Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis.

Transketolase, an enzyme of the pentose phosphate pathway, plays an important role in the functioning of mycobacteria. Using plasmid pET-19b carrying the Rv1449c gene of transketolase from Mycobacterium tuberculosis and an additional histidine tag, we isolated and purified recombinant transketolase and determined the conditions for obtaining the apoform of the protein. The Michaelis constants were evaluated for the thiamine diphosphate cofactor in the presence of magnesium and calcium ions. We found that the affinity of mycobacterial transketolase for thiamine diphosphate is by three orders of magnitude lower than that of the human enzyme. Analysis of the structural organization of the active centers of homologous enzymes showed that this difference is due to a replacement of lysine residues by less polar amino acid residues.

CRISPR Interference of Adenylate Cyclases from Mycobacterium tuberculosis.

This work describes a modification of the pRH2521 vector of the pRH2502/pRH2521 system for CRISPR-dCas9-mediated RNA interference. The modification enabled an increase in the cloning efficiency of guide RNA spacers. The ability of the modified pRH2502/pRH2521 system to suppress the transcription of certain genes was evaluated with the use of genes of Mycobacterium tuberculosis adenylate cyclases. The results revealed the limitations of the pRH2502/pRH2521 system for CRISPR interference associated with the probability of the detection of a protospacer adjacent motif (PAM) in the gene promoter region.

Expression of glyceraldehyde-3-phosphate dehydrogenase from M. tuberculosis in E. coli. Purification and characteristics of the untagged recombinant enzyme.

The goal of the present work was to produce glyceraldehyde-3-phospate dehydrogenase from M. tuberculosis in E. coli cells in soluble and catalytically active form and to elaborate a method for the purification of the recombinant enzyme. The His-tagged recombinant enzyme (Mtb-GAPDH_His) was shown to be inactive and insoluble. The untagged enzyme (Mtb-GAPDH) was catalytically active and exhibited higher solubility. Mtb-GAPDH was purified from the cell extract using ammonium sulfate fractionation and ion-exchange chromatography. The presence of glycerol was necessary for isolation of Mtb-GAPDH, presumably, to facilitate folding of the recombinant enzyme. The yield of Mtb-GAPDH constituted 1.3 mg per 10 g of the cell biomass. The specific activity of the purified Mtb-GAPDH was 55 ±â€¯5 µmol NADH/min per mg protein (pH 9.0, 22 °C) that exceeded the activity of the previously described preparation of His-tagged recombinant GAPDH from M. tuberculosis that was co-expressed with GroEL/ES chaperone by approximately 5-fold. The results suggest that the folding of the recombinant GAPDH is hindered by the His-tag, which may result in the production of insoluble protein or in isolation of the preparation with decreased specific activity.

[Whole-cell bacterial biosensors for the detection of aromatic hydrocarbons and their chlorinated derivatives].

The review summarizes the data on new directions in biosensor technologies based on whole bacterial cells. Biosensors for the monitoring of mono(poly)aromatic hydrocarbons and their chlorinated derivatives, which are constructed with genetically modified bacterial cells bearing a reporter gene fusion, are considered. The operating principle of these biosensors is based on the expression of reporter genes (luc, lux, gfp, rfp) under the control of a promoter and a regulator that specifically respond to a detected compound.

[VapC toxin inhibition as a method for prevention of the formation of resting forms of mycobacteria].

It has been shown that inactivation of the VapC toxin from the VapВС toxin-antitoxin system prevents mycobacterial cells from transitioning to an ovoid state that meets the criteria of dormancy. The results indicate a potential target for medicines that prevent the development of latent tuberculosis infection and provide a basis to obtain bacterial cells for the testing of compounds that are active towards dormant forms of mycobacteria.

[Adaptive functions of Escherichia coli polyamines in response to sublethal concentrations of antibiotics].

Escherichia coli exposure to sublethal antibiotic concentrations induced an increase in cell polyamine contents. Maximum accumulation of putrescine and spermidine in response to antibiotics-induced oxidative stress preceded the increment of cadaverine, the content of which was dependent on the rpoS expression level and reached the maximum in response to fluoroquinolones. The polyamine positive modulating effects on rpoS expression increased in the following order: cadaverine-putrescine-spermidine. The reason for cadaverine accumulation was the increase in activities of lysine decarboxylases CadA and Ldc. High cadaverine accumulation in the cells exposed to fluoroquinolones and cephalosporins resulted in the reduction of porin permeability; so it was considered as a response aimed at cell protection against antibiotic penetration into the cell. Netilmycin, unlike other antibiotics, did not substantially affect the lysine decarboxylase activity and cellular polyamine pools.

Role of polyamines in formation of multiple antibiotic resistance of Escherichia coli under stress conditions.

Under stress conditions, polyamines decreased the permeability of the outer membrane of Escherichia coli. This effect is caused by at least three mechanisms providing for an increase in the resistance to antibiotics transported through porin channels (fluoroquinolones, beta-lactams): a positive modulation of the gene micF transcription (its product antisense RNA inhibits the synthesis of porin proteins on the translational level); a positive effect on the cell content of the multiple stress resistance factor sigma(S) (it is accompanied by a decrease in the porin transport because of suppression of ompF transcription and induction of cadaverine synthesis); a direct inhibition of the transport activity of porin channels. The production of cadaverine in E. coli cells significantly increased in response to various antibiotics, and this was likely to be a manifestation of oxidative stress.

Putrescine as a modulator of the level of RNA polymerase sigma S subunit in Escherichia coli cells under acid stress.

Metabolites accumulated in the culture medium of Escherichia coli cells induce expression of the rpoS gene encoding the alternative sigmaS subunit of RNA polymerase, which controls adaptation of E. coli to acid stress during growth in glucose-mineral medium. The effect of acetate and succinate as end products of E. coli metabolism has been investigated on the levels of transcription, translation, and sigmaS protein stability. These end products mainly influenced the stability of the RNA polymerase sigmaS subunit. Under conditions of acid stress caused by acetate addition, the content of polyamines in the cells and medium decreased, whereas artificial rpoS gene switch-off by antisense RNA was accompanied by increase in polyamine level. Addition of polyamine to E. coli cells treated with acetate and especially with succinate caused a significant concentration-dependent stimulatory effect on rpoS expression. Thus, induction of the rpoS regulon depends on the combined action of the investigated metabolites determining adequate control of gene expression under conditions of acid stress. A scheme for metabolic pathways describing the role of putrescine in the maintenance of intracellular pH and polyamine pool homeostasis during E. coli adaptation to acid stress is proposed.

Role of putrescine in regulation of the sigmaS subunit of RNA polymerase in Escherichia coli cells on transition to stationary phase.

Expression of the rpoS gene encoding the sigmaS subunit of E. coli RNA polymerase was studied on transition to the stationary phase and under conditions of starvation. Polyamines, in particular putrescine, are involved in regulation of the rpoS expression by concentration-dependent stimulation at the levels of translation and stability of the protein sigmaS. The stimulatory effect of exogenous putrescine inversely depended on its content in the cell and was the most pronounced in the beginning of the exponential growth. Possible mechanisms of the polyamine effect on the rpoS expression on post-transcriptional and post-translational levels are discussed.