A synthetic strategy for the preparation of spiro[indoline-3,2'-pyrrolidine] derivatives has been developed, featuring a two-step sequence consisting of the reaction of 2-arylindoles with α,β-unsaturated ketones, followed by Fe(II)-catalyzed spirocyclization of readily accessible oxime acetates. The method exhibits a broad substrate scope and good functional group tolerance. The synthesized spirocyclic compounds showed no significant antimicrobial activity.

The present article is devoted to the method for comparative analysis of sliding colonies. This new method includes measuring the density of bacterial cells in a monolayer. Using this method, we studied the effect of biogenic polyamines and alarmone synthetase RelMsm on Mycobacterium smegmatis sliding motility and showed a multidirectional effect of spermidine and spermine on sliding, as well as an increase in sliding motility by a strain with a relMsm deletion. It is also shown that there is no dependence between the sliding of mycobacteria and their hydrophobicity.

The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm formation. The (p)ppGpp alarmones are synthesized by proteins of the RelA/SpoT homolog (RSH) superfamily, including long bifunctional RSH proteins and small alarmone synthetases. Here, we investigated enzyme kinetics and dose-dependent enzyme inhibition to elucidate the mechanism of 4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid (DMNP) action on the (p)ppGpp synthetases RelMsm and RelZ from Mycolicibacterium smegmatis and RelMtb from Mycobacterium tuberculosis. DMNP was found to inhibit the activity of RelMtb. According to the enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor of RelMsm and RelZ. Based on the results of molecular docking, the DMNP-binding site is located in the proximity of the synthetase domain active site. This study might help in the development of alarmone synthetase inhibitors, which includes relacin and its derivatives, as well as DMNP - a synthetic analog of the marine coral metabolite erogorgiaene. Unlike conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the bacterial stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may address challenges associated with antimicrobial resistance and bacterial persistence.

The article is devoted to construction of an Escherichia coli strain with overexpression of the cadA gene encoding lysine decarboxylase, as well as to assessment of its ability to convert lysine to cadaverine depending on the pH of the medium. The overexpressing strain was constructed on the base of E. coli BL21DE3 strain transformed with the pET19b plasmid carrying the cadA gene from E. coli MC4100. Bacteria were grown in 5 mL of LB broth at 37 °C without stirring, overexpression of cadA was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside (at OD600 = 0.6), after 2 hours the cells were washed and transferred to a medium with pH 7.4 or 4.0 with the addition 5 g/L L-lysine hydrochloride. The amount of synthesized cadaverine was quantified by TLC with preliminary derivatization with dansyl chloride. The maximum rate of bioconversion was observed when cells overexpressing cadA were cultivated in a medium with pH 4.0 (2.8 ± 0.7 mmol cadaverine/g dry weight per hour). The rate of bioconversion under basal conditions (pH 7.4, basal expression) was 6.8 times lower. The final concentration of cadaverine when cultivated in a medium with pH 7.4 and/or in the absence of overexpression of the cadA gene was 0.8-1 mM; under conditions of overexpression of the gene, up to 1.2 mM of cadaverine accumulated in a neutral medium, and 1.8 mM in an acidic medium.

Polyamines and indole are small regulatory molecules that are involved in the adaptation to stress in bacteria, including the regulation of gene expression. Genes, the translation of which is under the regulatory effects of polyamines, form the polyamine modulon. Previously, we showed that polyamines upregulated the transcription of genes encoding the ribosome hibernation factors RMF, RaiA, SRA, EttA and RsfS in Escherichia coli. At the same time, indole affected the expression at the transcriptional level of only the raiA and rmf genes. Ribosome hibernation factors reversibly inhibit translation under stress conditions, including exposure to antibiotics, to avoid resource waste and to conserve ribosomes for a quick restoration of their functions when favorable conditions occur. In this work, we have studied the influence of indole on the expression of the raiA and rmf genes at the translational level and regulatory effects of the polyamines putrescine, cadaverine and spermidine on the translation of the rmf, raiA, sra, ettA and rsfS genes. We have analyzed the mRNA primary structures of the studied genes and the predicted mRNA secondary structures obtained by using the RNAfold program for the availability of polyamine modulon features. We have found that all of the studied genes contain specific features typical of the polyamine modulon. Furthermore, to investigate the influence of polyamines and indole on the translation of the studied genes, we have constructed the translational reporter lacZ-fusions by using the pRS552/λRS45 system. According to the results obtained, polyamines upregulated the expression of the rmf, raiA and sra genes, the highest expression of which was observed at the stationary phase, but did not affect the translation of the ettA and rsfS genes, the highest expression of which took place during the exponential phase. The stimulatory effects were polyamine-specific and observed at the stationary phase, when bacteria are under multiple stresses. In addition, the data obtained demonstrated that indole significantly inhibited translation of the raiA and rmf genes, despite the stimulatory effect on their transcription. This can suggest the activity of a posttranscriptional regulatory mechanism of indole on gene expression.

