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1.
Biomolecules ; 11(6)2021 05 29.
Artículo en Inglés | MEDLINE | ID: mdl-34072355

RESUMEN

A broad spectrum of volatile organic compounds' (VOCs') biological activities has attracted significant scientific interest, but their mechanisms of action remain little understood. The mechanism of action of two VOCs-the cyclic monoterpenes (-)-limonene and (+)-α-pinene-on bacteria was studied in this work. We used genetically engineered Escherichia coli bioluminescent strains harboring stress-responsive promoters (responsive to oxidative stress, DNA damage, SOS response, protein damage, heatshock, membrane damage) fused to the luxCDABE genes of Photorhabdus luminescens. We showed that (-)-limonene induces the PkatG and PsoxS promoters due to the formation of reactive oxygen species and, as a result, causes damage to DNA (SOSresponse), proteins (heat shock), and membrane (increases its permeability). The experimental data indicate that the action of (-)-limonene at high concentrations and prolonged incubation time makes degrading processes in cells irreversible. The effect of (+)-α-pinene is much weaker: it induces only heat shock in the bacteria. Moreover, we showed for the first time that (-)-limonene completely inhibits the DnaKJE-ClpB bichaperone-dependent refolding of heat-inactivated bacterial luciferase in both E. coli wild type and mutant ΔibpB strains. (+)-α-Pinene partially inhibits refolding only in ΔibpB mutant strain.


Asunto(s)
Proteínas Bacterianas , Monoterpenos Bicíclicos , Daño del ADN , ADN Bacteriano , Escherichia coli K12 , Limoneno , Respuesta SOS en Genética/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Monoterpenos Bicíclicos/química , Monoterpenos Bicíclicos/metabolismo , Monoterpenos Bicíclicos/farmacología , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Limoneno/química , Limoneno/metabolismo , Limoneno/farmacología , Photorhabdus/genética
2.
Bioorg Med Chem ; 28(7): 115378, 2020 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-32089391

RESUMEN

A set of AT-specific fluorescent dimeric bisbenzimidazoles DBPA(n) with linkers of different lengths bound to DNA in the minor groove were synthesized and their genetic, virological, and biochemical studies were performed. The DBPA(n) were shown to be effective inhibitors of the histon-like protein H-NS, a regulator of the DNA transcription factor, as well as of the Aliivibrio logei Quorum Sensing regulatory system in E. coli cells. Their antiviral activity was tested in model cell lines infected with herpes simplex virus type I. Also, it was found that DBPA(n) could inhibit catalytic activities of HIV-1 integrase at low micromolar concentrations. All of the dimeric bisbenzimidazoles DBPA(n) manifested fluorescent properties, were well soluble in water, nontoxic up to concentrations of 200 µM, and could penetrate into nuclei followed by binding to DNA.


Asunto(s)
Bisbenzimidazol/química , Bisbenzimidazol/farmacología , ADN/química , Aliivibrio/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Secuencia de Bases , ADN/genética , Diseño de Fármacos , Escherichia coli/metabolismo , Colorantes Fluorescentes , Integrasa de VIH , Inhibidores de Integrasa VIH/farmacología , Ligandos , Estructura Molecular , Pirroles , Percepción de Quorum/fisiología , Relación Estructura-Actividad
3.
Curr Microbiol ; 76(11): 1374-1378, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31407052

RESUMEN

Anti-restriction proteins ArdB/KlcA specifically inhibit restriction (endonuclease) activity of restriction-modification (RM) type I systems. Molecular mechanisms of ArdB/KlcA-based anti-restriction remain unknown. In this study, we quantitate effects of ArdB on protection of unmodified λ phage DNA from EcoKI restriction. After UV irradiations, which produce significant amounts of unmodified chromosomal DNA in Escherichia coli K12 cells, the protective activity of ArdB decreases. Unlike ArdB, DNA-mimicking protein Ocr retains its ability to protect the unmodified λ phage regardless of UV dose. We hypothesize that the observed decrease in ArdB protective activity in UV-treated cells is due to its binding to unmodified chromosomal DNA, which decreases effective concentrations of free ArdB molecules available for λ phage protection against type I restriction enzymes.


