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1.
Appl Microbiol Biotechnol ; 104(10): 4577-4592, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32221691

RESUMO

New strategies to improve crop yield include the incorporation of plant growth-promoting bacteria in agricultural practices. The non-pathogenic bacterium Pseudomonas putida KT2440 is an excellent root colonizer of crops of agronomical importance and has been shown to activate the induced systemic resistance of plants in response to certain foliar pathogens. In this work, we have analyzed additional plant growth promotion features of this strain. We show it can tolerate high NaCl concentrations and determine how salinity influences traits such as the production of indole compounds, siderophore synthesis, and phosphate solubilization. Inoculation with P. putida KT2440 significantly improved seed germination and root and stem length of soybean and corn plants under saline conditions compared to uninoculated plants, whereas the effects were minor under non-saline conditions. Also, random transposon mutagenesis was used for preliminary identification of KT2440 genes involved in bacterial tolerance to saline stress. One of the obtained mutants was analyzed in detail. The disrupted gene encodes a predicted phosphoethanolamine-lipid A transferase (EptA), an enzyme described to be involved in the modification of lipid A during lipopolysaccharide (LPS) biosynthesis. This mutant showed changes in exopolysaccharide (EPS) production, low salinity tolerance, and reduced competitive fitness in the rhizosphere.


Assuntos
Proteínas de Bactérias/genética , Produtos Agrícolas/microbiologia , Desenvolvimento Vegetal , Raízes de Plantas/microbiologia , Pseudomonas putida/fisiologia , Estresse Salino , Produtos Agrícolas/crescimento & desenvolvimento , Etanolaminas/metabolismo , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Rizosfera , Tolerância ao Sal , Sementes/metabolismo , Cloreto de Sódio/metabolismo , Glycine max/metabolismo , Glycine max/microbiologia , Transferases/química , Transferases/genética , Zea mays/metabolismo , Zea mays/microbiologia
2.
PLoS One ; 11(6): e0157799, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27310257

RESUMO

Microorganisms produce siderophores to facilitate iron uptake and even though this trait has been extensively studied, there is growing evidence suggesting that siderophores may have other physiological roles aside from iron acquisition. In support of this notion, we previously linked the archetypal siderophore enterobactin with oxidative stress alleviation. To further characterize this association, we studied the sensitivity of Escherichia coli strains lacking different components of the enterobactin system to the classical oxidative stressors hydrogen peroxide and paraquat. We observed that strains impaired in enterobactin production, uptake and hydrolysis were more susceptible to the oxidative damage caused by both compounds than the wild-type strain. In addition, meanwhile iron supplementation had little impact on the sensitivity, the reducing agent ascorbic acid alleviated the oxidative stress and therefore significantly decreased the sensitivity to the stressors. This indicated that the enterobactin-mediated protection is independent of its ability to scavenge iron. Furthermore, enterobactin supplementation conferred resistance to the entE mutant but did not have any protective effect on the fepG and fes mutants. Thus, we inferred that only after enterobactin is hydrolysed by Fes in the cell cytoplasm and iron is released, the free hydroxyl groups are available for radical stabilization. This hypothesis was validated testing the ability of enterobactin to scavenge radicals in vitro. Given the strong connection between enterobactin and oxidative stress, we studied the transcription of the entE gene and the concomitant production of the siderophore in response to such kind of stress. Interestingly, we observed that meanwhile iron represses the expression and production of the siderophore, hydrogen peroxide and paraquat favour these events even if iron is present. Our results support the involvement of enterobactin as part of the oxidative stress response and highlight the existence of a novel regulation mechanism for enterobactin biosynthesis.


