RESUMEN

AIMS: To clarify whether an antibacterial surfactant, cetyltrimethylammonium bromide (CTAB), induces superoxide stress in bacteria, we investigated the generation of superoxide and hydrogen peroxide and expression of soxR, soxS and soxRS regulon genes in Escherichia coli cells with the treatment of CTAB. METHODS AND RESULTS: In situ oxidative stress analyses with BES fluorescent probes revealed that generation of both superoxide and hydrogen peroxide were significantly increased with the CTAB treatment at a sublethal concentration in wild-type strain OW6, compared with the CTAB-resistant strain OW66. The activity of manganese-superoxide dismutase (Mn-SOD), a member of the soxRS regulon proteins, was decreased by the CTAB treatment only in strain OW6. Furthermore, quantitative real-time PCR analyses revealed that expression of the soxRS regulon genes was not upregulated, although soxS was upregulated by the CTAB treatment in strain OW6. CONCLUSIONS: Cetyltrimethylammonium bromide treatment led E. coli cells to a generation state of superoxide and hydrogen peroxide. It was also suggested that superoxide generation was caused by inhibiting SoxS function and decreasing Mn-SOD activity. SIGNIFICANCE AND IMPACT OF THE STUDY: It was revealed that excess superoxide generation in bacterial cells play a key action of antibacterial surfactants.

Asunto(s)

Antibacterianos/farmacología , Compuestos de Cetrimonio/farmacología , Escherichia coli/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Superóxidos/metabolismo , Tensoactivos/farmacología , Cetrimonio , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Peróxido de Hidrógeno/metabolismo , Regulón , Superóxido Dismutasa/metabolismo

RESUMEN

AIMS: To clone and characterize the genes bisdA and bisdB, encoding Ferredoxin(bisd) (Fd(bisd)) and cytochrome P450(bisd) (P450(bisd)), respectively, from the bisphenol A (BPA) degrading Sphingomonas bisphenolicum strain AO1. METHODS AND RESULTS: The 3.7 kb region containing bisdA and bisdB was cloned by genome walking and colony hybridization. The deduced N-terminal amino acid sequences of bisdA and bisdB were consistent with those of Fd(bisd) and P450(bisd) proteins characterized in our previous report. Two transposase genes, tnpA1 and tnpA2, were also located upstream and downstream of bisdAB. From amino acid sequence analysis, P450(bisd) has two conserved regions corresponding to the oxygen and heme binding regions of the bacterial cytochrome P450 family. Fd(bisd) was similar to putidaredoxin-type [2Fe-2S] ferredoxins. Escherichia coli BL21 (DE3) cells bearing bisdB- and bisdAB-recombinant pET19b were able to degrade BPA. A spontaneous mutant, strain AO1L, which was unable to degrade BPA, was isolated from the stock culture, and it was confirmed that strain AO1L had no bisdAB region. CONCLUSIONS: P450(bisd) monooxygenase sytem, encoded by bisdAB, is one system required for BPA hydroxylation in S. bisphenolicum strain AO1. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results indicate that bisdAB are key genes for BPA degradation in S. bisphenolicum strain AO1.

Asunto(s)

Sistema Enzimático del Citocromo P-450/genética , Disruptores Endocrinos/metabolismo , Ferredoxinas/genética , Fenoles/metabolismo , Sphingomonas/genética , Contaminantes Químicos del Agua/metabolismo , Secuencia de Bases , Compuestos de Bencidrilo , Biodegradación Ambiental , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Genoma Bacteriano , Hidroxilación , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Sphingomonas/metabolismo , Transgenes

RESUMEN

AIMS: The predicted survival of Bacillus subtilis 168 spores from a polynomial regression equation was validated in milk. METHODS AND RESULTS: Bias factor suggested as an index of model performance was used to validate the polynomial model predictions in ultrahigh temperature (UHT) treated and sterilized whole and skim milk. Model predictions were fail safe, predicting higher D-values (decimal reduction times) in buffer than actually noted in milk. CONCLUSIONS: The D-values for spores were lower in milk as compared with those predicted in potassium phosphate buffer contrary to the popular expectation of better spore survival in complex food systems. The Bias factor, a quantitative measure of the model performance, indicated that on average the model predictions exceed the observations by 40% in the case of whole milk and by 60% in the case of skim milk. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work is an attempt to ascertain the extent of reliability that one can safely place in polynomial model predictions, without compromising on the safety or palatability of foods where it is eventually intended to be applied. The work has also highlighted the differences in the thermal inactivation pattern of spores in buffer and in milk with a possible influence of the various constituents of milk. The work will assist the dairy industry to better design thermal processes to ensure longer shelf life of dairy foods.