Asunto(s)
Bacteriófago lambda/fisiología , Enzimas de Restricción del ADN/metabolismo , Escherichia coli K12/metabolismo , Escherichia coli K12/virología , Proteínas de Escherichia coli/inmunología , Bacteriófago lambda/genética , Enzimas de Restricción del ADN/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/efectos de la radiación , Proteínas de Escherichia coli/genética , Rayos Ultravioleta
4.
Arch Microbiol ; 201(10): 1415-1425, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31392374

RESUMEN

Regulation of Aliivibrio logei luxR1 and luxR2 genes was evaluated in Escherichia coli cells with use of transcriptional fusions of luxR1 and luxR2 promoter/operator regions with the Photorhabdus luminescens luxCDABE reporter gene cassette. Expression of the luxR1 and luxR2 genes was shown to largely depend on the CRP as activator. The hns::kan mutation increases the expression of luxR2 gene by two to three orders of magnitude and luxR1 gene by two to threefold. The LuxR1 and LuxR2 proteins in the presence of autoinducer (N-acyl homoserine lactone, AI) separately as well as together considerably enhanced the transcription of the luxR2 gene. In contrast, the transcription of luxR1 gene decreases depending on AI concentration in the presence of the luxR1 and luxR2 genes combination. It was identified that the promoter region of luxR2 gene consists of two promoters: Pcrp is located downstream of the crp box and Plux-box is located between the crp box and the lux box.


Asunto(s)
Aliivibrio/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Mutación , Photorhabdus/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
FEMS Microbiol Lett ; 365(23)2018 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-30239714

RESUMEN

Antirestriction proteins of the ArdB group (ArdB, KlcA) specifically inhibit restriction (endonuclease) activity of restriction-modification (RM) type I systems. Antirestriction activity of KlcA and ArdB, encoded in transmissible plasmids RP4 (IncPα) and R64 (IncI1), respectively, has been determined. We show that the protein KlcA (RP4), an amino acid sequence identical to that of the protein KlcA (RK2), inhibits the activity of EcoKI when the klcA gene is located on the plasmid under the control of strong promoter. It was demonstrated that proteins KlcA (RP4) and ArdB (R64) are characterized by approximately equal antirestriction activity. Analysis of amino acid sequences of ArdB homologs revealed four groups of conserved amino acids located on the surface of the protein globule: (1) R16, E32, W51; (2) Y46, G48; (3) S84, D86, E132 and (4) N77, L140, D141. It was shown that substitution of polar amino acids to hydrophobic A and L leads to a significant decrease in the ArdB antirestriction activity level (approximately 100-fold). A conserved region forming a 'ring belt' on the globule surface consisting of E32, S84, E132, and both N77 and D141 as the 'key section' of ArdB/KlcA was identified.


Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Enzimas de Restricción del ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/metabolismo , Transferencia de Gen Horizontal , Plásmidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia Conservada , Escherichia coli/enzimología , Escherichia coli/genética , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/genética , Homología de Secuencia de Aminoácido
6.
Microbiol Res ; 207: 75-82, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29458871

RESUMEN

DNA sequence-specific fluorescent dimeric bisbenzimidazoles DBP(n) and DBPA(n), noncovalently interacting with A-T pairs in the minor groove of double-stranded DNA were used for studying and monitoring the expression of histone-like H-NS-dependent promoters. Histone-like H-NS selectively binds to AT-rich segments of DNA and silences a large number of genes in bacterial chromosomes. The H-NS-dependent promoters of Quorum Sensing (QS)-regulated lux operons of the marine bacteria mesophilic Aliivibrio fischeri, psychrophilic Aliivibrio logei were used. Escherichia coli lux biosensors were constructed by cloning fragments bearing QS-regulated promoters into the vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE genes. It was shown that the dimeric bisbenzimidazoles DBP(n) and DBPA(n) counteract the H-NS silencing activity. Thus, the presence of DBP(n) or DBPA(n) in the medium leads to an approximately 10-100-fold increase in the level of transcription of QS promoters in E. coli hns+. The largest decrease in the level of H-NS repression was observed using ligands containing a linker with a length of ca. 18Å, such as DBP(2) and DBPA(2). Ligands containing linkers with n=1 and 3 are an order of magnitude less active; ligands with n=4 are inactive. DBPA(2) exhibits activity starting with a concentration of 0.5µM; the minimum concentration of DBP(2) is 5-7 times higher. It is suggested that A-T pairs located at five nucleotide pair intervals, which correspond to the linker length in highly active ligands with n=2, play a key role in the structure of H-NS-binding sites in QS-regulated promoters.


