[Low-Molecular Thiols as a Factor Improving the Sensitivity of Escherichia coli Mutants with Impaired ADP-Heptose Synthesis to Antibiotics].

Low molecular-weight thiols as glutathione and cysteine are an important part of the cell's redox regulation system. Previously, we have shown that inactivation of ADP-heptose synthesis in Escherichia coli with a gmhA deletion induces the oxidative stress. It is accompanied by rearrangement of thiol homeostasis and increased sensitivity to antibiotics. In our study, we found that restriction of cysteine metabolism (ΔcysB and ΔcysE) and inhibition of glutathione synthesis (ΔgshAB) lead to a decrease in the sensitivity of the ΔgmhA mutant to antibiotics but not to its expected increase. At the same time, blocking of the export of cysteine (ΔeamA) or increasing import (Ptet-tcyP) into cells of the oxidized form of cysteine-cystine leads to an even greater increase in the sensitivity of gmhA-deleted cells to antibiotics. In addition, there is no correlation between the cytotoxic effect of antibiotics and the level of reactive oxygen species (ROS), the total pool of thiols, or the viability of the initial cell population. However, a correlation between the sensitivity to antibiotics and the level of oxidized glutathione in cells was found in our study. Apparently, a decrease in the content of low-molecular-weight thiols saves NADPH equivalents and limits the processes of protein redox modification. This leads to increasing of resistance of the ΔgmhA strain to antibiotics. An increase in low-molecular-weight thiols levels requires a greater expenditure of cell resources, leads to an increase in oxidized glutathione and induces to greater increase in sensitivity of the ΔgmhA strain to antibiotics.

Enhancement of the Bactericidal Effect of Antibiotics by Inhibition of Enzymes Involved in Production of Hydrogen Sulfide in Bacteria.

Counteraction of the origin and distribution of multidrug-resistant pathogens responsible for intra-hospital infections is a worldwide issue in medicine. In this brief review, we discuss the results of our recent investigations, which argue that many antibiotics, along with inactivation of their traditional biochemical targets, can induce oxidative stress (ROS production), thus resulting in increased bactericidal efficiency. As we previously showed, hydrogen sulfide, which is produced in the cells of different pathogens protects them not only against oxidative stress but also against bactericidal antibiotics. Next, we clarified the interplay of oxidative stress, cysteine metabolism, and hydrogen sulfide production. Finally, demonstrated that small molecules, which inhibit a bacterial enzyme involved in hydrogen sulfide production, potentiate bactericidal antibiotics including quinolones, beta-lactams, and aminoglycosides against bacterial pathogens in in vitro and in mouse models of infection. These inhibitors also suppress bacterial tolerance to antibiotics by disrupting the biofilm formation and substantially reducing the number of persister bacteria, which survive the antibiotic treatment. We hypothesise that agents which limit hydrogen sulfide biosynthesis are effective tools to counteract the origin and distribution of multidrug-resistant pathogens.

[Enhancement of the Bactericidal Effect of Antibiotics by Inhibition of Enzymes Involved in Production of Hydrogen Sulfide in Bacteria].

Counteraction of the origin and distribution of multidrug-resistant pathogens responsible for intra-hospital infections is a worldwide issue in medicine. In this brief review, we discuss the results of our recent investigations, which argue that many antibiotics, along with inactivation of their traditional biochemical targets, can induce oxidative stress (ROS production), thus resulting in increased bactericidal efficiency. As we previously showed, hydrogen sulfide, which is produced in the cells of different pathogens protects them not only against oxidative stress but also against bactericidal antibiotics. Next, we clarified the interplay of oxidative stress, cysteine metabolism, and hydrogen sulfide production. Finally, demonstrated that small molecules, which inhibit a bacterial enzyme involved in hydrogen sulfide production, potentiate bactericidal antibiotics including quinolones, beta-lactams, and aminoglycosides against bacterial pathogens in in vitro and in mouse models of infection. These inhibitors also suppress bacterial tolerance to antibiotics by disrupting the biofilm formation and substantially reducing the number of persister bacteria, which survive the antibiotic treatment. We hypothesise that agents which limit hydrogen sulfide biosynthesis are effective tools to counteract the origin and distribution of multidrug-resistant pathogens.

[Inactivation of Terminal Oxidase bd-I Leads to Supersensitivity of E. coli to Quinolone and Beta-Lactam Antibiotics].