Bacterial stress adaptive response is formed due to changes in the cell gene expression profile in response to alterations in environmental conditions through the functioning of regulatory networks. The mutual influence of network signaling molecules represented by cells’ natural metabolites, including indole and second messengers (p)ppGpp and cAMP, is hitherto not well understood, being the aim of this study. E. coli parent strain BW25141 ((p)ppGpp+) and deletion knockout BW25141ΔrelAΔspoT which is unable to synthesize (p)ppGpp ((p)ppGpp0) were cultivated in M9 medium supplemented with different glucose concentrations (5.6 and 22.2 mM) in the presence of tryptophan as a substrate for indole synthesis and in its absence. The glucose content was determined with the glucose oxidase method; the indole content, by means of HPLC; and the cAMP concentration, by ELISA. The onset of an increase in initially low intracellular cAMP content coincided with the depletion of glucose in the medium. Maximum cAMP accumulation in the cells was proportional to the concentration of initially added glucose. At the same time, the (p)ppGpp0 mutant showed a decrease in maximum cAMP levels compared to the (p)ppGpp+ parent, which was the most pronounced in the medium with 22.2 mM glucose. So, (p)ppGpp was able to positively regulate cAMP formation. The promoter of the tryptophanase operon responsible for indole biosynthesis is known to be under the positive control of catabolic repression. Therefore, in the cells of the (p)ppGpp+ strain grown in the tryptophan-free medium that were characterized by a low rate of spontaneous indole formation, its synthesis significantly increased in response to the rising cAMP level just after glucose depletion. However, this was not observed in the (p)ppGpp0 mutant cells with reduced cAMP accumulation. When tryptophan was added to the medium, both of these strains demonstrated high indole production, which was accompanied by a decrease in cAMP accumulation compared to the tryptophan-free control. Thus, under glucose depletion, (p)ppGpp can positively regulate the accumulation of both cAMP and indole, while the latter, in its turn, has a negative effect on cAMP formation.

The treatment of bacterial infections with antibiotics is significantly complicated because of the adaptive mechanisms employed by bacteria. Chronic and recurrent infections are often linked to bacterial persis-tence, biofilm formation, and antibiotic tolerance. These processes result in reduced metabolic activity, rendering bacteria insensitive to conventional antibiotics that primarily target actively growing cells. The stringent re-sponse, regulated by (p)ppGpp alarmone molecules, serves as a stress adaptation mechanism. It is conserved across numerous bacterial species and plays an important role in long-term survival under nutrient-depleted con-ditions. (p)ppGpp alarmones also play a significant role in bacterial persistence and the formation of biofilms. The pursuit of novel antibacterial agents that specifically target (p)ppGpp synthesis, thereby inhibiting the strin-gent response, presents a promising strategy in the battle against bacterial infections. In this context, alarmone synthetase inhibitors emerge as promising candidates for clinical application, as they have demonstrated their effectiveness in suppressing bacterial survival mechanisms, inhibiting biofilm formation, and reducing antibiotic tolerance and bacterial persistence.