Asunto(s)

Bacillus subtilis/crecimiento & desarrollo , Calor , Leche/microbiología , Animales , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/fisiología , Leche/química , Modelos Biológicos , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/fisiología

RESUMEN

AIMS: The inactivation of Bacillus subtilis 168 spores subjected to the combined stress of pH, temperature and sodium chloride in a buffer system was modelled. METHODS AND RESULTS: Bacillus subtilis 168 spore suspension in 50 mmol l-1 potassium phosphate buffer was heated in an open system using a block heater. A second order polynomial equation was used to describe the relationship between pH, temperature, sodium chloride concentration and the logarithm of the decimal reduction time (D-value) of the spores. Response surface graphs were constructed to predict the inactivation within the experimental domain. The data obtained were also compared with those reported for B. subtilis in different media and foods included in a large reference-based database of thermal inactivation (ThermoKill Database, TKDB R9100), which was constructed in the laboratory. CONCLUSIONS: All the variables studied seemed to have a significant effect on the inactivation of B. subtilis 168 spores in potassium phosphate buffer. The coefficient of determination, r2, and an analysis of the residuals from the model indicated the adequacy of the model to predict the inactivation of B. subtilis spores within the range of the experimental variables studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study will enable a better understanding of the inactivation of B. subtilis spores under the influence of the studied environmental variables. The model can be used by food industries to assess and monitor the shelf life of food products in the event of a chance contamination by B. subtilis spores.

Asunto(s)

Bacillus subtilis/fisiología , Mantequilla/microbiología , Microbiología de Alimentos , Bacillus subtilis/efectos de los fármacos , Calor , Concentración de Iones de Hidrógeno , Matemática , Modelos Biológicos , Cloruro de Sodio/farmacología , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/fisiología

RESUMEN

AIMS: In order to clarify the involvement of an energy-yielding system in the antibacterial action of surfactants, the effects of carbon source and anaerobiosis during the growth period on the surfactant sensitivity of Escherichia coli cells were investigated. METHODS AND RESULTS: Cetyltrimethylammonium bromide (CTAB) and N-dodecyl-N,N-dimethylglycine, at relatively low concentrations, caused a delay in growth of E. coli cells. Cells grown in M9 medium supplemented with glycerol, succinate or acetate as a carbon source were more sensitive to surfactants and had a higher respiratory activity than those grown with glucose. Cultivation under anaerobiosis made cells resistant to CTAB. CONCLUSIONS: Bacterial sensitivity to surfactants was affected by carbon source and anaerobiosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained should be helpful in determining suitable conditions of treatment in the practical use of surfactants for bacterial decontamination.

Asunto(s)

Carbono/metabolismo , Compuestos de Cetrimonio/farmacología , Escherichia coli/efectos de los fármacos , Tensoactivos/farmacología , Anaerobiosis/efectos de los fármacos , Cetrimonio , Medios de Cultivo , AMP Cíclico/farmacología , Farmacorresistencia Bacteriana , Metabolismo Energético/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Oxígeno/metabolismo , Oxígeno/farmacología

RESUMEN

AIMS: In order to investigate the mechanism of bacterial resistance to surfactants, a spontaneous mutant of Escherichia coli, OW66, resistant to a cationic surfactant cetyltrimethylammonium bromide (CTAB), was isolated and its physiological properties analysed. METHODS AND RESULTS: Strain OW66 grew in M9 medium containing CTAB at 45 micromol l(-1), whereas its parent strain, OW6, did not, even at 15 micromol l(-1). The mutant was also resistant to some other surfactants, antibiotics, heavy metals, organic solvents and oxidants examined. To determine the differences in physiology between strains OW66 and OW6, the compositions of their cell surface structures were analysed. In strain OW66, the relative content of OmpC in particular was higher than that of OmpF, whereas a reverse situation was seen in OW6 strain. The lipopolysaccharide (LPS) profile was different between these strains, and altered LPS in strain OW66 was suggested to be involved in the resistance to CTAB. CONCLUSIONS: A CTAB-resistant E. coli isolate possesses an altered outer membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment with a relatively low concentration of CTAB was found to introduce multi-drug resistance into bacterial cells. This acquired resistance should be taken into account with the frequent use of surfactants in industries and various environments.