In cells of Escherichia coli, terminal oxidase bd-I encoded by the cydAB gene catalyzes the reduction of O2 to water using hydroquinone as an electron donor. In addition to the cydAB operon, two other genes, cydC and cydD, encoding the heterodimeric ATP-binding cassette-type transporter are essential for the assembly of cytochrome bd-I. It was shown that inactivation of cytochrome bd-I by the introduction of cydB or cydD deletions into the E. coli chromosome leads to supersensitivity of the bacteria to antibiotics of the quinolone and beta-lactam classes. The sensitivity of these mutants to antibiotics is partially suppressed by introduction of a constitutively expressed gene katG under the control of the Ptet promoter into their genome. The increased level of hydrogen sulfide resulting from the introduction of the mstA gene, encoding 3-mercaptopyruvate sulfurtransferase, under the control of the Ptet promoter, leads to the same effect. These data demonstrate the important role of cytochrome bd-I in the defense of bacteria from oxidative stress and bactericidal antibiotics.

[Mechanisms and Origin of Bacterial Biolumenescence].

The origin of bioluminescence in living organisms was first mentioned by Charles Darwin (1859) and remains obscure despite significant success achieved over the past decades. Here we discuss the mechanisms of bacterial bioluminescence. We have the main results from structural and functional analysis of the genes of lux operons, enzymes (luciferase), and mechanisms of bioluminescence in several species of marine bacteria, which belong to three genera, Vibrio, Aliivibrio, and Photobacterium (A. fischeri, V. harveyi, P. leiognathi, and P. phosphoreum), and in terrestrial bacteria of the genus Photorhabdus (Ph. luminescens). The structure and mechanisms for the regulation of the expression of the lux operons are discussed. The fundamental characteristics of luciferase and luciferase-catalyzed reactions (stages of FMNH2 and tetradecanal oxidation, dimensional structure, as well as folding and refolding of the macromolecule) are described. We also discuss the main concepts of the origin of bacterial bioluminescence and its role in the ecology of modern marine fauna, including its involvement in the processes of detoxification of the reactive oxygen species and DNA repair, as well as the bait hypothesis.

Acadesine Triggers Non-apoptotic Death in Tumor Cells.

We studied the cytotoxicity of acadesine (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside) for tumor and normal cells of various species and tissue origin. In tumor cells, acadesine triggered non-apoptotic death; the potency of the compound to normal cells was substantially lower. Acadesine was toxic for tumor cells with multidrug resistant phenotypes caused by the transmembrane transporter Р-glycoprotein or lack of proapoptotic p53. Activity of adenosine receptors was required for acadesine-induced cell death, whereas functioning of АМР-dependent protein kinase was not required. A more pronounced cytotoxicity for tumor cells, as well as the non-canonical death mechanism(s), makes acadesine a promising candidate for antitumor therapy.

[1-butanol synthesis by Escherichia coli cells through butyryl-CoA formation by heterologous enzymes of clostridia and native enzymes of fatty acid beta-oxidation].

Anaerobic biosynthesis of 1-butanol from glucose is investigated in recombinant Escherichia coli strains which form butyryl-CoA using the heterologous enzyme complex of clostridia or as a result of a reversal in the action of native enzymes of the fatty acid beta-oxidation pathway. It was revealed that when the basic pathways of acetic and lactic acid formation are inactivated due to deletions in the ackA, pta, poxB, and ldhA genes, the efficiency of butyryl-CoA biosynthesis and its reduced product, i.e., 1-butanol, by two types of recombinant stains is comparable. The limiting factor for 1-butanol production by the obtained strains is the low substrate specificity of the basic CoA-dependent alcohol/aldehyde AdhE dehydrogenase from E. coli to butyryl-CoA. It was concluded that, in order to construct an efficient 1-butanol producer based on a model strain synthesizing butyryl-CoA as a result of a reversal in fatty acid beta-oxidation enzymes, it is necessary to provide intensive formation of acetyl-CoA and enhanced activity of alternative alcohol and aldehyde dehydrogenases in the cells of a strain.

[Butanol as a regulatory factor of ompC gene expression in E. coli cells].

The influence of butanol on the expression of ompC gene encoding synthesis of OmpC porin in the MG 1655 strain of E. coli and butanol-tolerant mutant ButR was studied. It was shown that in the case of wild bacteria, the addition of butanol to the growth medium results in an increased level of ompC transcription. However, OmpC porin is not detected in the membrane fraction of cells. ButR mutant exhibits a higher level of ompC gene expression. A direct correlation is observed between the level of OmpC porin expression and its content in the membrane fraction of ButR mutant cells. In the regulatory region of the ompC gene of the ButR mutant, three nucleotide substitutions located in the binding sites of OmpR and CpxR activator proteins were identified. It was shown that mutations in the regulatory region of the ompC gene in the ButR mutant are responsible for the decreased level of OmpC porin expression under normal growth conditions. However, these mutations lead to an increased level of OmpC porin synthesis in the presence of butanol. These data suggest an additional mechanism of ompC gene regulation with the participation of butanol as a positive transcription effector.