The effect of redox-cycling methyl viologen, which causes the production of superoxide and formation of oxidative stress in bacterial cells, on the expression of the cadA and ldcC genes was studied. Gene expression was assessed using Escherichia coli strains bearing reporter gene fusions of the promoters of the studied genes with the structural part of the lacZ gene; expression values are given in Miller units. Bacteria were grown in batch cultures in LB broth without agitation or with agitation at 100 r.p.m. We have shown that exposure to methyl viologen, which leads to the induction of the nfo gene included in the soxRS regulon of oxidative defense (a long-term increase in the expression level by 10 times), led to a slight and short-term increase in the expression of the cadA and ldcC genes (by a maximum of 1.4 times, no longer than 1 h). An increase in the strength of oxidative stress due to elevated agitation/aeration had no effect on the expression of the cadA and ldcC genes. Methyl viologen at the concentrations used (1-100 μg/mL) did not affect the number of colony-forming units in the culture. Thus, non-lethal superoxide stress caused by exposure of bacteria to methyl viologen had little effect on the expression of the ldcC and cadA genes compared to the genes included in the soxRS regulon, which were strongly induced under these conditions.

The treatment of many bacterial diseases remains a significant problem due to the increasing antibiotic resistance of their infectious agents. Among others, this is related to Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA) and Mycobacterium tuberculosis. In the present article, we report on antibacterial compounds with activity against both S. aureus and MRSA. A straightforward approach to 2-(1H-indol-3-yl)quinazolin-4(3H)-one and their analogues was developed. Their structural and functional relationships were also considered. The antimicrobial activity of the synthesized compounds against Mycobacterium tuberculosis H37Rv, S. aureus ATCC 25923, MRSA ATCC 43300, Candida albicans ATCC 10231, and their role in the inhibition of the biofilm formation of S. aureus were reported. 2-(5-Iodo-1H-indol-3-yl)quinazolin-4(3H)-one (3k) showed a low minimum inhibitory concentration (MIC) of 0.98 μg/mL against MRSA. The synthesized compounds were assessed via molecular docking for their ability to bind long RSH (RelA/SpoT homolog) proteins using mycobacterial and streptococcal (p)ppGpp synthetase structures as models. The cytotoxic activity of some synthesized compounds was studied. Compounds 3c, f, g, k, r, and 3z displayed significant antiproliferative activities against all the cancer cell lines tested. Indolylquinazolinones 3b, 3e, and 3g showed a preferential suppression of the growth of rapidly dividing A549 cells compared to slower growing fibroblasts of non-tumor etiology.

The treatment of many bacterial and fungal infections remains a problem due to increasing antibiotic resistance and biofilm formation by pathogens. In the present article, a methodology for the chemoselective synthesis of 2-(1H-indol-3-yl)-1H-benzo[d]imidazole derivatives is presented. We report on the antimicrobial activity of synthesized 2-(1H-indol-3-yl)-1H-benzo[d]imidazoles with significant activity against Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 43300 (MRSA), Mycobacterium smegmatis (mc(2)155/ATCC 700084), and Candida albicans ATCC 10231. High activity against staphylococci was shown by indolylbenzo[d]imidazoles 3ao and 3aq (minimum inhibitory concentration (MIC) < 1 µg/mL) and 3aa and 3ad (MIC 3.9–7.8 µg/mL). A low MIC was demonstrated by 2-(1H-indol-3-yl)-1-methyl-1H-benzo[d]imidazole (3ag) against M. smegmatis and against C. albicans (3.9 µg/mL and 3.9 µg/mL, respectively). 2-(5-Bromo-1H-indol-3-yl)-6,7-dimethyl-1H-benzo[d]imidazole (3aq) showed a low MIC of 3.9 µg/mL against C. albicans. Compounds 3aa, 3ad, 3ao, and 3aq exhibited excellent antibiofilm activity, inhibiting biofilm formation and killing cells in mature biofilms. Molecular docking analysis identified three potential interaction models for the investigated compounds, implicating (p)ppGpp synthetases/hydrolases, FtsZ proteins, or pyruvate kinases in their antibacterial action mechanism.