Asunto(s)

Compuestos de Cetrimonio/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Tensoactivos/farmacología , Proteínas Bacterianas/análisis , Cetrimonio , Escherichia coli/química , Escherichia coli/aislamiento & purificación , Lipopolisacáridos/análisis , Pruebas de Sensibilidad Microbiana , Fosfolípidos/análisis

RESUMEN

We purified and characterized a 39-kDa Bacillus subtilis 168 nuclease that has been suggested in this laboratory to be involved in chromosomal DNA degradation induced by lethal heat and cold shock treatments in vivo. The nuclease activity was inhibited in vitro by aurintricalboxylic acid but not by Zn(2+). By the mutant analysis, we identified the 39-kDa nuclease as a product of yokF gene. The yokF gene contained a putative lipoprotein signal peptide motif. After in vivo exposure to lethal heat and cold stresses, the chromosomal DNA fragmentation was reduced in the yokF mutant, which demonstrated about a 2-10-fold higher survival rate than the wild type. The yokF mutant was found to be more sensitive to mitomycin C than the wild type. The transformation efficiency of the yokF mutant was about 10 times higher than that of the wild type. It is suggested that when B. subtilis cells are exposed to a stressful thermal shock resulting in membrane perturbation, YokF nuclease consequently dislocates into the cytoplasm and then attacks DNA.

Asunto(s)

Bacillus subtilis/enzimología , Bacillus subtilis/genética , Proteínas Bacterianas , Desoxirribonucleasas/química , Desoxirribonucleasas/aislamiento & purificación , Endonucleasas/química , Endonucleasas/aislamiento & purificación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Ácido Aurintricarboxílico/metabolismo , Membrana Celular/enzimología , Cromosomas/metabolismo , Frío , Citoplasma/metabolismo , ADN/metabolismo , Fragmentación del ADN , Desoxirribonucleasas/biosíntesis , Desoxirribonucleasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Endonucleasas/metabolismo , Escherichia coli/metabolismo , Calor , Concentración de Iones de Hidrógeno , Mitomicina/farmacología , Datos de Secuencia Molecular , Mutación , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transformación Genética , Transgenes , Zinc/farmacología

RESUMEN

To investigate the function of Escherichia coli small heat shock proteins, IbpA and IbpB, we constructed ibpA-, ibpB- and ibpAB-overexpressing strains and also an ibpAB-disrupted strain. The ibpA-, ibpB- and ibpAB-overexpressing strains were found to be resistant not only to heat but also to superoxide stress. However, the ibpAB-disrupted strain was not more sensitive to these stresses than the wild-type strain. The heat sensitivity of a rpoH amber mutant was partially suppressed by the overexpression of plac::ibpAB. These results suggest that IbpA and IbpB may be involved in the resistances to heat and oxidative stress.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli/fisiología , Proteínas de Choque Térmico/fisiología , Calor , Estrés Oxidativo , Factor sigma , Escherichia coli/genética , Eliminación de Gen , Proteínas de Choque Térmico/genética , Peróxido de Hidrógeno/farmacología , Paraquat/farmacología , Plásmidos/genética , Superóxidos/farmacología , Factores de Transcripción/genética

RESUMEN

We constructed a sodA-disrupted mutant of Bacillus subtilis 168, BK1, by homologous recombination. The mutant was not able to grow in minimal medium without Mn(II). The spore-forming ability of strain BK1 was significantly lower in Mn(II)-depleted medium than that of the wild-type strain. These deleterious effects caused by the sodA mutation were reversed when an excess of Mn(II) was used to supplement the medium. Moreover, the growth inhibition by superoxide generators in strain BK1 and its parent strain was also reversed by the supplementation with excess Mn(II). We therefore estimated the Mn-dependent superoxide-scavenging activity in BK1 cells. Whereas BK1 cells have no detectable superoxide dismutase (Sod) on native gel, the superoxide-scavenging activity in crude extracts of BK1 cells grown in Mn(II)-supplemented LB medium (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter) was significantly detected by the modified Sod assay method without using EDTA. The results obtained suggest that Mn, as a free ion or a complex with some cellular component, can catalyze the elimination of superoxide and that both SodA and Mn(II) are involved not only in the superoxide resistance of vegetative cells but also in sporulation.