[Anaerobic synthesis of succinic acid by Escherichia coli strains with activated NAD+ reducing pyruvate dehydrogenase complex].

Effect of constitutive expression of the aceEF-lpdA operon genes coding for the enzymes of NAD+ reducing pyruvate dehydrogenase complex on the anaerobic production of succinic acids from glucose by recombinant Escherichia coli strains was studied. Basic producer strains were obtained by inactivation of the main pathways for synthesis of acetic and lactic acids by deletion of the genes ackA, pta, poxB, and ldhA (SGMO.1) in E. coli strain MG 1655 cells and additional introduction of the Bacillus subtilis pyruvate carboxylase (SG M0.1 [pPYC]). A constitutive expression of the genes aceEF-lpdA in derivatives of the basic strains SGM0.1 PL-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] was provided by replacing the native regulatory region of the operon with the lambda phage PL promoter. Molar yields of succinic acid in anaerobic glucose fermentation by strains SGM0.1 P(L)-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] exceeded the corresponding yields displayed by several control strains (exceeded considerably in the case of the strains with a pyruvate carboxylase activity). It is concluded that an increase in the succinic acid production by strain SGM0.1 PL-aceEF-lpdA [pPYC] as compared with the strains SGM0.1 and SGM0.1 [pPYC], which synthesize this substance in the reductive tricarboxylic acid cycle, is determined by activation of the glyoxylate shunt.

Reconstruction of Purine Metabolism in Bacillus subtilis to Obtain the Strain Producer of AICAR: A New Drug with a Wide Range of Therapeutic Applications.

AICAR is a natural compound, an analogue and precursor of adenosine. As activator of AMP-activated protein kinase (AMPK), AICAR has a broad therapeutic potential, since it normalizes the carbohydrate and lipid metabolism and inhibits the proliferation of tumor cells. The synthesis of AICAR inBacillus subtiliscells is controlled by the enzymes of purine biosynthesis; their genes constituting purine operon (pur-operon). Reconstruction of purine metabolism inB. subtiliswas performed to achieve overproduction of AICAR. For this purpose, the genepurH, which encodes formyltransferase/IMP-cyclohydrolase, an enzyme that controls the conversion of AICAR to IMP, was removed from theB. subtilisgenome, ensuring the accumulation of AICAR. An insertion inactivating the genepurRthat encodes the negative transcriptional regulator of the purine biosynthesis operon was introduced into theB.subtilischromosome in order to boost the production of AICAR; the transcription attenuator located in the leader sequence ofpur-operon was deleted. Furthermore, the expression integrative vector carrying a strong promoter of therpsFgene encoding the ribosomal protein S6 was designed. The heterologousEscherichia coligenepurFencoding the first enzyme of the biosynthesis of purines with impaired allosteric regulation, as well as the modifiedE.coligeneprsresponsible for the synthesis of the precursor of purines - phosphoribosyl pyrophosphate (PRPP) - was cloned into this vector under the control of therpsFgene promoter. The modifiedpurFandprsgenes were inserted into the chromosome of theB. subtilisstrain.B. subtilisstrain obtained by these genetic manipulations accumulates 11-13 g/L of AICAR in the culture fluid.

[The role of histidine in regulating the synthesis of purine nucleotides in Escherichia coli cells]. / Rol' gistidina v reguliatsii sinteza purinovykh nukleotidov v kletkakh Escherichia coli.

Coordination of GTP and 5-aminoimidazole-4-carboxamide riboside 5'-phosphate pools changes was studied. The CTP pool is an important component of Escherichia coli metabolism, while AICAR 5'-phosphate being one of alarmones controls the synthesis of GTP. Main attention was paid to histidine, the biosynthesis of which is connected with formation of purine nucleotides. The expression of the histidine operon and biosynthesis of histidine are shown to change the AICAR pool and help the formation of the GTP pool. The ribosomal antibiotics streptomycin and chloramphenicol may cause the temporary deficiency of GTP eliminated by the increase of alarmone AICAR pool. The latter event is concluded to cause the increase in GTP pool independent of the means of AICAR accumulation (C1-pholatedependent restriction of metabolization or, vice versa, the stimulation in the histidine biosynthesis pathway).

[Isolation of mutants with impaired retroinhibition of histidine biosynthesis]. / Poluchenie mutantov s narushennym retroingibirovaniem biosinteza gistidina.