Assuntos
Enterobactina/biossíntese , Escherichia coli/genética , Regulação da Expressão Gênica , Sideróforos/biossíntese , Estresse Fisiológico/genética , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Cloretos/farmacologia , Enterobactina/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Compostos Férricos/farmacologia , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Hidrólise , Ferro/metabolismo , Ligases/genética , Ligases/metabolismo , Mutação , Oxidantes/antagonistas & inibidores , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo , Paraquat/antagonistas & inibidores , Paraquat/farmacologia , Sideróforos/genética , Transcrição Gênica
3.
PLoS One ; 9(1): e84734, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24392154

RESUMO

Numerous bacteria have evolved different iron uptake systems with the ability to make use of their own and heterologous siderophores. However, there is growing evidence attributing alternative roles for siderophores that might explain the potential adaptive advantages of microorganisms having multiple siderophore systems. In this work, we show the requirement of the siderophore enterobactin for Escherichia coli colony development in minimal media. We observed that a strain impaired in enterobactin production (entE mutant) was unable to form colonies on M9 agar medium meanwhile its growth was normal on LB agar medium. Given that, neither iron nor citrate supplementation restored colony growth, the role of enterobactin as an iron uptake-facilitator would not explain its requirement for colony development. The absence of colony development was reverted either by addition of enterobactin, the reducing agent ascorbic acid or by incubating in anaerobic culture conditions with no additives. Then, we associated the enterobactin requirement for colony development with its ability to reduce oxidative stress, which we found to be higher in media where the colony development was impaired (M9) compared with media where the strain was able to form colonies (LB). Since oxyR and soxS mutants (two major stress response regulators) formed colonies in M9 agar medium, we hypothesize that enterobactin could be an important piece in the oxidative stress response repertoire, particularly required in the context of colony formation. In addition, we show that enterobactin has to be hydrolyzed after reaching the cell cytoplasm in order to enable colony development. By favoring iron release, hydrolysis of the enterobactin-iron complex, not only would assure covering iron needs, but would also provide the cell with a molecule with exposed hydroxyl groups (hydrolyzed enterobactin). This molecule would be able to scavenge radicals and therefore reduce oxidative stress.


Assuntos
Enterobactina/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Estresse Oxidativo , Carga Bacteriana , Meios de Cultura , Enterobactina/genética , Escherichia coli/genética , Hidrólise , Mutação , Espécies Reativas de Oxigênio
4.
J Bacteriol ; 195(23): 5352-61, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24078611

RESUMO

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Transporte Biológico/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica
5.
BMC Microbiol ; 13: 95, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23634875

RESUMO

BACKGROUND: Microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide produced by Escherichia coli (E. coli). MccJ25 enters into the sensitive E. coli strains by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD and SbmA. The resistance of Salmonella enterica serovar Typhimurium (S. Typhimurium) to MccJ25 is attributed to the inability of its FhuA protein to incorporate the antibiotic into the cell. RESULTS: In this work we demonstrate that S. Typhimurium becomes notably susceptible to MccJ25 when replicating within macrophages. In order to determine the possible cause of this phenomenon, we studied the sensitivity of S. Typhimurium to MccJ25 at conditions resembling those of the internal macrophage environment, such as low pH, low magnesium and iron deprivation. We observed that the strain was only sensitive to the antibiotic at low pH, leading us to attribute the bacterial sensitization to this condition. A MccJ25-resistant E. coli strain in which fhuA is deleted was also inhibited by the antibiotic at low pH. Then, we could assume that the MccJ25 sensitivity change observed in both E. coli fhuA and S. Typhimurium is mediated by a MccJ25 uptake independent of the FhuA receptor. Moreover, low pH incubation also sensitized S. Typhimurium to the hydrophobic antibiotic novobiocin, which does not affect enteric bacteria viability because it is unable to penetrate the bacterial outer membrane. This observation supports our hypothesis about low pH producing a modification in the bacterial membrane permeability that allows an unspecific MccJ25 uptake. On the other hand, MccJ25 inhibited S. Typhimurium when cells were preincubated in acidic pH medium and then treated at neutral pH with the antibiotic. CONCLUSIONS: Our results suggest that acidic condition does not alter MccJ25 hydrophobicity but irreversibly modifies bacterial membrane permeability. This would allow an unspecific antibiotic uptake into the cell.From our data it is possible to infer that intracellular pathogenic strains, which are in vitro resistant to MccJ25, could become susceptible ones in vivo. Therefore, the MccJ25 action spectrum would be broader than what in vitro experiments indicate.


Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Macrófagos/microbiologia , Salmonella typhimurium/efeitos dos fármacos , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Meios de Cultura/química , Citosol/microbiologia , Escherichia coli/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Camundongos , Testes de Sensibilidade Microbiana , Modelos Teóricos , Novobiocina/farmacologia
6.
PLoS One ; 7(10): e46754, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23071628

RESUMO

BACKGROUND: Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition. METHODS AND PRINCIPAL FINDINGS: Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity. CONCLUSIONS: We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.


Assuntos
Antibacterianos/farmacologia , Enterobactina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fenóis/farmacologia , Tiazóis/farmacologia , Enterobactina/fisiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Proteínas de Escherichia coli/fisiologia , Ferro/farmacologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Sideróforos/metabolismo , Sideróforos/fisiologia
7.
Biochem Biophys Res Commun ; 406(3): 366-70, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21329661

RESUMO

Microcin J25 (MccJ25) is a 21 amino acid lasso-peptide antibiotic produced by Escherichia coli and composed of an 8-residues ring and a terminal 'tail' passing through the ring. We have previously reported two cellular targets for this antibiotic, bacterial RNA polymerase and the membrane respiratory chain, and shown that Tyr9 is essential for the effect on the membrane respiratory chain which leads to superoxide overproduction. In the present paper we investigated the redox behavior of MccJ25 and the mutant MccJ25 (Y9F). Cyclic voltammetry measurements showed irreversible oxidation of both Tyr9 and Tyr20 in MccJ25, but infrared spectroscopy studies demonstrated that only Tyr9 could be deprotonated upon chemical oxidation in solution. Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H2O2 was demonstrated by Electron Paramagnetic Resonance Spectroscopy; this radical was not detected when the reaction was carried out with the MccJ25 (Y9F) mutant. These results show that the essential Tyr9, but not Tyr20, can be easily oxidized and form a tyrosyl radical.


Assuntos
Antibacterianos/química , Bacteriocinas/química , Tirosina/química , Espectroscopia de Ressonância de Spin Eletrônica , Ferricianetos , Peróxido de Hidrogênio/química , Oxirredução , Espectrofotometria Infravermelho , Vibração
8.
Appl Environ Microbiol ; 76(20): 6837-42, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20802073

RESUMO

Microcin J25 (MccJ25) is a plasmid-encoded, 21-amino-acid, antibacterial peptide produced by Escherichia coli. MccJ25 inhibits RNA polymerase and the membrane respiratory chain. MccJ25 uptake into E. coli-sensitive strains is mediated by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. This peptide is active on some E. coli, Salmonella, and Shigella species strains, while other Gram-negative bacteria, such as clinical isolates of Enterobacter cloacae, Citrobacter freundii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Moraxella catarrhalis, and Salmonella enterica serovar Typhimurium, are completely resistant. In the present work, we demonstrated that the membrane-permeabilizing peptide (KFF)3K made some resistant strains sensitive to MccJ25, among them S. Typhimurium, where the antibiotic inhibits in vitro cell growth and bacterial replication within macrophages. The results demonstrate that the membrane permeabilization induced by (KFF)3K allows MccJ25 penetration in an FhuA and SbmA-independent manner and suggest that the combination of both peptides could be considered as a therapeutic agent against pathogenic Salmonella strains.


Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacteriocinas/metabolismo , Bacteriocinas/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Peptídeos/farmacologia , Contagem de Colônia Microbiana , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Negativas/fisiologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Oxigênio/metabolismo
9.
FEMS Microbiol Lett ; 311(2): 185-92, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20735480

RESUMO

The SbmA protein is involved in the transport of MccB17-, MccJ25-, bleomycin- and proline-rich peptides into the Escherichia coli cytoplasm. sbmA gene homologues were found in a variety of bacteria. However, the physiological role of this protein still remains unknown. Previously, we found that a combination of sbmA and tolC mutations in Tn10-carrying E. coli K-12 strains results in hypersusceptibility to tetracycline. In this work, we studied sbmA expression in a tolC mutant background and observed an increased expression throughout growth. We ruled out the global transcriptional regulator RpoS and the small RNA micF as intermediates in this regulation. The tolC mutation induced the expression of other well-characterized strong σ(E) -dependent promoters in E. coli. We observed that the increase in σ(E) activity led to a greater sbmA expression, conversely eliminating σ(E) prevented expression of sbmA. We also observed that the sbmA upregulation in a tolC mutant context was abolished in an rpoE-null strain. These results suggest a σ(E) -dependent positive regulation on sbmA by the tolC mutation. We hypothesize that this mechanism might be part of a compensatory cell envelope stress response.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Fator sigma/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Fator sigma/genética
10.
FEMS Microbiol Lett ; 300(1): 90-6, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19758327

RESUMO

Escherichia coli microcin J25 (MccJ25) is a lasso-peptide antibiotic comprising 21 L-amino acid residues (G(1)-G-A-G-H(5)-V-P-E-Y-F(10)-V-G-I-G-T(15)-P-I-S-F-Y(20)-G). MccJ25 has two independent substrates: RNA-polymerase (RNAP) and the membrane respiratory chain. The latter is mediated by oxygen consumption inhibition together with an increase of superoxide production. In the present paper, the antibiotic MccJ25 was engineered by substituting Tyr(9) or Tyr(20) with phenylalanine. Both mutants were well transported into the cells and remained active on RNAP. Only the Y9F mutant lost the ability to overproduce superoxide and inhibit oxygen consumption. The last results confirm that the Tyr(9), and not Tyr(20), is involved in the MccJ25 action on the respiratory chain target.


Assuntos
Antibacterianos/química , Bacteriocinas/química , Escherichia coli/metabolismo , Superóxidos/metabolismo , Tirosina/química , Antibacterianos/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Escherichia coli/química , Escherichia coli/genética , Tirosina/genética , Tirosina/metabolismo
11.
Appl Environ Microbiol ; 75(17): 5734-8, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19592527

RESUMO

Microcin J25 (MccJ25) is a 21-residue ribosomally synthesized lariat peptide antibiotic. MccJ25 is active against such food-borne disease-causing pathogens as Salmonella spp., Shigella spp., and Escherichia coli, including E. coli O157:H7 and non-O157 strains. MccJ25 is highly resistant to digestion by proteolytic enzymes present in the stomach and intestinal contents. MccJ25 would therefore remain active in the gastrointestinal tract, affecting normal intestinal microbiota, and this limits the potential use of MccJ25 as a food preservative. In the present paper, we describe a chymotrypsin-susceptible MccJ25 derivative with a mutation of Gly(12) to Tyr that retained almost full antibiotic activity and efficiently inhibited the growth of pathogenic Salmonella enterica serovar Newport and Escherichia coli O157:H7 in skim milk and egg yolk. However, unlike the wild-type MccJ25, the MccJ25(G12Y) variant was inactivated by digestive enzymes both in vitro and in vivo. To our knowledge, our results represent the first example of a rational modification of a microcin aimed at increasing its potential use in food preservation.


Assuntos
Substituição de Aminoácidos/genética , Bacteriocinas/metabolismo , Quimotripsina/metabolismo , Conservantes de Alimentos/metabolismo , Bacteriocinas/genética , Bacteriocinas/farmacologia , Contagem de Colônia Microbiana , Escherichia coli O157/efeitos dos fármacos , Conservação de Alimentos/métodos , Conservantes de Alimentos/farmacologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Mutagênese Sítio-Dirigida , Salmonella enterica/efeitos dos fármacos
12.
Curr Med Chem ; 16(5): 538-49, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19199920