Suppression of the stringent response is a promising strategy for the treatment of persistent bacterial infections. A novel class of compounds having a mechanism of action based on alarmone synthetase inhibition and suppressing the synthesis of (p)ppGpp alarmones in bacteria may provide a more effective treatment for latent infections and resolve problems associated with bacterial persistence. Conventional antibiotics primarily act on actively growing bacteria, but they are inactive against persister cells with a slowed metabolism. Alarmone synthetase inhibitors have antipersister properties that may enhance conventional antibiotics’ antibacterial action. Two groups of RSH proteins are responsible for the synthesis of alarmones: long RelA/SpoT homologs and small alarmone synthetases. Many species of bacteria possess both types of enzymes. Despite the fact that a number of inhibitors of bifunctional long synthetases/hydrolases have been described to date, their properties with respect to monofunctional small alarmone synthetases have been studied poorly. This study investigated the effect of the alarmone synthetase inhibitor DMNP on the purified RelZ small alarmone synthetase protein from Mycolicibacterium smegmatis .

(p)ppGpp synthetase inhibitors constitute a new promising class of antibacterial compounds that target bacterial defense mechanisms. These compounds block the stringent response in bacteria, which regulates bacterial survival in stress conditions. The stringent response is mediated by (p)ppGpp alarmones, intracellular regulatory molecules synthesized and hydrolyzed by RSH superfamily enzymes. The majority of bacterial species have one or more of these enzymes encoded in their genomes, including the RelMtb enzyme in Mycobacterium tuberculosis. (p)ppGpp synthesis is associated with multiple cellular processes including long-term survival in nutrient limitation conditions, chronic infection, biofilm formation, antibiotic tolerance, and bacterial persistence. Inhibition of this evolutionarily conserved pathway by (p)ppGpp synthetase inhibitors can have high pharmaceutical potential in the treatment of recalcitrant bacterial infections, such as TB. In this study, we apply molecular docking methods to assess the ability of known (p)ppGpp synthetase inhibitors to bind RelMtb enzyme active sites.

In this work we constructed the translational stpA::lacZ reporter fusion. The expression of the stpA gene was studied on the translational level in Escherichia coli cells grown in rich and defined media upon the standard conditions, as well as upon both the osmotic shock and the nitrogen starvation. The expression of stpA was higher and more stable in defined medium, as compared to the rich one. This difference was more remarkable at the stationary phase. The expression of stpA decreased dramatically upon the osmotic shock and the nitrogen starvation. Furthermore, here we have investigated the polyamine effects on the stpA expression on the transcriptional, translational and posttranslational levels. The stpA expression was mostly upregulated by putrescine. The stimulation of the gene expression on the transcriptional level occurred primarily at the exponential phase, whereas the translational stpA stimulation was dominated at the exponential phase and, to a greater extent, during the cell transition to the stationary phase. The stpA expression on the posttranslational level was primarily affected by putrescine at the exponential and stationary phase. The effects of cadaverine and spermidine were less noticeable.

Background. Some stress responses contribute to the formation of bacterial antibiotic resistance, including the soxRS oxidative defense regulon. Elevation of reactive oxygen species production and oxidative stress was detected in bacterial cells exposed to various environmental stresses. It can be supposed that a stress-mediated increase in the level of reactive oxygen species will activate the expression of the soxRS regulon genes, which may provide pre-adaptation to antibiotics.The aim. To study changes in the expression of soxRS regulon genes in Escherichia coli cells exposed to NaCl, acetic acid, and heating.Materials and methods. Gene expression was measured in cells bearing reporter gene fusions (soxS::lacZ, nfo::lacZ). An overnight broth culture was diluted in fresh LB broth to OD600 = 0.1 and cultivated at 37 °C without stirring until OD600 = 0.3, then the stressors were applied.Results. Exposure to NaCl and acetic acid activated the expression of soxRS regulon genes, while heating caused a decrease in gene expression. An increase in the expression level was observed in cells subjected to stresses of low intensity (which did not cause a decrease in the number of colony-forming units (CFU) by the 4th hour of exposure compared to the beginning of the stress exposure) and medium intensity (which caused a 10-fold decrease in the number of CFU), whereas high-intensity stresses (which caused a decrease in the number of CFU by more than 10 times), regardless of their nature, were accompanied by a decrease in the expression of the soxRS regulon genes.Conclusion. Under the conditions studied, only the osmotic stress caused by the addition of NaCl was accompanied by a significant activation of the soxRS regulon genes. Sublethal exposure to NaCl, causing an increase in the expression of soxRS regulon genes by 2–2.5 times, may provide pre-adaptation of bacteria to the factors that this regulon is aimed at counteracting, including antibacterial drugs.