Asunto(s)

Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/fisiología , Proteínas Bacterianas/metabolismo , Manganeso/metabolismo , Superóxido Dismutasa/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Depuradores de Radicales Libres/metabolismo , Depuradores de Radicales Libres/farmacología , Genes Bacterianos , Manganeso/farmacología , Mutación , Oxidantes/farmacología , Estrés Oxidativo , Paraquat/farmacología , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/genética , Esporas Bacterianas/fisiología , Superóxido Dismutasa/genética , Superóxidos/metabolismo , Vitamina K/farmacología

RESUMEN

Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2, 600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designated sodA, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region of sodA. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutant Escherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B. subtilis sodA.

Asunto(s)

Bacillus subtilis/enzimología , Superóxido Dismutasa/genética , Secuencia de Aminoácidos , Bacillus subtilis/genética , Secuencia de Bases , Inducción Enzimática , Escherichia coli/genética , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Superóxido Dismutasa/aislamiento & purificación

RESUMEN

The behaviors of heat-induced translocation of cytoplasmic beta-galactosidase to periplasm across the inner membrane of Escherichia coli cells were investigated in order to apply such phenomena to the process for production and separation of intracellular biomolecules. The heat stress was found to induce translocation of cytoplasmic beta-galactosidase (beta-gal) together with reduction of the amounts of intracellular soluble proteins and formation of their inactive aggregates. The translocation of beta-gal was then analyzed using (a) the location factor of beta-gal (LFG), which meant enzyme location in the cells and could be determined from the kinetic analysis of enzyme release process, and (b) the percentage of beta-gal activity in periplasm after solublizing the outer membrane of E. coli cells by lysozyme/EDTA treatment. The LFG values were maximized when cells were stressed at the temperature of 42-47 degrees C. From the results on the surface properties of both beta-gal and cell membrane under the heat stress, it is suggested that (1) the conformational change of cytoplasmic oligomeric beta-gal to the partially dissociated and/or unfolded state with higher local hydrophobicity, (2) the increase in membrane fluidity of inner membrane, (3) the enhancement of hydrophobic interaction between lipid and protein, and (4) the inhibition of its translocation by GroEL restabilizing the proteins could underlie the heat-induced translocation of beta-gal across the inner membrane. The possibility to apply the heat-induced translocation of beta-gal for the enhancement of the target selectivity at the process upstream is finally presented.

Asunto(s)

Citoplasma/enzimología , Calor , Membranas Intracelulares/metabolismo , beta-Galactosidasa/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli , Proteínas de Choque Térmico/biosíntesis , Solubilidad , Propiedades de Superficie , Agua/química

RESUMEN

Hyperthermic treatment reduces protein synthesis and modifies amino acid transport in Escherichia coli. The present study examined the role of nutrient availability on these processes. Cultures of E. coli in log phase were aliquoted into growth medium with or without complete amino acid supplementation and exposed to 37, 44, or 48 degrees C for 10 min. Amino acid supplementation increased radiolabelled arginine uptake at 48 degrees C when compared with unsupplemented cells. Exposure to 48 degrees C also reduced protein synthesis in both groups by at least 50% as reflected by labelled arginine incorporation. Two-dimensional gel electrophoresis indicated that this heat-related decrement in synthesis was most apparent in basic proteins. Total density analysis of the fluorographs demonstrated reductions in basic proteins of 15% at 44 degrees C and 89% at 48 degrees C, while acidic proteins only showed an 80% reduction at 48 degrees C. Amino acid supplementation appears to raise the baseline, but not to modify the final results of hyperthermia-induced inhibition of protein synthesis. The sensitivity of basic protein synthesis seems to be a key event in this process.