RESUMO

Microcin J25 (MccJ25) is a plasmid-encoded peptide of 21 L-amino acids (G1-G-A-G-H5-V-P-E-Y-F10-V-G-I-G-T15-P-I-S-F-Y20-G), excreted to the medium by an Escherichia coli strain. MccJ25 is active on Gram-negative bacteria related to the producer strain, including some pathogenic strains. The four-plasmid genes mcjABCD, are involved in MccJ25 production: mcjA encodes a 58-residue precursor, mcjB and mcjC codify two processing enzymes required for the in vivo synthesis of the mature peptide and mcjD encodes the immunity protein (McjD), a member of the super family of ABC transporters. Immunity is mediated by active efflux of the peptide, keeping its intracellular concentration below a critical level. YojI, a chromosomal protein with ATP-binding-cassette-type exporter homology, is also able to export MccJ25. The E. coli outer membrane protein, TolC, is necessary for MccJ25 secretion mediated by either McjD or YojI. The uptake of MccJ25 is dependent on the outer-membrane receptor FhuA and the four inner-membrane proteins TonB, ExbD, ExbB and SbmA. At least two mechanisms described the action of MccJ25 on the target cells: (1) inhibition of the RNA-polymerase (RNAP) activity by obstructing the secondary channel, and consequently, preventing the access of the substrates to its active sites; and (2) operating on the cell membrane, MccJ25 disrupts the electric potential inhibiting the oxygen consumption in Salmonella enterica. MccJ25 also inhibits oxygen consumption and the respiratory chain enzymes in E. coli throughout the increasing of ROS concentration. Nevertheless the exact mechanism of this phenomenon must be elucidated. The MccJ25 exhibits a prolonged antimicrobial activity in a mouse infection model, suggesting a noteworthy potential for therapeutic uses.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Bacteriocinas/química , Bacteriocinas/farmacologia , Sequência de Aminoácidos , Antibacterianos/isolamento & purificação , Bacteriocinas/isolamento & purificação , Dados de Sequência Molecular , Estrutura Molecular , Relação Estrutura-Atividade
13.
J Bacteriol ; 191(4): 1343-8, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19074390

RESUMO

Many Escherichia coli K-12 strains display an intrinsic resistance to the peptide antibiotic microcin J25. In this study, we present results showing that the leucine-responsive regulatory protein, Lrp, is involved in this phenotype by acting as a positive regulator of YojI, a chromosomally encoded efflux pump which expels microcin out of cells. Exogenous leucine antagonizes the effect of Lrp, leading to a diminished expression of the pump and an increased susceptibility to microcin J25.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Bacteriocinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteína Reguladora de Resposta a Leucina/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Bacteriocinas/genética , Sequência de Bases , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Leucina/farmacologia , Proteína Reguladora de Resposta a Leucina/genética , Dados de Sequência Molecular
14.
J Bacteriol ; 190(12): 4328-34, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18408024

RESUMO

As Escherichia coli strains enter the stationary phase of growth they become more resistant to the peptide antibiotic microcin J25. It is known that starvation for nutrients such as amino acids or glucose leads to increases in guanosine 3',5'-bispyrophosphate (ppGpp) levels and that the intracellular concentration of this nucleotide increases as cells enter the stationary phase of growth. Therefore, we examined the effects of artificially manipulating the ppGpp levels on sensitivity to microcin J25. A direct correlation was found between ppGpp accumulation and microcin resistance. Our results indicate that the nucleotide is required to induce production of YojI, a chromosomally encoded efflux pump which, in turn, expels microcin from cells. This would maintain the intracellular level of the antibiotic below a toxic level.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriocinas/farmacologia , Escherichia coli/efeitos dos fármacos , Guanosina Tetrafosfato/metabolismo , Proteínas de Bactérias/genética , Bacteriocinas/metabolismo , Transporte Biológico/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ligases/genética , Ligases/metabolismo , Viabilidade Microbiana/efeitos dos fármacos
15.
J Bacteriol ; 190(4): 1491-4, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18083810

