Vol 55, No 6 (2019)

Persistence is a phenomenon in which bacteria avoid the lethal effects of antibiotics due to their transition to a physiological state, which enables tolerance to antibacterial drugs. Among the factors that contribute to persistence, a special role belongs to toxin–antitoxin (TA) systems, which are involved in the regulation of a wide range of physiological processes and have a significant effect on the results of antibiotic therapy. This review examines the mechanisms of the participation of TA systems in the formation of persistent bacteria, as well as the clinical importance of the phenomenon of persistence and methods for the control of phenotypically tolerant cells via regulation of the activity level of TA modules.

At biosynthetic production of hydrophobic recombinant proteins there is often a problem of their aggregation in so-called inclusion bodies, after which these proteins are difficult to renature. Obtaining such proteins in soluble forms is a formidable obstacle on the way of using them. One of possible solutions for this problem is the creation of fusion constructs with a leading protein. Previously, we have developed a system for biosynthetic production of insoluble hydrophobic proteins as a part of a fusion construct with a leader based on chaperone GroEL apical domain (GrAD, GroEL Apical Domain). Expressed as a part of a fusion with GrAD, two initially insoluble proteins were successfully obtained in soluble form. Still, such a system may have limitations in use, because supporting hydrophobic proteins in soluble state requires their interaction with GrAD substrate binding surface, which implies correct mutual orientation of proteins constituent parts of the construct, and for some target proteins such interaction may be sterically impeded. To enlarge the capability of using GrAD, the strategy of making permutations was used, which consists in linking original GrAD N- and C-termini with a linker and creating new N- and C-termini situated in closer proximity to the GrAD substrate binding surface. This work describes production and the study of physico-chemical properties of two permutated GrAD variants intended to be used as leaders in fusion constructs.

Solvent-tolerant lipase produced by Bacillus sp. DM9K3 has been isolated from the hypersaline area, White Rann of Kachchh, Gujarat, India. The strain initially showed lipase activity of 11.1 U/mL in a basal medium which increased to 52.0 U/mL under optimized culture conditions. Bacillus sp. DM9K3 exhibited stability at 7% salinity, pH 9.0 and 50°C. The extracellular lipase was partially purified by acetone precipitation followed by DEAE-cellulose resulting in 39-fold purification with 40% yield. Metals ions such as Mg²⁺, Ca²⁺ and K⁺ showed enhanced enzyme activity. EDTA did not have a significant effect on activity suggesting that lipase is not metalloenzyme. The lipase under study showed the highest activity when palmitate (C16) was used as a substrate and was also highly stable in organic solvents such as cyclooctane and benzene. The partially purified enzyme was immobilized for increasing the efficiency of the ethyl caprylate (an orange flavored ester) synthesis in the presence of cyclooctane. Additionally, lipase of Bacillus sp. DM9K3 was explored for biodegradation of polycaprolactone microspheres and showed promising results for potential applications in drug delivery system.

Ectoine is an osmoprotector that is synthesized by many halophilic and halotolerant bacteria in response to osmotic stress. The halotolerant methanotroph Methylomicrobium alcaliphilum 20Z was genetically modified to produce ectoine derivative hydroxyectoine by introduction of the gene encoding ectoine hydroxylase EctD from Pseudomonas stutzeri DSM5190^T. In the cells of the methanotroph transformed by the plasmid pMHA200 carrying ectD under control of either constitutive Pmxa promoter of methanol dehydrogenase or Pect promoter of the native ectABCask operon, most of intracellular ectoine was converted to hydroxyectoine. Cells of the methanotroph expressing the chromosomally located ectD gene accumulated ectoine and hydroxyectoine in almost equal ratio. Here we first showed that M. alcaliphilum 20Z can be appropriate strain to design hydroxyectoine producer from methane or methanol.

