosurfactant producer and could be applied in a variety of biotechnological and industrial processes, particularly in microbial enhanced oil recovery and the bioremediation of oil pollution.

The ability of the Alcaligenes faecalis 2 strain to utilize acrylamide and acrylic acid upon cultivation with these compounds as the only sources of carbon and energy has been investigated. Complete utilization of the acrylic acid present in the medium at concentrations below 0.113 g/L was observed by cultivation day 5, at a concentration of 0.225 g/L by day 7, and at a concentration of 0.45 g/L by day 17. Complete utilization of the acrylamide present in the medium at concentrations below 0.4 g/L was observed by day 5, at a concentration of 0.9 g/L by day 7, and at a concentration of 1.8 g/L by day 20. Importantly, bacterial growth did not start before complete transformation of acrylamide into acrylic acid. The rate of acrylamide transformation by growing bacteria and a cell suspension in the stationary growth phase amounted to 12.5 mg/L h at a cell concentration of 610 mg/L and 300 mg/L h, at a concentration of 1500 mg/L. A. faecalis 2 cells immobilized on BVV-22 basalt fibers and Carbopon-B-aktiv at concentrations of 3000 and 800 mg dry cells/L, respectively, transformed acrylamide at a rate of 1200 mg/L h.

The expression of two key genes of lovastatin (LOV) biosynthesis has been studied in two Aspergillus terreus strains characterized by a more than hundredfold difference in the LOV yield. As compared to the wild-type strain ATCC 20542, in the overproducing strain 43-16 significantly enhanced expression level of LOV biosynthetic genes (by 5–50 times), transcription factor lovE (by 3–20 times), and the acs gene, which encodes acetyl-CoA synthetase (by two times), was detected. To improve the efficiency of LOV biosynthesis, recombinant A. terreus strains constitutively expressing the ACS1 gene from Saccharomyce scerevisiae or the lovE gene from A. terreus have been designed by metabolic engineering methods. According to the obtained results, the expression of ACS1 in strain 43-16 results in the suppression of lovC and lovD production and a more than 25% reduction of LOV production. In the case of low-active ATCC 20542/ACS1 recombinants, the expression level of lov genes remains almost unchanged, while the expression of the endogenous asc1 gene is enhanced and the LOV yield increases by 117%. Constitutive overexpression of the lovE gene in the ATCC 20452 strain results in the increase of mRNA levels of biosynthetic lov-genes and is accompanied by a significant (6–10 times) increase in the LOV yield.

The effect of nitrogen sources in the fermentation medium and the cultivation conditions on the production of proteinases with plasmin-like and prekallikrein activation activity by micromycete Aspergillus terreus 2 was investigated. The highest secretion of proteinases was achieved when the micromycete was cultivated on a medium containing both amine- and mineral nitrogen sources at an initial pH of 5.5 and at 28°С. It was established that the extracellular micromycete proteinases are equally capable of hydrolyzing fibrin and fibrinogen.

A protein bifunctional inhibitor of endogenous α-amylase and subtilisin has been isolated from wheat grain and purified. The inhibitor specifically inactivates α-amylase isozymes with high isoelectric point values (group α-AMY1) and has almost no effect on the α-AMY2 isozymes with low isoelectric point values. This enzyme does not belong to glycoproteins and has a molecular weight of 21 kDa and an isoelectric point of 7.2. The protein displays a relatively high thermostability and pH optimum of 8.0; its inhibitory activity requires the presence of Ca²⁺ cations. The inhibition of excess α-amylase in wheat grain with a low falling number by the purified protein is studied.