RESUMO

Previously, we demonstrated that Escherichia coli tolC mutations reduce the high-level resistance to tetracycline afforded by the transposon Tn10-encoded TetA pump from resistance at 200 microg/ml to resistance at 40 microg/ml. In this study, we found that the addition of an sbmA mutation to a tolC::Tn10 mutant exacerbates this phenotype: the double mutant did not form colonies, even in the presence of tetracycline at a concentration as low as 5 microg/ml. Inactivation of sbmA alone partially inhibited high-level tetracycline resistance, from resistance at 200 microg/ml to resistance at 120 microg/ml. There thus appears to be an additive effect of the mutations, resulting in almost complete suppression of the phenotypic expression of Tn10 tetracycline resistance.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Elementos de DNA Transponíveis/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Resistência a Tetraciclina/genética , Antibacterianos/farmacologia , Antiporters/genética , Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Genes Bacterianos/genética , Genes Bacterianos/fisiologia , Testes de Sensibilidade Microbiana , Tetraciclina/farmacologia , Transposases/genética
16.
J Bacteriol ; 189(11): 4180-6, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17400747

RESUMO

Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, ExbB, and SbmA. MccJ25 appears to have two intracellular targets: (i) RNA polymerase (RNAP), which has been described in E. coli and Salmonella enterica serovars, and (ii) the respiratory chain, reported only in S. enterica serovars. In the current study, it is shown that the observed difference between the actions of microcin on the respiratory chain in E. coli and S. enterica is due to the relatively low microcin uptake via the chromosomally encoded FhuA. Higher expression by a plasmid-encoded FhuA allowed greater uptake of MccJ25 by E. coli strains and the consequent inhibition of oxygen consumption. The two mechanisms, inhibition of RNAP and oxygen consumption, are independent of each other. Further analysis revealed for the first time that MccJ25 stimulates the production of reactive oxygen species (O(2)(*-)) in bacterial cells, which could be the main reason for the damage produced on the membrane respiratory chain.


Assuntos
Bacteriocinas/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Superóxidos/metabolismo , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriocinas/farmacocinética , Catalase/genética , Catalase/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/genética , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Genótipo , Consumo de Oxigênio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
17.
J Antimicrob Chemother ; 59(4): 676-80, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17353221

RESUMO

OBJECTIVES: To study the possible therapeutic utility of microcin J25 (MccJ25), a peptide RNA polymerase inhibitor. METHODS: We subjected the antibiotic to two types of assays. First, with an ex vivo assay, we evaluated the stability and efficacy of MccJ25 in complex fluid biomatrices such as human whole blood, plasma and serum, compared with that in conventional laboratory media. Antimicrobial efficacy of MccJ25 was assessed by quantitative culture 2 h after inoculation of the biomatrices with a Salmonella Newport target organism and compared with that of MccJ25-free controls. Second, the antibiotic was tested in a mouse model of Salmonella infection. The latter was induced by intraperitoneal inoculation of 10(6) cfu of Salmonella Newport and the treatment with MccJ25 was initiated at 2 h post-infection. RESULTS: MccJ25 retained full activity after 24 h of incubation in whole blood, plasma or serum. In addition, it did not show any haemolytic activity. In whole blood, homologous plasma and serum, introduction of MccJ25 was associated with a significant reduction in cfu versus the respective peptide-free controls. The counts of viable bacteria in the spleen and liver of mice treated with MccJ25 at a total dosage of 3 mg/mouse during either 24 h (0.5 mg/mouse every 4 h) or 6 days (0.5 mg/mouse every 24 h) significantly decreased by two or three orders of magnitude (P

Assuntos
Antibacterianos/uso terapêutico , Bacteriocinas/uso terapêutico , Infecções por Salmonella/tratamento farmacológico , Ampicilina/uso terapêutico , Animais , Antibacterianos/farmacocinética , Bacteriocinas/farmacocinética , Contagem de Colônia Microbiana , Meia-Vida , Hemólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Fígado/microbiologia , Camundongos , Infecções por Salmonella/microbiologia , Baço/microbiologia
18.
J Antimicrob Chemother ; 58(1): 31-6, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16687460