The filamentous fungus Aspergillus terreus is the main industrial producer of a cholesterol-lowering drug, lovastatin, and simvastatin obtained on its basis. The biosynthesis of lovastatin in A. terreus is under the control of two major positive regulators, the LovE pathway-specific regulator and LaeA global regulator of the secondary metabolism of fungi. It is shown that laeA expression can be negatively regulated by LovE, the Zn₂Cys₆ transcription factor of lovastatin biosynthesis. The overexpression of lovE under the control of the gpdA constitutive promoter from Aspergillus nidulans, which results in a 10- to 30-fold increase in lovastatin production is accompanied by a decrease in laeA expression in the fermentation process. The observed negative interrelationship between the LovE and LaeA regulators consists of the downregulation of lov resistance and transport genes in A. terreus with an additional copy of lovE and phenotypically manifests itself as polyamine toxicity for lovastatin production. Polyamine addition into the medium in the course of fermentation of A. terreus OE::lovE leads to a decrease in the lovastatin production by 30–40% and inhibited the expression of the lov and laeA genes.

The ability of Azotobacter agile 12 to produce the polymers alginate (ALG) and poly(3-hydroxybutyrate) (PHB) and their physicochemical properties is studied in the work. It was shown that the strain A. agile 12 produces ALG and PHB simultaneously, thought predominantly ALG, under the conditions of high aeration levels. Infrared spectroscopy of bacterial ALG showed a prevalence of mannuronic over guluronic residues (M/G = 70/30) in the polymer chain and the presence of acetyl groups on the mannuronic monomers. No acetyl groups were found in seaweed ALG. Infrared spectroscopy showed that PHB is characterized by an absorption band in the region 1760 cm^–1. The chemical nature of PHB as a homopolymer consisting of resides of 3-hydroxybutyric acid was confirmed by ¹H-NMR analysis. The thermal behavior of bacterial ALG by thermogravimetric analysis is characterized by three stages (dehydration, first decomposition, and second decomposition). In contrast to ALG, PHB had only one stage of decomposition with a prior very low mass loss (1.25%) at the dehydration stage. The water absorption test showed that bacterial ALG has a higher capacity for water absorption in comparison with seaweed ALG, whereas PHB is a hydrophobic polymer. Thus, PHB and ALG synthesized simultaneously by A. agile 12 differ in their physicochemical properties: PHB is a hydrophobic, thermoplastic, mechanically strong polyester, while alginate is hydrophilic, hydrogel-forming, temperature unstable, elastic and mechanically destructible polysaccharide.

An efficient recombinant strain of the methylotrophic yeast Pichia pastoris was obtained; it produces active phospholipase A2 (PLA2) in a high yield. As a result of three stages of purification, a preparation of ~90% purity was obtained. The results of the evaluation of the reproducibility of chromatographic separation of PLA2 give grounds to consider the developed protocol acceptable for scaling; isolated phospholipase A2 can be used for industrial applications.

A simple HPLC technique for the quantitative determination of phytotoxins (stagonolide A and herbarumin I) in liquid culture of the fungus Stagonospora cirsii S-47 has been developed. Some examples of its application are given. The technique was tested in a study of effects of the concentration of refined sunflower oil and the cultivation duration on the production level of these toxins. At an oil concentration of 0.5% or higher in modified Czapek medium with vitamins, the accumulation of S. cirsii biomass and the concentration of stagonolide A increased, while the concentration of herbarumin I decreased. Maximal amounts of both phytotoxins were obtained on the tenth day of submerged S. cirsii cultivation in flasks. When S. cirsii was grown in the same medium supplied with 1% of oil in a laboratory bioreactor, the maximal concentration of stagonolide A (116 mg/L) in the liquid culture was achieved after 5 days of fermentation followed by its considerable reduction after 6 days, while after 7 days of the submerged growth the maximal content of herbarumin I (116 mg/L) increased sharply. The technique of the phytotoxins analysis was used to monitor them during their isolation, as well as for preparative HPLC.

Seven artificial mycobacterial antigens have been synthesized via the conjugation of synthetic carbohydrate epitopes of M. leprae phenolic glycolipid (PGL-1) and M. tuberculosis lipoarabinomannan (LAM) with bovine serum albumin (BSA). Each antigen has a different number of carbohydrate epitopes with a different structure and input sequence of the sugar residues. According to the serum reactivity, only three synthesized antigens can be useful for serodiagnosis of mycobacteriosis: (1) the antigen based on a disaccharide derivative of PGL-1: (DMG–Rha-AEP)-[BSA]; (2) the antigen based on a disaccharide fragment of PGL-1 and a hexasaccharide fragment of LAM: (DMG–Rha-AEP)-[BSA]–(Ara6-EG6); (3) the antigen based on the LAM diarabinofuranosyl fragment (Ara2-AEP)–[BSA].