Asunto(s)

Proteínas Bacterianas/biosíntesis , Escherichia coli/metabolismo , Calor , Aminoácidos/farmacología , Arginina/metabolismo , Arginina/farmacocinética , Proteínas Bacterianas/aislamiento & purificación , Transporte Biológico Activo/efectos de los fármacos , Medios de Cultivo , Electroforesis en Gel Bidimensional , Escherichia coli/efectos de los fármacos , Concentración de Iones de Hidrógeno

RESUMEN

Escherichia coli cells exposed to a sublethal heat treatment at 55 degrees C for 15s synthesized lipopolysaccharide during their recovery period after heat stress. As chloramphenicol at least partly inhibited the synthesis of lipopolysaccharide, it is suggested that its synthesis might be in part due to the lipopolysaccharide-synthesizing enzymes produced de novo. The results obtained coincided with our previous finding that the permeability barrier function was repaired by heat-stressed cells.

Asunto(s)

Escherichia coli/metabolismo , Calor/efectos adversos , Lipopolisacáridos/metabolismo , Adaptación Biológica , Proteínas Bacterianas/biosíntesis , Permeabilidad de la Membrana Celular , Cloranfenicol/farmacología , Medios de Cultivo , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Galactosa/metabolismo , Leucina/metabolismo

RESUMEN

The surfactants tested in this study lysed Bacillus subtilis 168 cells at the logarithmic growth phase. Results obtained with inhibitors and a mutant that had defective autolytic enzymes suggested that cell lysis resulted from the deregulation of autolysin activity. The addition of surfactants at sublytic concentrations produced twisted cells, filamented cells, or both. Autolysins extracted with 5 M LiCl from the cell wall fraction and lysozyme added to cells that were treated with surfactants restored the apparently normal cell rod morphology, suggesting that surfactants interfere with the role of autolysins in normal construction of the cell envelope. The rates of cellular autolysis and autolysin activity remaining in growing cells after exposure to a surfactant at a sublytic concentration decreased, although the rate of turnover of cell wall peptidoglycan was the same as that of control cells. Surfactants were suggested to interact with the regulatory system of autolysins and, thus, to affect the activities of autolysins in B. subtilis cells and to cause either morphological changes or cell autolysis, depending on the concentration of surfactants.

Asunto(s)

Bacillus subtilis/efectos de los fármacos , Tensoactivos/farmacología , Autólisis , Bacillus subtilis/ultraestructura , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Muramidasa/antagonistas & inhibidores , Peptidoglicano/metabolismo , Potasio/farmacología , Sodio/farmacología

RESUMEN

The fluorescent dye 1-N-phenylnaphthylamine permeated Escherichia coli cells after exposure to a heat stress at 55 degrees C in Tris/Mg2+ buffer, pH 8.0. The rate of dye permeation increased with time during heat treatment and decreased gradually during subsequent incubation at 37 degrees C in a minimal medium. The initial level of rapid adsorption of the dye also increased with heating time, although it remained roughly constant during post-heating incubation. The results obtained suggest that the permeability barrier to the dye in the outer membrane was damaged by heat stress and was repaired after sublethal heating. RNA, protein and lipid syntheses, as well as an energy-yielding process, appeared to be necessary for the repair of impermeability to the dye.

Asunto(s)

1-Naftilamina/metabolismo , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Colorantes Fluorescentes/metabolismo , Naftalenos/metabolismo , 1-Naftilamina/análogos & derivados , Adsorción , Antimetabolitos/farmacología , Permeabilidad de la Membrana Celular , Metabolismo Energético/efectos de los fármacos , Calor , Cinética

RESUMEN

The sensitivities of intact and heat-injured cells of Escherichia coli K-12 to several antibacterial compounds were measured by the prolongation of growth delay. Cells exposed to sublethal heat became more sensitive to various hydrophobic compounds, such as medium-chain fatty acids, alkyl esters of p-hydroxybenzoic acid, and some kinds of antibiotics or dyes, than unheated cells; but there was a smaller or no increase in sensitivity to short-chain fatty acids, chloramphenicol, and vancomycin. The destruction by heat of a permeability barrier of the outer membrane may have sensitized the cells to hydrophobic compounds. The sensitization was much lower for a strain defective in lipopolysaccharide, which is important as a barrier against hydrophobic compounds.