The Escherichia coli, strain possessing purF, deoD and add mutations converts exogenous adenine into guanine nucleotides exclusively by the pathway coupled with histidine biosynthesis. When grown on adenine, this strain demonstrated sensitivity to histidine, thus making it possible to select histidine-resistant hisGR mutants with ATP-phosphoribosyltransferase desensibilized for histidine. The hisGR mutations were obtained in two his operons introduced into the his operon-sensitive E. coli strain: his operon of Salmonella typhimurium incorporated in DNA and his operon of E. coli on the F'episome. In both cases, the hisGR mutants obtained were shown to excrete histidine.

[Cloning of the gpp gene of Escherichia coli and the use of recBC, sbcB cells for inserting its mutant allele into the chromosomal structure]. / Klonirovanie gena gpp Escherichia coli i ispol'zovanie kletok recBC, sbcB dlia vvedeniia ego mutantnogo allelia v sostav khromosomy.

The gpp gene involved in the pppGpp conversion into ppGpp in Escherichia coli cells was cloned and localized within the multicopy pBR322 plasmid. Amplification of the gpp gene leads to the decline of the intracellular level of pppGpp, which implies enhanced activity of the corresponding enzyme, guanosine pentaphosphatase. To inactivate the cloned gene, a fragment of the pUC4K plasmid containing the kan gene was inserted within the gpp gene. The functional chromosomal allele of the gpp gene was replaced by its inactivated gpp::kan allele, taking advantage of homologous recombination during the transformation of recBC, sbcB cells with the intact hybrid plasmid. This procedure is accompanied by plasmid elimination and may be used for the replacement of other loci of bacterial chromosome with appropriate cloned alleles.

[Metabolic regulation of the histidine operon in Escherichia coli and Salmonella typhimurium]. / Metabolicheskaia reguliatsiia gistidinovogo operona Escherichia coli i Salmonella typhimurium.

Expression of the histidine operon in Escherichia coli cells in contrast to the one in Salmonella typhimurium is changed proportionally to cells growth rate on the different carbon sources. The specific activity of histidinol-dehydrogenase is repressed by addition of 19 amino acids both in Escherichia coli and Salmonella typhimurium independent of the growth medium used. Using of Escherichia coli and Salmonella typhimurium strains containing the heterologous histidine operons made possible to demonstrate the dependence of the histidine operon metabolic regulation to be determined by the operon itself but not by the specificity of the recipient cells. ppGpp was shown to be a positive regulator of the histidine operon expression in Escherichia coli.

[Precise mapping of the gpp gene involved in guanosine tetraphosphate synthesis and ilvC-gpp deletion in the region of the Escherichia coli chromosome]. / Tochnoe kartirovanie gena gpp, uchastvuiushchego v sinteze guanozintetrafosfata, i poluchenie deletsii ilvC-gpp v oblasti khromosomy Escherichia coli.

Using the set of transducing lambda phages the gpp gene, responsible for pppGpp to ppGpp conversion, was localized between rep and trxA genes on 85 min of the Escherichia coli genetic map. Taking advantage of the Tn10 transposon inserted into the adjacent ilvY locus, we deleted the region of E. coli chromosome covering ilvC, rep and gpp genes. The metabolism of (p)ppGpp in the deletion-containing cells confirms that the product of the gpp gene, guanosine pentaphosphatase, is not the only enzyme, responsible for pppGpp degradation and ppGpp synthesis.

[Transcription of ribosomal protein genes rplKAJL and RNA-polymerase genes rpoBC in Escherichia coli cells: metabolic regulation of attenuation and the effect of rifampicin]. / Transkriptsiia genov rplKAJL ribosomnykh belkov i genov rpoBC RNK-polimerazy v kletkakh Escherichia coli: metabolicheskaia reguliatsiia attenuatsii i deistvie rifampitsina.

The E. coli genes rplKAJL specifying ribosomal proteins L11, L1, L10, L7/L12 are co-transcribed with the genes rpoBC encoding the beta- and beta'-subunits of RNA polymerase, but are separated by the site of attenuation. The efficiency of attenuation within rplKAJL-rpoBC operon was determined as a ratio of rplKAJL transcription frequency to the same of rpoBC genes. The efficiency of attenuation was found to be a growth-rate dependent parameter of E. coli cells. At growth rate 1.2 doublings per hour the attenuation is rare and simultaneously increases with the increase in the growth rate (at mu = 1.2 doublings per hour the efficiency of attenuation is 4). Rifampicin (10-30 micrograms/ml) inhibits the transcription of both rplKAJL and rpoBC genes in fast growing cells but paradoxically stimulates their transcription in slowly growing cells. The stimulatory effect of rifampicin on rplKAJL genes transcription is supposed to be based on its ability to repress the ppGpp synthesis. The possible role of ppGpp in the regulation of transcription attenuation in rplKAJL-rpoBC operon is discussed.