Asunto(s)
Aliivibrio fischeri/genética , Proteínas Bacterianas/genética , Bisbenzimidazol/farmacología , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Percepción de Quorum/genética , Proteínas Bacterianas/antagonistas & inhibidores , Composición de Base/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Escherichia coli/genética , Mediciones Luminiscentes , Photorhabdus/genética
7.
Appl Microbiol Biotechnol ; 101(14): 5765-5771, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28577028

RESUMEN

Many bacteria, fungi, and plants produce volatile organic compounds (VOCs) emitted to the environment. Bacterial VOCs play an important role in interactions between microorganisms and in bacterial-plant interactions. Here, we show that such VOCs as ketones 2-heptanone, 2-nonanone, and 2-undecanone inhibit the DnaKJE-ClpB bichaperone dependent refolding of heat-inactivated bacterial luciferases. The inhibitory activity of ketones had highest effect in Escherichia coli ibpB::kan cells lacking small chaperone IbpB. Effect of ketones activity increased in the series: 2-pentanone, 2-undecanone, 2-heptanone, and 2-nonanone. These observations can be explained by the interaction of ketones with hydrophobic segments of heat-inactivated substrates and the competition with the chaperones IbpAB. If the small chaperone IbpB is absent in E. coli cells, the ketones block the hydrophobic segments of the polypeptides and inhibit the action of the bichaperone system. These results are consistent with the data on inhibitory effects of VOCs on survival of bacteria. It can be suggested that the inhibitory activity of the ketones indicated is associated with different ability of these substances to interact with hydrophobic segments in proteins.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Cetonas/farmacología , Luciferasas de la Bacteria/química , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Calor , Luciferasas de la Bacteria/genética , Luciferasas de la Bacteria/metabolismo , Chaperonas Moleculares/metabolismo , Pliegue de Proteína/efectos de los fármacos , Compuestos Orgánicos Volátiles/farmacología
8.
Microbiol Res ; 192: 283-291, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27664747

RESUMEN

The antirestriction proteins ArdA ColIb-P9, Arn T4 and Ocr T7 specifically inhibit type I and type IV restriction enzymes and belong to the family of DNA-mimic proteins because their three-dimensional structure is similar to the double-helical B-form DNA. It is proposed that the DNA-mimic proteins are able to bind nucleoid protein H-NS and alleviate H-NS-silencing of the transcription of bacterial genes. Escherichia coli lux biosensors were constructed by inserting H-NS-dependent promoters into a vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE operon. It was demonstrated that the DNA-mimic proteins ArdA, Arn and Ocr activate the transcription of H-NS-dependent promoters of the lux operon of marine luminescent bacteria (mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio logei), and the dps gene from E. coli. It was also demonstrated that the ArdA antirestriction protein, the genes of which are located on transmissive plasmids ColIb-P9, R64, PK101, decreases levels of H-NS silencing of the PluxC promoter during conjugation in the recipient bacteria.