RESUMO

OBJECTIVES: Starting from the observation that Escherichia coli tolC mutations severely reduced the high-level resistance to tetracycline afforded by Tn10- and plasmid-encoded Tet(A) pumps, we studied the mechanism of this susceptibility. METHODS: The MIC of tetracycline for MC4100 tolC::Tn10 and several tolC mutants carrying the Tn10 in other sites on the chromosome (thr::Tn10) was determined. The effect of a tolC mutation on the level of expression of Tn10 tet(A) was examined by using a tet(A)::lacZ gene fusion. Influence of tolC mutations on tetracycline efflux and accumulation was quantified by spectrofluorometric assays. The contribution of the AcrAB multidrug efflux system to high-level tetracycline resistance was measured in a Tn10-carrying acrAB null mutant strain. RESULTS: Tn10- and plasmid-encoded Tet(A) conferred 5- to 6-fold lower levels of tetracycline resistance in tolC mutants, as compared with control strain tolC+. Spectrofluorometric analyses showed that this resulted from a decrease in drug efflux in tolC mutants. Chlortetracycline resistance was also compromised by loss of TolC. Mutational loss of the AcrAB multidrug efflux transporter had the same effect as tolC mutations on tetracycline resistance. This indicated that tolC mutations act through inactivation of the AcrAB system. CONCLUSIONS: Our results are compatible with the hypothesis that the AcrAB pump is an important component in the development of high levels of resistance to tetracycline in E. coli, perhaps by working in combination with Tet(A).


Assuntos
Antiporters/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Resistência a Tetraciclina/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras , Mutação
19.
J Bacteriol ; 188(9): 3324-8, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16621826

RESUMO

Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 L-amino acid residues (G1-G-A-G-H5-V-P-E-Y-F10-V-G-I-G-T15-P-I-S-F-Y20-G). E. coli RNA polymerase (RNAP) is the intracellular target of MccJ25. MccJ25 enters cells after binding to specific membrane transporters: FhuA in the outer membrane and SbmA in the inner membrane. Here, we studied MccJ25 mutants carrying a substitution of His5 by Lys, Arg, or Ala. The inhibitory effects on cellular growth and in vitro RNAP activity were determined for each mutant microcin. The results show that all mutants inhibited RNAP in vitro. However, the mutants were defective in their ability to inhibit cellular growth. Experiments in which the FhuA protein was bypassed showed that substitutions of MccJ25 His5 affected the SbmA-dependent transport. Our results thus suggest that MccJ25 His5 located in the lariat ring is involved, directly or indirectly, in specific interaction with SbmA and is not required for MccJ25 inhibition of RNAP.


Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Histidina/fisiologia , Proteínas de Membrana/metabolismo , Substituição de Aminoácidos , Antibacterianos/farmacologia , Bacteriocinas/genética , Bacteriocinas/farmacologia , Transporte Biológico , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Histidina/genética , Mutação , RNA Bacteriano/biossíntese
20.
J Bacteriol ; 187(10): 3465-70, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15866933

RESUMO

In the present study, we showed that yojI, an Escherichia coli open reading frame with an unknown function, mediates resistance to the peptide antibiotic microcin J25 when it is expressed from a multicopy vector. Disruption of the single chromosomal copy of yojI increased sensitivity of cells to microcin J25. The YojI protein was previously assumed to be an ATP-binding-cassette-type exporter on the basis of sequence similarities. We demonstrate that YojI is capable of pumping out microcin molecules. Thus, one obvious explanation for the protective effect against microcin J25 is that YojI action keeps the intracellular concentration of the peptide below a toxic level. The outer membrane protein TolC in addition to YojI is required for export of microcin J25 out of the cell. Microcin J25 is thus the first known substrate for YojI.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Peptídeos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Cromossomos Bacterianos , Clonagem Molecular , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana Transportadoras , Fases de Leitura Aberta/genética
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