Asunto(s)

Antibacterianos/farmacología , Escherichia coli/crecimiento & desarrollo , Calor , Permeabilidad de la Membrana Celular/efectos de los fármacos , Colorantes/farmacología , Escherichia coli/efectos de los fármacos , Ácidos Grasos/farmacología , Lipopolisacáridos/análisis , Pruebas de Sensibilidad Microbiana , Factores de Tiempo

RESUMEN

The lytic action of glycerol and sucrose esters of fatty acids with different carbon chain lengths on the exponentially growing cells of Bacillus subtilis 168 was investigated. Of each series of esters, glycerol dodecanoate and sucrose hexadecanoate were the most active. Lysis at 1 h after the addition of 0.1 mM glycerol dodecanoate or 20 mug of sucrose hexadecanoate per ml was 81 or 79%, respectively, as evaluated by the reduction in optical density. During this treatment a great loss of viability occurred that preceded lysis. The results that were obtained suggest that autolysis is induced by these esters. The esters caused morphological changes in the cells, but a seeming adaptation of the cells to esters was seen.

RESUMEN

Analysis of a mutant in fam, a pleiotropic gene affecting cell division in Escherichia coli, revealed that this gene is probably identical to the heat shock regulatory gene htpR. The fam-715 mutant and different htpR mutants were found to share the following three characteristics: temperature-sensitive growth, faulty cell division, and inability to induce the normal cellular heat shock response. These defects were all corrected in fam and htpR mutants by complementation with plasmids carrying intact htpR+ or by recombination between these mutant alleles and a plasmid carrying only a portion of htpR. These results implicate the E. coli heat shock system in the regulation of cell division and raise the question of a similar role in other organisms.

Asunto(s)

División Celular , Genes Reguladores , Proteínas de Choque Térmico/genética , Escherichia coli/genética , Lipoproteínas/genética , Mutación , Recombinación Genética

RESUMEN

Heat treatment of a wild-type Escherichia coli strain at 55 degrees C in 50 mM Tris-hydrochloride buffer with or without 10 mM magnesium sulfate or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 8.0 caused an increase in cell surface hydrophobicity. By determining the location of n-hexadecane droplets attached to cells by phase-contrast microscopy, the septal and polar regions of heated cells appeared to become the most frequently hydrophobic. Some of the lipopolysaccharide molecules in the outer membrane were released from heated cells, and the cells became susceptible to the hydrolytic action of added phospholipase C. Heat-treated cells also became permeable to the hydrophobic dye crystal violet, which was added externally. The release of part of the outer membrane by heat treatment appeared to bring about the disorganization of the outer membrane structure and, as a consequence, to result in the partial disruption of the permeability barrier function of the outer membrane. Tris was found to enhance damage to the outer membrane by heat.

Asunto(s)

Permeabilidad de la Membrana Celular , Escherichia coli/metabolismo , Fosfatasa Alcalina/metabolismo , Membrana Celular/metabolismo , Violeta de Genciana/metabolismo , Calor , Lipopolisacáridos/metabolismo , Sulfato de Magnesio/metabolismo , Propiedades de Superficie , Fosfolipasas de Tipo C/metabolismo

RESUMEN

The addition of saturated C6, C8, C10, and C12 fatty acids appeared to lyse actively growing cells of Bacillus subtilis 168, as judged by a decrease in the optical density of the culture. Of these fatty acids, dodecanoic acid was the most effective, with 50% lysis occurring in about 30 min at a concentration of 0.5 mM. These conditions also decreased the amount of peptidoglycan estimated by the incorporated radioactivity of N-acetyl-D-[1-14C]glucosamine. At concentrations above 1 mM, however, bacterial lysis was not extensive. Dodecanoic acid did not affect autolysis of the cell wall. The lytic action of dodecanoic acid was greatly diminished in cells in which protein synthesis was inhibited and in an autolytic enzyme-deficient mutant. The results suggest that fatty acid-induced lysis of B. subtilis 168 is due to the induction of autolysis by an autolytic enzyme rather than massive solubilization of the cell membrane by the detergent-like action of the fatty acids.

Asunto(s)

Amidohidrolasas/fisiología , Bacillus subtilis/efectos de los fármacos , Bacteriólisis/efectos de los fármacos , Ácidos Grasos/farmacología , N-Acetil Muramoil-L-Alanina Amidasa/fisiología , Pared Celular/metabolismo , Concentración de Iones de Hidrógeno , Octoxinol , Polietilenglicoles/farmacología