Asunto(s)
Proteínas Bacterianas/genética , Enzimas de Restricción del ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Transcripción Genética , Activación Transcripcional , Proteínas Virales/metabolismo , Conjugación Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo , Regulación Bacteriana de la Expresión Génica , Silenciador del Gen , Operón , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Virales/química
9.
Microbiology (Reading) ; 162(4): 717-724, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26847185

RESUMEN

The lux-operon of the psychrophilic bioluminescent bacterium Aliivibrio logei is regulated by quorum sensing (QS). The key components of this system are LuxI, which catalyses synthesis of the autoinducer (AI), and LuxR, which activates transcription of the entire lux-operon. The lux-operon of A. logei contains two copies of the luxR gene: luxR1 and luxR2. In the present study, lux-operon sequence analysis from 16 strains of A. logei, isolated from cold habitats of the White, Baltic, Okhotsk and Bering seas, was carried out. Phylogenetic analysis showed that all isolated strains of A. logei have both copies of luxR genes which are homologous to luxR genes of the related Aliivibrio salmonicida. Evaluation of LuxR1 and LuxR2 activity showed that LuxR2 remains active at significantly lower concentrations of AI (10- 9 M) than LuxR1, which is active only at high AI concentrations (10- 6 M). As the QS response is already prominent at AI concentrations as low as 10- 8 to 10- 9 M, we conclude that LuxR2 is the main activator of the lux-operon of A. logei. The thermolabilities of LuxR1 and LuxR2 are similar and exceed that of LuxR of the mesophilic bacterium Aliivibrio fischeri. In contrast to LuxR2, LuxR1 is not a substrate of Lon protease and does not require the chaperonin GroEL/ES for its folding. This study expands our current understanding of QS regulation in A. logei as it implies differential regulation by LuxR1 and LuxR2 proteins.

10.
FEMS Microbiol Lett ; 337(1): 55-60, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22967207

RESUMEN

The mercury-resistance transposon Tn5053 inhibits restriction activity of the type I restriction-modification endonuclease EcoKI in Escherichia coli K12 cells. This is the first report of antirestriction activity of a non-conjugative transposon. The gene (ardD) coding for the antirestriction protein has been cloned. The ardD gene is located within the tniA gene, coding for transposase, on the complementary strand. The direction of transcription is opposite to transcription of the tniA gene.


Asunto(s)
Enzimas de Restricción del ADN/antagonistas & inhibidores , Elementos Transponibles de ADN , Escherichia coli K12/enzimología , Escherichia coli K12/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Clonación Molecular , Proteínas de Escherichia coli/genética , Transcripción Genética , Transposasas/genética
11.
J Bacteriol ; 193(15): 3998-4001, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21665974

RESUMEN

Here we provide a molecular description of a new psychrophilic strain, KCh11, of marine luminescent bacteria classified as Aliivibrio logei. We sequenced the entire lux operon of A. logei KCh1 and showed that it is substantially similar to the lux operon of Aliivibrio salmonicida. It was demonstrated that the reduced production of bioluminescence in A. salmonicida is most likely defined by a specific defect in its luxD gene.


Asunto(s)
Proteínas Bacterianas/genética , Peces/microbiología , Proteínas Luminiscentes/genética , Operón , Agua de Mar/microbiología , Vibrionaceae/genética , Vibrionaceae/aislamiento & purificación , Animales , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Filogenia , Federación de Rusia , Vibrionaceae/clasificación
12.
J Bacteriol ; 192(20): 5549-51, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20729362

RESUMEN

Here we show that the C-terminal domain of LuxR activates the transcription of Aliivibrio fischeri luxICDABEG in Escherichia coli SKB178 gro(+) and E. coli OFB1111 groEL673 strains to the same level. Using affinity chromatography, we showed that GroEL binds to the N-terminal domain of LuxR, pointing to a GroEL/GroES requirement for the folding of the N-terminal domain of LuxR.


Asunto(s)
Chaperonina 60/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Vibrionaceae/genética , Vibrionaceae/metabolismo , Chaperonina 60/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Mutación , Estructura Terciaria de Proteína , Percepción de Quorum , Proteínas Represoras/genética , Estrés Fisiológico , Transactivadores/genética
13.
J Bacteriol ; 187(11): 3889-93, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15901718

RESUMEN

Citrobacter freundii cells produce L-methionine gamma-lyase when grown on a medium containing L-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded L-methionine gamma-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3'-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found.


Asunto(s)
Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Citrobacter freundii/enzimología , Citrobacter freundii/genética , Genoma Bacteriano , Secuencia de Bases , Liasas de Carbono-Azufre/aislamiento & purificación , Clonación Molecular , Evolución Molecular , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico
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