RaiA is one of the main ribosome hibernation factors in Escherichia coli. Like other ribosome hibernation factors, this protein reversibly inhibits translation under stress conditions. According to published data, being induced by indole, RaiA is involved in bacterial persistence, which is considered to play important role in the recalcitrance of chronic infections to antibiotics. Previously, we showed that the raiA expression on the transcriptional level is stimulated by polyamines, in addition to indole. In this work, we investigated the influence of polyamines on the raiA expression on the translational level. We obtained the predicted secondary structures of raiA mRNA, the analysis of which showed the presence of the bulged-out region in the initiation site with a high probability. This may be a sign of gene involvement in the polyamine modulon. We constructed translational raiA::lacZ reporter fusion. Using this genetic construct, we studied the effects of polyamines on the raiA expression through an addition of putrescine, cadaverine or spermidine at concentrations of 1 mM and 2 mM. According to the results, the raiA expression is primarily stimulated by cadaverine at the stationary phase.

Nowadays, sliding is the least investigated mode of bacterial motility. Sliding is a process of passive movement on the surface of semi-liquid mediums which was originally described for mycobacteria and other bacterial species deprived of the organelles specialized for movement. Some mycobacteria are able to colonize surfaces, including tissues of macro-organisms, using glycopeptidolipids localized in the cell envelope for this aim. This is a serious problem for effective therapy of mycobacteriosis caused by nontuberculosis mycobacteria. Furthermore, animal tissues contain biogenic polyamines, which can increase tolerance of microorganisms to stresses, including antibiotics, and modulate cell motility. Therefore, studying mutual effects of biogenic polyamines and antibiotics on the expansion of mycobacteria is important for medicine. Mycobacterial strains, including the parent Mycolicibacterium smegmatis mc2155 and strains containing single (ΔrelMsm) or double (ΔrelMsmΔrelZ) deletions, were used as the objects of this study. The content of glycopeptidolipids was determined using thin layer chromatography. Sliding motility was assessed by measuring the area of the sliding colony. The effectiveness of antibiotics was measured by comparison of the areas of sliding colonies in the presence of comparable concentrations of antibiotics. The polyamines spermidine and spermine had different effects on the sliding of mycobacteria through an increase or decrease in the colony areas. At the same time, polyamines had neither bactericidal nor bacteriostatic effects. The polyamines contained in the medium decreased the bactericidal effects of the antibiotics streptomycin or isoniazid, but enhanced the effects of DMNP, a synthetic analogue of the natural antibiotic erogorgiaene. Rifampicin was the most effective of all antibiotics investigated here. Moreover, we found that glycopeptidolipids are, apparently, not the only regulators of mycobacterial sliding.

Background. Indole and polyamines are involved in the regulation of physiological processes in bacteria associated with adaptation to stress, biofilm formation, antibiotic tolerance, and bacterial persistence. However, the molecular targets and mechanisms of action of these metabolites are still poorly understood. In this work, we studied the effect of polyamines and indole on the expression of such genes as: rpoS, relA, and spoT, encoding regulators of the general stress responses and starvation; hns and stpA, encoding global regulators of gene expression; rmf, yqjD, hpf, raiA, rsfS, sra, ettA, encoding ribosome hibernation factors.The aim. To study the regulatory effects of polyamines and indole on the expression of these genes, which are responsible for the adaptation of Escherichia coli to stress.Materials and methods. We used strains of E. coli in this study. The amount of polyamines was studied by thin layer chromatography. The indole concentration was determined by high performance liquid chromatography. Gene expression was studied using real-time RT-PCR.Results. The addition of polyamines putrescine, cadaverine and spermidine to the medium stimulated the expression of all the studied genes. The maximal stimulation was observed at the stationary phase mostly. Putrescine and spermidine had the most significant effect. At 24 h of cultivation, an equimolar conversion of exogenous tryptophan into indole was showed. At this time, the expression of two genes – rmf and raiA – increased.Conclusions. We have shown that polyamines upregulate the expression of all the studied genes at the transcriptional level. The stimulating effect is specific for the phase of the batch culture and the type of polyamine. Indole has a positive effect on the expression of the rmf and raiA genes.

Signaling molecules such as indole (product of tryptophan catabolism) and (p)ppGpp (stringent response regulator) are involved in regulation of physiological processes in bacterial cells aimed to adapt to antibiotics and stresses. However, question of existence of relationship between the stringent response and indole signaling requires more detailed investigation.The aim. To study effect of stringent response regulator (p)ppGpp on indole production in Escherichia coli depending on glucose content.Materials and methods. In this work, we studied the dynamics of indole accumulation in batch cultures of parent E. coli BW25141 ((p)ppGpp+ strain) and deletion mutant BW25141∆relA∆spoT ((p)ppGpp0 strain) in glucose-mineral tryptophan-free M9 medium, as well as with 2 mM tryptophan addition. In order to study effect of starvation stress on bacterial cell ability to synthesize indole, we used a model of growth limitation by carbon substrate at two glucose concentrations, 0.1 % and 0.4 %.Results. We have shown here that (p)ppGpp absence in E. coli cells reduces their ability to produce indole in the tryptophan-free medium and significantly slows down the rate of its accumulation in the tryptophan-containing one. Low glucose concentration (0.1 %) leads to decrease in indole production by (p)ppGpp+ cells in the tryptophan-free medium. The presence of indole synthesis precursor, tryptophan, in growth medium, on the contrary, increases the production of indole at lower glucose concentration in both (p)ppGpp+ and (p)ppGpp0 strains demonstrating direct dependence of delay time for onset of indole formation on glucose content, which is more pronounced in the culture of deletion mutant unable of synthesizing (p) ppGpp. The data obtained can be interpreted as result of complex regulatory effect of catabolic repression and the stringent response caused by alarmone (p)ppGpp action on expression level of tnaCAB operon responsible for indole biosynthesis.

This review concerns the mechanisms of bacterial cell survival based on the induction of hibernation factors (HFs) that suppress protein synthesis via the inhibition of ribosomal functions. These factors include ribosome-binding proteins that inhibit 70S ribosomes (RaiA), form their inactive 100S dimers (RMF and HPF), or act at different stages of the translation cycle (RsfS, YqjD, SRA, and EttA). In some cells of the population, HFs cause the formation of a dormant state, which is characterized by a low rate of metabolic processes. This results in the tolerance of these cells and, as a consequence, the ability to persist in the presence of antibiotics and under stress conditions. The role of metabolic factors (polyamines and indoles) in the regulation of the expression levels of hibernation genes is discussed.

This study demonstrated the role of cadaverine in the adaptation of Escherichia coli to hydrogen peroxide. A dose-dependent increase in the expression of the ldcC and cadA genes that encode cadaverine-synthesizing enzymes was shown in cells exposed to exogenous hydrogen peroxide. An inverse relationship between bacterial sensitivity and the level of intracellular cadaverine at the time of hydrogen peroxide addition was found. Sensitivity to hydrogen peroxide also depended on the ability to synthesize cadaverine (via the ldcC and cadA genes). In particular, the death rate of cells that are incapable of synthesizing cadaverine was higher compared to cadaverine-producing cells. Sensitivity to hydrogen peroxide increased in a series of isogenic strains with the genotype: wild type < ΔcadA < ΔldcC < ΔldcCΔcadA. The role of the accumulation of reactive oxygen species, including hydrogen peroxide, in the activation of the ldcC and cadA genes in cells exposed to the antibiotic levofloxacin was also shown.